Factors Affecting Water Deprivation Resistance in Bactrocera oleae (Olive Fruit Fly)

: The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), causes significant damage to olive crops worldwide. However, the factors affecting its survival under water deprivation have not been studied yet. In this study, the water deprivation resistance of male and female olive fruit flies was measured at three ages in virgin and mated adults fed either a full or a restricted diet. The experiments (24 treatments) were conducted under constant laboratory conditions, using insects collected in the wild and reared on olives. Additionally, a baseline experiment was conducted to provide data on the insects’ life expectancy under no-stress conditions. Our findings revealed that males showed much less resistance under water deprivation compared to females. Younger adults endured for longer than older ones, and adults fed a restricted diet endured water deprivation longer than those fed a full diet. Our results suggest that during periods of water scarcity, releasing sterile males is most effective, because the wild male population decreases. Since females of reproductive age are more resistant, this should ensure a higher number of matings with sterile males. These findings can be used to formulate improved pest control strategies that enhance olive product quality while relying less on insecticides.


Introduction
The olive fruit fly, known as Bactrocera oleae (Rossi) (Diptera, Tephritidae), is a significant pest that affects olive crops.Its distribution has now extended across the Mediterranean basin, North America, and Sub-Saharan Africa [1].The developing larvae of these flies feed on the mesocarp of the olives, leading to crop losses that can exceed 90% if their population is not effectively managed [2].Insecticides have traditionally been employed to control the olive fruit fly.However, field results have shown the development of resistance in the insects to these chemical treatments [3,4].Furthermore, there are growing concerns about the potential health risks posed to humans and other mammals due to the presence of pesticide residues, which are frequently detected in olive oil [5].In recent years, efforts have been made to adopt more sustainable and eco-friendly approaches, such as studying essential oils with insecticidal properties or oviposition deterrent activity on B. oleae [6][7][8].Additionally, there has been progress in the research on the use of genetic methods for pest management [9,10].
In olive groves, alternative biological control methods are available, such as the release of parasitoid hymenopterans belonging to the Braconidae family [11,12].Lately, there has been growing interest in investigating more sustainable pest control methods, one of which is the sterile insect technique (SIT) [13,14].Moreover, a technology focused on a genetically modified olive fruit fly strain with self-limiting characteristics has been proposed as a method for managing the olive fruit fly [15].The effectiveness of these methods hinges on the production of mass-reared male insects.When released into the wild in significant numbers, these sterile males are expected to outcompete their wild counterparts and to demonstrate better resilience in challenging environmental conditions, such as periods of water scarcity [16][17][18].The key to the success of these alternative methods of pest control lies in studying the physiology and biology of the target organisms.
Environmental conditions such as thermal stress, relative humidity, starvation, water deprivation, and desiccation are the most frequently encountered environmental challenges that insects may confront during their lifespans [19][20][21].A recent study on B. oleae showed that the population density of, and the economic damage caused by, this pest between the years of 2019 and 2021 in Iran were highly influenced by relative humidity in combination with temperature and orchard management practices [22], while another study reported that the insect's population was affected by local climate and weather conditions [23].According to the existing literature, desiccation refers to a state of very low relative humidity, leading to the dehydration of insect bodies [21,[24][25][26][27][28][29].On the other hand, water deprivation refers to a condition where water is unavailable for the insects to access [30,31].While desiccation resistance has been studied in Tephritid flies to prevent the spread of harmful species [21,25], water deprivation in constant relative humidity, and in B. oleae specifically, remains unexplored.
Both desiccation and water deprivation are conditions that adults of B. oleae are likely to encounter due to climate change and the periods of drought observed during the summer months.B. oleae is a species that requires access to water for survival; without it, the olive fruit fly perishes within a few hours or days, regardless of the relative humidity [32,33].In periods of extended drought in non-irrigated olive groves, the population of B. oleae declines due to decreased survival and reproduction [23,32,33].Moreover, it has been found that high summer temperatures constrain the population of B. oleae adults in California's Central Valley [32].Given the recent increase in mean temperatures and a decrease in mean relative humidity during the summer period [34][35][36], these factors are expected to negatively affect the biology of B. oleae.High temperatures, along with reduced humidity, hinder the ovarian maturation process in the olive fruit fly [37].The key factors considered to affect resistance under water deprivation and desiccation in insects usually include their age, sex, diet, and mating [38][39][40].In the current study, we examined how mating status (virgin and mated adults), age (15, 30, and 45 days old), sex (males and females), and diet (full and restricted) affect water deprivation resistance in B. oleae adults.Furthermore, a previous study conducted in our laboratory found that these factors affect food deprivation resistance in B. oleae [41].
Based on the existing literature, similar studies on Pachycrepoideus vindemmiae (Rondani) (Hymenoptera: Pteromalidae) have shown that water scarcity conditions induce behavioral changes [42], while Culex tarsalis (Coquillett) (Diptera: Culicidae) exhibits increased responsiveness to oviposition cues, predominantly laying their egg rafts around dusk [43].Recent studies on D. melanogaster revealed that thirst transforms water avoidance into active seeking and helps flies to associate smells with water rewards.This indicates that water seeking, learned water seeking, and water learning involve distinct neural circuits in dehydrated flies [44].Results from another study showed the identification of novel central brain circuit elements, showcasing their role in coordinating internal state drives to selectively regulate motivated seeking behavior [45].Hopefully, similar studies on B. oleae may yield insights applicable to mass-reared populations intended for sterile insect technique programs, enhancing the resistance of the released insects to water deprivation compared to their wild counterparts.Additionally, in sterile insect technique programs, factors such as age, diet, and mating are taken into consideration when rearing sterile males, to ensure their competitiveness with their wild counterparts [18].
Our study comprises basic biological research aimed at understanding how age, sex, diet, and mating affect olive fruit fly resistance to water deprivation.We investigate the following scientific questions: 1.
Do older adults exhibit the same resistance to water deprivation as younger ones? 2.
Do males and females exhibit the same resistance to water deprivation?3.
Does the adults' diet affect resistance to water deprivation?4.
Does mating affect resistance to water deprivation?5.
Do sex, diet, and mating affect insect life expectancy?
By prioritizing eco-friendly practices over widespread chemical applications, we align with global efforts to minimize environmental impact.Moreover, our research supports the development of integrated pest management (IPM) strategies, fostering a diverse approach that reduces the risk of resistance in insect populations.In a nutshell, our study represents a shift towards environmentally conscious methods, aiming to reveal the conditions under which B. oleae adults are more vulnerable to water deprivation.

