Journal of Insect Physiology 136 (2022) 104340

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Journal of Insect Physiology

journal homepage: www.elsevier.com/locate/jinsphys

Methoprene treatment increases activity, starvation and desiccation risk of

Queensland fruit fly
Saleh Mohammad Adnan a, c, *, Iffat Farhana a, Polychronis Rempoulakis b, Phillip W. Taylor a
a
Applied BioSciences, Macquarie University, Australia
b
Biosecurity and Food Safety, NSW Department of Primary Industries, Australia
c
Department of Primary Industries and Regional Development, Western Australia, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Juvenile hormone is an important regulator of sexual development in insects, and application of methoprene, a
Methoprene juvenile hormone analogue, together with access to a protein-rich diet, has been found to accelerate sexual
Food maturation of several tephritid fruit fly species including Queensland fruit fly Bactrocera tryoni (‘Q-fly’). Such
Water
accelerated development is a potentially valuable means to increase participation of released males in sterile
Stress
Desiccation
insect technique programs. However, there is a risk that benefits of accelerated maturation might be countered
Vulnerability by increased vulnerability to starvation and desiccation. The present study investigates this possibility. After
emergence, flies were treated with three levels of methoprene (0, 0.05%, and 0.5%) incorporated into a diet of
sugar and yeast hydrolysate for two days after emergence. Survival of groups and individual flies was assessed
under conditions of food stress, food and water stress, and ad libitum access to diet, and survival of individual flies
was also assessed under desiccation stress. Most flies provided ad libitum access to diet were still alive at 7 days,
whereas all stressed flies died within 4 days. Desiccation stressed flies had the shortest survival followed by food
and water stress, and then food stress. Methoprene supplements increased susceptibility of flies to each stress.
Flies subjected to food and water stress had the least lipid reserves at death, whereas flies subjected to desiccation
stress retained the least water reserves. To investigate mechanisms that might underlie reduced survival under
stress; we also quantified activity level of flies that were subjected to food and water stress and desiccation stress.
Activity level was greater for flies provided methoprene, but did not vary with stress type or sex, suggesting that
increased vulnerability of flies to stress is related to elevated metabolism associated with elevated activity.
Deleterious effects of methoprene supplements on stress tolerance indicate a need for careful consideration of the
conditions that will be encountered by flies in the field before deploying methoprene as a pre-release treatment
in Q-fly sterile insect technique programs.

1. Introduction affecting a vast range of commercial and non-commercial crops in

Tephritid fruit flies (Diptera: Tephritidae) present significant chal­
lenges to production and trade of fruit and vegetables by reducing the
yield of marketable crops and by triggering quarantine measures and
restrictions on international trade in most tropical and subtropical re­
gions, and in some temperate regions of the world (Christenson and
Foote, 1960; Hardy, 1969, 1983; Leblanc et al., 2013; Qin et al. 2015;
White and Elson-Harris, 1992). Amongst 11 economically important
fruit fly species that are currently present in Australia, Queensland fruit
fly (Bactrocera tryoni (Froggatt), or ‘Q-fly’) is the most difficult and
costly challenge to market access for Australia’s horticulture industries,
eastern Australian states and territories (Dominiak and Daniels, 2012;
Dominiak and Mapson, 2017; PHA, 2008).
The Sterile Insect Technique (SIT) is a sustainable technology that is
growing rapidly in prominence as a means of controlling Q-fly pop­
ulations. SIT has been deployed to manage some of the world’s most
economically important tephritid fruit fly pests, including the Mediter­
ranean fruit fly, or medfly, Ceratitis capitata (Wiedemann) (Reyes et al.
2007), melon fly, Zeugodacus cucurbitae (Coquillett) (Itô et al. 1993;
Kakinohana 1994), oriental fruit fly, Bactrocera dorsalis (Hendel)
(Orankanok et al. 2007), and Mexican fruit fly, Anastrepha ludens (Loew)
(Orozco-Dávila et al. 2015). SIT involves releasing millions of sterile

* Corresponding author at: Applied BioSciences, Macquarie University, Australia.

E-mail address: saleh-mohammad.adnan@mq.edu.au (S. Mohammad Adnan).

