Assessment of Compaction, Temperature, and Duration Factors for Packaging and Transporting of Sterile Male Aedes aegypti (Diptera: Culicidae) under Laboratory Conditions

Simple Summary The sterile insect technique (SIT) is a proven method to control some insect pests and is currently being tested to control some mosquito species, including Aedes aegypti. It is challenging to maintain the quality of sterile male mosquitoes in operational SIT during the packaging and transportation processes. The experiment presented in this manuscript was undertaken to investigate compaction, temperature, and duration factors during the packaging and transportation of gamma-sterilized male Ae. aegypti. The effects of packaging and transportation factors on the quality parameters of gamma-sterilized male Ae. aegypti—mortality, flight ability, induced sterility, and longevity—were assessed. The results of this experiment demonstrate appropriate packaging and transportation conditions for maintaining the quality of gamma-sterilized male Ae. aegypti. Abstract Optimized conditions for the packaging and transportation of sterile males are crucial factors in successful SIT programs against mosquito vector-borne diseases. The factors influencing the quality of sterile males in packages during transportation need to be assessed to develop standard protocols. This study was aimed to investigate the impact of compaction, temperature, and duration factors during packaging and transportation on the quality of gamma-sterilized male Ae. aegypti. Aedes aegypti males were sterilized at a dose of 70 Gy, compacted into Falcon tubes with densities of 40, 80, and 120 males/2 mL; and then exposed to temperatures of 7, 14, 21, and 28 °C. Each temperature setup was held for a duration of 3, 6, 12, 24, and 48 h at a 60 rpm constant vibration to simulate transportation. The parameters of mortality, flight ability, induced sterility, and longevity were investigated. Results showed that increases in density, temperature, and duration significantly increased mortality and reduced flight ability and longevity, but none of the factors significantly affected induced sterility. With a mortality rate of less than 20%, an escaping rate of more than 70%, considerable longevity, and the most negligible effect on induced sterility (approximately 98%), a temperature of 7 °C and a compaction density of 80 males/2 mL were shown to be optimized conditions for short-term transportation (no more than 24 h) with the minimum adverse effects compared with other condition setups.


Introduction
Aedes aegypti (Linnaeus) is considered one of the most dangerous animals on the planet due to its vectorial capacity for major human diseases, including dengue, chikungunya, yellow fever, and Zika [1,2]. Historically, this species originated from Africa (Aedes aegypti formosus) and then distributed worldwide (Aedes aegypti aegypti), especially in tropical

Gamma Irradiation Procedure
The gamma irradiator used in the experiments was a Gammacell model 220 (originally manufactured by Atomic Energy of Canada Ltd., Ottawa, Canada, in 1968, upgraded by Institute of Isotopes, Co., Ltd., Budapest, Hungary, in 2015) with a cobalt-60 (Co-60) source (current activity of 4870 Curie and dose rate of 3514 Gy/h on 14 July 2021) located in the Research Center for Radiation Process Technology-BRIN, Jakarta. Routine dosimetry calibration was conducted and resulted in approximately 3% uncertainty of the absorbed dose (certificate no. 19C-109B, accredited by DTU Nutech, Denmark, 2019; certificate no. N • ID C/ET 23-11/1075, accredited by Aerial, France, 2021). Male Ae. aegypti pupae were placed into a transparent plastic tube (14 cm in diameter and 3 cm high), excess water was removed (pupae remained damp), and they were irradiated at a dose of 70 Gy [40].

Temperature Regime in Packed Conditions
Irradiated male Ae. aegypti pupae were placed into the adult cage and supplied with a 10% (v/v) sucrose solution. One day post-emergence, Ae. aegypti males were anesthetized at a temperature of 4 • C for approximately 5 min [44], and then 40, 80, and 120 males were counted and transferred into 15 mL Falcon tubes (Biologix Plastic Changzhou Co., Ltd., Jinan, Shandong, China) and compacted by pressing a 1.5 cm × 1.5 cm × 1.5 cm sponge down to 2 mL to achieve individual densities of 40, 80, and 120 males/2 mL, respectively. The cap and the bottom end of the tube were drilled to create holes using a 1.5 mm spiral drill needle (Model DIN 338 R-N, Guhring, Germany) to allow for airflow [41]. Tubes were transferred into a 1 L beaker glass and then placed into a water bath shaker (Model OLS26, Grant Instruments, Ltd., Cambridge, UK) set to temperatures of 7, 14, 21, and 28 • C. Each temperature setup was performed for 3, 6, 12, 24, and 48 h at a constant vibration of 60 rpm to simulate transportation. Both unirradiated-unpacked and irradiatedunpacked specimens maintained under laboratory conditions were used as the control. Three replicates were carried out for each combination of factors. The studied parameters were mortality, flight ability, induced sterility, and longevity.