Materials and Methods
This study was conducted at the Laboratory of Applied Zoology and Parasitology, School of Agriculture, Aristotle University of Thessaloniki, Greece, during the period between October 2021 and February 2022.The experiment took place under constant and continuously controlled laboratory conditions: temperature (T) of 25 ± 2 • C, relative humidity (RH) of 45 ± 5%, and a photoperiod of L14:D10.The experiment consisted of three main stages (see Figure 1): (1) preparing materials and determining experimental conditions; (2) preparing experimental insects in cages; and (3) recording insect deaths due to water deprivation every 4 h daily.
Appl.Biosci.2024, 3, FOR PEER REVIEW 3 Our study comprises basic biological research aimed at understanding how age, sex, diet, and mating affect olive fruit fly resistance to water deprivation.We investigate the following scientific questions: 1. Do older adults exhibit the same resistance to water deprivation as younger ones? 2. Do males and females exhibit the same resistance to water deprivation?3. Does the adults' diet affect resistance to water deprivation?4. Does mating affect resistance to water deprivation?5. Do sex, diet, and mating affect insect life expectancy?By prioritizing eco-friendly practices over widespread chemical applications, we align with global efforts to minimize environmental impact.Moreover, our research supports the development of integrated pest management (IPM) strategies, fostering a diverse approach that reduces the risk of resistance in insect populations.In a nutshell, our study represents a shift towards environmentally conscious methods, aiming to reveal the conditions under which B. oleae adults are more vulnerable to water deprivation.

Materials and Methods
This study was conducted at the Laboratory of Applied Zoology and Parasitology, School of Agriculture, Aristotle University of Thessaloniki, Greece, during the period between October 2021 and February 2022.The experiment took place under constant and continuously controlled laboratory conditions: temperature (T) of 25 ± 2 °C, relative humidity (RH) of 45 ± 5%, and a photoperiod of L14:D10.The experiment consisted of three main stages (see Figure 1): (1) preparing materials and determining experimental conditions; (2) preparing experimental insects in cages; and (3) recording insect deaths due to water deprivation every 4 h daily.While conducting the experiments, we also performed a baseline experiment on the life expectancy of adults under the same constant laboratory conditions as the main experiments and without any stress applied (with access to food and water).In the baseline experiment, 100 adults were used in each treatment group (800 adults in total) and 8 treatments were considered: (sex: male, female) × (diet: full, restricted) × (mating status: virgin, mated).The total number of insects obtained in the main experiments was 1200 = (50 virgin males, 50 virgin females, 50 mated males, 50 mated females) × (2 diets) × (3 ages: 15, 30, 45 days old), and the main experiments included a total of 24 treatments: (age: 15, 30, While conducting the experiments, we also performed a baseline experiment on the life expectancy of adults under the same constant laboratory conditions as the main experiments and without any stress applied (with access to food and water).In the baseline experiment, 100 adults were used in each treatment group (800 adults in total) and 8 treatments were considered: (sex: male, female) × (diet: full, restricted) × (mating status: virgin, mated).The total number of insects obtained in the main experiments was 1200 = (50 virgin males, 50 virgin females, 50 mated males, 50 mated females) × (2 diets) × (3 ages: 15, 30, 45 days old), and the main experiments included a total of 24 treatments: (age: 15, 30, and 45 days old) × (sex: male, female) × (diet: full, restricted) × (mating status: virgin, mated).

Preparing Materials and Determining Experimental Conditions
Larvae were reared in non-infested olive fruit from pest-and disease-free trees (Figure 2).Initially, there were about 8000 adult insects, but this number decreased to 1200 flies due to mortality.Adults were transferred using an aspirator to avoid disturbing them.The aspirator allowed insects to move in and out without force.Any disturbed or injured insects were immediately removed and replaced.Survival of adults following water deprivation, serving as an indicator of water deprivation resistance, was evaluated under controlled laboratory conditions.During experiments, special care was taken to ensure that insects would not be affected by any type of disturbance (i.e., noise or other human activities).