https://doi.org/10.1016/j.jinsphys.2021.104340
Received 13 May 2021; Received in revised form 21 November 2021; Accepted 22 November 2021
Available online 25 November 2021
0022-1910/© 2021 Elsevier Ltd. All rights reserved.
S. Mohammad Adnan et al.
insects that reduce the reproductive capacity of pest populations by
inducing reproductive failure in females (Benelli et al. 2014; Knipling
1955). For successful SIT, a large proportion of the released male insects
must survive long enough to mature sexually and participate in mating.
Many fruit flies have long adult maturation phases and high mortality
rates in the field, such that a quite small proportion of the released flies
might survive to mature and contribute to SIT (Liedo et al. 2002; Pérez-
Staples et al. 2008; Reynolds et al. 2012; Weldon et al. 2008). Treat­
ments that increase the proportion of male insects contributing to SIT
can greatly increase the efficacy of management programs.
Like many other tephritid fruit flies (Drew and Yuval, 2000; Liedo
et al. 2013), Q-flies are anautogenous, needing to acquire nutritional
resources as adults, especially protein, to complete reproductive devel­
opment (Drew and Yuval, 2000). Yeast hydrolysate (YH) mixed with
sugar is a standard adult diet used to maintain fruit fly colonies,
providing a rich source of amino acids, micronutrients and carbohy­
drates (Fanson and Taylor 2012) that is effective at sustaining repro­
ductive development (Meats and Leighton 2004; Pérez-Staples et al.
2007; Vijaysegaran et al. 2002). When provided for even a 1–2 day pre-
release period, YH and sugar are effective for sustaining development of
male Q-flies over the following days (Pérez-Staples et al. 2011; Weldon
and Taylor 2011), increasing the prevalence of mature sterile flies in the
field (Reynolds et al. 2014). While providing pre-release nutritional
resources that support development is valuable, supplements that can
further reduce the delay between release and maturity remain of
particular interest (Adnan et al., 2018, 2020d; Akter et al., 2017).
Juvenile hormone (JH) functions in the endocrine system of insects
and regulates numerous physiological processes, including larval
development, metamorphosis, diapause, and reproduction (Dubrovsky,
2005; Gilbert et al., 2000; Riddiford and Ashburner, 1991; Vogel et al.,
1979; Wyatt and Davey, 1996). Considering the physiological role
played by JH in reproductive development, application of methoprene,
an analogue of JH, has been investigated as practical means of accel­
erating development of adult fruit flies in SIT programs (Teal et al. 2000,
2013). Treatment with methoprene has been effective in accelerating
sexual maturation in Z. cucurbitae (Haq et al., 2010a,b). A. fraterculus
(Segura et al., 2009, 2013), A. ludens (Gómez et al., 2013; Pereira et al.,
2013), A. suspensa (Pereira et al., 2009, 2010) and in Q-fly (Adnan et al.,
2018, 2019, 2020a,b).
While wild Q-flies have a quite wide bioclimatic range (Sultana et al.,
2017, Sutherst, 2000; Yonow and Sutherst, 1988), domesticated Q-flies
such as are used in SIT programs, can have greatly diminished ability to
tolerate starvation and desiccating conditions (Taylor et al., 2013;
Weldon et al., 2013). There is a risk that the changes in physiological
state induced by methoprene treatment might further exacerbate this
potential constraint on SIT (Haq et al., 2013). Treatments that accelerate
maturation at the cost of diminished environmental tolerance may be an
overall detriment to SIT. In the present study, we investigate whether
dietary methoprene treatment affects the survival of Q-flies challenged
with food stress, food and water stress, and desiccation stress under
laboratory conditions. Because sexing strains are not yet available for Q-
fly, we investigated the effect of pre-release treatment on both males and
females.
The ability of an insect to survive under prolonged food and water
stress is generally linked to its nutritional status (metabolic reserves) and
the rate at which stored reserves are utilised (metabolic rate) (Bennett
et al., 1990). In Drosophila melanogaster, reduced utilisation of lipids can
contribute to increased starvation resistance (Djawdan et al., 1997;
Harshman and Schmid, 1998; Hoffmann and Parsons, 1993), while
higher body water content and water storage in the haemolymph (Folk
et al., 2001; Gibbs et al., 1997), as well as increased metabolism of
stored carbohydrates that produce metabolic water, can increase
desiccation resistance (Marron et al., 2003). Understanding the mech­
anisms underlying stress resistance in insects can be important in
applied contexts, such as SIT, in which insects are required to perform
under sometimes challenging conditions. To gain insights to the role of
2
Journal of Insect Physiology 136 (2022) 104340
dry mass, lipid and water reserves on ability of Q-flies to survive under
food and water stress in the presence and absence of desiccation stress,
we quantified the effects of methoprene treatment on dry mass, water
content, and lipid content at the commencement of stress exposure and
at death. While fitness related traits including longevity, fecundity, body
mass and metabolic reserves have been well studied as stress resistance
characters in insects, links between activity level and stress tolerance are
much less known. Locomotor activity is directly or indirectly linked with
many fitness-related activities, including courtship (Cobb et al., 1987;
Greenspan and Ferveur, 2000), foraging and dispersal (Martin et al.,
1999), and predator avoidance (Heath and Wilkin, 1970; Lang et al.,
2006; Simmons, 1988). Daily activity levels may be linked to overall
energy reserves, metabolic state, or health (Catterson et al., 2010,
Dominiak et al., 2014; Fanson et al., 2013; Weldon et al. 2010b). To gain
insights to mechanisms mediating the effects of methoprene on survival
and reserves, we also investigated links between methoprene treatment
and activity level of flies subjected to food and water stress in the
presence and absence of desiccation stress.
2. Materials and methods
2.1. Insects
Pupae of B. tryoni were obtained from a colony maintained at Mac­
quarie University, New South Wales, Australia (22–25 generations from
wild). Larvae were reared on a gel larval diet (Moadeli et al., 2017) that
was based on the liquid diet formulation of Chang et al. (2006). All cages
were maintained in a controlled environment room at 25 ± 0.5 ◦ C,
65 ± 5% RH, and a 12:12 LD photoperiod with a simulated dawn and
dusk in which lights gradually ramped up or down over 30 min at the
beginning and end of each light phase. Approximately 4500 pupae were
placed in three 47.5 × 47.5 × 47.5 cm fine mesh cages (Megaview