Data Collection 2.4.1. Mortality Rate
To examine the effects of packaging and simulated transportation on mortality, irradiated male Ae. aegypti inside the Falcon tubes in each treatment were transferred into a cage (17.5 cm × 17.5 cm × 17.5 cm, Bugdorm-4M1515, MegaView Science Co., Ltd., Taichung, Taiwan) and provided with a 10% (v/v) sucrose solution. At 24 h post-treatment, the mortality rate was determined by dividing the dead specimens by the initial numbers of each treatment.

Flight Ability
Male Ae. aegypti in each treatment were tested for their flight ability according to the method of Bond et al. [37] with a slight modification. Briefly, all Ae. aegypti males inside each Falcon tube at each density and treatment were poured into a Petri dish (9 cm in diameter) equipped with a transparent plastic tube (8 cm in diameter and 25 cm high). This equipment was placed inside a 160 cm × 160 cm × 180 cm insect tent (Bugdorm-2960 insect Insects 2022, 13, 847 4 of 13 rearing tent, MegaView Science Co., Ltd., Taichung, Taiwan). Flight ability was determined according to the proportion of escapes over a 24 h period.

Induced Sterility
For each treatment, 20 treated sterile Ae. aegypti males were randomly selected and allowed to mate with 20 unmated females (1:1 ratio) in a cage (30 cm × 30 cm × 30 cm) with continuous access to a 10% (v/v) sucrose solution. Meanwhile, for the control, 20 unirradiated males were allowed to mate with 20 unmated females (1:1 ratio). After a 3-day mating period, females were provided a sheep's blood meal. Three days post-blood feeding, females were allowed to oviposit in a filter-paper-lined plastic cup. Egg paper was collected and slow-dried over four days under laboratory conditions for maturation before hatching. The egg hatching rate was determined by observing the detached operculum under a stereomicroscope (Model SMZ 745, Nikon Corp., Minato-ku, Tokyo, Japan). Residual fertility was determined as the percentage of control fertility, and induced sterility was determined by subtracting 100% from residual fertility [39].

Longevity under Laboratory Conditions
To investigate the effects of packaging and simulated transportation on longevity, 75 Ae. aegypti males were randomly selected from each treatment and evenly distributed into three cages (17.5 cm × 17.5 cm × 17.5 cm, Bugdorm-4M1515, MegaView Science Co., Ltd., Taichung, Taiwan). Each cage was continuously supplied with a 10% (v/v) sucrose solution. Control unirradiated-unpacked and irradiated-unpacked specimens were maintained under laboratory conditions. Longevity was determined by recording the survival (interval of 24 h) until all males succumbed to natural mortality.

Statistical Analysis
Prior to the statistical analysis, data were transformed using arcsine square root (sqrt) and tested for normality and homogeneity. General linear model (GLM) full univariate factorial followed by post hoc Tukey test was used to analyze the influence of the treatments on the parameters of mortality, flight ability, and induced sterility. A Kaplan-Meier survival analysis followed by Mantel-Cox log-rank test was used to analyze longevity in different treatments. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 22 for Windows (International Business Machine Corp., Armonk, NY, USA).

Mortality Rate
The statistical analysis showed that mortality of gamma-sterilized male Ae. aegypti was significantly affected by density, temperature, and duration as a single factor. In the twoway interaction, there was no interaction effect on the mortality rate between duration and density, while the effects of duration and temperature differed depending on the level of temperature and density, respectively. Furthermore, in the three-way interaction, the effects density, temperature, and duration differed from the simple sum of their effects ( Table 1). The range of mortality percentages, across the different levels of duration and density, varied among the four temperature levels: 5.0 ± 2.6% to 32.5 ± 6.29%, 13.33 ± 3.63% to 38.89 ± 2.82%, 7.5 ± 1.44% to 87.5 ± 6.29%, and 4.58 ± 0.83% to 86.94 ± 3.38% for the temperature levels of 7, 14, 21, and 28 • C, respectively ( Table 2). Interactions between the three factors could be seen at lower temperatures (7 and 14 • C), at which mortality showed no difference among all densities at durations from 3 to 24 h then significantly increased at a duration of 48 h. However, at higher temperatures (21 and 28 • C, mortality showed no difference among all densities at durations from 3 to 12 h and from 3 to 6 h at temperatures of 21 and 28 • C, respectively, then significantly increased with increasing duration. In general, mortality significantly increased with increasing density, temperature, and duration of the packaging and simulated transportation treatment. Additionally, the Insects 2022, 13, 847 5 of 13 effect of duration on the mortality rate did not differ across the levels of density (df = 8, F = 1.799, p = 0.082) ( Table 1); this phenomenon was mainly seen at lower temperatures (7 and 14 • C) ( Table 2); however, at higher temperatures (21 and 28 • C) and longer durations (12 to 48 h), mortality significantly increased at the highest density (120 males/2 mL).  Table 2. Mean mortality of gamma-sterilized male Ae. aegypti post-treatment by density, temperature, and duration factors.