Preparing the experimental insects in cages
Before the experiment, the following steps were taken to prepare the experimental insects (Figure 3):

•
Step 1: Pupae from infested olives were placed in Petri dishes in Plexiglass cages to ensure the required number of adults for the treatments.

•
Step 2: Upon emergence, adults were placed individually in plastic cup cages with water and either full or restricted diet until the 12th day of their life to prevent overcrowding.

•
Step 3: On the 13th day of their life, groups of 10 virgin males or females were placed in Plexiglass cages.Additionally, groups of 5 virgin males and 5 virgin females were placed in Plexiglass cages for one day to mate (Figure 3).This procedure was repeated for the two diets and the three ages.
On the 13th day, between 16:00 and 21:00, a human observer monitored insects in cages to verify mating.This time of day was chosen because mate searching and courtship in this species typically occur in the late evening [47,48].Individuals that had not mated were removed from the experiment and replaced with mated ones.

•
Step 4: On the 14th day of their life, insects were placed back in individual plastic cup cages before water deprivation, to study their resilience to water deprivation individually.The materials used in the experiments were as follows: (a) Three types of insect cage types: (A) BugDorm-type cages (30 × 30 × 30 cm) for rearing and maintaining insect colonies [46]; (B) Plexiglass transparent cages (20 × 20 × 20 cm) for obtaining experimental insects and facilitating mating or same-sex cohabitation on the 13th day of their life; and (C) plastic cup individual cages (6.5 × 8 × 9.5 cm) for studying insects individually.A total of 12 BugDorm-type cages, 120 plexiglass cages, and 2400 plastic-cup cages were used.The cages were supplied with either a full diet (water/sugar/yeast hydrolysate) or a restricted diet (sugar only).Water was provided via a wet cotton wick in all cages except for when insects were subjected to water deprivation.Ventilation was ensured in plastic cup cages by affixing a piece of plastic cloth with holes to the side of each cup.(b) Two types of diet: (A) Full diet, which was a laboratory-prepared mixture of hydrolyzed yeast (protein) with a ratio of 5:4:1 (water/sugar/yeast hydrolysate) commercially known as "Yeast Hydrolysate"; and (B) restricted diet, which consisted solely of commercial crystal sugar, without any protein.The food placed in each cage was maintained at a proper quality and quantity.Special care was taken to ensure that the food remained fresh and attractive to the insects.(c) Olive fruits: Non-infested and infested olive fruits were collected from olive trees in Chalkidiki, Northern Greece.Non-infested olives (approximately 100 kg) were handpicked to ensure that they were free from pests and diseases then stored in glass containers in the laboratory refrigerator.Infested olives were obtained using McPhail traps with attractants to monitor adult flights and infestation timing.Weekly collections of infested olives over three months ensured genetic diversity and maintained insect populations for experiments.Infested olives were harvested and hatched in wooden cages with water and protein-rich food and then transferred to fresh olives.After mating, females laid eggs in the olives, which were then placed into basins and covered with a piece of burlap to maintain optimal conditions.It should be mentioned that experiments with wild B. oleae are challenging due to the need for fresh, non-infested olives over the insect's life cycle, which must be promptly harvested and refrigerated.Pesticides and BVOCs can affect measurements, so olives should be rinsed and ideally sourced from organic groves.The limited preservation time of olives and the difficulty in obtaining large numbers of insects quickly complicate experiments.Maintaining consistent laboratory conditions and obtaining sufficient adults, especially older ones, are also challenging due to high mortality rates.In the experiment, efforts to reduce bias and obtain representative samples increased complexity and time requirements, highlighting the difficulty of conducting experiments on B. oleae.

Preparing the experimental insects in cages
Before the experiment, the following steps were taken to prepare the experimental insects (Figure 3):

•
Step 1: Pupae from infested olives were placed in Petri dishes in Plexiglass cages to ensure the required number of adults for the treatments.

•
Step 2: Upon emergence, adults were placed individually in plastic cup cages with water and either full or restricted diet until the 12th day of their life to prevent overcrowding.

•
Step 3: On the 13th day of their life, groups of 10 virgin males or females were placed in Plexiglass cages.Additionally, groups of 5 virgin males and 5 virgin females were placed in Plexiglass cages for one day to mate (Figure 3).This procedure was repeated for the two diets and the three ages.
On the 13th day, between 16:00 and 21:00, a human observer monitored insects in cages to verify mating.This time of day was chosen because mate searching and courtship in this species typically occur in the late evening [47,48].Individuals that had not mated were removed from the experiment and replaced with mated ones.

•
Step 4: On the 14th day of their life, insects were placed back in individual plastic cup cages before water deprivation, to study their resilience to water deprivation individually.

Recording the Longevity of the Insects Deprived of Water
The recording of insects' deaths as a measure of their resistance to water deprivation consisted of two main phases (Figure 4): (a) period during which insects had access to water, and (b) period during which no water was available, and the recording of deaths took place.More detailed information is as follows:

Recording the Longevity of the Insects Deprived of Water
The recording of insects' deaths as a measure of their resistance to water deprivation consisted of two main phases (Figure 4): (a) period during which insects had access to water, and (b) period during which no water was available, and the recording of deaths took place.More detailed information is as follows: • Phase 1-Period with access to water: upon reaching the 15th, 30th, and 45th day of their adult life, 50 individual adults from each treatment were transferred to new individual plastic cup cages with the corresponding diet and without access to water.