Bugdorm 4S4545, Taiwan) for adult emergence. Usually, few flies
emerge on the first day of emergence, and these were discarded. After
the following 24 h of emergence, all non-emerged pupae were removed
from the cages.
2.2. Treatments
Newly emerged adult males and females were provided ad libitum
access to a diet of sugar and yeast hydrolysate (MP Biomedicals LLC)
(3:1) containing methoprene at 0.05%, 0.5%, and 0% (control) for two
days (following Adnan et al., 2018, 2019). Methoprene was obtained as
NOMOZ® pellets (a trademark of Pacific Biologists, Brisbane, Queens­
land) that contain PROLINK® as active ingredient (40 g/kg (S)-metho­
prene). This product is locally available as a mosquito larvicide. NOMOZ
pellets were finely ground using a mortar and pestle. Then, the
powdered NOMOZ (as required on dry weight basis to achieve desired
methoprene concentrations) was added into diet of sugar and yeast
hydrolysate (w/w) using a blender (Adnan et al., 2018, 2019).
2.3. Survival assays
2.3.1. Group survival
Following exposure to methoprene treatments (see above), the
treated flies were immediately subjected to environmental stresses.
From each methoprene treatment (0.5%, 0.05%, and 0% methoprene),
two groups of 50 males and two groups of 50 female flies were trans­
ferred to separate 12-L cages. The cages were of clear plastic and had
three 100 mm-diameter mesh-covered openings for ventilation. Survival
of flies was assessed under the following stress conditions:
• Food stress: Flies had access to moistened cotton wool as a source of
water but no food.
• Food and water stress: Flies had no access to food or water.
S. Mohammad Adnan et al.
• Ad libitum access to sugar and water (Control): Flies had access to
sugar and water.
Dead flies were collected from the cage floor and counted every 6 h
(06:00, 12:00, 18:00, and 00:00 h AEST) for 7 days. The experiment was
replicated twice (i.e., total of 200 flies were tested for each sex,
methoprene treatment, and stress combination).
2.3.2. Individual survival
Application of methoprene treatments for individual survival assays
was similar to that of group survival assays. Following methoprene
treatment, 40 females and 40 males from each methoprene treatment
(0.5%, 0.05%, and 0% methoprene) were transferred individually to 5-
mL round bottom glass vials (Lab Australia Pty Ltd, Australia). Survival
of flies was assessed under the following stress treatments:
• Food stress: Flies were individually placed in glass vials with access
to a 1 g block of agar gel (prepared by boiling 10 g agar in 100 ml
water and cooling to set before cutting), thereby allowing access to
water but not food.
• Food and water stress: Flies were provided no access to food or water.
• Desiccation: Flies were exposed to two to three large granules of
desiccant (silica beads, Sigma Aldrich) to test survival under condi­
tions of food and water stress (above) with the added challenge of
desiccating conditions. A loose barrier of cotton wool was used to
separate the flies from the desiccant (following Weldon et al., 2013).
• Ad libitum access to Sugar and water (Control): The flies were placed
individually placed in glass vials with a 1 g block of sugar-agar diet
(30 g white sugar, 100 ml water, 10 g agar, 1.5 g Nipagin), thereby
allowing fly access to water and carbohydrate.
In all treatments, the glass vials were tightly sealed with plugs to
maintain conditions. Mortality was recorded by visually inspecting the
vials every 3 h for 7 days. Flies were considered dead when they were
incapable of holding onto the inner surface of the vial, and when no
movement of their legs or mouthparts was observed after the vials were
gently flicked with a finger. Three replicates of the experiment were
conducted.
2.4. Metabolic reserve analysis
2.4.1. Initial body water, dry mass, and lipid content
Following methoprene treatment for 48 hrs, 40 females and 40 males
from each methoprene treatment (0.5%, 0.05%, and 0%) not subjected
to stress resistance assay were separated. Flies were then subdued for
two minutes in a − 20 ◦ C freezer and weighed to the nearest 0.0001 g
using an analytical balance (Sartorius AG Microbalance ME5 24807564,
Germany) to measure initial wet weight. Then flies were stored at
− 30 ◦ C freezer for 2 – 4 weeks for later assessment of dry mass, water
content, and lipid content. The flies were then oven dried at 60 ◦ C for 48
hrs and re-weighed to calculate initial dry mass. Dry mass was sub­
tracted from initial wet mass to calculate initial body water. While we
weighed the samples, silica gel was used to prevent the flies from
absorbing moisture from the air. Lipids were then extracted from the
flies using three 24 h washes of chloroform (Sigma Aldrich) (Benelli
et al., 2019; Bligh et al., 1959; Folch et al., 1957; Nguyen et al., 2019). At
the end of the third chloroform wash, the vials were kept open in a fume
hood so that chloroform evaporated overnight. The samples were dried
again for 48 h and reweighed. The difference between the two dry
weights indicated the initial soluble lipid content (Ellers, 1996; Rau­
benheimer et al., 2007, Ponton et al., 2015).
2.4.2. Remaining body water, dry mass, and lipid content at death
Forty females and forty males from each methoprene treatment
(0.5%, 0.05%, and 0%) were transferred individually to 5 ml round
bottom glass vials (Lab Australia Pty Ltd, Australia). Flies were then
3
Journal of Insect Physiology 136 (2022) 104340
subdued for two minutes in a − 20 ◦ C freezer and weighed to the nearest
0.0001 g using an analytical balance (Sartorius AG Microbalance ME5
24807564, Germany) (initial wet weight). The flies were then returned
to their vials and were subjected to food stress, food and water stress, or
desiccation stress (see above). Mortality was recorded by visually
inspecting the vials every 3 h. Wet weight was recorded again at death.
After weighing, flies were stored at − 30 ◦ C for 2 – 4 weeks. The flies
were then oven dried at 60 ◦ C for 48 hrs and re-weighed to calculate dry
mass at the time of death. Dry mass was subtracted from wet mass at
death to calculate body water remaining at death. While we weighed the
samples, silica gel was used to prevent the flies from absorbing moisture
from the air. Finally, remaining lipids content at death was determined
following above-mentioned procedures (Ellers, 1996).
2.5. Activity under stress
Activity under stress was assessed using a Locomotor Activity
Monitor (LAM10, TriKinetics, MA, USA) (see Dominiak et al 2014;
Fanson et al. 2013). Flies were placed in glass tubes (8 mm internal
diameter) that were plugged at both ends under two environmental
conditions; (i) food and water stress under which flies were given no
