3. Biochemical mechanisms of insecticidal efficacy

Transcription

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Investigation of Metabolic-Stress Disinfection and
Disinfestation (MSDD) as An Alternative to Methyl
Bromide for Disinfestation
Felix Zulhendri
A thesis submitted in fulfilment of the requirements for the degree
of Doctor of Philosophy in Food Science,
School of Chemical Sciences,
The University of Auckland, 2012.
Acknowledgements
I would like to thank my supervisors Dr Allan Woolf and Lisa Jamieson of Plant and
Food Research and Prof Conrad Perera and Dr Siew-Young Quek of University of Auckland.
This work was supported by the University of Auckland Doctoral Scholarship and NZ
Foundation for Research, Science and Technology contract no. C06X0709.
I wish to specifically mention a couple of Allan’s wonderful ideas. Firstly, the
‘weekly meeting’ which was brilliant (and with Lisa’s presence in the meeting  never a dull
moment)! And I strongly recommend it to everyone who wishes to pursue a PhD; ask for a
weekly meeting with your supervisors. It definitely kept me on track. Secondly, Allan’s
insistence on getting as many people with different expertise as possible on board has been a
highlight for me. That’s what science is all about. And to Conrad, thank you for pushing me
to write, write and write from the start.
I also like to thank all the mentors that I met along the way. I would definitely not be
able to complete this work without your help and guidance. To John Curtain and Kevin
Reade, thank you for helping me construct the prototype, Rod McDonald, for engineering
advice and to Di Brewster, Janine Cooney, Dwayne Jensen and William Laing, for
introducing me to the amazing proteomics. To David Billing, Anne White, Jason Johnston
and Ringo Feng, for the invaluable advice on GC, postharvest handling and storage. To
Robert Simpson and Dave Greenwood, for the advice on entomology and biochemistry. To
Tim Holmes, for those sharp, focused and free-of-distortion photos. And to the statistics
whizzes, Nihal de Silva and Patrick Connolly.
Lastly, I wish to extend my appreciation to my wife, Erica, my parents, Ma and Pa,
my evil twin, Feli and my sister, Felicia, for their unconditional support.
This thesis is dedicated to my awesome and beloved grandfather, Akong (RIP).
i
Abstract
Metabolic-stress disinfection and disinfestation (MSDD) is a Methyl Bromide (MeBr)
alternative disinfestation technology that utilises the combination of pressure change,
hypercarbia, hypoxia and ethanol treatments. The thesis describes the investigation of the
insecticidal efficacy of MSDD against various insect pests; longtailed mealybug
(Pseudococcus longispinus), 5th instar light brown apple moth (LBAM, Epiphyas postvittana)
larvae and 5th instar codling moth larvae (Cydia pomonella). MSDD consists of 2 phases,
namely a physical phase and followed by a chemical phase. The physical phase is
characterised by cycles of pressure changes (90–110 kPa) carried out by drawing air from the
treatment chamber and replacing it with ballast gases (CO2 or N2). The physical phase usually
lasts for 30 min. At the end of the physical phase, the chemical phase is initiated by drawing
the pressure down to 10 kPa and ethanol is introduced into the chamber (held for 60 min).
This 90 min MSDD treatment protocol was shown to be effective in controlling surface pests.
Further investigation showed parameters associated to both the physical phase (the
length of the physical phase) and the chemical phase (the concentration of ethanol, the length
of chemical phase and the pressure of chemical phase) were required to ensure high mortality
of pests. The efficacy of MSDD was limited to surface pests. Inoculation of 5th instar codling
moth larvae into the apples severely limited the capacity of MSDD to disinfest. Low
mortality was shown to be correlated with the inability of ethanol to penetrate into apple
flesh. Ethanol only penetrated 1 cm (from the skin) into the apple flesh.
Investigation into the protein changes in the haemolymph of LBAM larvae revealed
that the insecticidal efficacy of MSDD is strongly linked to the chemical phase and its ability
to disrupt melanisation regulation. MSDD prevented the up-regulation of alaserpin and
induced the over-expression of serine protease. Serpin family has been shown to negatively
regulate melanisation, whereas serine protease has been shown to promote melanisation. By
disrupting this regulation, MSDD induced excessive melanisation in the larvae which was
evident in the physical appearance of the treated larvae. Melanisation is an innate immune
response against pathogens in most arthropods. However, excessive melanisation has been
shown to be cytotoxic to insects.
ii
Research into the effect of MSDD on fruit physiology and quality showed that MSDD
had adverse effect on ‘Hayward’ and ‘Hort 16A’ kiwifruit. The most significant result was
the increase in rot incidence in both cultivars. MSDD also resulted in disorders such as flesh
breakdown and uneven ripening in the ‘Hort 16A’ fruit. Ripening (and softening) was also
accelerated in the non-cool stored ‘Hayward’ kiwifruit. Conversely, MSDD treatments did
not have noticeable effect on the physiology and quality of ‘Cripps Pink’ apple and ‘Hass’
avocado. Ripening physiology and quality parameters, such as rot incidence, internal
browning (apple) and flesh greying (avocado) were not affected by MSDD treatments.
Packaging materials restricted the efficacy of MSDD. Materials with high ethanol
sorption such as cardboard (kiwifruit boxes) reduced the mortality of insects treated with
MSDD significantly. Exposure to ethanol was also demonstrated to be a critical factor to
ensure high mortality. ‘Bulk’ treatment using a harvest crate showed that MSDD only
managed to disinfest the first layer of fruit. Mortality steeply decreased in the subsequent
layers of fruit. Therefore, a single layer fruit tray was needed to ensure high mortality.
In conclusion, MSDD treatments were shown to be effective in controlling insect
pests. Both the physical and chemical phases were important in achieving high insect
mortality. The MSDD mode of action is proposed to be involving the disruption of the
melanisation regulation in the form of up-regulation and down-regulation of serine protease
and its inhibitor (serpin), respectively. The efficacy of MSDD was limited to surface pests,
non-absorbing and single layered packaging materials. ‘Cripps Pink’ apple and ‘Hass’
avocado were MSDD tolerant. Conversely, ‘Hayward’ and ‘Hort 16A’ were susceptible to
over- production of ethanol and flesh breakdown disorder in response to MSDD treatments,
respectively.
iii
Table of contents
Acknowledgements
I
Abstract
II
List of tables
VII
List of figures
IX
List of abbreviations
XIV
1. Introduction
1
1.1 Background information
1.2 Literature review
1.2.1 Effects of vacuum (low pressure) and controlled atmosphere on
insect mortality
1.2.2 Effect of ethanol on insect mortality
1.2.3 The insecticidal efficacy of metabolic-stress disinfestation and
disinfection (MSDD)
1.2.4 Uncovering the mechanism of MSDD using proteomics tools;
2-D difference gel electrophoresis (2-DIGE) and mass spectrometry
1.2.4.1 Two-Dimensional differential in-gel electrophoresis (2-DIGE)
1.2.4.2 Identification of protein of interest using mass spectrometry (MS)
1.2.5 The effect of physical and chemical components of MSDD treatments
on fruit physiology and quality
1.2.5.1 The effect of hypobaric storage and modified atmosphere/
controlled atmosphere on fruit physiology and quality
1.2.5.2 The effect of ethanol treatment on fruit physiology and quality
1.3 Aims of the thesis
1
3
3
8
10
13
15
19
22
22
26
31
2. Insecticidal efficacy of MSDD against longtailed mealybug
(Pseudococcus longispinus)
32
2.1 Overview
2.2 Materials and method
2.2.1 The construction of Plant & Food Research MSDD prototype
2.2.2 MSDD treatments
2.2.3 Longtailed mealybug (Pseudococcus longispinus)
2.2.4 Gas concentration measurement
2.2.5 Statistical analysis
2.3 Results
2.4 Discussion
32
33
33
35
36
37
37
38
43
3. Biochemical mechanisms of insecticidal efficacy of MSDD
45
3.1 Overview
3.2.1 Chemicals
45
45
45
iv
3.2.2 Lightbrown apple moth (Epiphyas postvittana) haemolymph protein
extraction and preparation for labelling
3.2.3 Two-DIGE for proteins labelled with CyDyes
3.2.4 Isoelectric focusing (IEF) (first dimension)
3.2.5 SDS PAGE (second dimension) and gel scanning
3.2.6 Image analysis using Delta 2D (Decodon)
3.2.7 In-gel trypsin digestion of identified proteins for ESI-MS/MS
3.2.8 ESI-MS/MS analysis of peptides
3.2.9 Database Searching and Data Interpretation
3.3 Results
3.3.1 Insect mortality and gross physical changes
3.3.2 Differential expression of proteins between the larvae treated with
different treatments
3.4 Discussion
3.4.1 MSDD disrupts melanization pathway
3.4.2 Other proteins affected by different phases of MSDD treatments
47
50
51
51
52
54
54
55
55
55
56
65
65
66
4. The effect of MSDD on fruit physiology and quality
69
4.1 Overview
4.2.1 ‘Hass’ avocado (Persea Americana)
4.2.2 ‘Hayward’ kiwifruit (Actinidia deliciosa) and ‘Hort 16A’ kiwifruit
(Actinidia chinensis)
4.2.3 ‘Cripps Pink’ apple (Malus domestica)
4.2.4 Respiration rate (apple, kiwifruit and avocado)
4.2.5 Ethylene production measurement (kiwifruit and avocado)
4.2.6 Internal ethylene concentration measurement (apple)
4.2.7 Ethanol and acetaldehyde measurement
4.2.8 Statistical analyses
4.3 Results
4.3.1 Avocado
4.3.2 Kiwifruit
4.3.2.1 First season
4.3.2.2 Second season
4.3.3 ‘Cripps Pink’ apple
4.4 Discussion
4.4.1 ‘Hass’ avocado
4.4.2 ‘Hayward’ and ‘Hort 16A’ kiwifruit
4.4.3 ‘Cripps Pink’ Apple
4.5 Conclusions
69
69
69
5. Investigation of the significance and optimisation of MetabolicStress Disinfection and Disinfestation (MSDD) parameters to
control longtailed mealybug Pseudococcus longispinus
5.1 Overview
5.2.1 Insects
5.2.3 Experimental design
70
72
73
73
74
74
74
75
75
80
80
86
95
98
98
99
101
101
102
102
102
102
103
103
v
5.2.4 Statistical models of the effect of varying parameters of MSDD on insect
mortality
5.3 Results
5.3.1 Insect mortality
5.3.2 Modelling of the effect of varying MSDD parameters on insect mortality
5.4. Discussion
6. Technological issues associated with MSDD: I. Internal pest
6.1 Overview
6.2.1 ‘Braeburn’ apple (Malus domestica) and ‘Pacific Rose’ apple
(Malus domestica) for 5th instar codling moth (Cydia pomonella) larvae
inoculation
6.2.2 Codling moth (Cydia pomonella) 5th instar larvae
6.2.3 Investigation of ethanol penetration into apple
6.3 Results
6.4 Discussion
7. Technological issues associated with MSDD: II. The effect of
loading on the efficacy of MSDD
7.1 Overview
7.2.1 Investigation of the effect of different types of packaging on longtailed
mealybug (Pseudococcus longispinus) mortality
7.2.2 The effect of loading type on ethanol concentration
7.3 Results
7.4 Discussion
8. General discussion, future work and conclusions
8.1 The efficacy of MSDD as a disinfestation treatment
8.2 The biochemical mechanisms of the insecticidal efficacy of MSDD
8.3 The effect of MSDD on fruit physiology and quality
8.4 Future work
8.5 Conclusions
104
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134
138
140
140
144
145
146
149
9. References
150
10. Appendix
161
10.1 A worked example of calculating ethanol condensation point.
10.2 Publications and conference proceedings resulting from this thesis
vi
161
161
List of tables
Table 1-1 Pressure units and the associated conditions.
3
Table 1-2 Typical experimental designs of 2-DIGE using 3 cyanine dyes.
16
Table 2-1 Concentration of ethanol introduced calculated in chamber as volatile and
mist/liquid. Based on the temperature profile, the maximum concentration of ethanol
vapour was 84.5 ±9.5 mg. L-1. The remaining ethanol existed as liquid or mist that
condensed on the product surface.
39
Table 2-2 Mean lethal doses (95% CI) for 50 and 90% mortality (LD50s and LD99s)
derived from dose-mortality data from three life stages of longtailed mealybug
(Pseudococcus longispinus) exposed to MSDD treatment with various concentrations of
ethanol vapour and mist (see Table 2-1 for details).
40
Table 3-1 Programme used to run the first dimension.
51
Table 3-2 Mean percentage mortality of 5th instar light brown apple moth (Epiphyas
postvittana) larvae 3 d after treatments (n=3).
56
Table 3-3 Proteins that were differentially expressed in the haemolymph of larvae
treated with PP, CP and full MSDD treatments when compared to controls.
61
Table 4-1 Experimental design for first season kiwifruit.
71
Table 4-2 Experimental design for second season kiwifruit.
72
Table 4-3 Ripe-fruit quality of early (November 2009) and late season (February 2010)
‘Hass’ avocado exposed to 90 min MSDD treatments with 371 mg. L-1 ethanol and in
air (untreated controls) (n=3).
79
Table 4-4 Kiwifruit quality exposed to 90 min MSDD treatments with 371 mg. L-1
ethanol and in air (untreated control), after being stored at 0°C and 0.5°C (16 weeks)
for ‘Hayward’ and ‘Hort 16A’ kiwifruit, respectively (n=3).
81
Table 4-5 Kiwifruit quality exposed to 90 min MSDD treatments with 1,113 mg. L-1
ethanol and in air (untreated control), after being stored at 0°C and 0.5°C
(0, 4 and 8 weeks) for ‘Hayward’ and ‘Hort 16A’ kiwifruit, respectively, n=3.
88
Table 4-6 ‘Cripps Pink’ apple quality exposed to 90 min MSDD treatments with
371 mg. L-1 ethanol and in air (untreated control), after being stored for 16 weeks at
0.5°C.
97
Table 4-7 ‘Cripps Pink’ apple quality exposed to 90 min MSDD treatments with
371 mg. L-1 ethanol and in air (untreated control) and after being stored for
16 weeks at 0.5°C. All measurements were taken after 7 d at 20°C.
97
vii
Table 5-1 Concentration of ethanol introduced into the chamber calculated as volatile
and mist/liquid. The maximum concentration of ethanol vapour was 137.7 ±15.3 mg. L-1
(at 22.5 ±2.5°C). The remaining ethanol existed as liquid or mist that condensed on the
product surface (n=3).
108
Table 5-2 The effect of CO2 or N2 during the physical phase of MSDD on the
percentage mortality (±SE) of longtailed mealybug (Pseudococcus longispinus).
Each treatment contained 300 insects of each lifestage, n=3.
109
Table 5-3 The effect of increasing length of physical phase of MSDD on the percentage
mortality (±SE) of longtailed mealybug (Pseudococcus longispinus), n=3.
110
Table 5-4 The effect of increasing length of chemical phase of MSDD on the percentage
mortality (±SE) of longtailed mealybug (Pseudococcus longispinus), n=3.
111
Table 5-5 The effect of increasing pressure during chemical phase (45 and 60 min)
of MSDD on the percentage mortality (±SE) of longtailed mealybug (Pseudococcus
longispinus), n=3.
112
Table 5-6 The parameter estimates obtained from logistical regression models and the
associated goodness-of-fit statistics and percent concordant of the predicted and
observed mortalities.
113
Table 5-7 The predicted optimal parameters of MSDD to cause 99% mortality of
longtailed mealybug (Pseudococcus longispinus) adults, 2nd/3rd instars and crawlers and
comparative validation experimental percentage mortalities (±SE).
115
Table 6-1 Experimental design for comparing the mortality of 5th instar codling moth
(Cydia pomonella) larvae in open and closed calyx apples.
126
Table 6-2 Control mortality of 5th instar codling moth larvae, n=3.
127
Table 7-1 The effect of kiwifruit boxes and placement of longtailed mealybug
(Pseudococcus longispinus) infested potatoes (n=3).
135
Table 7-2 Mortality data resulted from MSDD treatments using plastic-coated wire and
harvest crate (n=3).
136
Table 7-3 Parameter estimates, goodness-of-fit statistics and percent concordant of
logistical models in the harvest crate experiment.
viii
136
List of figures
Figure 1-1 LT99 of two different insects; Rhyzopertha dominica and Cadra cautella, in
response to different pressure and temperature conditions (Mbata et al., 2004).
5
Figure 1-2 A. 12 L MSDD Research Prototype, B. 250 L MSDD Research Prototype
(Lagunas-Solar et al., 2006).
11
Figure 1-3 A cascade of events from DNA to protein and its associated fields of study,
namely genomics (DNA), transcriptomics (mRNA) and proteomics (proteins)
(modified from www.nobelprize.org).
14
Figure 1-4 Chemical structure of cyanine dyes (Tonge et al., 2001).
15
Figure 1-5 The principle of 2-DIGE from protein mixture labelling for identification of
protein spots of interest (modified from Tonge et al., 2001).
17
Figure 1-6 The diagram illustrates the importance of Cy2 (internal standard) in
determining ‘real’ difference in quantifying protein spots. The top diagram shows the
difference among the four samples without normalising the volume against the internal
standard (increased expression level in gels 3 and 4). The samples that are quantified
against the internal standard produced a significantly different results (reduced
expression level in gel 3) (Marouga et al., 2005).
18
Figure 1-7 A false colour image depicting different expression level of proteins in a
mixture of 3 samples; red, green and blue spots represent samples that were labelled
with Cy5, Cy3 and Cy2, respectively. White spots represent proteins that are expressed
in all 3 samples. The intensity of the spots correlates with the expression level of
proteins.
19
Figure 1-8 The schematic diagram of ESI-MS and the desolvation process of charged
droplets to quasi-molecular ions (modified from Fenn et al., 1989).
20
Figure 1-9 The schematic diagram of MALDI-TOF (Patterson and Aebersold, 1996).
21
Figure 1-10 Hypobaric storage prevents climacteric respiration in ‘Golden Delicious’
apples (Bangerth, 1984).
24
Figure 1-11 Delaying the establishment of CA can reduce the CO2 related injury in
‘Fuji’ apples (Argenta et al., 2000).
25
Figure 1-12 Ethylene biosynthesis and methionine cycles. KMB:
2-keto-4-methylthiobutyric acid, Met: methionine, AdoMet: S-adenosyl-L-methionine,
ACC: 1-aminocyclopropane-1-carboxylic acid, MTA: 5’-methyladenosine, MTR:
5’methylthioribose, MTR-1-P: 5’-methylthioribose-1-phosphate
(Bleecker and Kende, 2000).
27
Figure 1-13 The proposed mechanisms of ethanol and acetaldehyde in inhibiting
ethylene biosynthesis and fruit ripening (modified from Podd and Van Staden, 1998).
28
ix
Figure 1-14 The relationships between concentrations of α-farnesene, conjugated trienes,
ethanol content and its consequential suppression of superficial scald in ‘Granny Smith’
apples (modified from Ghahramani and Scott, 1998b).
30
Figure 2-1 Adult longtailed mealybug.
32
Figure 2-2 Plant & Food Research MSDD prototype. A. MSDD prototype, B. Volatile
delivery system, C. Fan (1) and temperature sensors (2), D. Temperature control,
E. Vacuum pump. Solid arrow indicates parts that are visible in diagram A, striped
arrows indicate the positions of the parts that are not observable in diagram A.
34
Figure 2-3 MSDD operational protocol consists of the physical phase of 30 min
followed by the chemical phase of 60 min.
35
Figure 2-4 Mean (±SE) temperature of atmosphere, core and skin temperature of potato
during simulated runs of MSDD treatment to calculate the concentration of ethanol, n=3. 41
Figure 2-5 Oxygen and CO2 partial pressure (kPa) (mean ±SE) during a typical MSDD
run during the 30-min physical phase, n=3.
41
Figure 2-6 Percentage mortality of three different life stages of longtailed mealybug
(Pseudococcus longispinus) (mean ±SE, n = 3, each n=150 insects). A. Ambient control
(columns), B. Chemical-only control (125 mg. L-1 of ethanol treatment at 10 kPa for
90 min) (columns). MSDD treatments were carried out with increasing concentrations of
ethanol in the chemical (second) phase (lines). Based on the temperature data, maximum
ethanol vapour concentration was calculated to be 84.5 ±9.5 mg. L-1. Above that
concentration, ethanol existed as vapour and liquid which condensed on the product
surface. The bar represents standard error of ethanol condensation point based on the
standard error of temperature measured during MSDD treatments.
42
Figure 3-1 Three different treatments that were used to treating 5th instar lightbrown
apple moth (Epiphyas postvittana) larvae. Physical phase-only treatment (PP; A),
chemical phase-only treatment (CP; B) and full MSDD treatment (MSDD; C).
49
Figure 3-2 The working steps of CyDyes labelling from labelling the samples to pooled
samples. Each pooled sample contained 100 µg of total protein; 33.3 µg of internal
standard, 33.3 µg of control and 33.3 µg of treated sample. I: Internal standard
(contains equal proportion of protein from PP only, CP only, MSDD and the 3 controls).
P: PP treatment, C: CP treatment and M: MSDD treatment.
50
Figure 3-3 The flow chart of protein image analysis by Delta 2D (A–D); A. Linking the
groups of images, B. Warping and cleaning the image by eliminating artefact spots, C.
Statistical analyses of the resulting spots, D. Identification of significant spots prior to
LC-MS analysis.
53
x
Figure 3-4 Fifth instar lightbrown apple moth (Epiphyas postvittana) larvae treated with
3 different treatments at 0 h (A), 24 h (B), 72 h (C) and 96 h (D). Melanisation started
occurring after 24 h. The control and PP treated larvae started to pupate at 72 d and 96 h,
respectively. Melanisation was more pronounced in MSDD treated larvae than CP
treated larvae.
57
Figure 3-5 A typical gel scanning result (false colour overlay) from the experiments
using CyDyes. Cy3 (control), Cy5 (treatment-MSDD) and Cy2 (pooled sample) appear
green, red and blue, respectively. White spots represent proteins that are equally
expressed in all 3 samples. The intensity of the spots correlates with the expression
level of proteins.
59
Figure 3-6 A typical gel from the experiments with pI and molecular weight annotation
showing protein spots of interest across different treatments.
60
Figure 3-7 The melanisation pathway of arthropod. Red and green arrows indicate the
downregulation and upregulation of serpin and serine protease by MSDD, respectively,
which consequently increases the melanin synthesis (black arrow)
(modified from Cerenius and Söderhäll, 2004).
68
Figure 4-1 Carbon dioxide production (mg. kg-1. s-1) (mean ±SE) of early (A and B) and
late (C and D) season ‘Hass’ avocado from three orchards, untreated (A and C) and
MSDD treated (B and D), held at 20°C, n=3.
76
Figure 4-2 Ethylene production (µg. kg-1. s-1) (mean ±SE) of early (A and B) and late
(C and D) season ‘Hass’ avocado from three orchards, untreated (A and C) and MSDD
treated (B and D), held at 20°C, n=3.
77
Figure 4-3 Cumulative weight loss (±SE) of ‘Hass’ avocado, n=3.
78
Figure 4-4 Physical appearance of ‘Hass’ avocado straight after MSDD treatment (left)
v. untreated control (right). Browning of the peduncle was observed on the MSDD
treated fruit (arrow).
78
Figure 4-5 A. Flesh breakdown (water-soaked appearance) in MSDD treated
‘Hort 16 A’ kiwifruit. B. Normal fleshed ‘Hort 16A’ kiwifruit.
82
Figure 4-6 Respiration rate (mg. kg-1. s-1) (mean ±SE) (A and B) and ethylene production
(ng. kg-1. s-1) (C and D) of first season ‘Hayward’and ‘Hort 16A’ kiwifruit,
non-coolstored (A and C) and coolstored for 16 weeks (B and D).
83
Figure 4-7 Ethanol (A and B) and acetaldehyde (C and D) (ng. kg-1. s-1) (mean ±SE)
production of first season ‘Hayward’ and ‘Hort 16A’ kiwifruit, non-coolstored (A and C)
and coolstored for 16 weeks (B and D).
84
Figure 4-8 Acoustic firmness (Hz2 kg2/3) (mean ±SE) of first season ‘Hayward’ and
‘Hort 16A’ kiwifruit. Firmness during coolstorage (A and C) and firmness of
non-coolstored fruit (B and D).
xi
85
Figure 4-9 Uneven ripening observed in ‘Hort 16A’ kiwifruit; characterized by white
flesh appearance.
89
Figure 4-10 Respiration rate (mg. kg-1. s-1) (mean ±SE) of second season ‘Hayward’ and
‘Hort 16A’ kiwifruit, non-coolstored (A) and coolstored for 4 and 8 weeks (B and C), n=3.
90
Figure 4-11 Ethylene production (ng. kg-1. s-1) (mean ±SE) of second season ‘Hayward’
and‘Hort 16A’ kiwifruit, non-coolstored (A) and coolstored for 4 and 8 weeks (B and C),
n=3.
91
Figure 4-12 Ethanol production (ng. kg-1. s-1) (mean ±SE) of second season ‘Hayward’ and
92
Figure 4-13 Acetaldehyde production (ng. kg-1. s-1) (mean ±SE) of second season ‘Hayward’
and ‘Hort 16A’ kiwifruit, non-coolstored (A) and coolstored for 4 and 8 weeks (B and C),
n=3.
93
Figure 4-14 Acoustic firmness (106 Hz2 kg2/3) (mean ±SE) of second season ‘Hayward’
n=3.
94
Figure 4-15 CO2 production (mg. kg-1. s-1) (mean ±SE) (A and B) and internal ethylene
concentration (µg. L-1) (C and D) of ‘Cripps Pink’ apple, non-coolstored (A and C) and
coolstored (B and D) at 0.5°C for 16 weeks, n=3
96
Figure 4-16 Ethanol (A) and acetaldehyde (B) production (ng. kg-1.s-1) (mean ±SE) of noncoolstored ‘Cripps Pink’, n=3.
96
Figure 5-1 Mortality of 3 life stages of longtailed mealybug (Pseudococcus longispinus)
treated with a constant 30 min of physical phase using CO2 v. N2 with increasing
concentration of ethanol at 10 kPa for 60 min (A. Adult, B. 2nd/3rd instars. C. Crawler).
The line graphs are mortality models generated using binary logistical regression.
116
treated with an increasing physical phase with a constant chemical phase at 125 mg. L-1
at 10 kPa for 60 min (A. Adult, B. 2nd/3rd instars. C. Crawler).
117
treated with a constant 30 min of physical phase and varying conditions of chemical
phase; 125 mg. L-1 at 10 kPa for increasing length of chemical phase (A–C), increasing
concentration of ethanol at 10 kPa for 60 min (D–F) and 125 mg. L-1 for 60 min at
increasing pressure (G–I).
118
Figure 6-1 Damage by codling moth (Cydia pomonella) larvae
(courtesy of Dave Rogers, Plant and Food Research).
122
Figure 6-2 Closed and open calyx apples (Braeburn and Pacific Rose). Arrows indicate
the morphology of the calyx of both apples.
123
xii
Figure 6-3 Experimental work for investigating ethanol penetration into apple flesh.
A. Cork borer was used to obtain flesh sample. B. Flesh sample was divided into 3
one-cm sections (arrows indicate where apple flesh was divided into 3 one-cm sections).
C. A section was put into a rubber-capped 60 mL syringe. D. A low pressure condition
was created in the syringe to facilitate volatile extraction from the sample. E. A 1-mL gas
sample was obtained from the syringe.
125
Figure 6-4 The mortality of 5th instar codling moth (Cydia pomonella) larvae inoculated
into 2 different calyx types of apples, treated with ranges of ethanol concentration
(mg. L-1) of chemical phase of MSDD treatments, n=3.
128
Figure 6-5 The ethanol (top) and acetaldehyde (bottom) concentration of tissue at
different distances from the skin.
128
Figure 7-1 Experimental set up to investigate the effect of kiwifruit boxes on longtailed
mealybug (Pseudococcus longispinus) mortality. A. Potatoes in the kiwifruit boxes, B.
Kiwifruit and potatoes were lined with polyliner, C. Potatoes in the unlined box,
D. MSDD treatment set-up.
132
Figure 7-2 Plastic-coated wire experimental set up.
132
Figure 7-3 Experimental set up using the harvest crate. Top: potatoes were placed
randomly at different layers of kiwifruit. Bottom: MSDD treatment using the
harvest crate.
133
Figure 7-4 Logistical models and proportion mortality of longtailed mealybug
(Pseudococcus longispinus) in the harvest crate experiment.
137
Figure 7-5 Ethanol concentration (mg. L-1) (mean ±SE), during chemical phase with 371
mg. L-1, inside the MSDD chamber with various types of loading, n=3.
137
Figure 8-1 The conceptual designs of commercial scale in a commercial setting
143
Figure 8-2 Suggested experimental design to investigate the effect of different pressure
oscillation ranges on insect mortality; A. 90–110 kPa (used in this work), B. 10–110
(Lagunas-Solar et al., 2006), and C. 50–110 kPa.
147
xiii
List of abbreviations
ACC
ACP
ADH
AdoMet
AIC
ANOVA
ApoLp
ATP
BLAST
BSA
CA
CHAPS
CLL
CP
Cydyes
d
2-DIGE
DMF
DNA
DTT
EMA
ESI
FID
g
(x) g
GABA
GC
GLM
GLMM
GRAS
h
HCl
Hz
ID
IEF
IPG
kg
L
LBAM
LC
LD
LT
MA
MALDI
1-MCP
MDLC
1-aminocyclopropane-1-carboxylic acid
Anaerobic compensation point
Alcohol dehydrogenase
S-adenosyl-L-methionine
Akaike information criteria
Analysis of variance
Apolipophorine-like protein
Adenosine triphosphate
Basic Local Alignment Search Tool
Bovine serum albumin
Controlled atmosphere
3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
Complementary log-log
Chemical phase
Cyanine dyes
day
Two-dimensional in-gel electrophoresis
Dimethylformamide
Deoxyribonucleic acid
Dithiothreitol
Employers and Manufactures Association
Electrospray ionization
Flame ionization detector
gram
gravitational constant
γ-amino butyric acid
Gas chromatography
Generalized linear model
Generalized linear mixed model
Generally recognized as safe
hour
Hydrochloric acid
Hertz
Internal diameter
Isoelectric focusing
Immobilised pH gradient
kilogram
litre
Light brown apple moth
Liquid chromatography
Lethal dose
Lethal temperature
Modified atmosphere
Matrix-assisted laser desorption/ionization
1-methyl cyclopropene
Multidimensional LC system
xiv
MeBr
mg
µg
MHO
min
mRNA
MS
MSDD
MTA
MTR
MTR-1-P
NCBI
ng
PAGE
PFR
PP
RH
RPC
s
SDS
SH
SSC
TEMED
TOF
ULO
Methyl Bromide
Milligram
Microgram
6-methyl-5-hepten-2-one
minute
messenger Ribonucleic acid
Mass spectrometry
Metabolic-stress disinfection and disinfestation
5’-methyladenosine
5’methylthioribose
5’-methylthioribose-1-phosphate.
National Center for Biotechnology Information
nanogram
Polyacrylamide gel electrophoresis
Plant and Food Research
Physical phase
Relative humidity
Reverse phase chromatography
second
Sodium dodecyl sulphate
Sulfhydryl
Soluble solid content
Tetramethylethylenediamine
Time of flight
Ultra low oxygen
xv
1. Introduction
1.1 Background information
Horticultural produce is one of New Zealand’s main export earnings, which was
worth NZ$3.3 billion in 2009 (Anonymous, 2010a). Accidental introduction of exotic pests
and diseases have been known to inflict major (and sometimes irreversible) damage on
horticultural production in many countries. In order to maintain and improve horticultural
trade, effective pest and disease control procedures must be put in place. In USA alone, it was
estimated that introduced exotic pests cause US $100 billion worth of damage annually
(Pimentel et al., 2005). Exotic pests have also been recognized as one of the major causes in
the decline of native biodiversity in many places due to lack of natural enemies (Jenkins,
1996). The most significant and recent case in NZ to date is the discovery of kiwifruit
bacterial canker disease caused by Pseudomonas syringae pv. actinidiae, threatening the NZ
$1 billion industry (Anonymous, 2010b; Rees-George et al., 2010). This disease was reported
to have nearly eliminated NZ-owned ‘Hort16A’ kiwifruit crop in Italy (NZ-Herald, 2011).
Therefore, considering the environmental and economic impact of biosecurity breach, many
countries (including NZ trade partners) impose phytosanitary requirements on imported
produce.
Postharvest disinfestation treatments are one of the most important aspects in this
regard. The most common means of postharvest disinfestation has been Methyl Bromide
(MeBr) which has been used for fumigating fresh produce for over 70 years due to its ease of
application (Taylor, 1994). MeBr is an excellent fumigant with efficacy against a wide range
of pests such as insects, nematodes, weeds and soil-borne pathogens including fungi, virus
and bacteria (Taylor, 1994).
The mode of action of MeBr is attributed to its capacity of methylating sulfhydryl
(SH) groups of proteins. Lewis (1948) demonstrated that MeBr reduced the activity of SHcontaining enzymes. It was shown that the exposure, to 400–500 mg. L-1 for 1–2 h, of urease,
succinic dehydrogenase and papain resulted in 12–50%, 42–81% and 67–100% inhibition of
their activities, respectively. Methylation of free SH groups in insects and glutathione in
1
mammalian cells by MeBr has also been demonstrated (Winteringham et al., 1958;
Nishimura et al., 1980). Inhibition of SH groups of proteins has profound deleterious effects
on organisms, especially the disruption of oxidative metabolism such as glycolysis and
pyruvate oxidation which eventually results in depletion of energy molecules such as
adenosine triphosphate (ATP) and phosphoglycerate (Price, 1985).
The effects of MeBr on fruit physiology and quality vary across different fruit.
Treatment of 32–64 g. m-3 for 2–4 h induced internal disorders of apples of various cultivars
with 100% ‘Spartan’ apples affected (compared to 33% of untreated ‘Spartan’ apples) after
45 d of storage at 0°C in air (Meheriuk et al., 1990). Similar results were recorded in grapes
(Leesch et al., 2008). There was also notable reduction in flavour score of MeBr treated
apples. MeBr fumigation to control codling moth (Cydia pomonella) was shown to have no
negative effect on nectarines. However, the ripening of ’May Grand’ and ‘Firebrite’ cultivars
seemed to be delayed slightly (Hartsell et al., 1992). The quality of sweet cherries were also
shown to be unaffected by MeBr fumigation (Jessup, 1988).
Due to its efficacy, MeBr has been used for a wide range of applications such as soil
fumigation, fumigation of durable and perishable commodities such as fruit and vegetables
and quarantine treatments (Taylor, 1994). However, MeBr has significant ozone-depleting
properties and therefore, industrialized countries, that were assignees to the Montreal
protocol, have been slowly and consistently phasing out its use since 1st January 2005
(Taylor, 1994; Gullino et al., 2005). The cost of using MeBr as a fumigant has also increased
(Wang et al., 2006). It should be noted that MeBr is still permitted for quarantine purposes.
There have been concerted efforts in this area by researchers across the globe which
focuses on clean and green technologies with one of the most critical criteria being reduction
of toxic chemicals usage. Physical treatments such as removal, heat and cold treatments,
vacuum and modified (controlled) atmosphere fit perfectly with the notion. “Soft” chemical
treatments using Generally Recognized as Safe (GRAS)/ food additive chemicals such as
ethanol have also been examined.
2
1.2 Literature review
1.2.1 Effects of vacuum (low pressure) and controlled atmosphere on insect
mortality
A vacuum or negative pressure that is relative to atmospheric pressure has been
demonstrated to have insecticidal efficacy. Insect mortality under vacuum is predominantly
caused by low oxygen partial pressure (Mbata et al., 2005). Dehydration caused by
elimination of water vapour is also noted as one of the effects (Finkelman et al., 2003).
Generally, the lower the pressure the higher the mortality recorded with few exceptions
(Calderon and Navarro, 1968). Calderon and Navarro (1968) tested the efficacy of pressures,
ranging from 10–160 mmHg for 24 h at 26°C and 70% relative humidity (RH), on several
insects; adult Sitophilus oryzae Linnaeus, adult Callosobruchus maculates Fabricius and
Trogoderma granarium Everts larvae. It was found that insect mortality did not always
correlate with the level of pressure. Mortality rate of S. oryzae was demonstrated to be a bellshaped curve as pressure increased from 10–160 mmHg. T. granarium larvae were the most
resistant as 100% mortality was not reached at any pressure tested. Table 1-1 provides three
pressure units and the associated physical conditions that are commonly used in this thesis.
Table 1-1 Pressure units and the associated conditions.
Physical conditions
Pressure units
kPa
atm
mmHg
Positive pressure
>101.3
>1.0
>760.0
Atmospheric
101.3
1.0
760.0
<101.3
<1.0
<760.0
pressure
Negative pressure
(vacuum)
The main disadvantage of using low pressure or vacuum treatment as a means of
disinfestation is the length of treatments. Temperature modification has been demonstrated to
reduce the time of low pressure treatment on grain and cocoa pests. Increased in temperature
is not always desirable, especially on fruit and vegetables. Finkelton et al. (2003; 2004) did
two sets of experiments demonstrating the effect of temperature on insecticidal efficacy at
similar pressure levels (50–55 mmHg). By increasing the temperature treatment from 18°C to
3
30°C, LT99 values of eggs, larvae, pupae and adults of Tribolium castaneum Herbst were
reduced significantly from 96, 37, 72 and 30 h to 22, 6.5, 13 and 7 h, respectively. Whereas
LT99 values of eggs, larvae, pupae and adults of Ephestia cautella Walker were reduced from
77, 37, 128 and 164 h to 45, 10, 6.6 and 6 h, respectively. Another example is demonstrated
by Mbata et al. (2005); they investigated the effect pressure of 32.5 mmHg on cowpea weevil
mortality at 20°C to 35°C. Figure 1-1 illustrates the effect of temperature on reduction of
LT99 of cowpea weevil at 32.5 mmHg.
The increase in temperature during vacuum treatment does not always result in
decrease in LT99 values. The type of insects also plays a significant role, particularly whether
they are cold or heat tolerant. For example, T. castaneum eggs were the most resistant at
15°C and 300 mmHg, whereas at 200 mmHg the eggs were the most resistant at 23°C (Mbata
et al., 2004). Figure 1-1 illustrates the resistance of eggs of two different insects at different
level of temperature and pressure; Rhyzopertha dominica Fabricius eggs generally became
more susceptible to low pressure treatments as temperature increases, except at 300 mmHg
where LT99 increases from 5°C to 15°C (Mbata et al., 2004). Cadra cautella Walker eggs,
however, had an increased tolerance as temperature increased from 5°C to 15°C at different
pressure treatments, with a significant increase at 300 mmHg.
4
Figure 1-1 LT99 of two different insects; Rhyzopertha dominica and Cadra cautella, in
response to different pressure and temperature conditions (Mbata et al., 2004).
Another method to achieve insect kill is by modifying the atmosphere by increasing
CO2 concentration (hypercarbia). Soderstrom et al. (1991) demonstrated that hypercarbia can
be utilised to control Cydia pomonella Linnaeus eggs. It was demonstrated that >20% CO2
was sufficient to reach LT95 well below 4 days, with 60%, 80% and 100% CO2 resulting in
LT95 of 1.4, 1.6 and 1.4 d, respectively. Held et al. (2001) demonstrated that exposure to 99%
CO2 for 12–18 h can result in complete mortality of Myzus persicae Sulzer, Bemisia sp.,
Tetranychus urticae Koch and Frankliniella occidentalis Pergande. Mortality of >90% of E.
cautella pupae can be reached by exposure to 21%, 51% and 88% of CO2 for 3 d at 20–22%
RH or 54–55% RH.
5
Insects generally respond to hypercarbia in a similar way to a low oxygen
environment; by reducing the rate of respiration and metabolism (Mitcham et al., 2006). Zhou
et al. (2000) noted that reduced metabolism in Platynota stultana Walsingham pupae exposed