•
Phase 2-Recording resistance to water deprivation: deaths were recorded every four hours during the light period at 08:00, 12:00, 16:00, and 20:00 daily as a measure of water deprivation resistance.

Experimental Design
The dependent variable assessed in the experiments was the duration of survival for insects following water deprivation, specifically measured in hours as a measure of water deprivation resistance.A consistent human observer recorded insect deaths at 4 h intervals daily.The water deprivation resistance was calculated based on the recorded date and time of death.The initial fly count was at least sixfold greater than those used in the experiments to account for pre-experimental fly mortality.Each of the 24 treatments included 50 replications, consisting of insects of the same sex, mating status, and age and fed the same diet.A wild B. oleae individual matures for mating after the 7th day [49]; hence, the 13th day was chosen for mating.The selection of the three age classes was designed to explore the response of adults to water deprivation as they became older, to reveal potential vulnerabilities.

Statistical Analyses
The survival resistance of adult insects was evaluated using the analysis of variance (ANOVA) method within the methodological framework of general linear models.The ANOVA model included the main effects of four factors, six two-way interactions, four three-way interactions, and one four-way interaction.These factors included, as mentioned above, the mating status factor with two levels, the diet factor with two levels, the In cases where there were difficulties in discerning an insect's death, a fine paintbrush was employed to carefully manipulate the insect and verify its death.Daily rotation of the position of the plastic cages was implemented to mitigate possible experimental systematic errors.Water deprivation resistance was calculated as the difference between the recorded date and time of death and the corresponding date and time when the insects were subjected to water deprivation.

Experimental Design
The dependent variable assessed in the experiments was the duration of survival for insects following water deprivation, specifically measured in hours as a measure of water deprivation resistance.A consistent human observer recorded insect deaths at 4 h intervals daily.The water deprivation resistance was calculated based on the recorded date and time of death.The initial fly count was at least sixfold greater than those used in the experiments to account for pre-experimental fly mortality.Each of the 24 treatments included 50 replications, consisting of insects of the same sex, mating status, and age and fed the same diet.A wild B. oleae individual matures for mating after the 7th day [49]; hence, the 13th day was chosen for mating.The selection of the three age classes was designed to explore the response of adults to water deprivation as they became older, to reveal potential vulnerabilities.

Statistical Analyses
The survival resistance of adult insects was evaluated using the analysis of variance (ANOVA) method within the methodological framework of general linear models.The ANOVA model included the main effects of four factors, six two-way interactions, four three-way interactions, and one four-way interaction.These factors included, as mentioned above, the mating status factor with two levels, the diet factor with two levels, the sex factor with two levels, and the age factor with three levels.There was a total of 24 combinations of the four factor levels (3 × 2 × 2 × 2 = 24).The ANOVA method was used to estimate standard errors for differences between mean values of factor level combinations.Tukey's multiple-comparison procedure [50] assessed the significance of differences among these mean values.
Residuals from linear models were tested for normality and homoscedasticity.Normality was evaluated by inspecting histograms and boxplots, comparing residuals' median values with zero, assessing skewness and kurtosis, and using the Kolmogorov-Smirnov test for normality (p > 0.05).The homoscedasticity assumption was tested by visually inspecting the scatter plot of the residuals to compare it against the model's predicted values and by assessing the magnitude of Spearman's rho rank correlation coefficients between the absolute values of the residuals and the predicted values estimated by the model.No significant violations of these assumptions were detected.The data are presented as mean ± standard error (SE).
To further understand the impact of the independent variable, partial eta squared (n 2 partial ) was calculated.The "partial eta squared, η 2 " value indicates the effect size (ES) of an effect (main or interaction) after all other effects have been removed.It is calculated by the formula η 2 = SSA SSA+SSE , where SSA is the sum of squares of effect of interest, and SSE is the sum of squares of the error [51].The quantities SSA and SSE are provided via the correct ANOVA table.The partial eta squared values were 0.203 for sex and 0.232 for age, indicating that approximately 20.3% and 23.2% of the variance in the dependent variable could be attributed to these independent variables, respectively, when accounting for the effects of other variables in the model [50].Detailed values for partial eta squared for the main effects and two-way, three-way, and four-way interactions are provided in Supplementary Figure S1.
Additional descriptive statistical indices are provided in Supplementary Table S1.Given that only the terminal survival time of the insects was recorded, there was no specific need to employ a survival analysis model.All statistical analyses were conducted using IBM SPSS Statistics v.26.0 Software (IBM Corp., Armonk, NY, USA) [52].The significance level in all statistical hypothesis testing procedures was preset at a = 0.05 (p ≤ 0.05).
Finally, within each treatment, the percentage (%) of the population that survived above or below (hours of water deprivation endurance) the total survival median value (90 h) across all treatments was calculated.The factors considered in this calculation were age, sex, diet, and mating status.Additionally, the age of adults subjected to water deprivation and their observed resistance to water deprivation were calculated as a percentage (%) of their life expectancy, based on our baseline data and our study results.