access to food and water inside the glass tubes and (ii) desiccation stress
under which flies were exposed to the same nutritional conditions as
under food and water stress with the addition of four to six large gran­
ules of silica gel desiccant (Sigma-Aldrich, USA). A loose barrier of
cotton wool separated the flies from the desiccant. For each stress con­
dition, a total of 15 males and 15 female flies from two methoprene
treatments were assessed (0.5% and 0% methoprene; only the higher
methoprene dose was tested as this treatment increased mortality risk in
survival assays). For each LAM, two glass vials were kept empty to check
for errors in readings that were not generated by moving flies. Flies were
transferred to activity monitors ca. 4 h before recording started. Activity
was assessed over 24 h as the total number of times that each fly passed
through the tube mid-point. The experiments were repeated two times
using flies from two different batches of pupae, with a total of 480 flies
recorded.
2.6. Statistical analysis
All analyses were conducted using R v3.5.1 (R Core Team, 2018). For
the individual survival assay, two separate analyses were conducted. For
the flies with ad libitum access, data were censored, and a Cox-
proportional hazards model was used including dose and sex as main
effects using coxph() from the survival package (Therneau, 2015). Post-
hoc pairwise comparisons were performed with a Tukey correction
using emmeans package (Lenth, 2019). For the flies subjected to stress
treatments, data were not censored (all died) and a linear mixed model
was performed on survival times (log-transformed) including metho­
prene treatment, sex, and stress as fixed effects. Post-hoc pairwise
comparisons were performed using Tukey’s tests. In both models all
interactions were initially included, and a backward model selection
process was performed by removing non-significant interactions.
Similarly, for survival of the flies with ad libitum access in the group
survival assays, a frailty Cox proportional hazard model was used
including methoprene treatment and sex as main effects and cage as a
random effect. For the flies subjected to stress treatments, a linear mixed
model was performed including methoprene treatment, sex, and stress
as fixed effects and cage as a random effect. Again, post-hoc pairwise
comparisons were performed with a Tukey correction. In both models all
interactions were initially included, and a backward model selection
process was performed by removing non-significant interactions.
For analysis of metabolic reserves, separate linear models were used
for both initial and remaining water, dry mass, and lipid content. In case
of initial metabolic reserves for each response variable, sex, methoprene
treatment as well as their interactions were included as main effects.
Initial body mass centered at mean was also included as linear covariates
S. Mohammad Adnan et al.
to control for variable initial mass. Similarly, for remaining metabolic
content for each response variable, sex, methoprene treatment, and
stress were included as main effects as well as all interactions. Initial
body mass and survival time were also included as linear covariates to
control for variable initial mass as well as to control for differences in
survival times. Both covariates were centered at their mean. Post-hoc
pairwise comparisons were performed using Tukey’s tests. Model as­
sumptions were assessed using graphical analyses of the residuals.
A general linear model (GLM) was performed for total activity under
stress assuming a Gaussian distribution. Model assumptions were
assessed by graphical analyses of the residuals. Total activity was
square-root transformed to stabilize the variance. The model included
fixed main effects of methoprene treatment, stress condition and sex.
Post-hoc pairwise comparisons were performed with Tukey’s tests.
Initially all interactions were included in the model, and a backward
model selection process was performed by removing non-significant
interactions.
3. Results
3.1. Group survival
All flies that were subjected to food and water stress and food stress
died during the trial period, whereas 99.5% of the flies provided with ad
libitum food and water access were alive after 7 days. For the group with
full access to food and water, there was no effect of methoprene treat­
ment (χ2 ¼ 0.11, df = 2, P > 0.9) or sex (χ2 ¼ 0.11, df = 1, P > 0.9) on
survival (Fig. 1). Excluding the group with full access to food and water,
Fig. 1. Relationship between types of stress (food stress, food and water stress, ad libitum access) and survival of male and female Q-flies after being fed a diet of sugar
plus yeast hydrolysate combined with one of three levels of methoprene supplementation (0, 0.05, 0.5 %) for two days after emergence and then were subjected to
group survival assay in 12-L cages.
4
Journal of Insect Physiology 136 (2022) 104340
the survival of flies in stress treatments was significantly affected by
stress type (χ2 ¼ 27.4, df = 1, P < 0.001) and methoprene treatment
(χ2 ¼ 178.3, df = 2, P < 0.001), but not by sex (χ2 ¼ 25, df = 1, 1153,
P = 0.12). Flies subjected to food stress lived longer than those subjected
to food and water stress (estimate of differences = 0.11 ± 0.02,
P < 0.001) (Fig. 1). For both stressors, flies treated with methoprene had
a shorter survival than untreated flies (Fig. 1). The effects of methoprene
on survival time increased with the dose of methoprene; flies provided
0.5% methoprene had the shortest survival, followed by 0.05% metho­
prene and control (methoprene 0.05 vs. control estimate of
differences = -0.18 ± 0.02, P < 0.001; methoprene 0.5 vs. control esti­
mate of differences = -0.33 ± 0.02, P < 0.001; Methoprene 0.05 vs.
methoprene 0.5 estimate of differences = 0.14 ± 0.020, P < 0.001)
(Fig. 1).
3.2. Individual survival
Survival of flies provided ad libitum access to food and water did not
vary with sex (χ2 ¼ 0.82, df = 1, P = 0.37) or with methoprene treat­
ment (χ2 ¼ 0.60, df = 2, P = 0.74), whereas survival of the flies exposed
to stress conditions was significantly influenced by the stress type
(χ2 ¼ 297.4, df = 2, P < 0.001) and methoprene treatment (χ2 ¼ 297.4,
df = 2, P < 0.001) but not by sex (χ2 ¼ 0.11, df = 1, P = 0.74) (Fig. 2).
Among the stressed flies, survival of the flies that were subjected to
desiccation was the shortest, whereas flies subjected to food stress had
the longest survival. Flies subjected to food and water stress had inter­
mediate survival (desiccation vs. food stress difference of estimate (log-
scale) = -0.41 ± 0.017, P < 0.001; desiccation vs. food and water stress
S. Mohammad Adnan et al. Journal of Insect Physiology 136 (2022) 104340