to hypercarbia was observed even at 21% O2. At 5% CO2 and 21% O2, the percentage
decrease in the metabolic rate was 28%. By increasing the CO2 concentration to 20% and
79%, the metabolic rate was reduced by 59% and 79%, respectively. Leakage of body fluid of
P. stultana pupae was also observed in hypercarbia, suggesting that membrane systems of
pupae were affected by high CO2 level.
Controlled or modified atmosphere treatments are usually carried out by coupling
hypoxia and hypercarbia to increase the insecticidal efficacy. Hypoxia and hypercarbia
affects two different physiological functions of insects. Hypoxia affects metabolism of insects
by limiting the primary substrate of respiratory metabolism which is O2. Insects respond to
low O2 stress by reducing their metabolic rate; a mechanism called metabolic arrest.
Metabolic arrest allows the reduction of energy requirement works by lessening the pressure
to commence anaerobic metabolism and ATP production in insects. By doing so, insects
prevent the activation of glycolysis and consequently reduce the energy demand for cell
functions (Mitcham et al., 2006). A decrease in cell membrane permeability is also suggested
as one of the mechanisms used by insects. When O2 level is low and ATP production is
lessened, low membrane permeability helps maintain the concentration gradients of Na+ and
K+ across cell membrane and consequently reduces the pressure on ATP pumps (Hochachka,
1986). This mechanism cannot be maintained perpetually. There is a critical point when ATP
is needed to run primary cell functions; this is when hypoxic toxicity commences leading
eventually to insect death.
Insects also use the metabolic arrest mechanism to cope with hypercarbia.
Hypercarbia affects physiological functions of insects differently. Carbon dioxide toxicity
inhibits specific enzymes in oxidative phosphorylation, namely succinic dehydrogenase and
causes an accumulation of toxic waste such as lactate, pyruvate and succinic acid (Mitcham
et al., 2006). Under high CO2 environment, insects suffer a greater energy shortage.
Friedlander and Navarro (1979a) showed that ATP level and energy charge of E. cautella
pupae were lower at 80% and 90% CO2 than at 1% O2 after 24 h of treatment. Anaerobiosis
is induced at high CO2 concentration even with 20% oxygen present causing insect to be
prone to accumulation of toxic waste (Mitcham et al., 2006). High CO2 level also affects
6
membrane permeability which causes haemolymph leakage. It can be partly attributed to
reduced energy supply, but it also suggests that high CO2 might affect membrane systems of
insects (Zhou et al., 2001). High CO2 increases intercellular concentration of Ca2+ by
reducing the pH, consequently disrupting the cellular ionic balance (Hochachka, 1986;
Mitcham et al., 2006). Carbon dioxide has also been shown to keep insect spiracles open
which consequently increases insect dehydration (Burkett and Schneiderman, 1967).
Friedlander and Navarro (1979b) demonstrated that exposure to high CO2 concentration at
100% RH, 20% O2 and normal atmospheric pressure caused weight loss in E. cautella pupae.
At normal atmospheric condition (20% O2, 0% CO2) and 100% RH, the pupae lost 0.8–1.6%
whereas exposure to high CO2 (80%) at 20% RH caused 26–59% weight loss.
7
1.2.2 Effect of ethanol on insect mortality
Ethanol has been shown to have insecticidal effect. Ethanol immersion, at different
temperature and different length of immersion time, was shown to control two-spotted spider
mite (Tetranychus urticae Koch) (Dentener et al., 1998b). Two-spotted spider mite was
resistant to ethanol with the effective length of immersion time ranging from 600–1200 s
(100% v/v ethanol). Non-diapausing spider mite was more susceptible to ethanol immersion
at different temperature compared to diapausing mites. Dentener et al. (1998a) also
demonstrated that ethanol immersion could be utilised to control light brown apple moth
(LBAM) (Epiphyas postvittana Walker) larvae. Fifth instar LBAM larvae were shown to be
susceptible to immersion to various ethanol concentrations with varying degree of immersion
time. With 2–4 s immersion time, 5th instar LBAM larvae can be controlled with 73–74% v/v
ethanol.
Ethanol vapour is also effective in controlling insect pests. The length of treatment
time of ethanol vapour is predictably longer than ethanol immersion. Fifth instar LBAM
larvae can be controlled by exposure to ethanol vapour generated by 20% and 50% v/v
ethanol solution (Dentener et al., 1998a). A significantly longer exposure time was needed to
achieve high mortality (>95%); 48 h, compared to 2–4 s with immersion technique. The
efficacy of ethanol vapour was reduced in the presence of the commodity–apples (Dentener
et al., 2000). Dentener et al. (2000) showed that 20% v/v ethanol solution generated 3500 µL.
L-1 in the presence of apples compared to 10000 µL. L-1 without the presence of apples,
suggesting ethanol absorption by apples. Hence, the LT99 values were 93 and 27.9 h for
exposure to ethanol vapour with and without the presence of commodity (apples),
respectively. Increasing the base solution from 20% to 70% v/v ethanol solution also reduced
the LT99 significantly from 18.4 to 7.5 h, respectively (without commodity).
Jamieson et al. (2003) investigated the effect of ethanol vapour treatment, at ambient
(20°C) and cold temperature (0.5°C) derived from various concentration of ethanol solution
on 5th instar LBAM larvae inoculated on ‘Royal Gala’ apples. It was found that at ambient
(20°C), the most effective treatment was a 7 d exposure to ethanol vapour (2.44 x 107 mg. m3
) generated by 15% v/v ethanol solution which resulted in 87% mortality. At low
temperature (0°C), 12 d exposure to 53.30 x 105 mg. m-3 ethanol vapour generated by 40%
v/v ethanol solution resulted in complete kill of 5th instar LBAM larvae. The ethanol vapour
8
concentration at 0.5°C can only generate 3.2 x 104 mg. m-3 (based on vapour pressure
calculation) which means that substantial amount of ethanol condensation took place and
might potentially contribute to mortality.
Insecticidal efficacy of ethanol is contributed to its toxicity to central nervous system
which causes dehydration, ataxia, sedation and eventually mortality (Aston and Cullumbine,
1959; Rodan et al., 2002). Ethanol does not act on a specific receptor in cells. It has been
shown to affect several cell surface proteins such as γ-amino butyric acid (GABA) receptors
and ion channels, namely, potassium (Harris, 1999). Mazzeo et al. (1988) demonstrated that
ethanol affects membrane phospholipids of rabbit parietal cells, with greater ability to disrupt
or disorganize the hydrophobic moieties of the membrane than the hydrophilic parts. This
consequently inhibits the K+ induced phosphatase activity (Mazzeo et al., 1988). Gurtovenko
and Anwar (2009) demonstrated that ethanol (<30.5% v/v) induced the expansion of
phospholipid bilayer, reduced the thickness and disordering the interdigitation of lipid acyl
chains. When exposed to higher ethanol concentration (>30.5% v/v), transient defects in the
membrane became more prominent, leading to disassembly of the bilayer structure
(Gurtovenko and Anwar, 2009).
9
1.2.3 The insecticidal efficacy of metabolic-stress disinfestation and disinfection
(MSDD)
Metabolic-Stress Disinfestation and Disinfection (MSDD) is a patented technology
developed by scientists at the University of California at Davis (Lagunas-Solar et al., 2006;
Lagunas-Solar and Essert, 2011). MSDD was claimed to have potential applications for
control of insects as well as fungal and bacterial diseases of plants and humans (disinfection).
MSDD functions by generating cycles of expansion and compression forces in combination
with low vapour concentrations of ethanol. The insecticidal effect of this technology is
claimed as the result of modification of respiratory metabolisms, structural disruption and
chemical toxicity at the cellular level. The cyclic forces of compression and decompression
were thought to cause structural damage and removal of O2 reserves. Rapid expansion (due to
vacuum) and compression (pressurized ballast gases) forces are thought to:
1. Open spiracles and consequently remove air from the bodies of arthropods.
2. Displace dissolved O2 from cells and consequently cause changes of cell
chemistry such as O2-CO2 balance, hence lowering the pH of cells. The reduction
of cellular pH and equilibrium shifts of dissolved O2 induces anoxic toxicity.
3. Exert structural stress.
MSDD has two phases, namely physical and chemical. The physical phase employs a
cycle of rapid decompression (vacuum) and compression. Pressure in the chamber is reduced
to the lowest point (10 kPa) and maintained for a short interval (<1 min). Compression then
follows by feeding CO2 gas or other inert gases until positive pressure is reached. The
pressure is also maintained at the highest point for a short period (<1 min). The sequence is
then repeated for 10 cycles and lasts for 10–15 min. At the end of the process, the
environmental O2 is eliminated (<0.0001%) and displaced from the body of arthropods
subjected to the treatment. Upon completion of the physical phase, pressure in the chamber is
again reduced to the lowest point (10 kPa) once again. Volatile chemical(s) are then added
into the chamber for disinfection, which enhance the efficacy of the disinfestation property of
the process. The chemical phase is proposed to be rapid (<15 min). Thus the total
recommended treatment time is between 15–30 min, depending on target insects and fruits
(Pers. comm.: Dr Manuel Lagunas-Solar). Upon completion of the process, the chamber will
then be equilibrated to atmospheric pressure before flushing the chamber with N2 or CO2 gas
10
or filtered air. A recycling system could be employed to trap and reconstitute the chemicals.
Figure 1-2 (A and B) illustrates the schematic of 12 L and 250 L MSDD research prototypes.
Figure 1-2 A. 12 L MSDD Research Prototype, B. 250 L MSDD Research Prototype
MSDD has also been demonstrated to be effective in sterilizing fresh produce on both
surface and wounded areas which was thought to be due to GRAS chemicals. The
recommended GRAS chemical used for MSDD is ethanol. Operating temperatures are
maintained at 0–4°C and 22–23°C and absolute pressure must be lower than atmospheric
pressure. The optimum MSDD operating parameters under laboratory conditions are
suggested as follow (Lagunas-Solar et al., 2006):
1. Decompression pressure: 9.84 kPa
2. Compression pressure: 108.00 kPa
3. Sequence of 10 cycles
4. Chemical: absolute ethanol
11
5. Ethanol vapour pressure: 6.78 kPa at 22–23°C, 1.86 kPa at 0–4°C
Early studies by the UC Davis group demonstrated that MSDD was effective in
treating several arthropods. Mortalities of >90% of arthropods such as fruit flies, thrips,
aphids and mites were able to be achieved using physical treatments of MSDD alone. The
treatment times were 2–6 h. Combination of physical and chemical phases of MSDD was
proven to be more effective and efficient in achieving high mortality. The combined
treatments managed to reach 100% mortality of adult, pupae, larvae and eggs of Drosophila
melanogaster Meigen and Heliothis virescens Fabricius in 0.4–2.5 h depending on the life
stages. The cyclic forces of MSDD were claimed to cause permanent damage or
modifications on tracheal systems of larvae, implosion of abdominal cavities of adults, pupae
and larvae and modification of outer membrane and structure (chorion) of the eggs.
12
1.2.4 Uncovering the mechanism of MSDD using proteomics tools; 2-D difference
gel electrophoresis (2-DIGE) and mass spectrometry
As a relatively new technology, no biochemical mechanism of MSDD insecticidal
efficacy has been described. Therefore, there is a need for research to investigate the
underlying biological effects of MSDD on insects. There are three major areas of ’omics’,
namely genomics, transcriptomics and proteomics.
With ever increasing availability of DNA sequences (genomes), the challenge is to
understand what effects the biological or environmental factors might have in relation to
DNA expression which consequently affects biological functions. The information of the
presence of a gene (within a DNA molecule) does not necessarily explain the factors
affecting its transcription, translation (to a protein), post-translational modifications and
hence its functionality (Rabilloud and Humphery-Smith, 2000). Therefore, studies that
examine the downstream level of DNA sequences are needed. In this case, two branches of
‘omics’ were considered namely transcriptomics (mRNA) and proteomics (protein). Figure 13 illustrates the cascade from DNA to functional proteins and its related fields of study.
Transcriptomics was initially considered to be a useful tool to deduce the functionality of
expression of a gene. It was soon found that abundance of mRNA transcripts does not
frequently translate to functional proteins (Tew et al., 1996; Anderson and Seilhamer, 1997;
Anderson and Anderson, 1998; Haynes et al., 1998). A study examining relationship
between the relative abundance of mRNA transcripts and proteins expressed in human liver
found the correlation coefficient between the two was 0.5, suggesting post-translational
regulations were highly frequent in higher organisms (Anderson and Seilhamer, 1997). Gygi
et al. (1999) described that in mid-log phase yeast (Saccharomyces cerevisiae), quantitative
mRNA data could not predict protein expression levels due to low correlation between
mRNA and protein levels (R2=0.4). In some genes, the difference between protein expression
level and mRNA transcripts observed was 20–30 folds. Therefore, proteomics was chosen
here to investigate the mechanisms of MSDD on insect mortality.
13
Figure 1-3 A cascade of events from DNA to protein and its associated fields of study,
namely genomics (DNA), transcriptomics (mRNA) and proteomics (proteins) (modified from
www.nobelprize.org).
Proteome, coined by Wasinger et al. (1995) is an expressed protein complement of a
genome. In a cascade of events leading from expression of genes to active proteins,
proteomics can be considered as a study of end product of genes (Rabilloud and HumpherySmith, 2000). The aim of proteomics is to understand how particular biological events affect
aggregate protein expression patterns and subsequently identify the affected proteins (Ünlü et
al., 1997). Hence, proteomics was described by Anderson and Anderson (1998) as: ‘the use of
quantitative protein-level measurements of gene expression to characterise biological
processes (e.g. disease processes and drug effects) and decipher the mechanisms of gene
expression control’.
14
1.2.4.1 Two-Dimensional differential in-gel electrophoresis (2-DIGE)
Two-dimensional gel electrophoresis is a technique for separation of a complex
protein mixture by isoelectric focusing (IEF), which separates proteins according to their
isoelectric points (pI), followed by separation according to their molecular weight using
polyacrylamide gel (O'Farrell, 1975). Detection of proteins can then be carried out using one
of the 5 techniques; namely, detection by organic dyes, differential precipitation using salts,
metal ion reduction, fluorescence and radioactive isotopes (Rabilloud and Charmont, 2000).
Detection (and quantification) of protein spots are then typically followed by mass
spectrometric techniques (Corthals et al., 2000).
Figure 1-4 Chemical structure of cyanine dyes (Tonge et al., 2001).
15
The original two-dimensional gel format posses several problems in terms of
detecting and quantifying differences between protein samples because it relies on
comparison of at least 2 different gels, which are not identical due to variability of
polyacrylamide (PAGE) gels, electric and pH fields and thermal fluctuations during gel
electrophoresis (Unlu et al., 1997; Liley and Friedman, 2006). Ünlü et al. (1997) developed a
modified technique of 2-D gel called differential in-gel electrophoresis (2-DIGE) by
introducing two fluorescent cyanine dyes; namely propyl-Cy3(1-(5-carboxypentyl)-1’propylindocarbocyanine halide N-hydroxysuccinimidyl ester) and methyl-Cy5 (1-(5carboxypentyl)-1’-methylindodicarbocyanine halide N- hydroxysuccinimidyl ester) which
subsequently added the 3rd one which increases DIGE statistical power (Cy2; (3-(4carboxymethyl) phenylmethyl)-3’-ethyloxacarbocyanine halide N-hydroxy succinimidyl
ester)) (Figure 1-4). Figure 1-5 illustrates the flowchart of 2-DIGE (Tonge et al., 2001). Prior
to analysis, the samples, i.e. treatment and control, are labelled with 2 different cyanine dyes
(Cy3 and Cy5) and the internal standard, which consists of a pool of all the samples in the
experiment, is labelled with Cy2 (Table 1-2) (Alban et al., 2003). Figure 1-6 (taken from
Marouga et al., 2005) illustrates the importance of Cy2 in enhancing the quality of statistical
analysis of 2-DIGE by normalizing the protein spots against the internal standard.
Table 1-2 Typical experimental designs of 2-DIGE using 3 cyanine dyes.
Gel
Cy2
Cy3
Cy5
1
Pooled sample
Sample 1
Sample 2
2
Pooled sample
Sample 1
Sample 2
3
Pooled sample
Sample 3
Sample 4
4
Pooled sample
Sample 3
Sample 4
5
Pooled sample
Sample 5
Sample 6
6
Pooled sample
Sample 5
Sample 6
16
Figure 1-5 The principle of 2-DIGE from protein mixture labelling for identification of
protein spots of interest (modified from Tonge et al., 2001).
17
Figure 1-6 The diagram illustrates the importance of Cy2 (internal standard) in determining
‘real’ difference in quantifying protein spots. The top diagram shows the difference among
the four samples without normalising the volume against the internal standard (increased
expression level in gels 3 and 4). The samples that are quantified against the internal standard
produced a significantly different results (reduced expression level in gel 3) (Marouga et al.,
2005).
The mixture of samples is then subsequently run at the same time in the same gel.
Protein spots can then be detected using fluorescence imaging using different wavelengths
that excite different dyes. Cy2, Cy3 and Cy5 are excited at 488, 532 and 633 nm with
emission at 520, 580 and 670 nm, respectively (Unlu et al., 1997; Alban et al., 2003;
Marouga et al., 2005). Figure 1-7 shows a false colour images produced from the gel. The
images of protein spots can then be analysed using advance image analysis softwares. Image
processing procedure is often considered as the bottleneck of 2-DIGE due to being laborious
and user-variable (Marouga et al., 2005). Following image analysis, protein spots of interest
can then removed from the gel and analyzed using mass spectrometry.
18
Figure 1-7 A false colour image depicting different expression level of proteins in a mixture
of 3 samples; red, green and blue spots represent samples that were labelled with Cy5, Cy3
and Cy2, respectively. White spots represent proteins that are expressed in all 3 samples. The
intensity of the spots correlates with the expression level of proteins.
1.2.4.2 Identification of protein of interest using mass spectrometry (MS)
Mass spectrometry (MS) has now become one of the most important tools in
proteomics, especially for rapid protein and peptide identification from gel separated proteins
(Corthals et al., 2000; Pandey and Mann, 2000; Aebersold and Goodlett, 2001). MS
essentially has 3 main components to measure molecular mass of a particular compound,
namely an ion source, a mass analyzer and a detector (Biemann, 1963; Kiser and Sullivam,
1968; Patterson and Aebersold, 1996; Aebersold and Goodlett, 2001). Ions produced in the
ion source are separated by mass (m) to charge (z) ratio and subsequently detected by an
electron multiplier. The set of data is produced as spectra that illustrate the ion intensity v.
mass-to-charge ratio (m/z value) (Corthals et al., 2000). The breakthrough in protein
identification using MS came when two new methods were introduced; matrix-assisted laser
desorption/ ionization (MALDI) (Karas and Hillenkamp, 1988) and electrospray ionization
(ESI) (Fenn et al., 1989). Both techniques allow an accurate identification of a wide range of
proteins and peptides with molecular masses >100 kDa at picomole (10-12) and femtomole
(10-15) sensitivity (Andersen et al., 1996). MALDI is usually utilised to characterize simple
19
peptide mixtures whereas ESI is preferred for the analysis of more complex mixtures
(Aebersold and Mann, 2003).
ESI is usually combined with quadrupole mass analyzers capable of tandem mass
spectrometry (MS/MS). ESI-MS works by passing through a solution containing protein
mixture through a fine needle with high potential of about +5.0 kV for the generation of
positive ion spectra (Corthals et al., 2000). The resulting electric field at the tip charges the
surface and subsequently disperses the liquid into a fine spray of charged droplets (Figure 18) (Fenn et al., 1989). The charged droplets are then directed across the small inlet orifice in a
plate of lower potential (+0.5 ─ +1.0 kV) to the evacuated quadrupole mass analyzer. The
droplets are desolvated into a quasi-molecular ion (gas phase) in the zone between the spray
needle and orifice plate by dry inert gas or heat, which can subsequently be detected by the
mass analyzer (Fenn et al., 1989; Patterson and Aebersold, 1996). Instruments that are
capable of MS/MS can then select ions of a particular m/z ratio, fragment them by collision
induced dissociation (CID) and record the precise masses of the fragment ions.
Figure 1-8 The schematic diagram of ESI-MS and the desolvation process of charged
droplets to quasi-molecular ions (modified from Fenn et al., 1989).
20
MALDI, on the other hand, is usually combined with time-of-flight (TOF) tube as a
mass analyzer (Figure 1-9) (Corthals et al., 2000). MALDI is a desorption/ ionization method
where solid state proteins are impacted by photons generated by a laser to produce protein
ions (Karas and Hillenkamp, 1988). Protein mixture is co-crystallized and embedded with
excess matrix in large molar surplus. The matrices are usually weak aromatic acids that are
soluble in solvents used for polypeptides, can enhance protein and peptide solubility and
strongly absorb light of the ionizing laser (Andersen et al., 1996). The protein ions formed by
irradiating the sample with the laser which consequently extracted the formed ions into a gas
phase. The ionizing laser also simultaneously triggers the clock to measure the flight time of
ions to the ion detector (hence the name; ‘time-of-flight’ (TOF) MS). The resulting MS
spectra (of both techniques) can then be utilised to deduce the amino acid sequence by
comparison to the databases (Corthals et al., 2000). The main database used in this study is
the protein database in National Center for Biotechnology Information (NCBI). The amino
acid sequences obtained from MS spectra are then subjected to Basic Local Alignment
Search Tool (BLAST) to find regions of local similarity between sequences. These results
can be used to identify members of gene families and suggest functional and evolutionary
relationships (blast.ncbi.nlm.nih.gov/blast.cgi).
Figure 1-9 The schematic diagram of MALDI-TOF (Patterson and Aebersold, 1996).
21
1.2.5 The effect of physical and chemical components of MSDD treatments on
fruit physiology and quality
MSDD treatments were claimed to be highly effective for disinfestations purposes
while maintaining the sensory quality of the produce (Lagunas-Solar et al., 2006). Citrus
fruits showed the highest tolerance with no detectable sensory effect after 60–70 d of storage.
Fruit that showed the least tolerance were raspberries and blackberries due to their soft
texture. The texture of the berries was damaged due to ‘juicing’ of flesh (Lagunas-Solar et al.,
2006). This problem might be solved by modifying MSDD parameters such as lowering
pressure and increasing the cycles to compensate. Analyses of the effect of MSDD on
textural properties suggesting that fruits such as kiwifruit and apples will have high MSDD
tolerance due to their “tougher” structures. MSDD treatments could potentially cause
physiological stresses due to low O2, high CO2 and ethanol.
1.2.5.1 The effect of hypobaric storage and modified atmosphere/controlled
atmosphere on fruit physiology and quality
MSDD utilises combination of high CO2 and low O2 atmosphere, pressure
modification and ethanol vapour to disinfest. The first component of MSDD treatment
mimics hypobaric storage, controlled atmosphere (CA)/ modified atmosphere (MA)
treatments. The recommended atmosphere composition in CA/ MA storage is generally <5
kPa O2 and/or 0–20 kPa CO2 (Gorris and Peppelenbos, 1992). In MSDD system, partial
pressure of O2 is reduced to ultra low (ULO-ultra low O2) which is around 0.1–0.3 kPa and
partial pressure of CO2 reaches near saturation (99.7–99.9 kPa). This condition is considered
to potentially impose anaerobic stress for fruit. The treatment time of MSDD is relatively
short (<2 h) compared to MA/CA storage (days to months).
Upon exposure to controlled atmosphere or low pressure, the primary response of
fruit is to adapt by lowering their metabolic rate to cope with absence of oxidative substrate
(O2). Hypoxia/anoxia has been shown to delay the ethylene climacteric and consequently
retard the ripening rate of fresh produce (Zhen-guo et al., 1983; Bangerth, 1984; Kader et al.,
1989; Gorris and Peppelenbos, 1992).The physiological effect is dependent upon the type and
maturity of the fresh produce (Kader et al., 1989). Controlled atmosphere and hypobaric
storage treatments lower the metabolic rate and delay ripening and therefore prolong the
22
postharvest storage life of various fruit such as banana, apple, orange, peach and so on (Burg
and Burg, 1966; 1969; Dilley, 1977; Ke and Kader, 1990; Ke et al., 1991). Atmosphere
modification has also been shown to have beneficial secondary effects such as reduction in
chlorophyll degradation, cell wall degradation and phenolic oxidation (Beaudry, 2000). O2
concentration must not fall the tolerance limit to support aerobic respiration and cell
metabolism, of fresh produce. CO2 concentration should also not exceed the tolerance limits.
If the tolerance limit is exceeded, glycolytic conversion of pyruvate to acetaldehyde and
ethanol will occur and subsequently lead to tissue fermentation (off flavour), browning and
other undesirable physiological changes (Gran and Beaudry, 1993).
Burg and Burg (1966) demonstrated that ripening of ‘Gros Michel’ bananas can be
delayed to 15–40 d when stored at 20–48 kPa as opposed to 7–14 d at normal atmospheric
pressure (101.3 kPa). Hypobaric storage (1.3–10.7 kPa) retained firmness, of ‘Red
Delicious’, ‘Golden Delicious’, ‘Idared’, ‘Northern Spy’, ‘Red Rome’, ‘Stayman’ apples
(Dilley, 1977). Hypobaric storage also reduced sensitivity of apples to ethylene treatment
(Bangerth, 1984). Bangerth (1984) showed that ‘Golden Delicious’ apples did not reach
climacteric respiration (Figure 1-10) and autocatalytic ethylene production during 11 month
of hypobaric storage (6.6 kPa). Similar response was observed even when ethylene was
added during storage. No significant physiological response was observed on ‘Cavendish’
bananas when stored under similar conditions. Ke et al. (1991) investigated the effect of
insecticidal low O2 atmosphere on physiology and quality of peaches. It was found that
peaches, stored at 0 and 5°C with 0.25 kPa and 0.02 kPa O2 for 3 d, had a 21–48% and >94%
reduction in respiration rate and ethylene production, respectively. The eating quality was
maintained up until 14 d in storage. However, off flavour was noted in 40 d CA stored
peaches.
Argenta et al. (2000) demonstrated that delaying an exposure to CA or CO2 storage
can reduce the CO2 injury in ‘Fuji’ apples. Storage of ‘Fuji’ apples in 3 kPa CO2 and 1.5 kPa
O2 has been shown to delay firmness and titratable acidity (TA) loss. This storage condition
exacerbated brown-heart disorder (CO2 related) and slowed the disappearance of watercore
incidence. They demonstrated that brown-heart disorder was detected after 15 d upon CA
storage and the severity increased during 4 months of CA storage. It was also shown that
delaying the establishment of CA storage by 2 to 12 weeks resulted in much lower brown-
23
heart incidence (Figure 1-11). The delay in CA storage resulted in lower firmness and TA.
The firmness and TA were still significantly higher than apples stored in air.
Figure 1-10 Hypobaric storage prevents climacteric respiration in ‘Golden Delicious’ apples
(Bangerth, 1984).
Wang and Dilley (2000) demonstrated that hypobaric storage at 5 kPa and 1°C while
ventilating air prevented accumulation of volatiles associated with superficial scald incidence
in ‘Law Rome’ and ‘Granny Smith’ apples. They found that the accumulation of α-farnesene
and 6-methyl-5-hepten-2-one (MHO) was reduced when fruits were immediately stored
hypobarically after harvest. A 3-month delay resulted in superficial scald incidence that was
similar to storage in air. They also found that the accumulation of MHO in the epicuticular
wax was proportional to the delay in hypobaric storage. They did not find a strong correlation
between accumulation of MHO and scald incidence, suggesting MHO was not a limiting
factor in scald incidence.
24
Figure 1-11 Delaying the establishment of CA can reduce the CO2 related injury in ‘Fuji’
apples (Argenta et al., 2000).
CO2 has been shown to competitively inhibit the binding of ethylene to its receptors,
which are usually metal containing enzymes such as catalase (Burg and Burg, 1969). Zhenguo et al. (1983) showed that CO2 gas appeared to inhibit the production of ethylene in
ripening apples through inhibition of ACC (1-aminocyclopropane-1-carboxylic acid)
conversion to ethylene and ACC formation itself. Carbon dioxide treatments also reduced the
accumulation of two members of ACC oxidase gene families (PP-ACO1 and PP-ACO2) in
peach to the same level of 1-methylcyclopropene (1-MCP) treatments (Mathooko et al.,
2001). Elevated level of CO2 promotes volatile production especially alcohols and aldehydes
which are substrates for aroma compound (esters) production. Dixon and Hewett (2001)
demonstrated that exposure of various cultivars of apples to 100 kPa CO2 for 24 h at 20°C
resulted in increase of synthesis in various volatiles associated with aroma. It was found that
concentrations of acetaldehyde, ethanol, ethyl acetate and ethyl esters were increased while
25
acetate esters and aldehydes were reduced. However, this study did not discuss or present any
physiological disorders associated with the treatment.
CA storage with relatively lower CO2 concentration was shown to have an opposite
effect. Lara et al. (2003) found that long term CA storage (2 kPa O2 with 0.7, 2 or 5 kPa CO2)
at -1°C of ‘Doyenne du Comice’ pears for up to 7 months resulted in reduced volatile
production. Argenta et al. (2004) found that exposure of ‘Fuji’ apples to short-term CA
storage (10 kPa O2 and 20 kPa CO2 at 20°C for 12 d) resulted in different response in terms
of volatile production compared to long-term CA storage (0.5 kPa O2 and 0.05 kPa CO2 or
1.5 kPa O2 and 3 kPa CO2). Short term CA resulted in enhanced production of ethanol,
methyl and ethyl esters and aldehydes whereas the production of C3–C6 alcohols, butanal,
propyl, butyl, pentyl, and hexyl esters was decreased. On the other hand, the production of
most volatiles, from fruits that were exposed to long-term CA storage, was reduced when
compared to air-stored fruits. These results suggest that short term, not long term, exposure of
high CO2 and low O2 might be beneficial for aroma volatile production in some fruit.
1.2.5.2 The effect of ethanol treatment on fruit physiology and quality
The chemical phase of MSDD is characterised by ethanol treatment in a reduced
pressure condition (Lagunas-Solar et al., 2006). Ethanol is readily converted to acetaldehyde
by alcohol dehydrogenase (ADH) in fruit (Pesis, 2005; Podd and Van Staden, 1998).
Therefore, the effect of acetaldehyde in ripening physiology of fruit is thought to be
important and in most cases, it was shown to be more significant than ethanol (Mencarelli et
al., 1991; Beaulieu and Saltveit, 1992; Beaulieu et al., 1997). The role of ethanol and
acetaldehyde in fruit ripening can be either as an inducer or a retardant depending on maturity
and the type of fruit (Saltveit, 1989; Mencarelli et al., 1991; Beaulieu and Saltveit, 1997;
Ritenour et al., 1997).
The inhibitory effect of the treatments is attributed directly to the ability of ethanol
and acetaldehyde to inhibit ethylene synthesis (Saltveit, 1989). Ethylene is synthesised from
methionine through S-adenosyl-L-methionine (AdoMet) and 1-aminocyclopropane-1carbocylic acid (ACC) (Adams and Yang, 1981; Bleecker and Kende, 2000). The enzymes
responsible for catalysing the conversion of Adomet to ACC and of ACC to ethylene are
26
ACC synthase and oxidase, respectively (Adams and Yang, 1981). Figure 1-12 illustrates the
biochemical pathway of ethylene biosynthesis and its associated methionine cycle.
Figure 1-12 Ethylene biosynthesis and methionine cycles. KMB: 2-keto-4-methylthiobutyric
acid, Met: methionine, AdoMet: S-adenosyl-L-methionine, ACC: 1-aminocyclopropane-1carboxylic acid, MTA: 5’-methyladenosine, MTR: 5’methylthioribose, MTR-1-P: 5’methylthioribose-1-phosphate (Bleecker and Kende, 2000).
Acetaldehyde inhibits ACC oxidase activity in the presence of ACC, effectively
blocking ethyelene biosynthesis (Burdon et al., 1996). As well as blocking ethylene
production, ethanol can reduce the sensitivity of ACC synthase and oxidase to exogenous
ethylene. For example, ethanol treatment of broccoli reduced the activities of ACC synthase,
ACC oxidase and the expression of BO-ACO1, BO-ACO2 and BO-ACS1. Figure 1-13
27
illustrates the proposed inhibitory mechanism of ethanol and acetaldehyde in ethylene
biosynthesis and fruit ripening.
Methionine
Ethylene
binding
sites
Ethanol
SAM
Alcohol
dehydrogenase
ACC
synthase
ACC
Acetaldehyde
ACC
oxidase
Ethylene
Figure 1-13 The proposed mechanisms of ethanol and acetaldehyde in inhibiting ethylene
biosynthesis and fruit ripening (modified from Podd and Van Staden, 1998).
Kelly and Saltveit (1988) found that ripening of tomato was inhibited by either
exogenous ethanol (2–4 kPa) vapour or endogenous ethanol synthesis under anaerobic
conditions. They also reported that the respiration rate of the ethanol-treated tomato was
markedly reduced and the onset of climacteric rise was delayed by 3 d when compared to
controls. Fruits that were treated with highest level of ethanol (4 kPa) had a significantly
slower rate of ripening even when treated with ethylene continuously. This suggests that the
mode of action of ethanol in relation to ethylene might include inhibition of the action of
ethylene. Saltveit (1989) used lycopene synthesis of pericarp discs of mature green tomato to
illustrate that ethylene inhibition by ethanol was non-competitive (using Lineweaver-Burk
plot). The extent of inhibition seemed to be at specific ethanol concentration. At
concentration of ≤0.6 mmol. g-1 tissue, ethanol was shown to be directly inhibiting ethylene
action on lycopene synthesis. It is suggested that the mode of action of ethanol in inhibiting
ripening is thought to involve two mechanisms; namely reduction of ethylene production and
inhibition of ethylene action (Podd and Van Staden, 1998).
28
Ritenour et al. (1997) screened the ability of ethanol (≤6 mL. kg-1) to induce or inhibit
ripening in 8 different climacteric fruits. It was found that ripening of tomato and avocado
was reduced whereas banana, honeydew, muskmelon, nectarine, peach, pear and plum fruit
were not affected. Beaulieu et al. (1997) demonstrated that acetaldehyde (AA) accumulation
was the direct causal agent in ethanol-induced ripening inhibition. The accumulation of AA
as the result of ethanol or acetaldehyde vapour treatment was critical in determining the
ripening inhibitory effect. Mencarelli et al. (1991) demonstrated that ethanol or AA treatment
can induce ripening in kiwifruit. They also found that exposure to AA, instead of ethanol, had
a more pronounced effect in stimulating the ripening of kiwifruit. These results suggest that it
may not be ethanol that is the limiting factor but the subsequent conversion to acetaldehyde,
by alcohol dehydrogenase, that plays a crucial role in either inhibiting or stimulating ripening
in fruits (Podd and Van Staden, 1998).
Ethanol treatments have been shown to reduce superficial scald incidence in apples.
Superficial scald is commonly identified by browning of the skin of apples after being
coolstored (Scott et al., 1995). The oxidation of α-farnesene to its conjugated trienes is
proposed to be the cause of superficial scald (Huelin and Coggiola, 1970; Ghahramani and
Scott, 1998b; Ghahramani et al., 1999). The second proposed hypothesis is that scald
incidence is caused by oxidative stress (Whitaker, 2004) . Scott et al. (1995) demonstrated
that treatments of ethanol vapour of 0.5–1 g. fruit-1 at 0°C for 18 weeks controlled scald
incidence in four out of five seasons of ‘Granny Smith’ apples. They also found that ethanol
dipping was not as effective as vapour treatments, which is probably due to dissipation of
ethanol during long term storage. Ghahramani and Scott (1998b) found that there is an
inverse relationship between ethanol content and α-farnesene and conjugated trienes, and
consequently superficial scald incidence (Figure 1-14).
29
Figure 1-14 The relationships between concentrations of α-farnesene, conjugated trienes,
ethanol content and its consequential suppression of superficial scald in ‘Granny Smith’
apples (modified from Ghahramani and Scott, 1998b).
30
1.3 Aims of the thesis
There are various parameters associated with MSDD; namely the length of the
physical phase, the length of the chemical phase, pressure oscillation, ethanol concentration,
and the extent of vacuum during chemical phase and so on. Due to its infancy, there is a lack
of understanding of the effect of parameters on the insecticidal efficacy of MSDD. Therefore,
one of the aims of the thesis was to investigate mechanisms of insecticidal efficacy of
MSDD, using physical and biochemical (proteomics) tools. This knowledge could be utilised
to optimize or modify any existing disinfestation methods, particularly in MA/CA or
GRAS/food additive chemical treatments, in order to enhance the insecticidal efficacy. The
thesis also investigated the effect of MSDD on fruit physiology and quality, which could
potentially help determine the commercial feasibility of MSDD. The technical limitations of
MSDD were also studied.
The specific aims of the thesis were:

Construction and commissioning of the MSDD equipment.

Investigation of the insecticidal efficacy of MSDD.

Investigation of biochemical mechanisms of MSDD using proteomics tools.

Investigation of the effect of MSDD on fruit physiology and quality.

Investigation of the significance and optimisation of MSDD parameters.

Investigation of technical issues associated with MSDD; internal pest and the loading
effect.
31
2. Insecticidal efficacy of MSDD against longtailed
mealybug (Pseudococcus longspinus)
2.1 Overview
Longtailed mealybug, Pseudococcus longispinus (Targioni-Tozzetti), is polyphagus
and widely spread in tropical and subtropical regions. It is known as a pest which affects
many horticultural crops (Swirski et al., 1980). The adults are 3–4 mm in length (Figure 2-1).
Mealybugs are usually found in location of fruit that are difficult to access by physical and
chemical treatments. Mealybugs directly withdraw sap of plant and indirectly produces honey
dew that becomes substrate for sooty mould (Swirski et al., 1980); a black fungus that can be
a very significant cosmetic defect on fruit.
Figure 2-1 Adult longtailed mealybug.
The management of longtailed mealybug is particularly difficult due to lack of
monitoring techniques and insecticides that do not have high level of phytotoxicity. Due to its
status as a quarantine pest in some countries and the difficulty that quarantine inspectors have
in distinguishing immature life stages of a non-quarantine species of mealybug species from
another quarantine species, Methyl Bromide (MeBr) has been employed to treat mealybug
infested fruit.
The length of treatment times of MSDD reported by Lagunas-Solar et al. (2006) ranged
from 20 min to 4 h. In this work, a modified MSDD treatment of 90 min with 30 min of
physical phase followed with 60 min of chemical phase was used, with increasing dose of
ethanol vapour, to understand the efficacy of MSDD and develop a standard protocol for
32
subsequent work. The lethal doses of ethanol for 50 and 99% mortality (LD50s and LD99s), in
the MSDD chemical phase, against 3 life stages of longtailed mealybug are also reported. The
aim of this chapter is to establish an effective protocol to control longtailed mealybug.
2.2.1 The construction of Plant & Food Research MSDD prototype
A two hundred and fifty-litre pressure chamber was purchased from Genera Ltd
(Mount Maunganui South, NZ). All fittings were stripped and the internal surface was
commercially painted by Abrasive Blasting & Coatings Ltd (Onehunga, Auckland, NZ) with
food grade coatings; primer coat: Interzinc® 42, intermediate coat: Intergard® 345, and top
coat: Interthane 990 (AkzoNobel, Amsterdam, the Netherlands). The large capacity chamber
(250 L), as opposed to smaller chambers, was considered because it was thought to be better
suited for the studies of technical issues (related to this technology) that might arise in a
commercial setting, e.g. the effect of fruit volume during treatments. The Plant and Food
Research (PFR) MSDD prototype (Figure 2-2) was designed and constructed based on
Lagunas-Solar et al. (2006) and Lagunas-Solar and Essert (2011). A liquid ring vacuum
pump (Model TRMB 32-75, Travaini Pumps, USA) was connected to the chamber. The
choice of vacuum pump was governed by the ability to reduce pressure from atmospheric
pressure to 10 kPa within 1 min.
During initial trials, a standard CO2 gas regulator (V05-200-P45A) (Norgren Ltd,
Penrose, Auckland, NZ) was used to deliver CO2 gas into the chamber. The rate of increase
in pressure (from 10 kPa to 100 kPa) could not be achieved in 1 min. Therefore, a CO2
Cigweld COMET™ (supplied from BOC Gases NZ, Penrose, Auckland, NZ) regulator was
used and the rate of increase of pressure from 10 kPa to 110 kPa was achieved in 1 min. The
food grade CO2 gas (≥99.9% purity, BOC gases NZ, Auckland, New Zealand) was used for
treatments. A port for gas sampling was added and a 110 mm-diameter spark proof fan was
fitted inside the chamber to ensure uniformity of treatment conditions.
A compound gauge (WIKA EN-8371, 316 L) and a water bath-chemical heating
system (ethanol delivery system) were subsequently connected to the chamber. The ethanol
delivery system was slightly different from the UC Davis and USDA ARS Hilo MSDD
33
prototypes. In PFR system, a known amount of liquid ethanol is injected through a tubular
heat exchanger and consequently converted to vapour and mist. The ethanol (vapour and
mist) is then inherently delivered to the treatment chamber by the established low pressure
condition. In UC Davis and USDA Hilo systems, the ethanol liquid is mechanically delivered
and subsequently heated by a heating element inside the treatment chamber.
A temperature control system using a thermal blanket and temperature sensors (inside
the chamber) set to 20°C was also added. After the construction, a safety analyst (Paul Jarvie)
from Employers and Manufacturers Association (EMA) was contacted to perform hazard
analysis and subsequently approve and commission the use of the PFR MSDD prototype.
B
A
D
E
C
1
2
Figure 2-2 Plant & Food Research MSDD prototype. A. MSDD prototype, B. Volatile
delivery system, C. Fan (1) and temperature sensors (2), D. Temperature control, E. Vacuum
pump. Solid arrow indicates parts that are visible in diagram A, striped arrows indicate the
positions of the parts that are not observable in diagram A.
34
Initially, pressure oscillation between 10 and 110 kPa for 10 cycles, followed by a 10
min of chemical phase at 10 kPa with 75 mg. L-1 was used. This procedure was obtained from
Lagunas-Solar et al. (2006) and used to treat 5th instar larvae of NZ native leafrollers
(Planotortrix execanna Walker and Planotortrix octo Dugdale), greedy scale (Hemiberlesia
rapax Comstock) and longtailed mealybug (P. longispinus). The treatments only resulted in
10–40% mortality. Therefore, the procedure was modified with the focus of reducing the
extent of pressure oscillation from between 10 and 110 kPa to 90 and 110 kPa while
increasing the chemical phase treatment time from 10 min to 60 min. The treatment started
with drawing pressure down from 100 kPa to 90 kPa followed by immediate flow of CO2 gas
which increased the pressure to 110 kPa (Figure 2-3). This is considered as 1 physical cycle.
Vacuum time from 110 kPa to 90 kPa can be achieved in 10 s, whereas time of increasing
pressure from 90–110 kPa was about 20 s. Therefore, one physical cycle was approximately
30 s. The physical phase of each MSDD treatment was set to 30 min which generated about
50–60 physical cycles.
Figure 2-3 MSDD operational protocol consists of the physical phase of 30 min followed by
the chemical phase of 60 min.
The chemical phase was initiated after 30 min of physical phase by lowering the
pressure to 10 kPa and injecting a known amount of liquid ethanol into the heating system.
35
The chemical phase generally lasted for 60 min. The doses of ethanol used were specifically
ranging from 0–275 mg. L-1. Ethanol vapour was introduced into the treatment chamber by
heating a known amount of liquid ethanol by the heat exchanger. The amount of liquid
ethanol was calculated for the internal volume of the empty treatment chamber (250 L) in
order to achieve the target concentration. Based on the temperature profile during the
treatment period, the saturated vapour concentration of ethanol was calculated to be 84.5 ±9.5
mg. L-1. Below that concentration, ethanol existed as vapour and above that concentration as
84.5 ±9.5 mg. L-1 vapour and the remainder as mist (Table 2-1). The temperature of the heat
exchanger was set to 90°C to ensure the liquid ethanol was fully vapourised.
Three repeated runs were carried out for each dose with 60 min of purging between
treatments. Three types of controls were used: an untreated (handling) control, a chemical
phase-only control, where the mealybug-infested potatoes were treated at 10 kPa with 125
mg. L-1 ethanol (as vapour and mist) for 90 min; and a physical phase-only control, where the
mealybug-infested potatoes were treated by cycling pressure at 100 ±10 kPa for 30 min,
followed by 60 min at 10 kPa (without any ethanol).
2.2.3 Longtailed mealybug (Pseudococcus longispinus)
Longtailed mealybugs were obtained from a laboratory colony reared on sprouting
‘Desire’ potatoes (Solanum tuberosum). The colony was maintained at 20 ±2°C with 60%
RH, and a 16:8 h light:dark cycle. Clean sprouting potatoes were put into the colony two
weeks before each experimental treatment so that each potato carried ~50–150 mealybugs
(with mixed numbers of adults, 2nd/3rd instar nymphs and crawlers) by the time the insects
were required. Female longtailed mealybugs produce crawlers; therefore no egg life stage
was tested. On the day of treatment, 1–2 potatoes were placed inside a plastic container (650
mL) with the lid off during MSDD treatment. After treatment, mealybug-infested potatoes
were held at 20°C in a 16:8 h light:dark cycle and three days later, insects were recorded as
live (movement) or dead (no movement) after being gently prodded with a blunt instrument.
36
2.2.4 Gas concentration measurement
Gas samples were taken during 3 MSDD trials using 1-mL syringes at 0, 1.25, 2.3, 4,
8, 12, 20, 25 and 30 min during the physical phase to determine the concentrations of O2 and
CO2. Carbon dioxide and O2 were measured using a gas chromatograph (GC) fitted with an
infra-red CO2 transducer (Servomex 1505, Servomex Ltd., Sussex, UK) and an O2 sensor
(CiTicel oxygen cell, model C/2, City Technology Ltd., London) with O2-free N2 used as a
carrier gas with flow rate of 0.67 mL. s-1 at 20°C.
For O2 analysis, 1 mL of sample gas was injected directly into the GC. For CO2
analysis, a dilution step was required as the transducer was saturated at around 45% v/v CO2
and accurate measurement cannot be >10% v/v CO2. A 1-L glass jar equipped with a rubber
bung and an aluminium stirrer plate was used for the dilution step. An aluminium plate was
used to stir the headspace to ensure uniformity of gas inside the jar. The jar was initially
flushed with N2 until the CO2 concentration dropped to ~0.05% v/v. Three mL of the MSDD
gas sample was then injected into the jar and mixed well by stirring with the aluminium plate
for at least 1 min. One mL of gas was drawn and analyzed using the same gas analyzer. From
the difference between the initial and final CO2 readings, the concentration of CO2 gas inside
the MSDD chamber was calculated. This was carried out up to 8 min into the treatment. From
12 min on, CO2 concentration was calculated by using equation 2-1. Equation 2-1 was used
because the change in CO2 concentration was beyond the detection limit of the gas analyzer.
CO2 (%)
= 100% - (O2 (%) + N2 (%))
= 100% - (O2 (%) + 3.76 O2 (%))
(2-1)
2.2.5 Statistical analysis
For the lethal dose values (Table 2-2), mortality data for each replicate run were fitted
using the complementary log-log (clog-log) model (Preisler and Robertson, 1989), with dose
as the explanatory variable to derive estimated doses for 50 and 99% mortality (LD50 and
LD99). The clog-log model was used in this study because of its better approximation to a
linear response compared with other transformations. These estimates were calculated as the
dose to achieve a mortality of c + (1 - c) × m, where c was the control mortality and m the
estimated proportion of mortality. For each life stage, a geometric mean LD and its associated
37
standard error (SE) were estimated, from which a 95% confidence interval (CI) was
calculated. Non-overlap of the 95% confidence intervals is equivalent to a test for difference
at P <0.05.
2.3 Results
During MSDD treatment, temperature inside the chamber dropped as the atmosphere
was replaced by CO2 gas (Figure 2-4). On average, temperature dropped from 19.3 ±2.4 to
11.5 ±1.0°C during the physical treatment. Decrease in temperature was due to introduction
of cold CO2 gas into the chamber and low pressure condition during the physical phase.
Gradual increase was observed during the chemical treatment. As hot ethanol vapour was
introduced into the chamber, the temperature rose from 11.5 ±1.0 to 14.5 ±1.8°C. Carbon
dioxide concentration rose from 0.07 ±0.00 to 90.67 ±0.10% within 2 min, after 4 cycles
(Figure 2-5). The chamber was saturated with CO2 after 10 min (approximately 10–11
cycles). Oxygen partial pressure was reduced from 21.48 ±0.32 to 0.93 ±0.10 kPa within 10
min. Ultra low oxygen (ULO) atmosphere (0.1–0.3 kPa) was achieved after 12 min.
Mean percentage mortalities for untreated longtailed mealybugs held in ambient
conditions ranged amongst the life stages from 1 to 7% (Figure 2-6, Group A bars).
Longtailed mealybugs exposed to a medium concentration (125 mg. L-1) of ethanol vapour
and mist at 10 kPa for 90 min (chemical phase only) suffered 7 to 9 % mortality (Figure 2-6,
Group B bars). After exposure to the physical phase-only treatment (cycling pressure from
110–90 kPa for 30 min, followed by 60 min at 10 kPa with no ethanol) 8 to 17% of
mealybugs were killed. However, when exposed to the entire MSDD process (physical and
chemical phases), using the same ethanol concentration as the chemical phase-only control,
an average of 70–100% of the three mealybug life stages were killed. This indicated that the
chemical phase or physical phase alone was not effective and both phases were required to
achieve high levels of mortality. Second and 3rd instar nymphs were the life stages most
tolerant of MSDD treatment, followed by crawlers, and adults were the most susceptible
(Table 2-2, Figure 2-6). The lethal dose of ethanol predicted for 99% mortality of 2nd/3rd
instar nymphs, crawlers and adults were 371.0, 142.6 and 81.3 mg. L-1 for 90 min,
respectively.
38
Table 2-1 Concentration of ethanol introduced calculated in chamber as volatile and
mist/liquid. Based on the temperature profile, the maximum concentration of ethanol vapour
was 84.5 ±9.5 mg. L-1. The remaining ethanol existed as liquid or mist that condensed on the
product surface.
Total volume
Calculated1
Calculated
Total
of ethanol
concentration
concentration
concentration
introduced
ethanol vapour
ethanol mist or
of ethanol in
(mL) into
(mg. L-1)
liquid
the chamber
(mg. L-1)
(mg. L-1)
250-L MSDD
chamber
7.9
25
0
25
19.0
60
0
60
31.7
84.5
15.5
100
39.6
84.5
40.5
125
55.5
84.5
90.5
175
63.4
84.5
115.5
200
71.3
84.5
140.5
225
87.1
84.5
190.5
275
See Appendix 1 for worked example.
39
Table 2-2 Mean lethal doses (95% CI) for 50 and 90% mortality (LD50s and LD99s) derived
from dose-mortality data from three life stages of longtailed mealy bug (Pseudococcus
longispinus) exposed to MSDD treatments with various concentrations of ethanol vapour and
mist (see Table 2-1 for details).
Life stage
1
ntotal
Mean LD50 (95%
Mean LD99 (95%
CL), mg. L-1
CL), mg. L-1
Adult
2 462
21.8
(18.8–25.4)1
2nd & 3rd instars
2 453
72.9
(62.5–84.9)
371.0 (310.9–442.6)
Crawler
2 582
21.1
(18.1–24.6)
142.6 (119.5–170.1)
Values in the bracket represent 95% CL.
40
81.3
(68.1–97.0)
Figure 2-4 Mean (±SE) temperature of atmosphere, core and skin temperature of potato
during simulated runs of MSDD treatment to calculate the concentration of ethanol (n = 3).
.
Figure 2-5 Oxygen and CO2 partial pressure (kPa) (mean ±SE) during a typical MSDD run
during the 30-min physical phase (n = 3).
41
Figure 2-6 Percentage mortality (mean ±SE, n = 3, each n=150 insects) of three different life
stages of longtailed mealybug (Pseudococcus longispinus). A. Ambient control (columns), B.
Chemical-only control (125 mg. L-1 of ethanol treatment at 10 kPa for 90 min) (columns).
MSDD treatments were carried out with increasing concentrations of ethanol in the chemical
(second) phase (lines). Based on the temperature data, maximum ethanol vapour
concentration was calculated to be 84.5 ±9.5 mg. L-1. Above that concentration, ethanol
existed as vapour and liquid which condensed on the product surface. The bar represents
standard error of ethanol condensation point based on the standard error of temperature
measured during MSDD treatments.
42
2.4 Discussion
Results from this study indicate that both the physical and chemical phases of the
MSDD process are required to achieve high mortality of longtailed mealybug. MSDD
treatments using ethanol concentrations above the condensing point of ethanol vapour were
required to achieve >90% mortality of the most tolerant 2nd and 3rd instar nymph life stages.
The results also demonstrate that slight changes of pressure during the physical phase of
MSDD treatment (10% of normal atmospheric pressure) are effective (in combination with
ethanol treatments) and so equipment costs could potentially be reduced if this technique was
applied commercially. In contrast, Lagunas-Solar et al. (2006) used a pressure reduction of
90% of normal atmospheric pressure in both the physical and chemical phases.
The entire MSDD process uses hypoxia (low O2), hypercarbic (high CO2) and ethanol
toxicity to achieve high mortality of pests. The insecticidal efficacy of low pressure systems
has been attributed to low oxygen partial pressure or hypoxia (Navarro and Calderon, 1979;
Mbata et al., 2005). Since insects can tolerate hypoxia quite effectively, this system needs
longer treatment times to achieve high mortality (> 24 h; Navarro and Calderon 1979). In this
study the physical phase cycles alone (100 ± 10 kPa for 30 min) achieved only 7–16%
mortality of longtailed mealybugs. Lagunas-Solar et al. (2006) found that a more severe
physical treatment (110 to 10 kPa) alone required 6 to 12 h to achieve over 90% mortality of
Drosophila melanogaster. Hypoxia limits adenosine triphosphate (ATP) production by
eliminating environmental oxygen. Insects lower their rate of metabolism (and ATP
requirement) in response to this type of environment via a survival mechanism called
metabolic arrest. This coping mechanism cannot be maintained beyond a point where ATP
needed to run primary cell functions is not obtained; this is when hypoxia toxicity
commences (Hochachka, 1986).
In this study a 90-min exposure to 125 mg L-1 ethanol under low pressure resulted in
7–9% mortality of longtailed mealybug. Jamieson et al. (2003) found that a much longer 48-h
exposure to ethanol vapour from a 20–50% ethanol solution was required for 97–100%
mortality of lightbrown apple moth (Epiphyas postvittana) larvae. Ethanol toxicity effects are
a result of disorganization of the inner (hydrophobic) layer of the phospholipid bilayer of the
cell membrane, which consequently affects its fluidity. Ethanol also affects K+-stimulated pnitrophenyl phosphatase activity, which reduces the cell’s ability to produce ATP (Mazzeo et
43
al., 1988). Ethanol also reduces the thickness as well as disrupting the structural integrity of
the cell membrane (Gurtovenko and Anwar, 2009). Further studies are required to determine
if these cell membrane effects play a part in the toxicity of ethanol to longtailed mealybugs.
The percentage loading of the MSDD chamber in this study ranged from 8% to 10%,
as calculated based on the average volume of ‘Hass’ avocados. Ethanol appeared as both
vapour and mist during the chemical phase of MSDD treatments. Therefore, further research
on the effects of fruit loading is required in order to estimate commercially effective amounts
of ethanol.
44
3. Biochemical mechanisms of insecticidal efficacy of
MSDD
3.1 Overview
Due to its relative infancy, there is a lack of information on the biological
mechanisms of the insecticidal effect of MSDD. Proteomics tools such as 2-DIGE and
electrospray ionisation tandem mass spectrometry (ESI-MS/MS) were used to elucidate the
effect of MSDD on protein expression of 5th instar LBAM larvae. MSDD utilises the
combination of hypoxia, hypercabia and ethanol treatments. Hypoxia (and low pressure) was
shown to limit ATP production by reducing the availability of environmental oxygen
(Hochachka, 1986). Hypercarbia toxicity was attributed to its ability to inhibit ATP
production and oxidative phosphorylation enzyme, namely succinic dehydrogenase and
consequently results in accumulation of toxic wastes such as pyruvate and lactate (Mitcham
et al., 2006). Ethanol also works by disorganizing lipid bilayer, dehydrating and disrupting
phosphatise activity in relation to its role in ATP production pathway (Mazzeo et al., 1988).
The aim of this chapter was to understand the importance of each parameter of
MSDD in terms of its effect on insect physiology and biochemical functions using the
established MSDD protocol (Chapter 2). Fifth instar LBAM larvae were treated using
physical phase-only, chemical phase-only and full MSDD treatments. Analysis of protein
changes in haemolymph 5th intar LBAM larvae using 2-DIGE and ESI-MS/MS was then
carried out.
3.2 Materials and method (Barraclough et al., 2004; Knoch et al., 2010)
3.2.1 Chemicals
Solubilisation buffer. 7 M urea (BioRad, Hercules, CA, USA), 2 M thiourea
(Sigma), 40 mM Tris (Invitrogen, Carlsbad, CA, USA), 4% CHAPS {3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate}(BioRad), made up to 2 ml in water.
45
Immobilised pH gradient (IPG) buffer. 8 M urea, 2% CHAPS, 2% IPG DryStrip
buffer*(GE Healthcare, Uppsala, Sweden), trace of Bromophenol blue (Sigma, St Louis, MO,
USA), 375 mg DeStreak* reagent (GE Healthcare), made up to 2 mL in water. *trade secret
CyDyes sample buffer. 7 M urea, 2 M thiourea, 130 mM dithiothreitol (DTT)
(Sigma), 4% v/v CHAPS, 2% v/v IPG Buffer, made up to 1 mL in water.
CyDyes. 1 µL of stock solution in 4 µL dimethylformamide (DMF) (Sigma).
Gel running buffer. 3M Tris (pH 8.8-adjusted with concentrated hydrochloric acid
(HCl) (BDH, Darmstadt, Germany)), made up to 200 mL in water
SDS (sodium dodecyl sulphate) equilibration buffer. 50 mM Tris, 6 M urea, 30%
(w/v) glycerol (BDH), 2% (w/v) SDS (Applichem, Darmstadt, Germany), bromophenol blue
(trace), made up to 200 mL in water.
SDS tank buffer. 25 mM Tris, 192 mM glycine (Sigma), 0.1% SDS (made overnight
prior to use), made up to 700 mL in water.
10% PAGE gel. 10% (v/v) acrylamide solution (BioRad), 12.5% (v/v) running
buffer, 20% (v/v) sucrose solution (BDH), 0.005 M sodium thiosulphate (BDH), 0.05% (v/v)
ammonium persulphate (BioRad), 0.05% (v/v) Tetramethylethylenediamine (TEMED)
(BioRad). The gel solution was poured into the settling plates, overlaid with 50% (v/v)
isopropanol (BDH) and let sit in a humidity chamber overnight prior to use.
Colloidal coomassie (G250). 170 g ammonium sulphate (Sigma), 36 mL phosphoric
acid (BioRad), 1 g Coomassie G-250 (Invitrogen), 34% v/v methanol (BDH), made up to 1 L
in water.
Gel rehydration solution. 25 mM ammonium bicarbonate (Sigma) in 50% v/v
acetonitrile (BDH).
Trypsin solution. 0.1 mg. L-1 trypsin (Roche, Basel, Switzerland) in HCl.
46
Digestion solution. 8.6% (v/v) trypsin solution, 87.0% v/v ammonium bicarbonate
(BDH), 4.4% v/v acetonitile (BDH), made up to 50 mL in water
Peptide recovery solution. 5% v/v formic acid (BDH) in 50% v/v acetonitrile, made
up to 50 mL in water
3.2.2 Light brown apple moth (Epiphyas postvittana) haemolymph protein
extraction and preparation for labelling
Fifth instar LBAM larvae were obtained from a laboratory colony on artificial diet
(Singh, 1983; Clare et al., 1987) and maintained at 20  2°C with 60% RH, and a 16:8 h
light:dark cycle. Prior to MSDD treatments, larvae were transferred to plastic container (650
mL). MSDD treatments and subsequent mortality assessments were carried out in the same
manner as longtailed mealybug. In all experiments, one hundred 5th instar larvae were treated
for each replicate. There were 4 treatment groups, namely control (untreated), physical-phase
only treatment (PP) (30 min of physical phase, followed by 1 h of low pressure condition (10
kPa) without ethanol treatment), chemical phase only treatment (CP) (low pressure condition
(10 kPa) for 90 min with 125 mg. L-1 ethanol) and MSDD treatment with 125 mg. L-1ethanol
(Figure 3-1). Upon treatment, insect were held at 20°C in a 16:8 h light:dark cycle and three
days later, insects were recorded as live (movement) or dead (no movement) after being
prodded with a blunt instrument. Each treatment was replicated three times.
For haemolymph analysis, 10─15 larvae from each treatment were collected 1 h after
treatment. LBAM haemolymph was extracted from larvae by exsanguinations (100─200
µL). Two samples from each treatment group were collected. Each sample will be run in a
separate 2-DIGE gel (resulting in 8 gels in total). Approximately 10 mg of phenylthiourea
was added to haemolymph samples to inhibit tyronsinase activity which can lead to
melanisation of haemolymph (Phalaraksh et al., 1999). The sample was stored at -80°C
before being processed. Protein concentration measurement was carried out using Protein
Assay kit (Biorad), which was based on Bradford assay (Bradford, 1976), with bovine serum
albumin (BSA) as the standard. The calibration curve was constructed using 0, 0.05, 0.1, 0.2,
0.3, 0.4, 0.5, 0.75, 0.1 mg. L-1 BSA. Two hundred microlitres of Bradford reagent was then
added to standard and sample solutions and held at room (20°C) temperature for 15 min. The
47
absorbance was then read at 595 nm. The amount of protein in the sample was calculated
using the calibration curve.
The haemolymph samples were then subjected to 2-D Clean-Up procedure (GE
Healthcare, Uppsala, Sweden). Three hundred microlitres of precipitant* was added to 100
µL of haemolymph. The mixture was then vortexed and incubated on ice for 15 min. Three
hundred microlitres of co- precipitant* was subsequently added to the mixture. The mixture
was then centrifuged at 12 000 g for 5 min. The supernatant was then removed. Another 40
µL of co-precipitant was added to the pellet. The mixture was then rested for 5 min on ice.
Twenty five microlitres of deionized water was added to the pellet. One millilitre of chilled
wash buffer and 5 µL of wash additive were added to the pellet mixture. The mixture was
then vortexed for 20─30 s every 10 min for at least 30 min. The mixture was then centrifuged
at 12 000 x g for 5 min. Supernatant was then removed and the pellet was allowed to air dry.
The pellet was then suspended in 200 µL solubilisation buffer. The amount of protein in the
mixture was then determined using Bradford assay before being labelled with CyDyes. *trade
secret
48
Figure 3-1 Three different treatments that were used to treating 5th instar LBAM larvae. Physical phase-only treatment (PP; A), chemical phaseonly treatment (CP; B) and full MSDD treatment (MSDD; C).
49
3.2.3 Two-DIGE for proteins labelled with CyDyes
The CyDyes used in this work were Cys 2, 3 and 5. A microlitre of stock solution was
mixed with 4 µL of dimethylformamide (DMF). This working solution contained 200 pmol.
µL-1. The amount of dyes was then adjusted to the amount of protein in the samples; 1 µL
working solution for 50 µg protein. Protein samples from the control, treated (PP, CP and
MSDD) and pooled sample (control + PP + CP + MSDD- equal amount of protein from each
sample) larvae were labelled with Cys 3, 5 and 2, respectively. The mixtures were then
centrifuged briefly and incubated on ice in dark for 30 min. The reaction was stopped by
addition of 1 µL of 10 mM lysine. The procedure was duplicated for each sample (technical
replicate). The mixtures were then incubated on ice for 10 min in dark. An equal amount of
sample buffer was added to the solutions followed by incubation on ice in dark for another 10
min. The Cy-labelled samples that were separated in one gel were then pooled (Figure 3-2).
Figure 3-2 The working steps of CyDyes labelling from labelling the samples to pooled
samples. Each pooled sample contained 100 µg of total protein; 33.3 µg of internal standard,
33.3 µg of control and 33.3 µg of treated sample. I: Internal standard (contains equal
proportion of protein from PP only, CP only, MSDD and the 3 controls). P: PP treatment, C:
CP treatment and M: MSDD treatment.
50
3.2.4 Isoelectric focusing (IEF) (first dimension)
The IPG strips used for this work were 3─10 NL strips (GE Healthcare, Uppsala,
Sweden). Prior to sample loading, strips were rehydrated with IPG buffer (3─10 NL) (with
trace of bromophenol blue). The re-swelling tray was cleaned and loaded with 300 µL of IPG
buffer. The strips were then placed in the re-swelling tray. Paraffin oil (Shell Ondina 15
medicinal paraffin oil, Houston, Texas) was then added to ensure the strips were properly
rehydrated. The strips were rehydrated overnight at room temperature. MultiphorTM (GE
Healthcare, Uppsala, Sweden Life Sciences) system was used to run the first dimension. The
voltage gradient used was as Table 3-1. The procedure was carried out according to
ReadyStrip IPG Strip Instruction Manual (Biorad). The volume containing 100 µg of protein
was loaded into each strip through sample-cup loading at the cathodic end of the strips.
Table 3-1 Programme used to run the first dimension.
Step
Volts
µAmps
Watts
Volt-Hours
1
200
2
5
0.01
2
200
2
5
1.00
3
3 500
2
5
1.00
4
3 500
2
5
50.00
3.2.5 SDS PAGE (second dimension) and gel scanning
SDS PAGE gels were made 24 h prior to IEF process finished. After IEF, the strips
were then equilibrated for the second dimension run. Equilibration was a two step process; 12
min equilibration in 10 mL of SDS equilibration buffer I (SDS equilibration buffer + 0.10 g
dithiothreitol) followed by 6 min in 10 mL SDS equilibration buffer II (SDS equilibration
buffer + 0.40 g iodoacetamide). The volume of solution used in the equilibration step was
used for every 2 strips. The weight markers used for second dimension was Biorad Precision
Plus Protein Standard plugs. The strips and markers were then separated by weight using a
SDS-PAGE Protean II unit (Biorad) with the following power gradient programme; 900 V,
48 mA for 4.5─5.0 h (2 gels) and 900 V, 96 mA for 4.5─5.0 h (4 gels). Prior to separation
procedure, the tank of the Protean unit was filled with the tank buffer. Following the
51
separation, the Cygels were washed and rinsed in MilliQ water for 5 min on gentle shaker.
The gels were then scanned using TyphoonTM 9400 imager (GE Healthcare, Uppsala,
Sweden); the excitation wavelengths used were 520, 580 and 670 nm. When gels were
scanned satisfactorily, the gels were overstained with colloidal coomassie. Colloidal
coomassie staining was carried out to assist the manual identification of analysed spots for
ESI-MS/MS analysis. The gels were then wrapped in cellophane to dry and stored at room