Results
The results of the Tukey post hoc analysis that was applied to examine whether statistically significant differences existed between the treatments are given in Figures 5 and 6 and in Supplementary Table S3.Descriptive statistics and the detailed ANOVA results are available in Supplementary Tables S1 and S2 S2).No statistically significant differences were observed for mating status (p = 0.763, Supplementary Table S2).
0.020; F(1, 23) = 6.41, p = 0.012,  = 0.008; F(2, 23) = 3.95, p = 0.020,  = 0.010; F(2, 23) = 17.94, p < 0.001,  = 0.043, respectively).Consequently, the focus is mainly on examining these interactions using the simple-simple effects analysis approach [53].Specifically, the two-way interactions between the three ages and the two sexes were examined within each diet [53] (Figures 5 and 6).The comparison between ages is based on the mean water deprivation resistance of the insects, measured in hours until death.Age and sex have the greatest effect sizes ( values), followed by the interactions of mating status × sex, sex × age, diet, and the interaction of mating status × age (Supplementary Figures S1 and S2).The percentage (%) of virgin and mated individuals that survived beyond or equal to and below the total survival median value (90 h) after water deprivation is presented in Figure 7.Following water deprivation, the percentage (%) of individuals surviving beyond or equal to the total survival median value (90 h) was over 50% in the following cases: (a) all virgin females, regardless of age and diet consumed, (b) all 15-day-old mated individuals, regardless of sex and diet consumed, and (c) all 30-day-old mated females, regardless of the diet consumed.
The results indicate that the percentage (%) of individuals surviving beyond or equal The water deprivation resistance (in hours until death) for virgin males and females at the three different ages is depicted in Figure 5, for both those fed the full diet and those fed the restricted diet.The resistance for mated males and females is shown in Figure 6.
The mean values for water deprivation resistance (hours to death) for the 24 treatments (2 diets × 3 ages × 2 sexes × 2 mated status) in Figures 5 and 6 are comparable with each other.Based on Figures 5 and 6 and on Supplementary Table S3, we can conclude that 15-day-old mated females fed the restricted diet endured water deprivation longer compared to those fed the full diet, while 15-and 30-day-old virgin females endured water deprivation better than their male counterparts, regardless of the type of food consumed.Similar differences can be observed in mated individuals fed the restricted diet (sugar).However, for mated individuals fed with the full diet (protein), only 30-day-old females endured water deprivation better than males.Regardless of diet, 45-day-old virgin females endured for a shorter time than their 15-day-old counterparts.Additionally, 45-day-old virgin females fed the restricted diet endured for a shorter time than their 30-day-old counterparts.Also, regardless of diet, 45-day-old mated females endured for a shorter time than their younger counterparts.No differences with aging are observed in virgin males in any treatment, while 30-and 45-day-old mated males endured for a shorter time than their 15-day-old counterparts, regardless of diet.
Additionally, significant two-way interactions were observed between mating status and sex, mating status and age, diet and sex, diet and age, and sex and age, as indicated by ANOVA (F(1, 23) = 37.79, p < 0.001, n 2 partial = 0.045; F(2, 23) = 8.15, p < 0.001, n 2 partial = 0.020; F(1, 23) = 6.41, p = 0.012, n 2 partial = 0.008; F(2, 23) = 3.95, p = 0.020, n 2 partial = 0.010; F(2, 23) = 17.94, p < 0.001, n 2 partial = 0.043, respectively).Consequently, the focus is mainly on examining these interactions using the simple-simple effects analysis approach [53].Specifically, the two-way interactions between the three ages and the two sexes were examined within each diet [53] (Figures 5 and 6).The comparison between ages is based on the mean water deprivation resistance of the insects, measured in hours until death.Age and sex have the greatest effect sizes (n 2 partial values), followed by the interactions of mating status × sex, sex × age, diet, and the interaction of mating status × age (Supplementary Figures S1 and S2).