Fig. 2. Relationship between types of stress (food stress, food and water stress, desiccation, and ad libitum access) and survival of male and female Q-flies after being
fed a diet of sugar plus yeast hydrolysate combined with one of three levels of methoprene supplementation (0, 0.05, 0.5 %) for two days after emergence and then
were subjected to survival assay in 5 ml individual vials.

difference of estimate (log-scale) = -0.13 ± 0.016, P < 0.001; food stress

Table 1
vs. food and water stress, estimate of differences (log-
Linear model analysis of initial body water, dry mass, and lipid content at 48hrs
scale) = 0.28 ± 0.076, P < 0.001). Survival of both sexes declined
of age in relation to fly sex (male, female), and methoprene treatment (0, 0.05%,
significantly as dose of methoprene increased. For all stressors, flies that and 0.5%).
were provided methoprene had shorter survival compared to the flies
Metric Variable df F P
provided no methoprene and flies provided 0.5% methoprene had
shorter survival than the flies provided 0.05% methoprene (methoprene Water Treatment 2, 299 0.3087 0.7346
Sex 1, 299 50.5055
0.05% vs. control estimate of differences = -0.14 ± 0.016, P < 0.001; <0.001
Treatment × sex 2, 299 0.1469 0.8635
methoprene 0.5% vs. control estimate of differences = -0.20 ± 0.016,
P < 0.001; methoprene 0.05% vs. methoprene 0.5% estimate of Dry mass Treatment 2, 299 24.0613 <0.001
differences = 0.06 ± 0.016, P = 0.001). Sex 1, 299 59.7299 <0.001
Treatment × sex 2, 299 2.4341 0.0894
Lipid Treatment 2, 299 17.9559 <0.001
3.3. Initial metabolic reserves after 48hrs of methoprene treatment Sex 1, 299 81.9162 <0.001
Treatment × sex 2, 299 7.1717 <0.001
3.3.1. Initial water content
Initial water content after 48hrs of methoprene treatment was
significantly affected by sex, but not by methoprene treatment (Table 1). 3.3.2. Initial dry mass
Females contained significantly more water than males Initial dry mass varied significantly with sex and methoprene treat­
(males = 7.723 ± 0.071, females = 8.440 ± 0.071). ment (Table 1). Females had significantly greater dry mass than males