temperature.
3.2.6 Image analysis using Delta 2D (Decodon)
Analysis of gel images using Delta 2D (Decodon, Griefswald, Germany) started off
by grouping the images into 5 groups; internal standard, control, PP only, CP only and
MSDD (Figure 3-3). The images were then linked to one another using ‘exact’ and ‘identical’
linkages. ‘Exact’ means the images were from the same gel and ‘identical’ means the images
were the replicate of the same treatment (but from different gels). The images were then
analysed and protein spots were ‘warped’ from one gel to another. After warping
satisfactorily, a ‘fused’ image’ from all 18 gels was then created. A ’fused’ image contained
the spots from all gel images which had been warped correctly to one another. The ‘fused’
image was then used to detect the protein spots across the gels- one way to ‘lock in’ the
position of spots and ensure that they do not differ across the gels. Spot detection parameters
were as follow: sensitivity: 20%, average spot size: 5, local background: 17. The process of
matching and detecting spots was also verified manually. Once the spot position had been
confirmed, the differential expression was then quantified across the gels- by division of the
‘treatment’ spots against the ‘control’ spots. T-test analysis was used to filter insignificant
spots (α =0.05). Up-regulation of ≥1.5 and downregulation of ≤0.8 of protein expression
compared to controls were considered as biologically important (Knoch et al., 2010).
52
A
B
D
C
Figure 3-3 The flow chart of protein image analysis by Delta 2D (A–D); A. Linking the groups of images, B. Warping and cleaning the image
by eliminating artefact spots, C. Statistical analyses of the resulting spots, D. Identification of significant spots prior to LC-MS analysis.
53
3.2.7 In-gel trypsin digestion of identified proteins for ESI-MS/MS
The identified protein spots from 2D analysis were excised from the PAGE gels (~1
3
mm ). A spot-free gel piece of the same size was also cut for a control digestion. One
hundred microlitres of MilliQ water was added to the pieces and left for 30 min to rehydrate
the gels and separated from the cellophane. The rehydrated gels were then separated from the
cellophane. Approximately 100 µL of gel rehydration solution I was added to each gel piece,
followed by incubation in a Thermomixer (20 x g at 22°C for 10 min). The wash/rehydration
step was repeated 3 times. The blue solution (coomassie) was then removed and this
wash/rehydration step was repeated 3 times. The gel particles were then dried in a vacuum
centrifuge (Speedyvac) (12000 x g for 10 min). The particles were then subjected to tryptic
digestion. Fifteen microlitres of digestion solution was added to each gel particle and
followed by incubation in the Thermomixer (4 x g, 37°C for 18 h). Following the digestion
process, 30 µL of peptide recovery solution was then added. The mixture was then sonicated
for 5 min followed with a quick centrifuge (12000 x g for 1 min). The supernatant was
transferred to a new tube. Two additional extractions were performed. The recovered peptides
were then concentrated in vacuum centrifuge (12000 x g for 10 min) to 20 µL and stored at 20°C before being subjected to ESI-MS/MS analysis.
3.2.8 ESI-MS/MS analysis of peptides
LC-MS (Liquid chromatography-mass spectrometry) analysis was carried out using
an LTQ (linear trap quadrupole) linear ion trap mass spectrometer fitted with a nanospray ESI
interface (ThermoQuest, Finnigan, San Jose, CA, USA) and coupled to an Ettan™ MDLC
(Multidimensional LC system) (GE Healthcare, Uppsala, Sweden). The MDLC was used in
the high-throughput configuration with two RPC (reverse phase chromatography) trap
columns 300 m ID (internal diameter) x 5 mm (Zorbax 300-SB C18) for on-line desalting
and sample clean-up, followed by two nanoscale RPC analytical columns 75 m ID x 150
mm, 3m, (LC Packings, San Francisco, CA, USA) for high-resolution separation. One set of
RPC trap/analytical columns was equilibrated while the second set separated the sample. The
mobile phases were A: 0.1% aqueous formic acid and B: 84% acetonitrile and 0.1% formic
acid. Two microlitre of the digested protein sample was injected and tryptic peptides were
separated at a flow rate of 280 nL. min-1 with a linear gradient from 0 to 60% B over 50 mins.
54
The nanospray interface was used with a 30m ID fused-silica standard coated PicoTipTM
(New Objective, Woburn, MA, USA) and voltage (1.6 kV) was applied via a liquid-liquid
junction at the base of the spray needle. The mass spectrometer was operated in the positive
ion mode and the mass range acquired was m/z: 300–1800. The heated capillary temperature
was set at 170 C. Data was acquired using a top 3 experiment in data-dependent mode with
dynamic exclusion enabled.
3.2.9 Database Searching and Data Interpretation
MS and MS/MS data were analysed using TurboSEQUEST, a computer programme
that allows the correlation of experimental data with theoretical spectra generated from
known protein sequences (Eng et al., 1994; Yates et al., 1995). Spectra were searched against
NCBI and PFR insect databases, with differential oxidation modification of 16 Da to Met was
selected and a static carboxyamidomethylation modification of 57 Da to Cys was selected.
The criteria used for a positive peptide identification for a doubly-charged peptide were a
correlation factor (XCorr) >2.0, a delta cross-correlation factor (dCn) >0.1 (indicating a
significant difference between the best match reported and the next best match) and a high
preliminary scoring (Sp). For triply-charged peptides the correlation factor threshold was set
at 2.5. All matched peptides were confirmed by visual examination of the spectra and BLAST
searches.
3.3 Results
3.3.1 Insect mortality and gross physical changes
The mean percentage mortalities of LBAM larvae exposed to the four treatments are
given in Table 3-2. None of the untreated larvae died. PP, CP and MSDD treatments recorded
approximately 3%, 100% and 100% mortality (assessed 3 d after treatments), respectively.
Melanization of the larvae treated with CP and MSDD treatments started to be observed 24 h
after treatment (Figure 3-4, A and B). At 72 and 96 h considerable melanization was evident
in the CP and MSDD treated larvae (Figure 3-4, C and D), with greater melanization in
MSDD treatments than CP. No melanization was observed in control and PP samples even
after 72 h. The larvae treated with PP and untreated controls began pupating after three days.
55
3.3.2 Differential expression of proteins between the larvae treated with different
treatments
There were 1868 protein spots detected with 20 differentially expressed spots. Sixteen
identifiable proteins were observed to be differentially expressed between treatments (Figures
3-5 and 3-6, Table 3-3). PP treatment increased the expression of proteins associated with
lipid transport and immune response (Apolipophorin-I and III like proteins), storage protein
(arylphorin), melanization regulation (alaserpin), apoptosis regulation (gelsolin),
metamorphosis/development regulation (juvenile hormone binding protein), iron
transport/storage protein (ferritin light chain) and lipid metabolism (transmembrane like-195
protein). CP treatments increased the expression of Apolipophorin-III like protein and
ommochrome binding protein (tryptophan metabolism). MSDD treatments elevated the
expression of ATPase subunit alpha-2 (energy metabolism), serine protease like-2
(melanization regulation), 23 kDa haemolymph protein, arylphorin and C-type mannose
receptor. There were 4 spots that could not be identified, possibly due to the inability to
manually spot pick (due to low intensity of the coomassie stain) and the database used for
identification which was derived from midgut and antennal tissue instead of haemolymph
(Simpson et al., 2007a; Jordan et al., 2008). MSDD up-regulated spots a and b and downregulated spot c, while PP treatment up-regulated spots b, c and d (Figure 3-6, Table 3-3).
Table 3-2 Mean percentage mortality of 5th instar light brown apple moth (Epiphyas
postvittana) larvae 3 d after treatments (n =3).
Treatments
Total insects
Mortality (±SE) (%)
Control
155
0 (0)
Physical phase only (PP)
146
2.87 (1.92)
Chemical phase only (CP)
145
100 (0)
MSDD
145
100 (0)
56
A
B
57
C
D
Figure 3-4 Fifth instar light brown apple moth (Epiphyas postvittana) larvae treated with 3 different treatments at 0 h (A), 24 h (B), 72 h (C) and
96 h (D). Melanisation started occurring after 24 h. The control and PP treated larvae started to pupate at 72 d and 96 h, respectively.
Melanisation was more pronounced in MSDD treated larvae than CP treated larvae.
58
Figure 3-5 A typical gel scanning result (false colour overlay) from the experiments using
CyDyes. Cy3 (control), Cy5 (treatment-MSDD) and Cy2 (pooled sample) appear green, red
and blue, respectively. White spots represent proteins that are equally expressed in all 3
samples. The intensity of the spots correlates with the expression level of proteins.
59
Figure 3-6 A typical gel from the experiments with pI and molecular weight annotation
showing protein spots of interest across different treatments.
60
Table 3-3 Proteins that were differentially expressed in the haemolymph of larvae treated with PP, CP and full MSDD treatments when
compared to controls.
Treatments
PP-only
Spot
Accession
no
no
71
24144
Genbank no
Protein ID
Calculated
Apparent
Number of unique
Sequest
Fold
MW (kDa)
pI
peptides matched
Pro
change
EV810725
Gelsolin
213.36
7.2
6
6.11e-12
2.5
EV810725
Gelsolin
213.36
7.1
2
4.60e-04
2.8
-
Ferritin light chain
155.46
7.1
2
1.42e-04
2.8
-
Arylphorin
188.89
4.6
8
2.22e-15
1.6
-
Juvenile hormone
65.19
4.6
4
2.66e-14
1.6
77.26
7.3
3
1.28e-08
3.2
(PFR)
PP-only
8
24144
(PFR)
PP-only
8
UnP 2
(PFR)
PP-only
10
UnP
(PFR)
PP-only
10
UnP
(PFR)
PP-only
11
UnP
(PFR)
binding protein
-
Apolipophorin-1
like protein
61
PP-only
11
22733
EV807025
Alaserpin
195.24
7.3
3
2.07e-06
3.2
AAG34698
Apolipophorin-3
20.42
6.9
8
1.11e-16
1.6
20.42
6.3
7
2.98e-11
2
60.64
5.0
4
3.88e-09
4.3
60.64
4.8
4
4.24e-08
2.8
(PFR)
PP-only
12
gi|113454
17
like protein
(NCBI)
PP-only
13
UnP
-
(PFR)
PP-only
15
455
like protein
EV803362
(PFR)
PP-only
16
455
Apolipophorin-3
Transmembrane195 like protein
EV803362
(PFR)
Transmembrane195 like protein
PP-only
c
-
-
Unidentified
-
7.2
-
-
1.6
PP-only
b
-
-
Unidentified
-
5.3
-
-
3.5
PP-only
d
-
-
Unidentified
-
5.3
-
-
2.2
62
CP-only
5
UnP
-
(PFR)
CP-only
14
gi|113454
Ommochrome
63.06
4.5
3
8.76e-07
3.7
20.42
5.1
3
2.62e-08
2.4
168.85
5.4
11
6.66e-15
2.5
168.85
5.3
13
1.00e-30
3.8
168.85
5.2
12
5.55e-15
5
168.85
5.1
7
4.44e-15
2.6
291.81
5.1
4
3.50e-11
2.6
binding protein
AAG34698
17
Apolipophorin-3
like protein
(NCBI)
MSDD
1
UnP
-
subunit α-2
(PFR)
MSDD
2
UnP
-
3
UnP
-
4
UnP
-
4
UnP
(PFR)
ATP synthase
subunit α-2
(PFR)
MSDD
ATP synthase
subunit α-2
(PFR)
MSDD
ATP synthase
subunit α-2
(PFR)
MSDD
ATP synthase
-
C-type mannose
receptor
63
MSDD
6
23330
EV810024
(PFR)
MSDD
9
UnP
9
UnP
106.27
5.8
6
5.14e-09
1.8
72.60
5.2
5
2.85e-10
2.2
protein
-
(PFR)
MSDD
Serine protease-like
Haemolymph 23
kDa protein
-
Arylphorin
188.89
5.2
4
2.52e-06
2.2
(PFR)
1
2
MSDD
a
-
-
Unidentified
-
7.1
-
-
2.7
MSDD
b
-
-
Unidentified
-
5.3
-
-
3.6
MSDD
c
-
-
Unidentified
-
7.2
-
-
0.6
Protein spot number from the gel (Figure 3-6).
Unpublished sequences from Plant & Food Research insect database.
64
3.4 Discussion
The mortality data indicated that chemical phase is the most important component in
MSDD treatment of LBAM larvae. This is at variance to a previous study on the longtailed
mealybug which found that both physical and chemical phases of MSDD, i.e. complete
MSDD treatment, were required to achieve high mortality (Chapter 2, section 2.3). While
both CP and MSDD resulted in complete mortality, the symptoms of melanization were
greater in MSDD than in CP, indicating that the PP also played a part in effectiveness of the
treatment.
Using 2-DIGE we are able to detect changes in protein levels associated with the
different treatments. The fact that the majority of protein spots were unchanged between the
control and the treatments illustrates the reliability of the method despite the possibility that
proteins could be modified by intermediate products of the melanization pathway. The PP
treatment, despite having a very low mortality, changed levels of a number of proteins not
altered in other treatments. It is possible that these proteins are more involved in combating
the effects of the treatment than the effects causing mortality. The different treatments had
different effects on the haemolymph proteome, yet it has been possible to postulate the role of
some of these on the effectiveness of the treatments.
3.4.1 MSDD disrupts melanization pathway
The physical appearance and protein analyses suggest that the insecticidal efficacy of
MSDD may be primarily due to its ability to disrupt the regulation of melanization. A
previous study by Lagunas-Solar et al. (2006) also showed occurrence of excessive
melanization in MSDD treated Drosophila larvae. Melanization is an important part of
immune response in most arthropods including insects (Kanost, 1999; Gettins, 2002;
Cerenius and Söderhäll, 2004). It is triggered by injury to cuticle or detection of microbial
cell wall components such as peptidoglycan, β-1,3 glucan or lipopolysaccharide which
consequently induces serine protease cascade (Wilson et al., 2001). Serine protease cascade
cleaves the pro-form of prophenoloxidase-activating enzyme to its active form which
consequently cleaves pro-phenoloxidase to phenoloxidase. Phenoloxidase then catalyzes the
oxidation of mono- and diphenols to toxic orthoquinones which are polymerized nonenzymatically to melanin (De Gregorio et al., 2002). The serine protease cascade must be
65
tightly controlled by serine protease inhibitors (serpins) to avoid a systemic activation that
can lead to over melanization and result in cytoxicity and mortality (De Gregorio et al., 2002;
Scherfer et al., 2008; Tang, 2009). MSDD treatments were shown to up-regulate serine
protease-2 like protein with no detection of serpin up-regulation which consequently led to
excessive melanization after three days. The proteomics suggest that MSDD promoted the
serine protease cascade while preventing the expression of critical regulatory mechanism of
melanization. Tang et al. (2008) demonstrated that in the absence of melanization regulatory
mechanism, small injuries caused over-melanization and inevitably death. Therefore it further
supports the notion that over-melanization is the result of MSDD treatments, not just an
artefact (i.e. melanin expression occurred not because the larvae were moribund).
PP treatments up-regulated the expression of alaserpin, but with no evidence of upregulation of serine proteases. CP treatment was not shown to have any effects on serine
protease cascade in melanization pathway; however, the physical appearance suggested
otherwise. CP was shown to up-regulate ommochrome binding protein which is involved in
tryptophan metabolism (Martel and Law, 1991; Nappi and Christensen, 2005). Tryptophan is
one of the substrates for melanin production (Nappi and Christensen, 2005). It was also
thought that CP might affect unidentified melanin associated proteins.
3.4.2 Other proteins affected by different phases of MSDD treatments
During the physical phase, insects were exposed to hypercarbic and hypoxic condition
within 12 min (Chapter 2, section 2.3). This condition was then held for further 18 min before
a further hour of low pressure. This treatment condition was shown to increase the expression
of two proteins associated lipid transport and storage (ApoLp-I and III-like proteins). CP was
also shown to up-regulate an ApoLp-III like protein. The up-regulation of these lipidassociated proteins may be a protective response against desiccation. Hypercarbia, low
pressure and ethanol had been shown to impart stresses related to water loss (Haapala, 1973;
Friedlander and Navarro, 1979b; Navarro and Calderon, 1979). A study on desiccationresistance in Drosophila showed that individuals that were desiccation-resistant tended to
have higher lipid content compared to susceptible (Harshman et al., 1999). Apart from the
roles of lipid transport and storage protein, ApoLp-III is also associated with immune
response of insects (Wiesner et al., 1997; Adamo et al., 2008; Zdybicka-Barabas and
Cytryńska, 2011). ApoLp-III has been shown to have antimicrobial activity in lepidopteran
66
insects such as Galleria mellonella and Manduca sexta (Dettloff et al., 2001) and also
observed to be stress-induced in crickets (Gryllus texensis) (Adamo et al., 2008). PP upregulated ferritin, an iron storage and transport protein, which was involved in immune
response in Drosophila either as antimicrobial peptides or inducers (Vierstraete et al., 2004b;
Vierstraete et al., 2004a; Arosio et al., 2009).
PP treatments also increased the expression of proteins associated with growth and
development; arylphorin (a hexamerin storage protein) and juvenile hormone binding protein
(Telfer et al., 1983; Pan and Telfer, 1996; Hakim et al., 2007; Zalewska et al., 2009). Juvenile
hormone binding protein has been shown to facilitate the transport of ApoLp and arylphorin
(Zalewska et al., 2009). The roles of these proteins in relation to stress conditions related to
PP, i.e hypoxia, hypercarbia and low pressure, are poorly understood.
Under PP, there was increased expression of gelsolin, an actin regulatory protein,
which has been shown to inhibit apoptosis (Stella et al., 1994; Ohtsu et al., 1997;
Kwiatkowski, 1999). Gelsolin was shown to be up-regulated in hypoxic condition as part of
survival mechanism in mouse fibroblasts cell culture (Greijer et al., 2006). The up-regulation
of storage, immune and apoptosis related proteins is likely to be the contributing factor to
high survival rate in larvae treated with PP treatments.
MSDD up-regulated C-type mannose receptor which is involved in innate immune
response of insects (Van Die et al., 2004). ATPase subunit alpha-2 (energy metabolism) and a
23 kDa haemolymph protein were also up-regulated by MSDD. The roles of these proteins in
relation to MSDD were not understood. ATPases were shown to be down-regulated in
hypoxic conditions to offset low oxygen tension (Li and Brouwer, 2009; Staples and Buck,
2009)- an opposite response compared to our results, suggesting that MSDD treatments
might affect energy balance of the larvae differently compared to hypoxia alone.
In conclusion, MSDD has been shown to be effective against 5th instar LBAM larvae.
The chemical phase was the most important part in terms of insecticidal efficacy, with 100%
mortality recorded (the same as full MSDD treatment). LBAM larvae treated with full MSDD
treatments were shown to have higher melanization rate than CP-only treated larvae. The
MSDD mode of action was thought to be attributed to its ability in disrupting melanization
pathway by inducing serine protease cascade while preventing the up-regulation of its
67
regulatory mechanism (Figure 3-7). PP treatment without the CP phase found in MSDD
resulted in the up-regulation of a number of storage, immune system and apoptosis related
proteins which may explain the low mortality of this treatment.
Figure 3-7 The melanisation pathway of arthropod. Red and green arrows indicate the
downregulation and upregulation of serpin and serine protease by MSDD, respectively,
which consequently increases the melanin synthesis (black arrow) (modified from Cerenius
and Söderhäll, 2004).
68
4. The effect of MSDD on fruit physiology and quality
4.1 Overview
Low O2 and high CO2 conditions in MSDD treatments are thought to have similar
condition to MA/CA storage. These conditions have been shown to reduce metabolic rate of
the fruit and reduce the ripening rate of fresh produce by delaying of ethylene biosynthesis
onset (Zhen-guo et al., 1983; Bangerth, 1984; Kader et al., 1989; Gorris and Peppelenbos,
1992). Ethanol treatment in the chemical phase of MSDD is thought to be the most
influencing factor in affecting the physiology of fresh commodity, based on the length of the
treatment and high concentration during MSDD treatments. Ethanol treatments have either
ripening promoting or inhibiting effect depending on the commodity and maturity (Kelly and
Saltveit, 1988; Ritenour et al., 1997). Acetaldehyde biosynthesis as the result of ethanol
treatment is the causal agent in ethanol-induced effects (Beaulieu et al., 1997).
Lagunas-Solar et al. (2006) showed that fruit that have ‘strong’ texture such as citrus
and banana had high tolerance towards MSDD and ‘weak’ textured fruit such as raspberry,
strawberry and blackberry had low tolerance towards MSDD, with pronounced textural
damage (juicing) after MSDD treatments. There was no comprehensive data on physiology
and quality of fruit treated with MSDD. Therefore, in this chapter, the effect of MSDD on 3
major NZ export commodities; kiwifruit, apple and avocado.
4.2.1 ‘Hass’ avocado (Persea Americana)
During 2009–2010, early (November 2009) and late season (February 2010) unripen
‘Hass’ avocados were harvested from three commercial orchards in South Auckland, New
Zealand. After harvest, fruit were held at 20°C overnight prior to MSDD treatments.
Approximately 80–100 fruit from each orchard were treated at each time, and each treatment
was replicated three times (i.e. three separate treatment runs). MSDD procedure used was 30
69
min of physical phase (100 ±10 kPa), followed by 1 h of chemical phase with 371 mg. L-1 at
10 kPa. The volume of the loading was 8–10% v/v of the MSDD chamber, calculated based
on the average volume of the avocados. Volume of the fruit was measured by immersing the
fruit in a water-filled container. The volume of the water displaced by the immersion was
recorded as the volume of the fruit.
Early season ‘Hass’ avocado. After MSDD treatment, 30 fruit from each replicate
run were held at 20°C and 25°C until ripe for eating. The higher temperature of 25°C was
chosen because it was shown to induce higher incidence of rots (Hopkirk et al., 1994).
Firmness was determined using an Anderson firmometer (Anderson Manufacturing and
Toolmaking, Arataki, New Zealand), as described by White et al. (2009). Five fruit per
replicate were weighed before and after treatment and during the ripening period at 20°C, to
determine mass loss. When fruit had reached a fully ripe stage (108 on Firmometer scale), it
was assessed for disorders based on the International Avocado Quality Manual (White et al.,
2009). The disorders were assessed using a 9 -point scale (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5), which
corresponded to 0, 5, 10, 15, 25, 33, 50, 75 and 100% of the fruit surface area being affected,
respectively. Data were expressed as% incidence, which is the proportion of fruit with any
expression of the disorder (i.e. a rating of ≥0.5). A fruit was considered ‘sound’ when the
severity of disorders did not exceed 15% of the surface area (White et al., 2009).
Late season ‘Hass’ avocado. After MSDD treatments, 30 fruit from each replicate
run were held at 20°C and 25°C until ripe for eating. Another 30–40 fruit from each replicate
run were stored at 5.5°C for 6 weeks to assess the effect of MSDD on long-term storage of
‘Hass’ avocado (particularly on long-term storage disorders such as diffuse flesh
discoloration). After 6 weeks of cold storage, fruit were ripened at 20°C. Five fruit were
weighed before and after treatment and during the ripening period at 20°C to determine mass
loss.
4.2.2 ‘Hayward’ kiwifruit (Actinidia deliciosa) and ‘Hort 16A’ kiwifruit
(Actinidia chinensis)
First season fruit. Approximately 1 020 kiwifruit each of ‘Hayward’ and ‘Hort16A’
cultivars (from one grower) were obtained from a commercial fruit supplier in South
Auckland, New Zealand in June 2010. The fruit had been harvested and coolstored at
70
approximately 0–2°C for 5 weeks. Kiwifruit were held overnight at 20°C prior to treatment.
Half of the fruit were untreated controls and the other half were MSDD treated.
Approximately, 170 fruit of each cultivar were MSDD treated (physical phase of 30 min
using CO2 as a ballast gas, followed with 60 min of chemical phase with 371 mg. L-1 of
ethanol). The volume of the loading was 7–8% v/v of the MSDD chamber, calculated based
on the average volume of the kiwifruit (volume measurement was carried out the same way
as in avocado). Upon treatment, half of the fruit were coolstored for 16 weeks at 0–0.5°C and
the other half were shelf-life tested. The untreated controls were also divided according to the
MSDD treated fruit. Table 4-1 shows the first season kiwifruit experimental design.
Table 4-1 Experimental design for first season kiwifruit.
Treatments
‘Hayward’
‘Hort 16A’
Control
MSDD treated
Control
MSDD treated
Non-coolstored
170 x 3 rep
170 x 3 rep
170 x 3 rep
170 x 3 rep
Coolstored for
170 x 3 rep
170 x 3 rep
170 x 3 rep
170 x 3 rep
16 weeks
Respiration rate and volatiles (ethylene, ethanol and acetaldehyde) production rates of
5 individual fruit from each replicate of control and MSDD treatment were measured during
every 2 d during shelf-life. Firmness was measured using Aweta® acoustic firmness
measurement (AFS, Nootdorp, the Netherlands) with a setting of microphone gain and tick
power of 80 and 16, respectively. The conversion formula of Acoustic firmness to N
according to Feng et al. (2010) is shown below:
(4-1)
Soluble solid content (SSC) and pH level were measured at the end of shelf-life using
an ISFET pH meter (Model IQ120, Scientific Instruments, Carlsbad, CA, USA) and a digital
refractometer (Atago, model PAL-1, Tokyo, Japan) (Johnston et al., 2009). Quality
assessment of rots and other apparent disorders were also made. The coolstored fruit were
also subjected to the same shelf-life and analysis procedure after the storage period.
71
Second season fruit. Approximately 1 080 kiwifruit each of ‘Hayward’ and
‘Hort16A’ cultivars (one grower) were obtained from the same commercial supplier in April
2011. The fruit had been harvested and coolstored at approximately 0–2°C for a week. The
fruit were treated the same way as the first season fruit. The ethanol concentration was
increased to 1113 mg. L-1. The higher ethanol concentration was used to compensate the
effect of loading on insect mortality as the worst case scenario (see Chapter 7). The volume
of the loading was 5–6% v/v of the MSDD chamber, calculated based on the average volume
of the kiwifruit. The treated and control fruit were divided into 3 categories; non-coolstored,
coolstored for 4 weeks and 8 weeks. At the end of each coolstorage period, the fruit were
subjected to shelf-life testing; physiology and quality measurements as in the first season.
The colour of the flesh was also measured using a chromameter (Minolta, model CR-300,
Tokyo, Japan). Table 4-2 shows the experimental design for 2nd season of kiwifruit.
Table 4-2 Experimental design for second season kiwifruit.
Treatments
‘Hayward’
‘Hort 16A’
Control
MSDD treated
Control
MSDD treated
Non-coolstored
60 x 3 rep
60 x 3 rep
60 x 3 rep
60 x 3 rep
Coolstored for
60 x 3 rep
60 x 3 rep
60 x 3 rep
60 x 3 rep
60 x 3 rep
60 x 3 rep
60 x 3 rep
60 x 3 rep
4 weeks
Coolstored for
8 weeks
4.2.3 ‘Cripps Pink’ apple (Malus domestica)
‘Cripps Pink’ apples were harvested from an orchard in Hawke’s Bay, New Zealand
in March 2010, and cool stored at 0.5°C for 1 d prior to treatments. The fruit were then held
at 20°C for overnight before being treated. Approximately 100 fruit were treated at each time,
and each treatment was replicated three times (i.e. three separate treatment runs). The volume
of the loading was 8–12% v/v of the MSDD chamber, calculated based on the average
volume of the apples. After being treated, 15 fruit from each replicate were held at 20°C for 7
d, while the remaining fruit were stored at 0.5°C for 16 weeks. Respiration rate, ethanol and
acetaldehyde production of 5 fruit from each replicate were then analysed as shown in 4.2.4
72
and 4.2.7. Internal ethylene concentration, instead of ethylene production, of 15 fruit from
each replicate was also measured as in section 4.2.5. Firmness was then measured according
to Johnston et al. (2009) using a Texture Analyser TAXT plus (Stable Microsystems, UK)
fitted with a 7.9 mm Effegi penetrometer probe. The speed of the probe driven into the flesh
was 4 mm. s-1 to a depth of 9 mm. The maximum force recorded was considered as the flesh
firmness. After being coolstored for 16 weeks, the same assessment was carried out with the
same number of fruit. Quality assessment of background skin colour, SSC and pH level was
carried out using similar instruments as in kiwifruit. Background skin colour was measured
using the Minolta chromameter with 2 readings on the blush-free area of skin. Disorder
incidence, such as superficial scald, internal browning and greasiness, was rated. Superficial
scald incidence was measured as slight (0–25% surface area affected), moderate (25–50%)
and severe (>50%). Internal browning and greasiness incidence was measured as percent
incidence without any severity scale.
4.2.4 Respiration rate (apple, kiwifruit and avocado)
Respiration rate was measured on 5 individual fruit from each replicate run. Weighed
fruit were sealed in a 1.2-L plastic container equipped with a septum and held for 1 h at 20°C.
A 1-mL headspace sample was removed and CO2 was measured using the GC system (see
section 2.2.4).
4.2.5 Ethylene production measurement (kiwifruit and avocado)
Ethylene production was measured on 5 individual fruit from each replicate run.
Weighed fruit were sealed in a 1.2-L plastic container equipped with a septum and held for 1
h at 20°C. A 1-mL headspace sample was removed and ethylene was measured using the GC
system. The GC is fitted with a flame ionization detector set at 140°C with H2 and air flow
rates of 0.5 and 5 mL. s-1, respectively. For separation and quantification of ethylene, the GC
is equipped with an activated alumina column set at 100°C using nitrogen as the carrier gas at
0.5 mL. s-1 and a Hewlett Packard integrator (model 3390A) that was calibrated with external
ethylene standards (1 uL. L-1) prior to usage.
73
4.2.6 Internal ethylene concentration measurement (apple)
Internal ethylene concentration was measured in 15 individual fruit from each
replicate run and control for 7 d on non-stored fruit. A 1-mL sample was drawn from core