The percentage (%) of virgin and mated individuals that survived beyond or equal to and below the total survival median value (90 h) after water deprivation is presented in Figure 7.Following water deprivation, the percentage (%) of individuals surviving beyond or equal to the total survival median value (90 h) was over 50% in the following cases: (a) all virgin females, regardless of age and diet consumed, (b) all 15-day-old mated individuals, regardless of sex and diet consumed, and (c) all 30-day-old mated females, regardless of the diet consumed.
The results indicate that the percentage (%) of individuals surviving beyond or equal to the total survival median value (90 h) decreased with age in all treatments, in both virgin and mated adults.Moreover, in most treatments, the percentage (%) of females surviving beyond or equal to the total survival median value (90 h) was higher than that of males.The highest percentage (%) of individuals surviving beyond or equal to the total survival median value (90 h) after water deprivation was 96.67%.This percentage (%) pertained to 15-day-old virgin females fed the full diet and mated females of the same age fed the restricted diet.On the other hand, the lowest percentage (%) of individuals that survived beyond or equal to the median value (90 h) was 13.04%, and this percentage (%) pertained to 45-day-old virgin males fed the restricted diet.A baseline experiment was conducted to determine the life expectancy of adults under constant, stress-free laboratory conditions with access to food and water (Table 1).Each treatment group included 100 adults to establish a baseline dataset (800 adults in total for all treatments).Virgin males on a full diet live 49.8% longer than mated males on a restricted diet, while virgin females on a full diet live 35.9% longer than mated females on a restricted diet.The life expectancy of virgin males and females fed the full diet is approximately the same (Table 1).However, females exhibit greater water deprivation resistance than males as 15-and 30-day-old adults (Table S3 and Figure 5).It can be deduced that diet and mating status greatly affect longevity in both sexes.Figure 8 depicts the age of adults subjected to water deprivation (as a percentage % of their life expectancy) and the corresponding observed resistance to water deprivation (also as a percentage % of life expectancy) based on the baseline data.This is shown for virgin and mated males and females at three different ages (as 15-, 30-, and 45-day-old adults) fed either a full or restricted diet.Figure 8 shows that 15-day-old mated females fed the restricted diet exhibited the highest resistance to water deprivation (9.5% of their total life expectancy), while the lowest resistance was observed in 45-day-old virgin males fed the full diet (2.5% of their total life expectancy).Detailed data regarding Figure 8 are given in Supplementary Table S4.A baseline experiment was conducted to determine the life expectancy of adults under constant, stress-free laboratory conditions with access to food and water (Table 1).Each treatment group included 100 adults to establish a baseline dataset (800 adults in total for all treatments).Virgin males on a full diet live 49.8% longer than mated males on a restricted diet, while virgin females on a full diet live 35.9% longer than mated females on a restricted diet.The life expectancy of virgin males and females fed the full diet is approximately the same (Table 1).However, females exhibit greater water deprivation resistance than males as 15-and 30-day-old adults (Table S3 and Figure 5).It can be deduced that diet and mating status greatly affect longevity in both sexes.Figure 8 depicts the age of adults subjected to water deprivation (as a percentage % of their life expectancy) and the corresponding observed resistance to water deprivation (also as a percentage % of life expectancy) based on the baseline data.This is shown for virgin and mated males and females at three different ages (as 15-, 30-, and 45-day-old adults) fed either a full or restricted diet.Figure 8 shows that 15-day-old mated females fed the restricted diet exhibited the highest resistance to water deprivation (9.5% of their total life expectancy), while the lowest resistance was observed in 45-day-old virgin males fed the full diet (2.5% of their total life expectancy).Detailed data regarding Figure 8 are given in Supplementary Table S4.