5
S. Mohammad Adnan et al. Journal of Insect Physiology 136 (2022) 104340

(males = 3.112 ± 0.038, females = 3.526 ± 0.038). Flies provided Table 2

0.05% and no methoprene had significantly greater dry mass than flies Linear model analysis of remaining body water, dry mass, and lipid content at
provided 0.5% methoprene. However, there was no significant differ­ death in relation to fly sex (male, female), stress (food stress, food and water
ences in dry mass of provided 0.05% and no methoprene (methoprene stress, and desiccation) and methoprene treatment (0, 0.05%, and 0.5%).
0.5 = 3.060 ± 0.046, methoprene 0.05 = 3.409 ± 0.046, Metric Variable df F P
control = 3.489 ± 0.046). Remaining water at Treatment 2, 2.5 0.08
death 704
3.3.3. Initial lipid content Sex 1, 7.6 0.059
Initial lipid reserves varied significantly with sex and methoprene 704
Stress 2, 136.4
treatment including significant treatment × sex interaction (Table 1).
<0.001
704
Male flies provided 0.5% and 0.05% had initial lipid content that was Treatment × sex 2, 0.7 0.49
not significantly different from each other, but both had significantly 704
lower initial lipid content than the untreated control flies (Fig. 3). Treatment × stress 4, 2.9 0.022
704
However, in females there was no significant differences in lipid content
Sex × stress 2, 1.5 0.23
amongst the flies provided 0.05%, 0.5%, and no methoprene (Fig. 3). 704
Treatment × sex × stress 4, 5.14 0.265
704
3.4. Remaining metabolic reserves
Dry mass at death Treatment 2, 9.9 <0.001
700
3.4.1. Remaining water at death Sex 1, 37.9 <0.001
The remaining water content of the flies at death varied significantly 700
with stress condition along with significant stress type × methoprene Stress 2, 21.7 <0.001
700
treatment interaction, but not with sex (Table 2, Fig. 4). Flies that were
Treatment × sex 2, 12.3 <0.001
exposed to desiccation conditions had the lowest remaining body water 700
at death whereas food stressed flies had the highest remaining body Treatment × stress 4, 4.4 0.0015
water at death. Flies subjected to food and water stress had intermediate 700
remaining water content at death (desiccation vs. food stress difference Sex × stress 2, 13.9 <0.001
700
of estimate = 2.6 ± 0.14, P < 0.001; desiccation vs. food and water stress
Treatment × sex × stress 4, 3.2 0.012
difference of estimate = 1.4 ± 0.12, P < 0.001; food stress vs. food and 700
water stress difference of estimate = 1.1 ± 0.13, P < 0.001). Effects of
methoprene treatment on remaining water at death were evident for Remaining lipid at Treatment 2, 6.6 0.0014
death 699
only the flies that were subjected to desiccation conditions; flies pro­
Sex 1, 0.1 0.0077
vided 0.5% methoprene retained more water at death than control flies 699
under desiccation conditions (Fig. 4). Flies provided 0.05% methoprene Stress 2, 127.8 <0.001
had intermediate water content at death (Fig. 4). 699
Treatment × sex 2, 4.4 0.013
699
3.4.2. Dry mass at death
Treatment × stress 4, 23.6 <0.001
Dry mass content at death was significantly affected by sex, stress, 699
and dose of methoprene treatment including all possible significant in­ Sex × stress 2, 17.1 <0.001
teractions (Table 2, Fig. 5). Under desiccation stress, remaining dry mass 699
Treatment × sex × stress 4, 8.6
was greater for control males compared to males treated with either <0.001
699
methoprene dose, whereas females provided 0.5% and no methoprene
had greater dry mass than females provided 0.05% methoprene (Fig. 5).
Food stressed males that were provided 0.5% methoprene had lower
remaining dry mass content at death compared to untreated males while
males provided 0.05% methoprene were intermediate. However, in
males and females exposed to food and water stress, and females

Fig. 3. Initial lipid (mg) content (Mean ± SE) in male and female Q-flies that
were provided a diet of sugar plus yeast hydrolysate combined with one of three
levels of methoprene supplementation (0, 0.05, and 0.5 %) for two days after
emergence. Different letters indicate significant differences among treatments.
Fig. 4. Body water (mg) remaining at death (Mean ± SE) in male and female Q-
flies that were provided a diet of sugar plus yeast hydrolysate combined with
one of three levels of methoprene supplementation (0, 0.05, 0.5 %) for two days
after emergence and then were subjected to survival assay in 5 ml glass vial
under three types of stresses (food stress, food and water stress, and desicca­
tion). Different letters indicate significant differences among levels of metho­
prene supplementation within assays.