cavity of fruit and ethylene concentration was measured using the GC system for ethylene
(Johnston et al., 2009) (see section 4.2.5).
4.2.7 Ethanol and acetaldehyde measurement
Ethanol and acetaldehyde production was measured on 5 individual fruit from each
replicate run. Weighed fruit were sealed in a 1.2-L plastic container equipped with a septum
and held for 1 h at 20°C Ethanol and acetaldehyde were measured using Phillips PU4500 GC
(Pye Unichem, Cambridge, UK) equipped with a flame ionization detector (FID) and N2 as
carrier gas. A packed stainless steel column (2 m x 2 mm ID,Carbograph 1 AW 20, 80/100)
was set isothermally at 180°C. Standard gases of 50 µL. L-1 of ethanol and 25 µL. L-1 of
acetaldehyde (BOC gases NZ) were used for calibration.
4.2.8 Statistical analyses
Analysis of variance on quality data was carried out using Origin 7.5 (OriginLab,
Northampton, MA) and means were separated post hoc using Duncan’s New Multiple Range
Test and significant level was tested at 95% confidence limit (P <0.05). Grower effect and
treatment x grower interaction on section 4.3.1 were analyzed using two-way ANOVA.
Statistical analyses of early and late season fruit were carried out separately.
74
4.3 Results
4.3.1 Avocado
Generally, avocado respiration rate and ethylene production were not significantly
affected by MSDD treatments (Figures 4-1 and 4-2). MSDD treatment decreased the peak
ethylene production for orchard 3 (early season; Figures 4-2A v. B). MSDD treatment also
appeared to result in a synchronisation of ethylene production of late harvest fruit (Figures 42C v. D). MSDD treatments were shown to reduce variability of ethylene production of late
season fruit (Figure 4-2D). No difference in weight loss was observed in treated and control
fruit (Figure 4-3).
MSDD treatments generally did not reduce the quality of early and late season ‘Hass’
avocado. The quality parameters that were not significantly affected were the incidence of
stem and body rots, vascular browning, flesh greying and proportion of sound fruit (Table 43). One novel physical damage symptom was observed after MSDD treatment, namely
damage to the stem peduncle (or ‘button’) due to ethanol (Figure 4-4). MSDD treatment was
shown to have no effect on external appearance (no noticeable damage to skin even after
ripening) or weight loss (data not shown). MSDD-treated early season fruit, when ripened at
25°C, had higher incidence of vascular browning compared with untreated controls. Neither a
grower effect nor an interaction between grower and treatment was found (P >0.05).
75
Figure 4-1 Carbon dioxide production (mg. kg-1. s-1) (mean ±SE, n=3) of early (A and B) and
late (C and D) season ‘Hass’ avocado from three orchards, untreated (a and c) and MSDD
treated (B and D), held at 20°C.
76
Figure 4-2 Ethylene production (µg. kg-1. s-1) (mean ±SE, n=3) of early (A and B) and late
(C and D) season ‘Hass’ avocado from three orchards, untreated (A and C) and MSDD
treated (B and D), held at 20°C.
77
Figure 4-3 Cumulative weight loss (mean ±SE, n=3) of ‘Hass’ avocado.
Figure 4-4 Physical appearance of ‘Hass’ avocado straight after MSDD treatment (left) v.
untreated control (right). Browning of the peduncle was observed on the MSDD treated fruit
(arrow).
78
Table 4-3 Ripe-fruit quality of early (November 2009) and late season (February 2010) ‘Hass’ avocado exposed to 90 min MSDD treatments
with 371 mg. L-1 ethanol and in air (untreated controls) (n=3).
Treatments
Rots
Vascular
Flesh greying
Soundness (%)
Days to ripe
Stem (%)
Body (%)
browning (%)
(%)
20°C air control
65.3a1
18.0a
39.1ab
0
95.5a
10.3a
25°C air control
46.9a
28.1a
20.3ac
0
96.9a
9.0a
20°C MSDD treated
79.0a
15.4a
54.5ab
0
94.2a
10.2a
25°C MSDD treated
80.9a
20.5a
71.5b
0
81.3a
8.7a
20°C air control
85.9a
25.9a
29.3a
0
89.6a
11.8a
25°C air control
94.0a
26.2a
48.8a
0
80.7a
10.8a
5.5°C for 6 weeks air
98.3a
39.1ab
85.5b
72.7a
49.4ab
7.4b
20°C MSDD treated
89.4a
36.3ab
43.3a
0
83.0a
11.6a
25°C MSDD treated
90.5a
38.1ab
46.4a
0
69.5ab
10.4a
5.5°C for 6 weeks MSDD treated
92.6a
68.4b
81.6b
82.5a
20.2b
7.4b
Early season
Late season
1
Values within a column followed by the same letter are not significantly different (P >0.05).
79
4.3.2 Kiwifruit
4.3.2.1 First season
No significant difference was observed in terms of SSC and expressed juice pH for
both coolstored ‘Hayward’ and ‘Hort 16A’ fruit (Table 4-4). MSDD treatments increased rot
incidence by more than 2 and 4.5-fold for ‘Hayward’ and ‘Hort 16A’ fruit respectively. In
addition, MSDD treated ‘Hort 16A’ fruit developed a water-soaked, appearance (flesh
breakdown) after being coolstored for 16 weeks (Figure 4-5).
The respiration rate of non-coolstored ‘Hayward’ fruit treated with MSDD was
consistently higher than control fruit (Figure 4-6a). During the 7 d shelf-life analysis,
respiration rate was between 0.025 and 0.032 mg. kg-1. s-1 for the treated fruit, compared to
0.017 and 0.018 mg. kg-1. s-1 for the control fruit. Whereas treated ’Hort 16A’ fruit did not
show any significant difference compared to controls (Figure 4-6a). There was a marked
increase in respiration rate at day 7 for control ‘Hort 16A’ fruit (none observed in treated
fruit). Ethylene production of treated ‘Hayward’ fruit increased from from day 1 to day 6, and
then decreased by day 7 (Figure 4-6c). There was no increase in ethylene production
observed in control fruit. An opposite trend was recorded in ‘Hort 16A’ fruit; ethylene
production increased steadily and peaked at day 7 for control fruit. No ethylene production
was measurable in the treated fruit.
MSDD treatments caused a dramatic increase in ethanol and acetaldehyde production
(especially from day 3); from 35.00–222.46 and 0–20.97 ng. kg-1. s-1 after 7 d for ethanol and
acetaldehyde, respectively (Figures 4-7a and b). The softening rate of treated fruit was also
higher compared to control fruit; the Aweta index dropped from 39.5 to 25.2 (106 Hz2 kg2/3)
and 39.0 to 28.4 (106 Hz2 kg2/3) for treated and control fruit, respectively (Figure 4-8a). The
Aweta index of 39.0, 28.0 and 25.0 (106 Hz2 kg2/3) equates to ~56.3, 29.0 and 23.4 N,
respectively.
After storage at 0.5°C for 16 weeks, treated fruit had lower CO2 production compared
to control fruit (although it levelled out after day 9; Figure 4-6b). There was no significant
difference in ethylene production, although the control fruit reached an earlier climacteric
peak (Figure 4-6d). No ethanol and acetaldehyde production could be detected in control
80
fruit. Ethanol and acetaldehyde production in treated fruit were not detected until day 9 and
13, respectively. The increase of production was from 4.80–64.85 and 0–12.61 ng. g-1. s-1,
for ethanol and acetaldehyde, respectively. During 16 weeks of coolstorage, the softening of
treated fruit was delayed compared to control fruit. The Aweta index of treated fruit dropped
from 39.5 to 15.7 (106 Hz2 kg2/3) compared to 39.0 to 10.7 (106 Hz2 kg2/3) for control fruit
(Figure 4-8). An Aweta index of 15.0 and 10.0 (106 Hz2 kg2/3) equates to ~11.3 and 8.2 N,
respectively.
Table 4-4 Kiwifruit quality exposed to 90 min MSDD treatments with 371 mg. L-1 ethanol
and in air (untreated control), after being stored at 0°C and 0.5°C (16 weeks) for ‘Hayward’
and ‘Hort 16A’ kiwifruit, respectively (n=3).
Treatments
Rot
High
Flesh
incidence
severity rot
breakdown
(%)
incidence
(%)
‘Hayward’
Brix (±SE)
pH (±SE)
(%)
Control
23.71a
0a
0a
15.35 (0.20)a
4.33 (0.08)a
MSDD
54.07b
0.74b
0a
14.99 (0.22)a
4.43 (0.08)a
Control
7.80a
2.83a
0a
16.89 (0.31)a
5.19 (0.04)a
MSDD
31.75b
4.95b
27.43b
15.41 (0.45)a
5.14 (0.05)a
treated
‘Hort 16A’
treated
81
A
B
Figure 4-5 A. Flesh breakdown (water-soaked appearance) in MSDD treated ‘Hort 16 A’
kiwifruit. B. Normal fleshed ‘Hort 16A’ kiwifruit.
82
Figure 4-6 Respiration rate (mg. kg-1. s-1, mean ±SE) (A and B) and ethylene production (ng.
kg-1. s-1, mean ±SE) (C and D) of first season ‘Hayward’and ‘Hort 16A’ kiwifruit, noncoolstored (A and C) and coolstored for 16 weeks (B and D), n=3.
83
Figure 4-7 Ethanol (A and B) and acetaldehyde (C and D) (ng. kg-1. s-1, mean ±SE)
production of first season ‘Hayward’ and ‘Hort 16A’ kiwifruit, non-coolstored (A and C) and
coolstored for 16 weeks (B and D), n=3.
84
Figure 4-8 Acoustic firmness (Hz2 kg2/3, mean ±SE) of first season ‘Hayward’ and ‘Hort
16A’ kiwifruit. Firmness during coolstorage (A and C) and firmness of non-coolstored fruit
(B and D), n=3.
85
4.3.2.2 Second season
Hue angle (flesh), brix, pH level of both ‘Hayward’ and ‘Hort 16A’ fruit did not
change significantly over coolstorage for control and MSDD treated (Table 4-5) and there
were no significant differences recorded between control and MSDD treated fruit for both
cultivars. MSDD treatment increased the rot incidence of both cultivars; 0–5% and 0–10%
recorded in ‘Hayward’ and ‘Hort 16A’ controls, respectively compared to 50–75% and 0–
10% for treated ‘Hayward’ and ‘Hort 16A’, respectively. Another symptom observed in
treated ‘Hort 16A’ fruit was uneven ripening which was characterized by hard flesh with soft
patches and white-yellow coloured flesh (Figure 4-9).
The respiration rate of treated ‘Hayward’ fruit was consistently higher than control
fruit at week 0 (Figure 4-10). There was no noticeable difference after 4 and 8 weeks of
coolstorage. Conversely, respiration rate of treated ‘Hort 16A’ fruit was lower at week 0,
especially after 7 d at 20°C. There was no difference observed after 4 weeks of coolstorage.
The same trend was observed in ethylene production. MSDD treated ‘Hayward’ fruit reached
earlier ethylene climacteric peak than controls (Figure 4-11), whereas control ‘Hort 16 A’
fruit had earlier ethylene climacteric peak. There was no significant difference observed in
‘Hort 16A’ fruit coolstored after 4 weeks as the ethylene production was low. Volatile
production of ‘Hort 16A’ fruit at week 8 was not measured as the fruit had reached eating
firmness. MSDD treatments stimulated the endogenous production of ethanol and
acetaldehyde of ‘Hayward’ kiwifruit (Figures 4-12 and 4-13). The ethanol and acetaldehyde
production ranged from 72.08–203.69 and 0–23.79 ng. kg. s-1, respectively. No detectable
levels of both volatiles were found in controls of both cultivars. No ethanol and acetaldehyde
was detected in treated fruit after 4 and 8 weeks of coolstorage.
The softening rate of the treated ‘Hayward’ fruit was significantly higher than that of
the control at week 0 (Figure 4-14a). The firmness of treated fruit reached eating firmness in
11 d from 38.7–8.7 (106 Hz2 kg2/3) ~56.01–7.52 N, whereas the firmness of control fruit did
not reach eating firmness even after 15 d. The Aweta index of control fruit decreased from
38.6–23.8 (106 Hz2 kg2/3) approximately 55.3–21.5 N in 15 d. The high softening rate of
treated fruit compared to controls was also evident after 4 weeks of coolstorage (Figure 414b). The fruit had similar softening rate after 8 weeks of coolstorage (Figure 4-14c).
86
MSDD treated ‘Hort 16A’ fruit softened at the lower rate compared to controls at
week 0 (Figure 4-14a). The Aweta index of control fruit decreased from 32.6–8.4 (106 Hz2
kg2/3), approximately 39.3–7.4 N (eating firmness) in 9 d, whereas the treated fruit reached
eating firmness in 11 d from 34.4–10.8 (106 Hz2 kg2/3) ~43.9–8.6 N. However after 4 and 8
weeks of coolstorage, the softening rate was not significant (Figures 4-14b and c). Although
at week 8, the control fruit reached eating firmness 2 d earlier than treated fruit.
87
Table 4-5 Kiwifruit quality exposed to 90 min MSDD treatments with 1113 mg. L-1 ethanol
and in air (untreated control), after being stored at 0°C and 0.5°C (0, 4 and 8 weeks) for
‘Hayward’ and ‘Hort 16A’ kiwifruit, respectively, n=3.
Treatments
Week in
Rot
coolstorage
incidence
‘Hayward’
Control
MSDD
Brix (±SE)
pH (±SE)
Hue angle
(±SE)
(%)
0
0.65a
13.04 (0.44)a
3.80 (0.13)a
102.51 (0.98)a
4
0a
13.54 (0.54)a
3.95 (0.15)a
103.25 (1.01)a
8
5.01b
13.36 (0.57)a
3.92 (0.25)a
101.27 (1.14)a
0
75.00c
13.14 (0.56)a
3.79 (0.24)a
103.21 (1.15)a
4
65.25c
13.25 (0.75)a
3.85 (0.17)a
101.24 (1.35)a
8
50.26d
13.37 (0.63)a
3.95 (0.24)a
99.12 (1.74)a
Week in
Rot
Uneven
Flesh
Brix
pH
Hue angle
coolstorage
incidence
ripening
breakdown
(±SE)
(±SE)
(±SE)
(%)
(%)
(%)
0a
0a
0a
16.35
4.45
102.73
(0.19)a
(0.15)a
(0.99)a
16.45
4.35
95.25
(0.25)a
(0.14)a
(0.85)b
16.56
4.26
95.47
(0.21)a
(0.15)a
(0.98)b
16.29
4.56
101.66
(0.25)a
(0.12)a
(1.68)a
16.52
4.36
96.25
(0.26)a
(0.16)a
(1.23)b
16.45
4.30
95.36
(0.35)a
(0.17)a
(1.15)b
treated
Treatments
‘Hort 16A’
Control
0
4
8
MSDD
0
0a
3.1b
10.1c
0a
0a
15.1b
0a
0a
0a
treated
4
8
0a
10.5c
2.3c
0a
0a
9.1b
88
Figure 4-9 Uneven ripening observed in ‘Hort 16A’ kiwifruit; characterized by white flesh
appearance.
89
Figure 4-10 Respiration rate (mg. kg-1. s-1, mean ±SE) of second season ‘Hayward’ and ‘Hort
16A’ kiwifruit, non-coolstored (A) and coolstored for 4 and 8 weeks (B and C), n=3.
90
Figure 4-11 Ethylene production (ng. kg-1. s-1, mean ±SE) of second season ‘Hayward’ and
91
Figure 4-12 Ethanol production (ng. kg-1. s-1, mean ±SE) of second season ‘Hayward’ and
92
Figure 4-13 Acetaldehyde production (ng. kg-1. s-1, mean ±SE) of second season ‘Hayward’
n=3.
93
Figure 4-14 Acoustic firmness (106 Hz2 kg2/3, mean ±SE) of second season ‘Hayward’ and
94
4.3.3 ‘Cripps Pink’ apple
MSDD treatments did not affect respiration rate and internal ethylene concentration of
non-coolstored apples (Figure 4-15). MSDD treatments reduced the ethanol and acetaldehyde
production from 31.65 and 2.25 ng. kg-1. s-1, respectively, at day 1, to an undetectable level at
day 7 (Figure 4-16). No ethanol production was detected in control fruit. Acetaldehyde was
detected at control fruit (0.49 ng. kg-1. s-1) only at day 1. After 7 d of shelf-life, MSDD
treatments reduced the firmness of the fruit; 84.3 (±1.7) N compared to 89.2 (±2.0) N
recorded for the control fruit (Table 4-6).
After storage, no difference in respiration rate and internal ethylene concentration was
observed between control and treated fruit. There was also no difference in terms of firmness
(57.8 N), SSC (13.0%) and pH (3.6) (Table 4-6). No significant difference was recorded in
terms of superficial scald (~47%), internal browning ~1─2%) or greasiness incidence
(~17─21%). MSDD treatments also had no effect on severity of superficial scald (Table 47).Treated fruit had a higher hue angle value of background colour of 94.8 (±1.1) (greener)
compared to 91.1 (±1.2) (yellower) for control fruit.
95
Figure 4-15 CO2 production (mg. kg-1. s-1, mean ±SE) (A and B) and internal ethylene
concentration (µg. L-1, mean ±SE) (C and D) of ‘Cripps Pink’ apple, non-coolstored (A and
C) and coolstored (B and D) at 0.5°C for 16 weeks, n=3.
Figure 4-16 Ethanol (A) and acetaldehyde (B) production (ng. kg-1.s-1, mean ±SE) of noncoolstored ‘Cripps Pink’, n=3.
96
Table 4-6 ‘Cripps Pink’ apple quality exposed to 90 min MSDD treatments with 371 mg. L-1
ethanol and in air (untreated control), unstored and after being stored for 16 weeks at 0.5°C,
n=3.
Treatments
Control
Firmness
Firmness (N)
pH (± SE)
Brix (± SE)
hue angle
(N) (±SE)
(±SE)
(stored)
(stored)
(±SE)
(unstored)
(stored)
89.57
58.21 (0.01)a
3.63 (0.02)a
13.15
91.05
(0.14)a
(1.24)a
13.39
94.76
(0.17)a
(1.12)b
(0.88)a*
MSDD treated
84.08 (0.78)b
57.92 (0.02)a
3.64 (0.02)a
*(±SE); Values within a column followed by the same letter are not significantly different (P
>0.05).
Table 4-7 ‘Cripps Pink’ apple quality exposed to 90 min MSDD treatments with 371 mg. L-1
ethanol and in air (untreated control) and after being stored for 16 weeks at 0.5°C. All
measurements were taken after 7 d at 20°C, n=3.
Treatments
Superficial scald incidence (%)
Total scald
Internal
Greasiness
Slight
Moderate
Severe
incidence
browning
(%)
(0-25%)
(25-50%)
(>50%)
(%)
incidence
(%)
Control
25.67a
16.67a
2.67a
47.67a
0a
20.78a
MSDD
30.00a
13.33a
4.00a
47.33a
1.33a
17.67a
treated
97
4.4 Discussion
4.4.1 ‘Hass’ avocado
MSDD treatments did not affect respiration rate and ethylene production of early and
late season fruit. The quality of early and late season was not significantly reduced by MSDD
treatments. The only damage symptom observed was the browning of the ‘button’, which was
not distinguishable from that of an untreated fruit when ripe. MSDD treatments generally
were found to have no adverse effect on external and internal fruit quality based on
assessment of stem and body rot, vascular browning, flesh greying and overall fruit quality
(% soundness). A high incidence of rots was detected across all treatments; however, the
soundness (overall acceptability) of fruit was not significantly affected, because of low
severity of the disorders (mostly <15% of surface area affected). Treated early season fruit
when ripened at 25°C had higher incidence of vascular browning than controls, as found by
Hopkirk et al. (1994).
MSDD treatments were shown to reduce variability in respiration rates of late season
fruit. MSDD treatments mimic aspects of hypobaric, controlled and modified atmosphere
treatments by reducing the level of oxygen and saturating the treatment chamber with CO2.
Long-term exposure (>24 h) to high CO2 atmosphere has been shown to retard ripening by
lowering the metabolic rate and inhibiting ethylene synthesis (Bangerth, 1984; Ke et al.,
1991; Gorris and Peppelenbos, 1992; Graell and Recasens, 1992). Valle-Guadarrama et al.
(2004) showed that anaerobic compensation points (ACP) of ‘Hass’ avocado were 1.44% and
1.81% O2 at 5.5°C and 20°C, respectively. The O2 concentration during MSDD treatment
was 0.1–0.3%, which was below the ACP points. There was no significant physiological
effect (in terms of ethylene production and respiration rate), most likely due to the relatively
short MSDD treatment time of 1.5 h.
Ritenour et al. (1997) found that ripening of ‘Hass’ avocado was delayed when treated
with 80% ethanol saturated air for up to 4 d. Browning of skin and flesh was observed upon
ripening. MSDD treatments with significantly higher ethanol concentration than Ritenour et
al. (1997) were found to have little effect on ethylene production and respiration rate of
‘Hass’ avocado; this is likely to be attributed to shorter treatment time. Coolstorage of 6
weeks was carried out on control and MSDD-treated avocados to investigate whether MSDD
98
can have a positive effect by increasing storage potential (delaying ripening) while
maintaining fruit quality (i.e. reducing internal chilling injury – flesh greying). There was no
significant difference between MSDD-treated and control fruit in terms of incidences of rots,
flesh greying and days to ripen. Both control and MSDD-treated fruit had high incidences of
chilling injury (flesh greying) and reduced soundness (overall acceptability), as is expected
after prolonged storage times (Woolf et al., 2005).
4.4.2 ‘Hayward’ and ‘Hort 16A’ kiwifruit
Generally, MSDD treatments did not affect quality parameters such as pH, brix and
hue angle of kiwifruit (both cultivars, both seasons). The most significant effect on MSDD
observed was the increase in rot incidence. MSDD increased rot incidence in “Hayward’ in
both seasons. Rot incidence was consistently recorded on >50% of the fruit. The same trend
was also shown in ‘Hort 16A’ fruit. Rot incidence was increased by 4–10 times after MSDD
treatment. Higher rate of rot incidence in the second season fruit could possibly due to several
factors such as higher ethanol concentration (3 x first season treatment) usaege and
grower/orchard effect. CA storage has been shown to have similar effect on’Hayward’
kiwifruit. Increased in rot incidence in ‘Hayward’ kiwifruit (4–6 fold compared to controls)
was observed in 2–3 weeks of CA (60–80% CO2) stored (Irving, 1992). It was not clear
whether the increase was due to high CO2 or the consequential accumulation of ethanol
and/or acetaldehyde during the CA storage period, as Irving (1992) did not investigate the
volatiles production of CA stored fruit.
MSDD was shown to promote the endogenous production of ethanol and
acetaldehyde in non-coolstored ‘Hayward’ kiwifruit from both seasons. MSDD treatments
also increased the respiration rate and stimulated the ethylene climacteric of ‘Hayward’
kiwifruit which consequently increased the rate of softening. Increased in ethanol
concentration during MSDD treatment in second season fruit did not seem to increase the
volatiles production, suggesting that there might be a concentration limit of ethanol affecting
‘Hayward’ kiwifruit ripening metabolism. Conversely, the long-term coolstored fruit showed
a different trend in softening rate. MSDD treatment reduced respiration rate and delayed
ethylene peak (by 2 d), upon removal from coolstorage. In addition, MSDD treatments also
reduced the softening rate during 16 weeks of coolstorage. Ethanol and acetaldehyde
production were detected at day 9 and 11 after removal from coolstorage, respectively in first
99
season fruit. This result suggests that the effect of MSDD treatment on ethanol and
acetaldehyde production was not transient and that significant physiological changes had
occurred. Ethanol and acetaldehyde production was not detected after 4 weeks and 8 weeks
coolstorage in second season fruit. It was thought to be due to shorter shelf-life testing days;
7 d as opposed to 13 d in the first season.
The effect of ethanol/acetaldehyde production on ripening of MSDD treated nonstored ‘Hayward’ kiwifruit reflected a similar trend observed by Mencarelli et al. (1991). It
was shown that 0.02–0.18% ethanol and 0.02–0.04% acetaldehyde vapour treatments for 14 d
induced ripening of ‘Hayward’ kiwifruit by increasing the ethylene production by 23 fold.
Irving (1992) showed that ‘Hayward’ kiwifruit stored at 60% and 80% CO2 atmosphere for 2
weeks at 5 and 10°C had higher respiration rate, earlier ethylene climacteric peak and
significantly reduced firmness (compared to air storage), upon removal to 20°C air (shelflife). Increased softening could possibly be due to accumulation of ethanol and/or
acetaldehyde in the flesh during storage which was not investigated by Irving (1992).
An opposite trend to ‘Hayward’ was recorded in ‘Hort 16A’. MSDD treatments
reduced ripening in non-stored fruit in both seasons. MSDD treatments also reduced
respiration rate and retarded the ethylene climacteric peak in treated fruit of both seasons. In
addition, it was shown that MSDD treatments did not induce any ethanol and acetaldehyde
production of non-stored fruit of both seasons. MSDD treatments resulted in water-soaked
appearance in the first season and in 8 week-coolstored fruit of second season. The disorder
was seen to be confined to the fruit that had been coolstored. Conversely, MSDD treatments
resulted in uneven ripening in freshly harvested fruit (second season). Similar to MSDD
treated ‘Hayward’ kiwifruit, MSDD treatments also increased the rot incidences in ‘Hort
16A’ fruit in both seasons.
100
4.4.3 ‘Cripps Pink’ Apple
Generally, MSDD treatments did not affect the physiology and quality attributes of
‘Cripps Pink’ apple. No differences were observed in terms of respiration rate, internal
ethylene concentration, SSC and pH level in both non-coolstored and coolstored fruit during
7 d of shelf-life assessment. The firmness of non-coolstored MSDD-treated fruit was reduced
by 5.5 N (a similar trend to that of ‘Hayward’ kiwifruit).
MSDD treatments did not affect the incidence of any postharvest disorders such as
superficial scald, internal browning or greasiness. Ethanol treatments over long time periods
(up to 16 weeks) have been shown to control superficial scald incidence in ‘Granny Smith’
apples by reducing the level of α-farnesene and its conjugated trienes (Ghahramani and Scott,
1998a; b). Our results showed that ethanol diffused out of the MSDD treated fruit to
undetectable level after 7 d. Consequently, the ethanol level required to control superficial
scald was most likely not sufficient to be effective.
In addition, Jamieson et al. (2003) showed that prolonged exposure (7 d) to ethanol
vapour might result in off-flavour in apples. MSDD treatment could potentially overcome the
problem as the fruit were only exposed to 90 min ethanol treatments. In conclusion, MSDD
might potentially be able to be utilised for treatments for apples. Further studies are required
to assess the effect of MSDD across various maturity stages and harvest conditions as well as
the effect on sensory properties of fruit.
4.5 Conclusions
MSDD treatments were shown to be unsuitable for treating kiwifruit. The most
prominent effect was the increase in rot incidence in both cultivars. MSDD treatments
induced ethanol and acetaldehyde production in ‘Hayward’ kiwifruit which would impart offflavour and accelerated ripening and consequently softening. On the other hand, ripening and
softening of ’Hort 16A’ were retarded by MSDD treatments. However, it induced two
physiological disorders, namely flesh breakdown (water-soaked appearance) and uneven
ripening. MSDD treatments were shown to have minimal or negligible effect on ripening
physiology of ‘Hass’ avocado and ‘Cripps Pink’ apples. Quality parameters of both fruit were
also unaffected by MSDD treatments.
101
5. Investigation of the significance and optimisation of
Metabolic-Stress Disinfection and Disinfestation (MSDD)
parameters to control longtailed mealybug Pseudococcus
longispinus
5.1 Overview
MSDD treatments consist of at least 4 main parameters; the length of physical phases,
the length of chemical phase, concentration of ethanol and pressure at chemical phase. All of
which were thought to contribute to insect mortality. The significance of each parameter had
not been provided in the first publication and patent (Lagunas-Solar et al., 2006; LagunasSolar and Essert, 2011). The knowledge of the importance of each parameter may lead to
improvement and optimisation of MSDD treatments, i.e. reduction of treatment time,
reduction of extent of vacuum and ultimately cost. In addition, reduction of the extent of the
parameters was thought to potentially reduce the negative effects that MSDD might impart on
fruit physiology and quality. This chapter investigated the effect of reducing four main
parameters of MSDD on longtailed mealybug mortality (P. longispinus). The results were
then modelled using binary logistic regression. The optimum parameters to achieve 99%
mortality from the resulting models were then produced and validated.
5.2.1 Insects
Longtailed mealybugs were obtained from a laboratory colony which was maintained
at 20 ±2°C with 60% RH, and a 16:8 h light:dark cycle, and reared on sprouting ‘Desire’
potatoes (Solanum tuberosum). Cleaned sprouting potatoes were put into the colony two
weeks before each experimental treatment which enabled c. 50–150 mealybugs (with mixed
numbers of adults, 2nd/3rd instar nymphs and 1st instar crawlers) to establish on each potato.
No egg lifestage was tested as female longtailed mealybugs produce crawlers. On the day of
treatment, 2–4 potatoes were placed inside plastic containers (650 mL each) with the lid off
during MSDD treatment. Three replicates (separate trials) of each treatment were carried out.
102
After treatment, mealybug-infested potatoes were held at 20°C in a 16:8 h light:dark
cycle for further three days. Insects were recorded as live (movement) or dead (no
movement) after being gently prodded with a blunt instrument. One hundred insects of each
life stage were counted from randomly selected sections of treated potatoes, as representative
of each replicate treatment. The total numbers of insects counted were 8 400 for each
lifestage (adults, 2nd/ 3rd instar nymphs and crawlers).
The standard MSDD equipment and treatment procedure were used as described in
Chapter 2. The procedure started by reducing the pressure down from ambient to 90 kPa,
followed by immediate rapid flow of CO2 or N2 gas which increased the pressure to 110 kPa.
This was considered one physical cycle. Vacuum time from 110 to 90 kPa was achieved in 10
s; whereas the time of increasing pressure from 90 to 110 kPa was about 20 s (thus one
physical cycle was approximately 30 s). The physical phase of each MSDD treatment was
carried out for 30 min (~50–60 physical cycles). The chemical phase was initiated at the end
of the physical phase by lowering the pressure to 10 kPa, followed by injection of a known
amount of liquid ethanol into the heat exchange system (at 90°C). The chemical phase lasted
for 60 min. The ethanol condensation point was calculated to be 137.7 ±15.3 mg. L-1, based
on the temperature profile during MSDD treatments (22.5 ±2.5°C). At or below that
concentration, ethanol existed as vapour and above that concentration as mist or condensate
(Table 5-1).
5.2.3 Experimental design
The following parameters were altered to determine their impact on mealybug
mortality: ballast gas used (CO2 or N2), length of physical phase, length of chemical phase,
concentration of ethanol, and the pressure of the chemical phase. In this study, the design
points for runs were selected in a partially adaptive way where the observations on response
of past runs were used to decide on the design point for the next run. The first set of
experiments is to investigate the efficacy of N2 v. CO2 as the ballast gas (with a constant
physical phase and a range of ethanol concentrations) (Table 5-2). This was followed by a
second experiment that investigated the impact of length of physical phase with CO2 on
mealybug mortality followed by a constant chemical phase consisting of 125 mg. L-1 ethanol
103
for 60 min at 10 kPa (Table 5-3). The third experiment studied the effect of the length of
chemical phase (on mortality) with a constant concentration of ethanol - 125 mg. L-1 for 60
min at 10 kPa and physical phase of 30 min (Table 5-4). The fourth experiment investigated
the effect of different pressures during chemical phase (125 mg. L-1 ethanol for 45 and 60
min) on mealybug mortality (Table 5-5). All treatments were replicated three times.
5.2.4 Statistical models of the effect of varying parameters of MSDD on insect
mortality
Let pi be the probability of insect mortality of the Ti –the treatment combination in
the chemical phase; where i =1:26 are the experimental design points including the controls.
If we assume mortality of individual insects are independent Bernoulli trials, the number of
insects killed yij in the j-th replicate run of the i-th treatment in a sample size of nij follows a