Discussion
In our study, we contribute to the global efforts to glean insights into the development of more efficient pest control strategies, emphasizing eco-friendly practices over widespread chemical applications, following recent trends in more sustainable pest management techniques [13,14].Accordingly, we focused on understanding the impact of age, sex, diet, and mating on B. oleae adults' resistance to water deprivation, to facilitate targeted interventions exploiting vulnerabilities in the adult stage of the insect.Furthermore, this study represents the first comprehensive assessment of baseline data for B. oleae adult life expectancy under constant laboratory conditions, demonstrating that diet and mating status greatly influence longevity in both sexes.
Improving sterile male performance has been a longstanding concern among scientists [54].According to our findings, age emerges as a crucial factor, with younger adults exhibiting higher resistance to water deprivation across all treatments.This implies that aging renders the olive fruit fly population more vulnerable to water deprivation.These results are consistent with other studies on Tephritid flies, such as Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) [24] and Ceratitis cosyra (Walker) (Diptera: Tephritidae) [40], which also show that younger individuals endure longer periods of desiccation than older ones.In our experiments, larvae were fed olives, which have a high-water content (about 70% of their weight) [55].This high-water content likely explains why younger flies endured water deprivation better than older ones, due to their water reserves.However, in

Discussion
In our study, we contribute to the global efforts to glean insights into the development of more efficient pest control strategies, emphasizing eco-friendly practices over widespread chemical applications, following recent trends in more sustainable pest management techniques [13,14].Accordingly, we focused on understanding the impact of age, sex, diet, and mating on B. oleae adults' resistance to water deprivation, to facilitate targeted interventions exploiting vulnerabilities in the adult stage of the insect.Furthermore, this study represents the first comprehensive assessment of baseline data for B. oleae adult life expectancy under constant laboratory conditions, demonstrating that diet and mating status greatly influence longevity in both sexes.
Improving sterile male performance has been a longstanding concern among scientists [54].According to our findings, age emerges as a crucial factor, with younger adults exhibiting higher resistance to water deprivation across all treatments.This implies that aging renders the olive fruit fly population more vulnerable to water deprivation.These results are consistent with other studies on Tephritid flies, such as Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) [24] and Ceratitis cosyra (Walker) (Diptera: Tephritidae) [40], which also show that younger individuals endure longer periods of desiccation than older ones.In our experiments, larvae were fed olives, which have a high-water content (about 70% of their weight) [55].This high-water content likely explains why younger flies endured water deprivation better than older ones, due to their water reserves.However, in the study of C. cosyra, no evidence was found to suggest that metabolic water from stored nutrients increased desiccation resistance.Our findings also align with a study on Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) [56], which reported that desiccation resistance decreased with age.Given that younger male olive fruit flies exhibit higher mobility [57] and endure water deprivation better than older males, this insight could update sterile insect techniques.
Additionally, based on the findings of our study, males exhibited less water deprivation resistance than females at reproductive age (15 days old).These findings suggest that releasing sterile males during periods of water scarcity is most effective, as it reduces the wild male population while females of reproductive age exhibit greater resistance, ensuring higher mating rates with sterile males.Thus, drought periods and arid regions are likely more suitable for applying sterile insect techniques.The higher endurance in females underscores a potential biological difference between the sexes in response to water deprivation, a phenomenon that could be further explored through genetic experiments.Moreover, our results are consistent with a study on D. melanogaster regarding desiccation resistance, which also found that males exhibit lower resistance to water deprivation compared to females [58].However, a study on Lucilia eximia (Wiedemann) (Diptera: Calliphoridae) within forensic entomology reported no differences between males and females in treatments after water deprivation [30].Furthermore, resistance to desiccation differs between males and females in some Aedes species [59], while a study on B. tryoni found that males endure longer periods of desiccation than females [21].
Regarding the effect of diet on insect water deprivation, our study revealed that 15day-old females fed a restricted diet endured water deprivation longer compared to those fed a full diet.This difference may be attributed to the fact that Tephritidae adults require a protein-rich diet for maturation and reproduction [60].Specifically, it has been reported that Tephritidae females consuming protein-rich foods experience increased fertility, resulting in greater egg production [61][62][63][64][65][66].Therefore, female B. oleae on a restricted diet may produce fewer eggs than those on a full diet.Consequently, our findings indicate that increased egg production is associated with higher water consumption requirements in females [37].The results of our study align with previous research on C. cosyra, where newly emerged adults exhibited higher desiccation resistance when unfed [40].Similarly, a study on Aedes aegypti (Linnaeus) (Diptera, Culicidae) reported that combining a sugar-based diet with controlled exposure to water deprivation appears to be a promising strategy, as it encourages more matings between sterile males and wild females, ultimately reducing the pest population over time [67].
In our study, no differences in water deprivation resistance were observed between virgin and mated adults.A study on B. tryoni adults reported that the continual decline in desiccation resistance with aging cannot be attributed to the onset of maturity, reproductive processes, or sexual activity [24].Furthermore, according to the results of our study, the simultaneous effect of mating status and diet is not statistically significant in water deprivation endurance.However, based on our baseline data, diet and mating status greatly influence longevity in both sexes.Previous studies on Tephritid flies and D. melanogaster indicate that larval diet and environmental conditions are more critical factors than adult diet in the reproductive process of flies [40,68,69].
Future research could focus on isolating and thoroughly examining the genes associated with increased resistance to water deprivation for potential use in mass rearing programs for sterile insect techniques [15].To this end, further studies should explore natural olive grove conditions, where factors affecting insect water deprivation resistance may vary.A study on C. cosyra adults found that those fed a protein-rich diet as larvae exhibited the highest body water content [40].Therefore, investigating whether larval diet in B. oleae affects water deprivation resistance could be informative.Additionally, it is important to study how the size of B. oleae adults affects their resistance to water deprivation.Further research is crucial in exploring the effects of food and water deprivation under different environmental conditions, such as extreme temperatures, in B. oleae.A study on C. capitata [70] found that limited access to food and water reduced female resistance to high temperatures by 54%, while males were unaffected.This knowledge could contribute to developing strains capable of tolerating water stress, which could be employed in sterile insect technique programs [27,29].

Conclusions
Our study provides insights into water deprivation resistance in B. oleae adults, highlighting their response to conditions of water scarcity in natural settings, such as during droughts.Additionally, the study demonstrates how age, sex, diet, and mating, as well as their interactions, impact water deprivation resistance in B. oleae.
The main conclusions drawn from this research are as follows: • Based on the results of the main experiments, we can deduce the following: • Age: Age significantly influences an individual's ability to withstand water deprivation, with younger adults consistently exhibiting higher resistance across all treatments compared to older ones.

•
Sex: Sex plays a critical role in water deprivation resistance, with females generally enduring longer periods than males.During drought periods, releasing sterile males is recommended as wild male populations decline, while wild females seem to be less vulnerable to water scarcity.This strategy can enhance mating feasibility between wild females and sterile males, thereby improving the effectiveness of sterile insect technique programs.

•
Diet: Adults fed a full diet exhibit lower resistance to water deprivation, highlighting the significant impact of diet on B. oleae.This finding underscores the importance of diet composition in sterile insect technique programs, where a protein-based diet is crucial for the insect, but a sugar-based diet enhances competitiveness.

•
Mating status: Mating does not significantly affect the water deprivation resistance of B. oleae.