6
S. Mohammad Adnan et al.
Fig. 5. Dry mass (mg) at death (Mean ± SE) in male and female Q-flies that
were provided a diet of sugar plus yeast hydrolysate combined with one of three
levels of methoprene supplementation (0, 0.05, 0.5 %) for two days after
emergence and then were subjected to survival assay in 5 ml glass vial under
three types of stresses [food stress (c), food and water stress (b), and desiccation
(a)]. Different letters indicate significant differences among levels of metho­
prene supplementation within each sex.
exposed to food stress, there was no significant effect of dose on
remaining dry mass (Fig. 5).
3.4.3. Lipid level at death
Lipid reserves remaining at death varied significantly with sex, stress
treatment, and methoprene treatment including all possible significant
interactions (Table 2, Fig. 6). Flies that were subjected to desiccation
stress retained more lipid at death than those subjected to food stress
(desiccation vs. food stress difference of estimate = 0.99 ± 0.07,
P < 0.001) and food and water stress (desiccation vs. food and water
stress difference of estimate = 0.93 ± 0.06, P < 0.001). However, there
was no evidence of difference between flies exposed to food stress and
food and water stress (food stress vs. food and water stress difference of
estimate = 0.06 ± 0.06, P = 0.66). Significant sex × stress × methoprene
dose interaction reveals several patterns. For males that were subjected
to desiccation and food stress, there was no significant effect of
methoprene dose on lipid reserves at death, whereas under food and
7
Journal of Insect Physiology 136 (2022) 104340
Fig. 6. Lipid content (mg) at death (Mean ± SE) in male and female Q-flies that
were provided a diet of sugar plus yeast hydrolysate combined with one of three
levels of methoprene supplementation (0, 0.05, 0.5 %) for two days after
emergence and then were subjected to survival assay in 5 ml glass vial under
three types of stresses [food stress (c), food and water stress (b), and desiccation
(a)]. Different letters indicate significant differences among levels of metho­
prene supplementation within each sex.
water stress males treated with 0.5% methoprene had more lipid re­
serves remaining at death than those treated with 0.05% methoprene or
no methoprene (Fig. 6). Females treated with both methoprene doses
had significantly less lipid reserves at death than untreated flies when
exposed to desiccation, whereas under food stress females provided both
methoprene doses had significantly greater lipid reserves at death than
untreated females. However, under food and water stress, as in males,
females treated with 0.5% methoprene had significantly more lipid re­
serves at death than those treated with 0.05% methoprene or no
methoprene (Fig. 6).
3.5. Activity under stress
Fly activity was significantly greater for the flies provided metho­
prene than for the flies provided no methoprene (F1, 467 = 17.13,
P < 0.001; Fig. 7). However, there was no evidence of significant dif­
ferences between males and females (F1, 467 = 2.20, P = 0.13) or be­
tween the two stress conditions (F1, 467 = 0.72, P = 0.41; Fig. 7).
S. Mohammad Adnan et al.
Fig. 7. Effect of methoprene treatment (0 or 0.5%), sex (male or female), and
stress type (food and water stress or desiccation) on total activity level
(Mean ± SE). Means separated by lower case letters indicates significant dif­
ferences between levels of methoprene supplementation within assays for
each sex.
4. Discussion
Methoprene supplements provided in a protein-rich diet for two days
following emergence have previously been found to significantly
accelerate sexual maturation of Q-flies (Adnan et al., 2018, 2020c;
Collins et al., 2014). Potentially countering the benefits of this rapid
maturation for SIT programs, we here find that methoprene treatment
diminishes the ability of Q-flies to tolerate nutritional and water stress.
To gain insights to the underlying physiological mechanisms mediating
the increased vulnerability of methoprene-treated flies, we considered
the effects on locomotor activity that might deplete water and energy
reserves, and the specific role of reserves.
The deleterious effects of methoprene on survival under stress are
likely related to elevated metabolic state that drives accelerated devel­
opment. The effects of dietary protein on ability to withstand dietary
stress provides a salient parallel. Like many fruit flies, Q-flies are
anautogenous, requiring dietary protein as adults to complete devel­
opment (Drew and Yuval, 2000). Although able to survive long periods
without access to protein, once access to protein has been acquired
development accelerates sharply (Meats and Leighton 2004) and
reproduction-associated demographic aging commences (Yap et al.
2015). However, reproductive development is associated with substan­
tial metabolic demands, and this may lead to increased vulnerability of
the flies when they are subjected to nutritional deprivation. Compared
with Q-flies maintained only with access to sugar, Q-flies that have
received protein for two days in simulation of pre-release feeding have
greatly increased mortality when subsequently denied access to food
(Taylor et al., 2013). While methoprene treatment has little effect on Q-
flies that lack access to protein, it substantially increases the already
8
Journal of Insect Physiology 136 (2022) 104340
high rate of development in flies that have access to protein (Adnan
et al., 2018; Gómez et al., 2013; Haq et al., 2010a, Segura et al., 2009;
Pereira et al., 2013) and in doing so is expected to substantially elevate
metabolic rate and physiological demands. Just as the development-
promoting effects of dietary yeast hydrolysate increase vulnerability to
starvation, the accelerated development of methoprene treated male Q-
flies appears to come at a physiological cost of even further diminished
stress tolerance (Aceves-Aparicio et al., 2021). While methoprene
application for first 48 h after emergence reduced the survival of Q-flies
under nutritional and desiccation stress, effects of methoprene under
nutritional stress were not evident in studies of Z. cucurbitae (Haq et al.,
2013). The reasons for this species difference may relate not only to
biological differences between the species but also to the differences in
method of methoprene application, and experimental design.
We did not find any evidence of sex differences in survival under any
of the tested stress conditions. This is consistent with findings of Taylor
et al. (2013), who also found no differences in starvation resistance of
male and female Q-flies in group cage assays that resembled those of the
present study. However, unlike the present study, Weldon et al. (2010a,
2013) found that male Q-flies survived for longer than females under
desiccating conditions. Differences between studies in responses of male
and female flies to desiccation stress may reflect differences in the ages
at which the flies were tested, differences in the domestication status of
the colonies used, or differences in the rearing conditions used. Weldon