Binomial distribution, i.e. yij ~ Bin(nij , pi ) , in thus case sample size was fixed at n =100. In
a general form, the relationship between the probability of mortality p and predictor variables,
x can be specified as:
p(x)    (1   ) F (x; θ)
(5-1)
where  =probability of natural mortality; F (x; θ) =a sigmoid curve defined by a cumulative
distribution function (cdf) which is asymptotically bounded by (0, 1), and x and θ are
respectively the vector of predictor variables and its corresponding parameter vector. The
commonly used cdf are that of logistic and Weibull. If the natural mortality parameter is
ignored, the above model can be fitted as a typical generalised linear model (GLM), i.e. the
relationship p and predictors can be made linear by the use of the link functions logit or
complementary log-log (CLL), respectively for the logistic and Weibull cdf: logit =log (p /
(1-p) ) and CLL =log(-log(1-p)). In this study, logit link was used. Using appropriate subsets
of design points, three separate analysis were carried out to ascertain effects on mortality: (a)
effect of ethanol concentration in CO2 and N2 gas environments; (b) effect of increasing
length of physical phase in CO2 gas; (c) effects of three factors that comprised the chemical
phase treatment, i.e. x1 =duration of chemical phase (min), x2 =concentration of ethanol (mg.
L-1), x3 =pressure (kPa) by using response surface methodology. The appropriate statistical
model for estimating a response surface can be specified as:
104
log
pi
  0  1 x1i   2 x2i  3 x3i
1  pi
(5-2)
where x1, x2 and x3 are the predictors as specified earlier by the vector x and 1 ,.3 are the
corresponding regression coefficients. The odds ratio between two treatments a and b can be
estimated by exponentiation of the difference of their fitted values on the logit scale, i.e.
OR  e
log
pa
p
 log b
1 pa
1 pb
(5-3)
An OR >1 (i.e. a positive difference in logits) indicates that treatment a is more likely to
impact on response variable (in this case cause higher mealybug mortality) than treatment b.
The above statistical model can be fitted as a generalised linear model (GLM) available in
most commonly used statistical software. The model including the offset for natural
mortality, however, can be only fitted within a generalised linear mixed model (GLMM)
framework where the linear predictor can be constrained iteratively.
Limitations in the spread of design points put boundaries on the complexity of model
that can be specified. As candidate models for model selection, first order response surface
models with different link functions, with and without the quadratic terms, were employed.
These were fitted as a GLM using Minitab (Minitab Inc., State College, PA, USA) and SAS
(SAS, Cary, NC, USA). Several criteria, including deviance, Akaike information criterion
(AIC), plot of deviance residuals v. predicted, concordant percentage and goodness-of-fit
statistics were used to evaluate and select the best models. The graphs were generated using
Origin 7.5 (OriginLab, Northampton, MA, USA).
Only three out of 4 parameters of MSDD were used as independent variables; the
length of chemical phase (x1), the concentration of ethanol (x2), and the pressure of chemical
phase (x3). The physical phase was not used as an independent variable because the results
showed that 30 min of physical phase was required to achieve maximum mortality (see Table
5-3). The mortalities of the three life stages of longtailed mealybug were dependent
variables.
105
The model equations estimated were then used to optimize the parameters. The
optimized parameters for LD99 for 3 life stages were obtained using Microsoft Excel 2007
Solver function, with the following constraints; 45 ≤x1 ≤60 min, x2 =275 mg. L-1, 10 ≤x3 ≤30
kPa.
5.3 Results
5.3.1 Insect mortality
Ambient, physical only and chemical only controls did not result in high mortalities
of longtailed mealybugs (Table 5-2; controls). Carbon dioxide as ballast gas in the physical
phase of MSDD was shown to cause higher mortality than N2 in the 50–225 mg. L-1 ethanol
range (Table 5-2). Logit models showed that CO2 had steeper mortality curves compared to
N2 (Figure 5-1). However, at 275 mg. L-1 ethanol concentration, there was no significant
difference between the mortalities of mealybugs exposed to CO2 and N2.
The length of the physical phase (5 to 30 min) was shown to have a significant effect
on mortality at a moderate ethanol concentration (Table 5-3, Figure 5-2) with 30 min of
physical phase resulted in the highest mortality. Second and third instars nymphs were not
affected by the length of the physical phase. An increase in the length of chemical phase
resulted in increasing mortality, with a plateau in mortality at ≥45 min (Table 5-4, Figures 53 A–C).
The atmospheric pressure (10 to 90 kPa) during the chemical phase was also shown to
have a significant effect on mortality (Table 5-5, Figure 5-3 G–I), with lower pressure
resulted in higher mortality. The mortalities were not significantly different between 10 kPa
and 30 kPa at the same ethanol concentrations (Table 5-5).
5.3.2 Modelling of the effect of varying MSDD parameters on insect mortality
Binary logistical regression was carried out for the three life stages to obtain
appropriate models (Figures 5-1 to 5-3). Based on design points available at the end of the
experiment, the estimation of a response surface for mortality through traditional higher order
106
models involving interactions of predictor variables were deemed to be inappropriate.
Instead, first order response surface models both in linear and quadratic form were employed
on the condition that the fitted model is limited to predictions to near or within observed
design points.
Table 5-6 shows the parameter estimates and the associated goodness-of-fit tests and
percent concordant between the predicted and observed mealybug mortalities. Using the
predicted models, it was calculated that the parameters for optimum conditions to achieve
99% mortality for longtailed mealybug were; chemical phase=45 min, concentration of
ethanol=275.0 mg. L-1, pressure=30 kPa (Table 5-7). Three replicate validation experiments
using the optimized parameters were carried out and resulted in 99% mortality of three life
stages.
107
Table 5-1 Concentration of ethanol introduced into the chamber calculated as volatile and
mist/liquid. The maximum concentration of ethanol vapour was 137.7 ±15.3 mg. L-1 (at 22.5
±2.5°C). The remaining ethanol existed as liquid or mist that condensed on the product
surface (n=3).
Total volume
Calculated1
Calculated
Total
of ethanol
concentration
concentration
concentration
introduced
ethanol vapour
ethanol mist or
of ethanol in
liquid
the chamber
(mg L-1)
(mg L-1)
(mL) into
-1
(mg L )
250-L MSDD
chamber
1
15.8
50
0
50
39.6
125
0
125
63.4
137.7
62.3
200
71.3
137.7
87.3
225
87.1
137.7
137.3
275
Ethanol concentration calculated at temperature during MSDD treatments at 22.5°C.
108
Table 5-2 The effect of CO2 or N2 during the physical phase of MSDD on the percentage mortality (±SE) of longtailed mealybug. Each
treatment contained 300 insects of each lifestage (n=3).
Treatment
Physical phase
Chemical phase
Total
treatment
% mortality
Adult
time (min)
Control handling
Control chemical phase
Control physical phase
-
2nd & 3rd
Crawler
instars
-
-
2.67 (0.67)
6.33 (2.03)
6.00 (1.00)
125 mg. L , 90 min, 10 kPa
90
12.33 (1.45)
11.67 (2.03)
23.33 (1.67)
-1
-1
CO2, 30 min
0 mg. L , 60 min, 10 kPa
90
4.67 (0.88)
5.33 (1.76)
8.33 (3.53)
Control physical phase N2
N2, 30 min
0 mg. L-1, 60 min, 10 kPa
90
5.00 (0.58)
4.33 (1.76)
8.33 (1.76)
1
N2, 30 min
50 mg. L-1, 60 min, 10 kPa
90
16.67 (1.67)
18.33 (5.24)
10.00 (1.15)
2
N2, 30 min
125 mg. L-1, 60 min, 10 kPa
90
29.67 (2.91)
34.67 (5.78)
41. 67 (4.91)
CO2
-1
3
N2, 30 min
200 mg. L , 60 min, 10 kPa
90
80.29 (2.89)
85.00 (2.89)
78.33 (8.82)
4
N2, 30 min
225 mg. L-1, 60 min, 10 kPa
90
88.33 (2.40)
87.33 (1.76)
86.00 (4.93)
5
N2, 30 min
275 mg. L-1, 60 min, 10 kPa
90
99.33 (0.33)
99.67 (0.33)
99.67 (0.33)
6
CO2, 30 min
50 mg. L-1, 60 min, 10 kPa
90
54.33 (4.70)
44.72 (5.53)
43.33 (11.67)
7
CO2, 30 min
125 mg. L-1, 60 min, 10 kPa
90
92.33 (1.45)
51.67 (12.02)
90.00 (2.87)
8
CO2, 30 min
200 mg. L-1, 60 min, 10 kPa
90
94.00 (4.51)
93.33 (2.40)
94.00 (1.00)
-1
9
CO2, 30 min
225 mg. L , 60 min, 10 kPa
90
97.33 (1.20)
95.00 (1.53)
95.00 (1.15)
10
CO2, 30 min
275 mg. L-1, 60 min, 10 kPa
90
99.00 (0.58)
99.67 (0.33)
99.00 (0.58)
109
Table 5-3 The effect of increasing length of physical phase of MSDD on the percentage mortality (±SE) of longtailed mealybug (n =3).
Treatment
Physical phase
Chemical phase
Total
treatment
% mortality
Adult
time
2nd & 3rd
Crawler
instars
(min)
1
CO2, 5 min
125 mg. L-1, 60 min, 10 kPa
65
46.67 (4.37)
51.00 (2.89)
58.33 (7.69)
2
CO2, 10 min
125 mg. L-1, 60 min, 10 kPa
70
43.67 (4.67)
54.00 (3.61)
66.67 (1.76)
3
CO2, 20 min
125 mg. L-1, 60 min, 10 kPa
80
71.00 (3.51)
65.67 (1.45)
81.00 (1.00)
4
CO2, 30 min
125 mg. L-1, 60 min, 10 kPa
90
92.33 (1.45)
51.67 (12.02)
90.00 (2.87)
(Same as no.7 in Table
5.2)
110
Table 5-4 The effect of increasing length of chemical phase of MSDD on the percentage mortality (±SE) of longtailed mealybug (n=3).
Treatment
Physical phase
Chemical phase
Total
treatment
% mortality
Adult
time
2nd & 3rd
Crawler
instars
(min)
1
CO2, 30 min
125 mg. L-1, 10 min, 10 kPa
40
21.33 (4.91)
20.67 (2.96)
16.67 (6.01)
2
CO2, 30 min
125 mg. L-1, 20 min, 10 kPa
50
40.00 (3.61)
29.33 (3.84)
35.33 (6.17)
3
CO2, 30 min
125 mg. L-1, 45 min, 10 kPa
75
92.33 (1.76)
57.33 (2.67)
90.00 (2.89)
4
CO2, 30 min
125 mg. L-1, 60 min, 10 kPa
90
92.33 (1.45)
51.67 (12.02)
90.00 (2.87)
(Same as no.7 in Table
5.2)
111
Table 5-5 The effect of increasing pressure during chemical phase (45 and 60 min) of MSDD on the percentage mortality (±SE) of longtailed
mealybug (n=3).
Treatment
Physical
Chemical phase
Total
phase
treatment
% mortality
Adult
time (min)
1
2
3
CO2, 30 min
CO2, 30 min
125 mg. L-1, 45 min, 30 kPa
2nd & 3rd
Crawler
instars
75
92.00 (0.58)
61.33 (7.17)
89.67 (1.20)
-1
75
68.00 (2.31)
39.00 (5.57)
67.00 (5.13)
-1
125 mg. L , 45 min, 50 kPa
CO2, 30 min
125 mg. L , 60 min, 10 kPa
90
92.33 (1.45)
51.67 (12.02)
90.00 (2.87)
4
CO2, 30 min
125 mg. L-1, 60 min, 30 kPa
90
90.67 (1.76)
61.67 (6.01)
90.33 (3.93)
5
CO2, 30 min
125 mg. L-1, 60 min, 50 kPa
90
69.00 (2.65)
43.00 (4.62)
69.00 (3.21)
6
CO2, 30 min
125 mg. L-1, 60 min, 70 kPa
90
40.00 (4.04)
27.00 (2.08)
65.00 (5.77)
(Same as no.7 in Table 5.2)
112
Table 5-6 The parameter estimates obtained from logistical regression models and the associated goodness-of-fit statistics and percent
concordant of the predicted and observed mortalities.
Experiments
Life stages
Parameter estimates (±SE)
Intercept
CO2 v. N2
Adult (CO2)
-2.47 (0.22)
x1
x2
x3
x12
z
0.05 (0.00)
x22
x32
z2
-0.00009
Goodness-of-fit
Percent
statistics
concordant
Deviance
Pearson
(P)
(P)
0.000
0.000
91.4
0.000
0.000
91.4
0.000
0.000
90.0
0.000
0.000
90.0
0.000
0.000
90.0
0.000
0.000
90.0
0.000
0.000
76.2
0.000
0.000
59.3
(0.00002)
Adult (N2)
-2.50 (0.22)
0.01 (0.00)
0.00006
(0.00002)
nd
2 /3
rd
-2.15 (0.24)
0.03 (0.00)
-0.00001
instars
(0.00002)
(CO2)
2nd/3rd
-2.57 (0.24)
0.01 (0.00)
0.00005
instars (N2)
Crawler
(0.00002)
-2.30 (0.27)
0.04 (0.01)
0.0001
(CO2)
Crawler
(0.0000)
-2.51 (0.27)
0.01 (0.00)
0.00005
(N2)
Length of
Adult (CO2)
(0.00002)
-0.10 (0.24)
physical
-0.04
-0.0001
(0.04)
(0.0000)
0.03
-0.00003
(0.03)
(0.0010)
phase (CO2)
2nd/3rd
instars
-0.08 (0.23)
113
(CO2)
Crawler
0.79 (0.26)
(CO2)
Concentration
Adult
-6.93 (0.30)
0.12 (0.02)
0.05 (0.00
, duration and
-0.04
-0.004
(0.04)
(0.001)
-0.04
-0.0007
-0.0001
-0.00002
(0.01)
(0.0002)
(0.0000)
(0.00013)
0.04
-0.0001
-0.000002
-0.0002
(0.01)
(0.0002)
(0.000010)
(0.0001)
-0.03
-0.001
-0.0001
-0.00001
(0.01)
(0.000)
(0.0000)
(0.00013)
0.000
0.000
70.8
0.000
0.000
84.6
0.000
0.000
80.4
0.000
0.000
84.3
pressure
during
chemical
2nd/3rd
phase
instars
Crawler
-4.45 (0.27)
-7.20 (0.31)
0.06 (0.01)
0.15 (0.02)
0.02 (0.00)
0.05 (0.00)
x1: the length of chemical phase
x2: concentration of ethanol in the chemical phase
x3: pressure at chemical phase
z: the length of physical phase
114
Table 5-7 The predicted optimal parameters of MSDD to cause 99% mortality of longtailed mealybug (Pseudococcus longispinus) adults, 2nd/3rd
instars and crawlers and comparative validation experimental percentage mortalities (±SE).
Dependent
variables
Y1 (Adult)
Independent variables
Predicted
Experimental mortality (±SE) (%)
x1 (min)
x2 (mg. L-1)
x3 (kPa)
mortality (%)
Adults
2nd &3rd instars
Crawlers
45
275
30
99.00
99.67 (0.33)
99.67 (0.33)
100 (0)
115
Figure 5-1 Mortality of 3 life stages of longtailed mealybug treated with a constant 30 min of
physical phase using CO2 v. N2 with increasing concentration of ethanol at 10 kPa for 60 min
(A. Adult, B. 2nd/3rd instars. C. Crawler). The line graphs are mortality models generated
using binary logistical regression.
116
Figure 5-2 Mortality of 3 life stages of longtailed mealybug treated with an increasing
physical phase with a constant chemical phase at 125 mg. L-1 at 10 kPa for 60 min (A. Adult,
B. 2nd/3rd instars. C. Crawler).
117
Figure 5-3 Mortality of 3 life stages of longtailed mealybug treated with a constant 30 min of
physical phase and varying conditions of chemical phase; 125 mg. L-1 at 10 kPa for
increasing length of chemical phase (A–C), increasing concentration of ethanol at 10 kPa for
60 min (D–F) and 125 mg. L-1 for 60 min at increasing pressure (G–I).
118
5.4. Discussion
MSDD treatments have been shown to be effective in controlling external pests in
relatively short treatment times (Lagunas-Solar et al., 2006; Arevalo-Galarza and Follett,
2011; Zulhendri et al., in Press). There was a lack of understanding of the mechanisms of its
insecticidal efficacy, i.e. the relative importance of each parameter. This study demonstrated
that four MSDD parameters had significant effects on longtailed mealybug mortality. Use of
the physical or chemical phases alone resulted in low (5–23%) mealybug mortality. Both
phases were required to achieve high mortality of mealybugs. The logit models generated
were shown to have good fit as deviance and Pearson p-values were <0.05. The concordant
percentages were mostly >80%, with exception of the length of physical phase experiments;
which ranged from 60–70%, suggesting that the logit models of MSDD parameters are
appropriate to predict longtailed mealybug mortalities.
Using CO2 as the ballast during the physical phase was more effective than N2 in
lower ethanol (sublethal) concentrations. In the lower to medium range of ethanol
concentration (50–125 mg. L-1), the odds ratio of CO2 to N2 in terms of adult mortality
increased from 7.6 to 27.7, which means that CO2 is 7.6 to 27.7 times more likely to result in
mortality than N2. Conversely, as the concentration of ethanol increases from 125 to 275 mg.
L-1, the odds ratio decreased to almost 1 which means that using either CO2 or N2 did not
matter at 275 mg. L-1. The effect of ballast gases in 2nd/3rd instars and crawler was
considerably less dramatic. The odds ratios ranged from 3.1–4.4 and 4.0–10.0 at 50–125 mg.
L-1, for 2nd/3rd instars and crawlers, respectively.
Overall, it was observed that CO2 was more effective as a ballast gas compared to N2.
Hence, the subsequent experiments were carried out using CO2. Several studies have
demonstrated that high level of CO2 can be utilised to control insect pests in long-term
storage (Navarro and Calderon, 1974; Friedlander and Navarro, 1979b; Soderstrom et al.,
1991; Locatelli and Daolio, 1993; Agnello et al., 2002). High CO2 has also been shown to
increase efficacy of other insecticides, such as ozone and propylene oxide (Navarro et al.,
2004; Hollingsworth and Armstrong, 2005).
119
The length of physical phase had a significant effect on mortality (Figure 5-2). Shorter
exposure to physical phase (<30 min) caused lower mealybug mortality of adult and crawler
life stages. The length of physical phase had no significant effect on 2nd & 3rd instars nymphs.
Mealybug mortality increased significantly as the concentration of ethanol in the
chemical phase was increased (Figure 5-1). Ethanol applied under vacuum alone (without
physical phase) did not achieve high mortality. Ethanol has been shown to have insecticidal
efficacy (Dentener et al., 1998a; Dentener et al., 1998b; Jamieson et al., 2003). The length of
exposure required, and risk of associated explosion proved to be significant limiting factors.
MSDD can be utilised as a better ethanol delivery system by removing O2 and saturating the
chamber with ballast gases such as N2 or CO2 in the physical phase (prior to introducing
ethanol).
Mealybug mortality increased significantly when the chemical phase was lengthened
from 10 to 45 min, but was unchanged by further increases Pressure at which chemical phase
was initiated also had a significant effect on mortality. Generally, lower pressure MSDD
treatments, at 10 and 30 kPa, resulted in higher mortality compared to 50 and 70 kPa
treatments. Optimisation (using logit models) and validation treatments confirmed that
MSDD treatments with 45 min chemical phase at 30 kPa produced similar results as the
corresponding MSDD treatments with a 60 min chemical phase using 275 mg. L-1 and 30 min
of physical phase.
Low pressure treatments alone or in combination with insecticidal compounds, have
been shown to be effective in controlling insect pests and the insecticidal efficacy of low
pressure has been attributed to lack of O2 rather than structural damage on the body of insect
(Calderon and Navarro, 1968; Mbata and Phillips, 2001; Finkelman et al., 2003; 2004;
Isikber et al., 2004; Navarro et al., 2004; Mbata et al., 2005; Davenport et al., 2006;
Finkelman et al., 2006). We also did not observe any physical damage on the longtailed
mealybug attributed to changes in pressure during MSDD treatments. Similar observation
was also made in a previous study (Arevalo-Galarza and Follett, 2011).
120
In conclusion, all four parameters of MSDD had significant effects on insecticidal
efficacy, i.e. the length of physical phase and chemical phase, ethanol concentration and the
extent of vacuum during chemical phase. The optimized parameters using logistical
regression resulted in reduction of the total treatment time by 15 min, and reduction of the
extent of vacuum by 20 kPa, yet maintained 99% mortality of all life stages of longtailed
mealybug. Furthermore, the effect of this treatment on fruit would presumably result in better
quality retention.
121
6. Technological issues associated with MSDD: I. Internal
pest
6.1 Overview
Codling moth (Cydia pomonella Linnaeus) is considered as one of the main pests of
pipfruit in the world. It is a fruit boring pest that managed to spread from natural apple forests
in Central Asia to most apple growing regions in the world, except some parts of Asia
including Taiwan and Japan (Aliniazee et al., 1999; Mills, 2005; Wearing et al., 2010).
Taiwan and Japan are two premium markets for NZ apples. And the development of reliable
postharvest quarantine treatments for controlling codling moth is highly desirable. Codling
moth is one of the most damaging pests for apples, where significant loss of crops is
unavoidable in the absence of pest management (Mills, 2005). Codling moth can cause
significant economic loss with low density population, for example a single mated female can
lay up to 300 eggs on, or within a few centimetres of fruit, limiting the ability of fruit growers
to prevent entry to the fruit (Wearing et al., 2010). Management of codling moth has been
largely dependent on pesticide usage which, directly or indirectly, contributes to secondary
pests and increased resistance of the pest (Croft et al., 1987; Sauphanor et al., 1997; Aliniazee
et al., 1999; Simon et al., 2007). The biggest challenge in controlling codling moth is the
access of pesticides to the larvae themselves. Figure 6-1 shows the typical damage of a
codling moth larva where it has bored into the core of an apple from the skin. Once inside the
fruit, they are unlikely to be within reach of most management techniques (Wearing et al.,
2010).
Figure 6-1 Damage by codling moth (Cydia pomonella) larvae (courtesy of Dave Rogers,
Plant and Food Research).
122
The rationale of the work in this chapter was MSDD might provide additional control
measure for codling moth. The pressure changes and/or ethanol treatments under vacuum
might facilitate ethanol delivery deeper inside the fruit. Little work has been carried out to
investigate the efficacy of MSDD on internal insects. Arevalo-Galarza and Follett (2011)
investigated the effect of MSDD on several species of fruit flies namely Mediterranean fruit
fly (Ceratis capitata Wiedemann), oriental fruit fly (Bactrocera dorsalis Hendel) and melon
fly (Bactrocera cucurbitae Coquillett). However, they found that MSDD treatments only
resulted in low mortality rate suggesting ethanol penetration into the fruit were the limiting
factor.
Therefore the primary aim of this chapter was to investigate the effect of MSDD
against 5th instar codling moth larvae inoculated inside closed and open calyx apples,
‘Braeburn’ and ‘Pacific Rose’, respectively (Figure 6-2). Secondly, the extent of ethanol
penetration to the apple was also investigated.
Braeburn
Pacific Rose
Figure 6-2 Closed and open calyx apples (Braeburn and Pacific Rose). Arrows indicate the
morphology of the calyx of both apples.
6.2.1 ‘Braeburn’ apple (Malus domestica) and ‘Pacific Rose’ apple (Malus
domestica) for 5th instar codling moth (Cydia pomonella) larvae inoculation
Insecticide residue free ‘Braeburn’ and ‘Pacific Rose’ apples were used to investigate
the efficacy of MSDD against internal pest, i.e 5th instar codling moth larvae. Apples were
123
sourced from Plant & Food Research in Hawkes Bay and stored at 0.5°C until needed. Fruit
were then left at 20°C overnight before codling moth larvae inoculation.
6.2.2 Codling moth (Cydia pomonella) 5th instar larvae
Fifth instar codling moth larvae were obtained from a laboratory colony on artificial
diet (Singh, 1983; Clare et al., 1987) and maintained at 20  2°C with 60% RH, and a 16:8 h
light:dark cycle photoperiod for non-diapause condition. Fifth instar codling moth larvae
were inoculated into closed calyx apples (Braeburn) and open calyx apples (Pacific Rose).
Three holes were punched on either calyx or stem of the apples. Three larvae were then
placed into the holes and sealed off with agar, followed by 2–3 mm of candle wax and tape.
This ensured that the artificial hole did not become an easy path for ethanol to enter the
apples. Each treatment contained 17 inoculated apples and replicated three times. Total
insects per treatment were 150 larvae. After MSDD treatments, inoculated apples were then
stored at 20°C for 3 d before being assessed for insect mortality.
6.2.3 Investigation of ethanol penetration into apple
The measurement of ethanol penetration was carried out in open and closed calyx
apples ‘Pacific Rose’ and ‘Braeburn’. The method was carried out according to Burdon et al.
(2007). Cylinders of tissue (skin to core) were removed from the equator of the MSDD
treated apple with a 10 mm-diameter cork borer (Figure 6-3A). The tissue was then cut to 1
cm pieces longitudinally from the skin to the core and weighed (Figure 6-3B). The tissue was
placed into a 60 mL syringe. The syringe plunger was then pushed to the 10 mL mark and
sealed with a rubber septum (Figure 6-3C). A vacuum was subsequently created by
withdrawing the plunger to the 60 mL mark and held for 1 min (Figure 6-3D). Subsequently,
the vacuum was released and 1 mL gas sample was taken (Figure 6-3E). The ethanol and
acetaldehyde concentration was analysed by gas chromatography. The result was expressed
in the amount of present in 1 mL per g of fresh weight.
124
A
B
C
D
E
Figure 6-3 Experimental work for investigating ethanol penetration into apple flesh. A. Cork
borer was used to obtain flesh sample. B. Flesh sample was divided into 3 one-cm sections
(arrows indicate where apple flesh was divided into 3 one-cm sections). C. A section was put
into a rubber-capped 60 mL syringe. D. A low pressure condition was created in the syringe
to facilitate volatile extraction from the sample. E. A 1-mL gas sample was obtained from the
syringe.
125
MSDD treatments. The experimental design is described in Table 6-1. Each
inoculated apple cultivar was subjected to 3 MSDD treatments with physical phase of 30 min,
followed by the chemical phase with ethanol concentration of 50, 125 and 275 mg. L-1. These
concentrations were chosen as they achieved low, intermediate and high mortalities in
longtailed mealybug, respectively. The controls were ambient control, chemical phase only
(with 275 mg. L-1 ethanol for 90 min at 10 kPa), physical phase only control (30 min of
physical phase at 90 and 110 kPa, followed with 10 kPa with 0 mg. L-1 for 60 min) and full
MSDD treatments of uninoculated larvae.
Table 6-1 Experimental design for comparing the mortality of 5th instar codling moth (Cydia
pomonella) larvae in open and closed calyx apples.
Treatments
Closed calyx
Open calyx
Naked
(Braeburn)
(Pacific Rose)
Untreated ambient control
50 larvae x 3
50 larvae x 3
-
Chemical phase control
50 larvae x 3
50 larvae x 3
-
50 larvae x 3
50 larvae x 3
-
50 larvae x 3
50 larvae x 3
-
MSDD with 125 mg. L-1
50 larvae x 3
50 larvae x 3
-
50 larvae x 3
50 larvae x 3
50 larvae x 3
Total larvae
900 larvae
900 larvae
150 larvae
(no physical phase)
Physical phase control (no
chemical phase)
ethanol
6.3 Results
Table 6-2 shows ambient control and chemical phase only control had mortality rate
ranged from 2–4%. Mortality of codling moth larvae in close and open calyx apple ranged
from ~2–5% and 2–20%, respectively, when treated with 50–275 mg. L-1, as opposed to
98.67% (±0.67) when the naked larvae were treated with 275 mg. L-1 (Figure 6-4). Ethanol
level in the flesh decreased according to the depth from the apple skin. The ethanol
concentration in treated ‘Braeburn’ dropped from 29,000 to 700 ng. kg. s-1, from 1 to 3 cm
depth from the skin, respectively (Figure 6-5). Ethanol concentration in treated ‘Pacific Rose’
126
dropped from 26,000 to 3,000 ng. kg. s-1, from 1 to 3 cm depth from the skin, respectively.
The ethanol concentration of the control fruit ranged from 60–90 ng. kg. s-1 from 1–3 cm
depth from the skin, respectively.
A different trend was observed in acetaldehyde concentration (Figure 6-5). The
control fruit had the highest concentration of acetaldehyde on the flesh furthest from the skin
or closest to the core (13,000 ng. kg. s-1 at 3 cm from the skin), whereas thosee 1 cm and 2
cm from the skin recorded 2,600 and 2,800 ng. kg. s-1, respectively. The treated fruit also
showed similar trend. Acetaldehyde level in treated ‘Braeburn’ was recorded as 7,000, 3,600
and 4,000 ng. kg. s-1, at 1, 2 and 3 cm of flesh from the skin, respectively. Whereas in ‘Pacific
Rose’, the acetaldehyde level was at 8,500, 4,700 and 13,700 ng. kg. s-1, at 1, 2 and 3 cm
flesh from the skin, respectively.
Table 6-2 Control mortality of 5th instar codling moth larvae (n=3).
Treatments
Closed calyx (Braeburn)
Open calyx (Pacific Rose)
Ambient
2.09 (2.04)a
4.17 (4.28)a
Chemical phase only (90
1.50 (1.30)a
2.27 (3.93)a
min at 10 kPa with 275
mg. L-1 ethanol)
Values followed by the same letter in columns and rows do not have significant difference (P
<0.05)
127
Figure 6-4 The mortality of 5th instar codling moth larvae inoculated into 2 different calyx
types of apples, treated with ranges of ethanol concentration (mg. L-1, mean ±SE) of chemical
phase of MSDD treatments, n=3.
Figure 6-5 The ethanol (top) and acetaldehyde (bottom) concentration (mean ±SE) of tissue
at different distances from the skin, n=3.
128
6.4 Discussion
MSDD treatments using parameters which are effective at controlling longtailed
mealybug (Chapter 2) were shown to be ineffective in controlling 5th instar codling moth
larvae inoculated inside apples. Mortality of the larvae, using 275 mg. L-1 of ethanol at the
chemical phase, was 20% and 5% for open and close calyx apples, respectively. The result
showed that open calyx apple might provide a better ethanol penetration into the apples. The
notion was also supported by ethanol data which showed that at 3 cm from the skin, the flesh
in treated ‘Pacific Rose’ contained ~5 times ethanol compared to ‘Braeburn’. MSDD was
shown to effectively control naked larvae, suggesting that ethanol penetration would be the
limiting factor in controlling internal pests. These results support the finding of ArevaloGalarza and Follett (2011). It was shown that MSDD repetitions effectively controlled naked
adult and larvae 3 fruit fly species; C. capitata, B. dorsalis and B. cucurbitae. The mortality
of the insects was significantly decreased and did not reach the level required for postharvest
disinfestation treatment, when they were inoculated inside the fruit. In conclusion, MSDD
treatments are not effective in controlling internal pests; limited by the ability (or lack of) of
ethanol to penetrate the flesh.
129
7. Technological issues associated with MSDD: II. The
effect of loading on the efficacy of MSDD
7.1 Overview
In order to be considered as an effective disinfestation treatment, several criteria such
as toxicity, sorption and reactivity with products (Dudley and Neal, 1942; Coggiola and
Huelin, 1964; Derrick et al., 1990), associated with MSDD, needs to be addressed. Sorption
of active ingredients of fumigants with materials or products has been shown to reduce the
efficacy of disinfestation treatments to the sublethal levels (Lindgren et al., 1962; Daglish and
Pavic, 2008; Darby, 2008). For example, it was demonstrated that phosphine and methyl
bromide irreversibly bound to the whole grain kernels and treatment time had to be prolonged
or concentration had to be increased to compensate. It was also shown that types of materials
had significant impact of fumigant performance as an effective disinfestation treatment
(Darby, 2008). Therefore, the information on the effect of loading and packaging materials on
the efficacy of MSDD is critical for implementation of MSDD as an effective disinfestation
treatment. This chapter investigates the possible sorption of ethanol by packaging materials
and fruit which might potentially reduce the efficacy of MSDD. The types of materials used