•
Based on our baseline experiment under constant laboratory conditions, diet and mating greatly influence longevity in both sexes.Specifically, virgin adults fed a full diet exhibit the highest longevity, while mated adults fed a restricted diet exhibit the lowest.
Overall, while the life expectancy of virgin males and females fed a full diet is approximately the same, females demonstrate greater water deprivation resistance than males as 15-and 30-day-old adults.Age, sex, and diet significantly influence water deprivation resistance in B. oleae.These findings should guide the development of more sustainable sterile insect techniques.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/applbiosci3030021/s1,Table S1: Additional descriptive statistical indices for each treatment combination for water deprivation resistance (in hours) for olive fruit flies; Table S2: Tests of between-subject effects (ANOVA) for testing the effects (main effects and interactions) of age, sex, diet, and mating status on water deprivation resistance; Figure S1 In each case (A, B, and C), bars with different lower-case letters above them correspond to differences in mean values that are statistically significant at a significance level of a = 0.05 (p ≤ 0.05), according to the results of Tukey's test in case (A) and ANOVA'S F-test in cases (B) and (C), with significance p-value (p < 0.001) in all cases (A, B, and C).Error bars correspond to standard errors of the mean values.The "partial eta squared, η 2 " indicates the effect size of the three main factors (age, sex, and diet); Table S3: Statistically significant differences (SSDs) between the different ages, sexes, and diets, with different lower-case letters corresponding to statistically significant differences among mean values at a significance level of a = 0.05, according to the results of Tukey's test; Table S4: Mean life expectancy based on the baseline experiment and the corresponding mean deprivation resistance as a result of the main experiments, age subjected to water deprivation (as percentage % of their life expectancy) and mean deprivation resistance (as percentage % of their life expectancy).

Figure 1 .
Figure 1.The three stages of the experiment: (1) preparing materials and determining experimental conditions; (2) preparing the experimental insects in cages; (3) recording insect deaths due to water deprivation.

Figure 1 .
Figure 1.The three stages of the experiment: (1) preparing materials and determining experimental conditions; (2) preparing the experimental insects in cages; (3) recording insect deaths due to water deprivation.

5 Figure 2 .
Figure 2. Gathering infested and non-infested olives for the experiments.

Figure 2 .
Figure 2. Gathering infested and non-infested olives for the experiments.

Figure 4 .
Figure 4. Schematic representation of the experimental design: (a) Phase A, water is available; (b) Phase B, recording of insect deaths every four hours daily as a measure of water deprivation resistance.

Figure 4 .
Figure 4. Schematic representation of the experimental design: (a) Phase A, water is available; (b) Phase B, recording of insect deaths every four hours daily as a measure of water deprivation resistance.

Figure 5 .
Figure 5. Water deprivation resistance (in hours to death) for virgin males (♂) and females (♀) at three different ages (15, 30, and 45-days old), in those fed the full diet and those fed the restricted diet.Lower-case letters above bars correspond to differences in mean values that are statistically significant at a significance level of a = 0.05 (p ≤ 0.05), according to the results of Tukey's test.Error bars correspond to standard errors of the mean values.

Figure 5 .
Figure 5. Water deprivation resistance (in hours to death) for virgin males (♂) and females (♀) at three different ages (15, 30, and 45-days old), in those fed the full diet and those fed the restricted diet.Lower-case letters above bars correspond to differences in mean values that are statistically significant at a significance level of a = 0.05 (p ≤ 0.05), according to the results of Tukey's test.Error bars correspond to standard errors of the mean values.Appl.Biosci.2024, 3, FOR PEER REVIEW 10

Figure 6 .
Figure 6.Water deprivation resistance (in hours to death) for mated males (♂) and females (♀) at three different ages (15, 30, and 45 days old), in those fed the full diet and those fed the restricted diet.Lower-case letters above bars correspond to differences in mean values that are statistically significant at a significance level of a = 0.05 (p ≤ 0.05), according to the results of Tukey's test.Error bars correspond to standard errors of the mean values.

Figure 6 .
Figure 6.Water deprivation resistance (in hours to death) for mated males (♂) and females (♀) at different ages (15, 30, and 45 days old), in those fed the full diet and those fed the restricted diet.Lower-case letters above bars correspond to differences in mean values that are statistically significant at a significance level of a = 0.05 (p ≤ 0.05), according to the results of Tukey's test.Error bars correspond to standard errors of the mean values.

Figure 7 .
Figure 7. Percentage (%) of virgin and mated individual males (♂) and females (♀) fed the full or restricted diet at three different ages (15, 30, and 45 days old) that survived water deprivation beyond or equal to and below the total survival median value (90 h).

Figure 7 .
Figure 7. Percentage (%) of virgin and mated individual males (♂) and females (♀) fed the full or restricted diet at three different ages (15, 30, and 45 days old) that survived water deprivation beyond or equal to and below the total survival median value (90 h).

Figure 8 .
Figure 8. Age of adults when subjected to water deprivation (blue color) as percentage of the life expectancy of adults and the corresponding observed water deprivation resistance (orange color) of virgin and mated individual males and females fed the full or restricted diet at three different ages (15, 30, and 45 days old) based on the baseline data.

Figure 8 .
Figure 8. Age of adults when subjected to water deprivation (blue color) as percentage of the life expectancy of adults and the corresponding observed water deprivation resistance (orange color) of virgin and mated individual males and females fed the full or restricted diet at three different ages (15, 30, and 45 days old) based on the baseline data.
: Partial eta squared (n 2 partial ) values and significance (p-value) of the main effects and interactions; Figure S2: Water deprivation resistance (in hours to death) in (A) three different ages (D 15 = 15 days old, D 30 = 30 days old, and D 45 = 45 days old), in (B) two sexes (males (♂) and females (♀)), with (C) two diets (restricted and full).

Table 1 .
Baseline laboratory data regarding the expected life expectancy of adults in each treatment.

Table 1 .
Baseline laboratory data regarding the expected life expectancy of adults in each treatment.