et al. (2010a) used flies from a mass-reared colony using lucerne chaff
larval diet; Weldon et al (2013) used wild type flies from naturally
infested hosts, whereas the present study used flies reared on a high
performing gel larval diet.
Resistance to both starvation and desiccation can be explained by
two distinct physiological mechanisms: amounts of resources (e.g., en­
ergy, water) that are available for somatic maintenance, and consump­
tion rate (which is expected to be linked to metabolic rate) (Hoffmann
and Parsons, 1991). In the present study, flies that died under food stress
retained the least lipid reserves. On the other hand, flies that died under
desiccation stress retained the least body water. Therefore, the effects of
methoprene on vulnerability of Q-fly to stress are likely explained
largely by increased utilisation of lipid and water reserves. Reduced
metabolic rate is a common mechanism for stress resistance (Blows and
Hoffmann, 1993; Hoffmann and Parsons, 1989a, b, 1991). Decreased
metabolic rate increases how long insects can survive under food and
water stress and reduces the need to open spiracles, and consequently be
exposed to loss of water, in desiccating conditions (Addo-Bediako et al.,
2001; Lighton, 1994, 1996; Zachariassen, 1996). Similar to our results,
in D. melanogaster survival under food and water stress particularly relies
on availability and use of lipid reserves (Arrese and Soulages, 2010;
Herrewege and David, 1997; Hoffmann and Harshman, 1999). Simi­
larly, A. serpentina and A. ludens deplete lipid reserves to survive under
food and water stress (Jacome et al., 1995; Tejeda et al., 2014). Accu­
mulation of lipids and dry mass is also associated with increased star­
vation resistance in Drosophila (Chippindale et al., 1996), and this is
likely also important in Q-fly. However, by testing flies at a stage when
resources are directed largely toward reproductive development rather
than the accumulation of reserves, this being the context for SIT releases
conducted when flies are 2 – 3 days of age, the ability of flies to accu­
mulate reserves may have been quite limited. The ability to tolerate
stress depends largely on the types and amounts of energy flies metab­
olised (Marron et al., 2003). In D. melanogaster no significant difference
has been found in the rate of lipid and protein metabolism during food
and water stress and desiccation stress, but carbohydrate metabolism
has been found to be greatly elevated under desiccation stress (Marron
et al., 2003). While lipids provide over twice as much energy per gram as
carbohydrates (Withers, 1992), and are appropriate fuels to counter
starvation, carbohydrate reserves are more effective for countering
desiccating conditions, and may be key to understanding some effects
observed in the present study.
Methoprene-treated flies made the least use of water and lipid stores
S. Mohammad Adnan et al.
under desiccation and nutritional stress respectively even though flies
provided methoprene contained greater amounts of lipid prior to
commencement of stress assays. A possible explanation is that lipid
storage provides fuel to survive under food stress only if methoprene-
treated flies are able to adequately up-regulate lipid metabolism under
these conditions. If methoprene-treated flies have greater need to access
reserves but lack ability to upregulate access quickly enough, then they
would succumb with more resources remaining unexploited. Similarly,
if methoprene treated flies could better metabolize carbohydrates under
desiccation stress, metabolic water could be released to increase sur­
vival. Inability of the methoprene-treated flies to adequately utilize the
available metabolic reserves under stress appears to at least partly
explain the increased vulnerability of methoprene-treated flies.
Dietary provision of methoprene resulted in increased activity when
two day old flies were exposed to nutritional and desiccation stress.
Increased activity of methoprene-treated flies might be linked to at­
tempts to escape unfavorable conditions. Elevated activity has also been
reported in nutritionally stressed Drosophila and mosquitoes (Bross
et al., 2005; Hagan et al., 2018; Kim et al., 2014), which might be
explained by increased demand to search for nutrients (Williams et al.,
2004; Ye et al., 1994). It is likely that elevated locomotor activity
induced by methoprene treatment imposes a substantial physiological
burden that exposes Q-flies to elevated energetic and nutritional de­
mands and renders them less able to tolerate adverse environmental
conditions.
In conclusion, while the incorporation of methoprene into pre-
release diet significantly accelerates sexual development of Q-fly
males (Adnan et al., 2018, 2020c) and is very promising as a pre-release
treatment for SIT, the present study indicates significant risks if flies are
released in conditions where food and water are scarce. Methoprene
treated flies have increased vulnerability to food stress, food and water
stress, and desiccation stress, and there is a need to consider such risks
when deploying methoprene as a pre-release supplement in operational
SIT programs. It may be that methoprene treatments are suitable in areas
and seasons with more benign environmental conditions, but not in
other areas and seasons. However, the present study has demonstrated
the deleterious effects of methoprene supplementation in a laboratory
context which may not reflect the real scenarios of operational SIT in the
field. It is expected that sterile flies released in SIT would manage to
locate fruiting trees as a source of shelter and food, which would
certainly minimise their vulnerability to nutritional and desiccation
stress upon release in the fields. Accordingly, by tailoring pre-release
treatments to likely field conditions, pre-release treatments could be
applied when advantageous by minimising the adverse effects of
increased stress vulnerability.
CRediT authorship contribution statement
Saleh Mohammad Adnan: Conceptualization, Methodology,
Investigation, Formal analysis, Writing – original draft, Review & Edit­
ing, Visualization. Iffat Farhana: Conceptualization, Methodology,
Investigation, Writing – review & editing. Polychronis Rempoulakis:
Conceptualization, Methodology, Investigation, Writing – review &
editing. Phillip W. Taylor: Conceptualization, Formal analysis, Writing
– review & editing, Supervision.
Acknowledgements
This research was conducted as part of the SITplus collaborative fruit
fly program. Project Raising Q-fly Sterile Insect Technique to World
Standard (HG14033) is funded by the Hort Frontiers Fruit Fly Fund, part
of the Hort Frontiers strategic partnership initiative developed by Hort
Innovation, with co-investment from Macquarie University and contri­
butions from the Australian Government. SMA was supported by Mac­
quarie University Research Excellence Scholarships. We thank Francisco
Díaz Fleischer and Chris Weldon for their valuable comments on the
9
Journal of Insect Physiology 136 (2022) 104340
manuscript.
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