in this chapter were cardboard (kiwifruit boxes), plastic-coated wire and plastic (harvest
crate).
7.2.1 Investigation of the effect of different types of packaging on longtailed
mealybug (Pseudococcus longispinus) mortality
The first set of experiments was carried out to investigate the effect of kiwifruit boxes
and different placement of longtailed mealybug-infested potatoes (refer to section 2.2.3 for
details), in the boxes, on the mortality. Eight single layered kiwifruit boxes (240 fruit) were
used as the treatment load. Three different placement types were used; inside the boxes and
lined with polyliner, inside the boxes (without polyliner), and naked insects (Figure 7-1). All
three types of placement were treated in the same MSDD treatment run and replicated three
130
times. The MSDD parameters used were 30 min of physical phase with CO2 as the ballast
gas, followed with a chemical phase at 10 kPa with 371 mg. L-1 of ethanol for 60 min.
The second set of experiments was to investigate the effect of non-absorbing
materials; plastic-coated wire and harvest crate, and the efficacy of MSDD to treat fruit in
‘bulk’. The fruit and potatoes were arranged in one layer in the tray experiment (Figure 7-2)
and 5 layers in the harvest crate experiment (Figure 7-3). The MSDD parameters used were
30 min of physical phase with CO2 as the ballast gas, followed with a chemical phase at 10
kPa with 371 or 742 (2 x 371) mg. L-1 of ethanol for 60 min, for the tray experiment, and
1113 mg. L-1 of ethanol for the ‘bulk’ experiment. Binary logistical models were used to
analyze the effect of placement of potatoes in the harvest crate on mortality (see Chapter 5).
The mortality of longtailed mealybug (100 insects for each replicate; 300 in totals for each
experiment) of both sets of experiments was assessed 3 d after treatments.
131
C
D
Figure 7-1 Experimental set up to investigate the effect of kiwifruit boxes on longtailed
mealybug (Pseudococcus longispinus) mortality. A. Potatoes in the kiwifruit boxes, B.
Kiwifruit and potatoes were lined with polyliner, C. Potatoes in the unlined box, D. MSDD
treatment set-up.
Figure 7-2 Plastic-coated wire experimental set up.
132
Figure 7-3 Experimental set up using the harvest crate. Top: potatoes were placed randomly
at different layers of kiwifruit. Bottom: MSDD treatment using the harvest crate.
133
7.2.2 The effect of loading type on ethanol concentration
The effect of different types of loading on ethanol concentration inside the chamber
during MSDD treatments was also investigated. The amount of fruit used in this experiment
were 240 fruit regardless of the type of packaging used. The types of packaging used were;
kiwifruit box, plastic-coated wire, harvest crate and empty chamber as the control. The
ethanol concentration injected into the chamber was 371 mg. L-1. The ethanol concentration
reading was taken at 0, 10, 25, 45 and 60 min during the chemical phase of MSDD.
7.3 Results
Table 7-1 shows the effect of loading (fruit and kiwifruit boxes) on mortality of
longtailed mealybug. As expected, the mortality of insects inside the lined kiwifruit boxes
was the lowest, ranges from 3–4%. Mortality inside the unlined boxes and naked were not
different; ranged from 26–32% across three life stages. The treatment control, where the
potatoes were treated in an empty chamber, resulted in 100% mortality as found previously
(section 2.3).
Mortality of longtailed mealybug treated with 371 mg. L-1 in a single layer manner
(plastic-coated wire) resulted in 87–94% mortality (Table 7-2). MSDD managed to achieve
100% mortality when the concentration of ethanol was doubled to 742 mg. L-1. The effect of
MSDD efficacy on ‘bulk’ product was tested using the harvest crate with 1113 mg. L-1 of
ethanol. There were approximately 5 layers of fruit and potatoes placed in a random manner
in each plastic bin. Mortality decreased from the 1st to 5th layer, from 100 down to 1–2%.
Logistical regression was used to model the decrease in mortality in relation to position of the
potatoes in the harvest crate (Figure 7-4). The models were shown to be good fit statistically
for 2nd/3rd instars and crawler (Table 7-3), Deviance and Pearson’s p-value and concordance
percentage). The goodness-of-fit statistics for adult showed that the model was not a good
fit. However, concordance percentage was 94.7%; therefore the model was still considered to
be appropriate. The high p-value for goodness-of-fit statistics of the adult mortality data was
thought to be due to the higher deviance recorded in the second layer as shown in the Table
7-3.
134
Figure 7-5 shows the concentration of ethanol inside the chamber using different
types of loading. The concentration ethanol applied into the chamber was 371 mg. L-1. Based
on the temperature during treatment (20.0 ±1.0°C), the ethanol vapour concentration was
approximately 120 mg. L-1. In the empty chamber (control), ethanol concentration rose from
3.57–117.88 mg. L-1 in 60 min of the chemical phase. However when kiwifruit boxes were
used (with or without fruit), ethanol concentration ranged from 0.53–2.08 mg. L-1 during the
chemical phase, which clearly indicated that the ethanol was absorbed by the packaging.
Ethanol concentration increased from 77.38–120.81 mg. L-1 and 85.25–120.24 mg.L-1 in the
chamber filled with fruit in white plastic-coated wire and harvest crate, respectively.
Table 7-1 The effect of kiwifruit boxes and placement of longtailed mealybug (Pseudococcus
longispinus) infested potatoes (n=3).
Ethanol concentration
Placement
-1
(mg. L )
Adult
2nd/3rd instars
Crawler
Untreated control
0.67 (0.67)a
0.67 (0.67)a
0.67 (0.33)a
3.51 (1.78)b
4.16 (1.24)b
4.25 (1.40)b
25.55
29.23 (2.57)c
26.25
371
Inside kiwifruit boxes,
layered with polyliner
sheet
371
inside kiwifruit boxes
(1.47)c
371
naked
(4.91)c
28.66(1.98)c 29.60 (4.30)c
32.42
(8.71)c
371 (Treatment
empty chamber
100 (0)d
100 (0)d
100 (0)d
control)
<0.05).
135
Table 7-2 Mortality data resulted from MSDD treatments using plastic-coated wire and
harvest crate (n=3).
Ethanol
Packaging
Mortality (% ± SE)
concentration (mg.
Adult
2nd/3rd instars
Crawler
94.00
86.33 (2.33)a
87.67
L-1)
371
Plastic-coated wire
(0.58)a
742
Plastic coated wire
(1.45)a
100
100 (0)b
100
(0)b
1 113
Harvest crate-layer 1
(0)b
100
100 (0)b
100
(0)b
1 113
65.00
(0)b
41.67 (8.82)c
(8.66)c
1 113
1.00
(5.21)c
1.67 (1.67)d
(0.58)d
1 113
1.00
1.00
6.00
(3.06)d
2.33 (1.45)d
(0.58)d
1 113
39.33
3.33
(3.33)d
1.33 (1.33)d
(1.00)d
2.33
(1.45)d
<0.05).
Table 7-3 Parameter estimates, goodness-of-fit statistics and percent concordant of logistical
models in the harvest crate experiment.
Life stages
Parameter estimates (± SE)
Intercept
Adult
x
x2
19.15
-12.31
1.52
(2.19)
(1.54)
(0.23)
2nd/3rd
13.25
-9.01
1.12
instars
(1.38)
(0.97)
(0.15)
Crawler
10.36
-7.05
0.86
(0.88)
(0.62)
(0.09)
136
Goodness-of-fit
Percent
statistics
concordance
Deviance
Pearson’s
(P)
(P)
0.134
0.065
94.7
0.004
0.001
92.6
0.000
0.001
90.4
x: layer from the top of the harvest crate
Figure 7-4 Logistical models and proportion mortality of longtailed mealybug
(Pseudococcus longispinus) in the harvest crate experiment.
Figure 7-5 Ethanol concentration (mg. L-1, mean ±SE), during chemical phase with 371 mg.
L-1, inside the MSDD chamber with various types of loading, n=3.
137
7.4 Discussion
Types of packaging. Types of packaging had a significant impact on the efficacy of
MSDD in controlling longtailed mealybug. The kiwifruit-box experiment showed that the
mortality of longtailed mealybug inside the polylined box was very low (~3–4%). It was
thought to be due to the inability of ethanol to penetrate the polyliner. The mortality of
longtailed mealybug placed inside the box (without polyliner) and outside the box (naked)
was not different, suggesting that kiwifruit boxes could potentially reduce the efficacy of
MSDD significantly. The kiwifruit boxes reduced the efficacy of MSDD by more than 3 fold,
when compared to that of using the empty chamber. Reduction of efficacy was attributed to
ethanol absorption by kiwifruit boxes.
The ethanol vapour concentration with kiwifruit boxes as the loading was 100 fold
lower compared to the empty chamber and other non-absorbing materials. Ethanol
concentration in the chamber filled with kiwifruit boxes only was not different from the
chamber filled with fruit and kiwifruit boxes. It suggests that the boxes absorbed most of the
ethanol during treatment and ethanol absorption by fruit might be negligible. The ethanol
vapour in the empty chamber took one hour to reach the maximum concentration due to the
sheer volume of the chamber and considered as one of the experimental errors associated
with ethanol measurement from the MSDD chamber.
Following the results, plastic-coated wires were used as the medium for treatments.
Fruit were placed as a single layer where the surface of fruit and potatoes was fully exposed.
The mortality of mealybug on a single layer ranged 86–94% with 371 mg. L-1, indicating that
the presence of a single layer of fruit only slightly reduced the efficacy of MSDD from the
100% mealybug mortality in the empty chamber. This was overcome by increasing the
ethanol concentration; doubling the amount of ethanol achieved 100% mortality.
Bulk loading. Exposure to ethanol was thought to be the critical part of insecticidal
efficacy of MSDD. This was reflected by the mortality results when the harvest crate was
used as the loading container. MSDD treatments with 1113 mg. L-1 resulted in 100%
mortality in the first layer. However, the mortality dropped significantly as the potatoes were
placed deeper in the bin. The mortality dropped to 40–65% in the second layer and further to
1–6% in the 3rd–5th layers. The odds ratio calculation using the models showed that the odds
138
of MSDD resulting in mortality in the first layer were 2318, 288 and 87 for adult, 2nd/3rd
instars and crawler, respectively when compared to the mortality in the second layer. The
odds were even higher when mortality in the first layer when compared to the 3rd, 4th and 5th
layers; ranged from 65–456548. It suggests that it is highly unlikely mortality can be
achieved in the layers beyond the 2nd layer. In conclusion, the packaging materials and the
placement of the infested fruit in regards to ethanol exposure are critical to the efficacy
MSDD as a disinfestation treatment. A single layer, non-absorbing material with full
exposure of the surface of fruit is therefore recommended for MSDD treatments.
139
8. General discussion, future work and conclusions
8.1 The efficacy of MSDD as a disinfestation treatment
MSDD is effective in controlling various surface pests. Lagunas-Solar et al. (2006)
demonstrated that the pressure oscillation (physical phase) between 10 and 100 kPa with CO2
as a ballast gas followed by 10 min of ethanol treatment at 10 kPa (chemical phase) managed
to control various life stages of representative species of fruit fly (D. melanogaster), thrips
(F. occidentalis), aphid (M. persicae), mites (T. urticae, A. cucumeris) and moth (H.
virescens). Various repetitions of MSDD procedure also controlled larvae and adults of C.
capitata (Mediterranean fruit fly), B. cucurbitae (melon fly), B. dorsalis (oriental fruit fly)
(Arevalo-Galarza and Follett, 2011). In this work, MSDD protocols were modified, various
treatment conditions optimised and mode of actions examined. The physical phase employed
was at 90–110 kPa cycle with CO2 as a ballast gas and followed by a 60 min chemical phase
at 10 kPa and ethanol treatments. The ethanol concentration needed to achieve high mortality
was generally above saturation point at which ethanol existed in vapour and mist (liquid). It
was shown that this protocol could potentially control various life stages of longtailed
mealybug (P. longispinus) (Chapter 2), 5th instar lightbrown apple moth (LBAM) larvae (E.
postvittana) (Chapter 3) and 5th instar codling moth larvae (C. pomonella). Nitrogen was
shown to be a less efficacious ballast gas as compared to CO2 in terms of insect mortality at
ethanol levels ranging from in the 50–225 mg. L-1. But the efficacy of MSDD with N2 was
similar to MSDD with CO2 at the higher level of ethanol 275 mg. L-1 (Chapter 5).
MSDD treatments use a combination of hypercabia, hypoxia, pressure changes and
ethanol treatments to disinfest. Physical phase of MSDD reduced the O2 level to very low
level (1–3%) - hypoxia within 12 min and increased the ballast gas concentration (CO2) to
saturation level (97–99%) - hypercarbia (Chapter 2). It established an inert condition, with
regards to explosion hazards, inside the chamber prior to chemical phase in which ethanol
was used. Ethanol treatments have been shown to be an effective disinfestation treatments
(Dentener et al., 1998a; Jamieson et al., 2003). However, the length of treatment (>7 d) and
explosion hazards were thought to be limiting factors for ethanol treatments. Chapter 2
(Figure 2-5) showed that MSDD could potentially overcome these barriers by eliminating air
and introducing inert ballast gases, and reducing treatment times.
140
MSDD utilises at least 4 major parameters to disinfest, namely, the length of physical
phase, the length of chemical phase, the concentration of ethanol in the chemical phase and
the pressure during the chemical phase. The significance of the each parameter on longtailed
mealybug mortality was investigated in Chapter 5. It was shown that each parameter
contributed significantly in achieving in high mortality. Generally, the mortality increases as:
1. The length of physical phase increases
2. The length of chemical phase increases
3. The concentration of ethanol increases
4. The pressure during chemical phase decreases
There was a stage where a plateau is reached for all the parameters with the exception of the
length of the physical phase. Logistical regression was then utilised to find the optimum
levels of the parameters. Consequently, it was found that an ethanol concentration at 275 mg.
L-1, the duration of the chemical phase and the extent of vacuum can be reduced by 25% and
22% to 45 min and 30 kPa, respectively while still maintaining 99% mortality of longtailed
mealybug.
This work showed that there are at least 3 major issues, which relate to ethanol
penetration and exposure that limited the efficacy of MSDD as an effective disinfestation
technology. Firstly, MSDD is not effective in treating internal pests, even with the use of
vacuum. Arevalo-Galarza and Follett (2011) demonstrated that MSDD effectively controlled
various larvae and pupae of fruit fly species when treated in petri dish (“naked”). However,
when the insects were inoculated inside papayas, the efficacy of MSDD was greatly reduced.
This was confirmed by the codling moth in apple work (Chapter 6). MSDD only managed to
control 10–20% larvae when they were inoculated inside the apples. The significant reduction
in MSDD efficacy was shown to be strongly correlated with the reduction in ethanol
exposure. Ethanol concentration was greatly reduced to sublethal concentration in the apple
flesh. It was found that the ethanol only penetrated into 1 cm of flesh from the skin and the
ethanol concentration was reduced by 5.9–11.0 fold in the subsequent 1 cm depth of the flesh.
Therefore, MSDD treatments cannot be used to treat internal pests.
Secondly, the types of packaging materials were shown to limit the efficacy of MSDD
against mealybugs (Chapter 7). The mortality was reduced to 20–30% when kiwifruit boxes
were used as the packaging material. Comparatively, MSDD managed to achieve 87–94%
mortality of mealybugs, with the same concentration of ethanol and same amount of fruit as
141
the kiwifruit boxes experiment, when plastic-coated wires were used. The ethanol absorption
by the packaging materials was shown to be the main limiting factor. Ethanol concentration
measurements showed that kiwifruit boxes (cardboard) reduced the ethanol concentration in
the atmosphere by 100-fold compared to empty chambers and other non-absorbing materials.
Therefore, packaging materials that minimally absorb ethanol are recommended for use
during MSDD treatments.
Thirdly, the extent of exposure of insects to ethanol had significant effect on the
efficacy of MSDD. The difference in mortality in different layers of fruit in a harvest crate
was investigated in Chapter 7. It was shown that high efficacy of MSDD against mealybugs
was limited to the first layer. The mortality was significantly reduced to 40–65% in the 2nd
layer and further reduced to 1–6% in the subsequent layers. This consequently demonstrated
that exposure to ethanol is the critical factor to ensure effective disinfestation by MSDD.
Lagunas-Solar et al. (2006) suggested conceptual designs of MSDD for commercial use
(Figure 8-1). This design will be unlikely to succeed if ethanol is used as the primary
disinfestation chemical and particularly if cardboard is used as packaging material
(commonly used in commercial export situations). Drawing from the results of this thesis, the
main limitation for commercial application of MSDD treatments, using ethanol is the
inability for the ethanol vapour and/or mist to penetrate into air spaces between commodities
if containers for bulk treatments are used. The lack of a powerful fan in the design to ensure
the circulation of the chemical also might reduce its efficacy. Therefore, in order to overcome
these limitations, it is suggested that a fan should be a critical part of the design. In addition,
the packaging materials should be made of non-absorbing materials such as metal or plastic
trays which can accommodate single layers of fruit (Figure 7.2). All of these issues make
commercial implementation as proposed by Lagunas-Solar et al. (2006) unlikely to succeed.
142
Figure 8-1 The conceptual designs of commercial scale in a commercial setting (LagunasSolar et al., 2006).
143
8.2 The biochemical mechanisms of the insecticidal efficacy of MSDD
As a relatively new technology, there is no information on biochemical mechanism of
MSDD to date. Initially, the MSDD mode of action was thought to mainly involve the
disruption of biochemical pathways and enzymes which are related to oxidative
phosphorylation and energy balance, such as ATP synthase and succinic dehydrogenase
(Navarro and Calderon, 1979; Mitcham et al., 2006). Insecticidal efficacy of ethanol
treatments as a part of MSDD was thought to be due to ethanol toxicity to central nervous
systems (Rodan et al., 2002). Ethanol also causes dehydration, ataxia, sedation which can
consequently lead to mortality (Aston and Cullumbine, 1959). Ethanol was also shown to
inhibit energy balance in cells by disrupting the hydrophobic moieties of the cell membrane
which led to inhibition of K+-induced phophatase activity (Mazzeo et al., 1988).
Investigation of the proteome of 5th instar LBAM (E. postvittana) larvae (Chapter 3)
showed that the insecticidal efficacy of MSDD was primarily attributed to its ability to
disrupt the regulation of melanisation. Melanisation is an innate immune response of most
arthropods (Nappi and Christensen, 2005; Scherfer et al., 2008; Tang, 2009). Excessive
melanisation however has been shown to be cytotoxic to insects (Nappi and Christensen,
2005). Two-DIGE was utilised to analyze different expression of haemolymph proteins of
LBAM larvae treated with different phases of MSDD; physical phase-only (PP), chemical
phase-only (CP) and full MSDD treatments. It was found that only PP treatments induced the
expression of alaserpin. Alaserpin (or serpin family) is responsible for negatively regulating
melanisation (De Gregorio et al., 2002; Ligoxygakis et al., 2002; Tang et al., 2008; Ragan et
al., 2010; Silverman et al., 2010).
On the other hand, serine protease-2 like protein was up-regulated in the MSDD
treated larvae. Serine proteases are critical in inducing melanisation in response to pathogens
(De Gregorio et al., 2002; Tang, 2009; Gubb et al., 2010). The comparison of physical
appearance of LBAM larvae treated with PP, CP and MSDD treatments also suggested that
MSDD treatments induced excessive melanisation. Excessive melanisation was evident on
larvae treated with CP and MSDD, with the latter being more prominent after 2–3 d after
treatments. In conclusion, MSDD was shown to disrupt melanisation regulation which
potentially leads to excessive melanisation and consequently death.
144
8.3 The effect of MSDD on fruit physiology and quality
Knowledge of the effect of MSDD on physiology and quality of fresh produce is
critical in order to assess the feasibility of MSDD to be implemented as a disinfestation
treatment for fresh produce. The extent of information on the effect of MSDD on fruit
physiology and quality was limited to mere observation of physical characteristics (LagunasSolar et al., 2006). Lagunas-Solar et al. (2006) listed that citrus fruit, table grape, asparagus,
blueberry and banana as having high tolerance to MSDD. Whereas, it was shown that
strawberry, lettuce, raspberry and blackberry had medium to low MSDD tolerance. Studies
have shown that ethanol (and acetaldehyde) treatments have either ripening inhibition or
ripening promotion effects depending on types of fruit and/or maturity (Saltveit, 1989;
Beaulieu and Saltveit, 1992; Ritenour et al., 1997). In general, ethanol treatments inhibit fruit
ripening. However, ethanol treatments were observed to induce ripening in kiwifruit
(Mencarelli et al., 1991). Beaulieu and Saltveit (1997) found that the inhibitory or inducing
effect of ethanol and acetaldehyde depends on the concentration and maturity of the fruit. The
atmospheric conditions during the physical phase of MSDD treatments were similar to
CA/MA storage; elevated level of CO2 and reduced O2. These conditions have been shown to
reduce metabolic rate and retard ripening of fruit (Harman and McDonald, 1989; Kader et al.,
1989; Ke et al., 1990; Ke et al., 1991; Gorris and Peppelenbos, 1992; Graell and Recasens,
1992). Conventional CA/MA storage treatments usually last for >24 h and whereas the length
of MSDD treatments last only 90 min. Therefore, this work leads to examining the effect of
ethanol component of MSDD as the limiting factor in MSDD treatments.
This work investigated the effect of MSDD treatments on 3 major NZ fruit crops;
kiwifruit, apple and avocado (Chapter 4). Ripening was induced in MSDD treated ‘Hayward’
kiwifruit and it was thought to be promoted by ethanol and acetaldehyde (Mencarelli et al.,
1991). MSDD treatments were also shown to promote endogenous ethanol and acetaldehyde
production. The effect of MSDD on ‘Hayward’ kiwifruit ripening was consistent with studies
on the effect of ethanol treatments on ‘Hayward’ kiwifruit (Mencarelli et al., 1991). MSDD
treatments did not affect the ripening of ‘Hort 16A’ fruit, suggesting that ‘Hayward’ and
‘Hort 16A’ fruit have different ripening physiology. It could be because the fruit were
harvested at different maturity and firmness levels. In terms of fruit quality, it was found that
MSDD treatments resulted in an elevated level of rots in ‘Hayward’ (50–75%) and ‘Hort
16A’ (10-30%) kiwifruit, compared to 0–5% rots in untreated fruit. MSDD treatments also
145
resulted in disorders such as flesh breakdown (first and second season) and uneven ripening
(second season) in ‘Hort 16A’ fruit. Flesh breakdown seemed to be expressed in ‘soft’ fruit
that has been coolstored and uneven ripening was expressed in ‘firm’ (freshly harvested)
fruit.
MSDD treatments generally had little physiological effect on ‘Cripps Pink’ apple and
‘Hass’ avocado. Ripening rate and ethylene production of both treated fruit were not different
from those of untreated controls. The quality indices of treated fruit, i.e. rot incidence,
physiological disorders such as flesh greying (avocado) and internal browning (apple), were
also indifferent from the untreated controls. Ethanol treatments have been shown to control
superficial scald in apples (Ghahramani and Scott, 1998a; b; Ghahramani et al., 1999). The
work in this thesis failed to observe any beneficial effect of MSDD on the reduction of
superficial scald in ‘Cripps Pink’. It was thought to be due to the diffusion of ethanol out of
the flesh after MSDD treatments. Therefore, the amount of ethanol was not sufficient to
prevent the occurrence of superficial scald. In conclusion, MSDD was shown to be a
promising candidate as an alternative disinfestation treatment for apple and avocado from a
fruit physiology point of view, if exposure issues could be overcome.
8.4 Future work
The area for future research which is likely to be the most productive is the
investigation of the use of GRAS/food additive chemicals other than ethanol, which have
higher vapour pressure and insecticidal activity, such as ethyl formate. The limitation of
ethanol as a disinfestation chemical is the relatively low vapour pressure and toxicity to
insects. As it has been demonstrated in Chapter 1, the concentration of ethanol required to
achieve disinfestation level was significantly above its vapour pressure which means ethanol
existed as vapour and mist/liquid. It consequently limits the ability of liquid ethanol to
penetrate deeper into the air spaces between fruit or materials. The problem was
demonstrated in Chapter 7. It was shown that the efficacy of MSDD was reduced
significantly when bulk loads of fruit were used. The mortality of insects dropped as the
infested potatoes were placed deeper in the bin. Studies on other GRAS/food additive
compounds such as ethyl formate and propylene oxide showed that the concentrations needed
to control insect pests were far below its saturation point with relatively short treatment times
(1–2 h) (Isikber et al., 2004; Navarro et al., 2004; Simpson et al., 2007b; Jamieson et al.,
146
2011). Therefore, they should be examined as the possible chemical for the chemical phase
of MSDD.
In this thesis, the oscillation of pressure used in the physical phase was between 90
and 110 kPa with 50–60 cycles. The rationale for the use of this range was to increase the
feasibility of MSDD for commercial use. Low pressure oscillation can lead to the use of a
smaller vacuum pump which significantly reduces the capital cost of an MSDD unit. The
difference between different ranges of pressure oscillation in terms of mortality was not
investigated in this thesis. The numbers of cycles might also affect insect mortality.
Therefore, it might be useful to investigate the relationship between different oscillation
ranges, numbers of cycles and the length of the physical phase in terms of insect mortality
and the rate of O2 depletion/ CO2 increase (Figure 8-2).
B
A
C
Figure 8-2 Suggested experimental design to investigate the effect of different pressure
oscillation ranges on insect mortality; A. 90–110 kPa (used in this work), B. 10–110
(Lagunas-Solar et al., 2006), and C. 50–110 kPa.
147
Proteomics study in this thesis suggested that, in addition to melanisation, energy
balance of the larvae was affected by MSDD treatments. It was evident in the upregulation of
ATP synthase in the haemolymph of the LBAM larvae treated with MSDD. Studies have
shown that hypoxia and hypercarbia mainly affect the enzymes related to oxidative
phosphorylation and consequently exert oxidative stresses on insects (Mitcham et al., 2006).
Oxidative stresses related to hypercarbia and hypoxia could also be the reason why MSDD is
more effective when compared to ethanol treatments alone. Therefore, the knowledge of the
fate of energy sources, such as trehalose and glucose and the associated waste compounds,
such as lactate in the haemolymph of MSDD treated larvae would be helpful in understanding
the mode of action of MSDD. Advance analytical techniques such as high resolution nuclear
magnetic resonance (NMR) spectroscopy have been successfully used to analyse and
quantify low molecular weight organic metabolites in insect haemolymph (Phalaraksh et al.,
1999; Lenz et al., 2001) and would be a useful tool in this study.
The work related to fruit physiology and quality in this thesis has been restricted, due
to limitations in resources, to one growing area in one season for all crops, with an exception
for kiwifruit (two seasons). Consequently, it is difficult to draw a definitive conclusion on the
effect of MSDD on fruit physiology and quality, for commercial use, without taking into
account the effect of climate differences and growing areas into the experimental design.
Therefore, further studies on the effect of MSDD on kiwifruit, apple and avocado across
different seasons, regions and growers are needed to investigate the uniformity of MSDD on
the effect on physiology and quality.
148
8.5 Conclusions
The aim of the research was to evaluate the insecticidal efficacy of MSDD as a
potential candidate to replace MeBr. The work presented in this thesis has produced an
MSDD procedure to control longtailed mealybug (P. longispinus) and 5th instar LBAM (E.
postvittana) larvae. It has also demonstrated that the insecticidal efficacy of MSDD is
attributed to the chemical phase and its ability to disrupt melanisation regulation, which
consequently results in excessive melanisation. Furthermore, this work also evaluated
technological issues related to the feasibility of MSDD as an effective disinfestation
technology. It was found that MSDD was limited to surface pests and level of ethanol
exposure. Packaging materials with high sorption capability, such as cardboard, reduces the
efficacy of MSDD. A single layer medium is recommended for MSDD treatments as ‘bulk’
treatments were shown to significantly reduce the exposure of produce to MSDD and
consequently limit its insecticidal efficacy. These limitations pose significant barriers to
effective commercialisation of MSDD using ethanol. Furthermore, MSDD treatments were
shown to have adverse effect on the physiology and quality of ‘Hayward’ and ‘Hort 16A’
kiwifruit but not of ‘Cripps Pink’ apple and ‘Hass’ avocado.
149
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160
10. Appendix
10.1 A worked example of calculating ethanol condensation point.
T = 15.5 deg C
Ethanol vapour pressure (mmHg):
P = 10^(8.04494 – (1554.3/(222.65 + T))- Lange’s Handbook of Chemistry 10th Ed.
= 33.00 mmHg
= 4.40 kPa
Using ideal gas law:
PV=nRT
4.40 x 1 L = (mass/ Mr ethanol) x 8.314 x (273.1 + 15.5)
Mass = 84.5 mg/L
10.2 Publications and conference proceedings resulting from this thesis
Peer reviewed publications

Zulhendri F, Jamieson L E, J Feng, C. O. Perera, SY. Quek and A.B. Woolf. The
effect of metabolic stress disinfection and disinfestations (MSDD) on ripening
physiology and quality of kiwifruit and apple. Postharvest Biology and Technology
(2012) 63: 50-54. Chapter 4 of this thesis.

Zulhendri F, Jamieson L E, R. M. McDonald, C. O. Perera, SY. Quek and A.B.
Woolf. The effect of metabolic stress disinfection and disinfestation (MSDD) on
‘Hass’ avocado and mortality of longtailed mealybug (Pseudococcus longispinus).
Postharvest Biology and Technology (2012) 64: 138-145. Chapters 2 and 4 of this
thesis.

Zulhendri F, Jamieson LE, C. O. Perera, SY. Quek and A.B. Woolf. Optimization of
metabolic stress disinfection and disinfestations (MSDD) against longtailed mealybug
(Pseudococcus longispinus). Journal of Economic Entomology. Accepted with
revisions, Chapter 5 of this thesis.

Zulhendri F, Brewster D., R. Simpson, J. Cooney, L.E. Jamieson, C. O. Perera, SY.
Quek and A.B. Woolf. Uncovering the mechanisms of MSDD using proteomics tools;
2-D differential gel electrophoresis (2-DIGE) and ESI-MS/MS. Journal of Economic
Entomology. Submitted, Chapter 3 of this thesis.
161
Conference proceedings

Zulhendri F, Jamieson L.E, Brewster D, Simpson R, Cooney J, Jensen D, Perera C. O,
Quek S. Y, Woolf A. B. Metabolic stress disinfection and disinfestation (MSDD): An
alternative to fumigants for disinfestation treatment. APHC/AuSHS/NZIAHS,
Horticulture for Future Conference, 18 – 22 September 2011, Lorne, Victoria,
Australia.

Zulhendri F, Brewster D, Simpson R, Cooney J, Jensen D, Jamieson L. E., Quek S.
Y., Perera C. O, Woolf A. B. Uncovering the mechanisms of metabolic stress
disinfection and disinfestation (MSDD) using proteomics tools. New Zealand Institute
of Food Science & Technology, Science to Reality New Zealand and Beyond
Conference, 29 June – 1 July 2011, Rotorua, NZ.

Zulhendri F, Jamieson L, R. M. McDonald, C. O. Perera, SY. Quek and A.B. Woolf,
2010. The effect of Metabolic-Stress Disinfestation and Disinfection on physiology of
apple and avocado. Poster presentation at the Gordon Research Conference, 27th June
- 2nd July 2010. Tilton, New Hampshire, USA.

Zulhendri F, Jamieson L, R. M. McDonald, C. O. Perera, SY. Quek and A.B. Woolf,
2010. A Novel Disinfestation Technology (Metabolic-Stress Disinfestation and
Disinfection)- fruit physiology perspective. Poster presentation at NZ Institute of
Food Science and Technology conference, 23rd - 25th June 2010. Auckland, New
Zealand.

Zulhendri F, Jamieson L, Curtain J, Reade K, McDonald R, Perera C, Quek SY,
Woolf AB, 2009. Can we replace Methyl Bromide? - MSDD against long-tailed
mealybug (Pseudococcus longispinus Targioni-Tozzetti). Poster presentation at the
Postharvest Pacifica, 15th – 19th November 2009. Napier, New Zealand.
162

3. Biochemical mechanisms of insecticidal efficacy

Transcription

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