Treatments (Density and Duration)
Mean Mortality ± SE (%)  * Control 1 and control 2 were not packed and exposed to temperature treatment. The same lowercase, uppercase, and symbol indicate no significant difference within the same density and temperature, the same duration and density, and the same duration and temperature, respectively (one-way ANOVA post hoc Tukey, p = 0.05).

Flight Ability
The flight ability of gamma-sterilized male Ae. aegypti showed a significant reduction compared with both unirradiated-unpacked and irradiated-unpacked controls, except for the duration of 3 h at lower temperatures (7 and 14 • C) ( Table 3). The flight ability parameter was significantly affected by a three-way interaction between density, temperature, and duration (df = 24, F = 2.239, p = 0.001) ( Table 1). Mean flight ability ranged from 23.44 ± 3.8% to 100.0 ± 0.0%, 34.06 ± 3.76% to 93.13 ± 3.59%, 23.13 ± 4.75% to 82.5 ± 5.1%, and 28.54 ± 0.02% to 80.31 ± 0.04% at temperatures of 7, 14, 21, and 28 • C, respectively (Table 3). Lower temperatures (7 and 14 • C) could maintain the flight ability of more than 70% in all durations except for 48 h. However, keeping the males at 21 • C for, at most, 12 h, and at 28 • C for 3 h resulted in escape rates of more than 70% (Table 3). Generally, flight ability was significantly reduced with increasing density, temperature, and duration. Table 3. Mean flight ability of gamma-sterilized male Ae. aegypti post-treatment by density, temperature, and duration factors.

Treatments (Density and Duration)
Mean Flight Ability ± SE (%) 28.54 ± 0.02 aAα * Control 1 and control 2 were not packed and exposed to temperature treatment. The same lowercase, uppercase, and symbol indicate no significant difference within the same density and temperature, the same duration and density, and the same duration and temperature, respectively (one-way ANOVA post hoc Tukey, p = 0.05).

Induced Sterility
The density, temperature, duration, and their interaction were negligible factors affecting induced sterility (df = 20, F = 0.474, p = 0.972) ( Table 1). The mean induced sterility in all treatments ranged from 95.34 ± 2.27% to 99.52 ± 0.24% and indicated no significant differences (Table 4). Table 4. Mean induced sterility of gamma-sterilized male Ae. aegypti post-treatment by density, temperature, and duration factors.

Treatments (Density and Duration)
Mean Induced Sterility ± SE (%) 99.08 ± 0.6 aAα 97.41 ± 0.57 aAα n/a n/a * Control 1 and control 2 were not packed and exposed to temperature treatment. The same lowercase, uppercase, and symbol indicate no significant difference within the same density and temperature, the same duration and density, and the same duration and temperature, respectively (one-way ANOVA post hoc Tukey, p = 0.05). No data were applicable for the duration of 48 h and temperatures of 21 and 28 • C in all densities due to the high mortality rate (>80%).

Longevity under Laboratory Conditions
The survival probability of gamma-sterilized male Ae. aegypti was reduced by all factors of the treatments, i.e., density, temperature, and duration ( Figure 1). The mean longevity of the treatment groups was significantly reduced compared with both unirradiated-unpacked and irradiated-unpacked controls. Among the packaging and simulated transportation treatments, the mean longevity ranged from 3.05 ± 0.3 to 6.87 ± 0.43 days, 3.23 ± 0.39 to 6.72 ± 0.49 days, 1.48 ± 0.14 to 7.77 ± 0.49 days, and 1.43 ± 0.14 to 8.04 ± 0.73 days at temperatures of 7, 14, 21, and 28 • C respectively (Table S1). In general, longevity was significantly reduced with increasing density, temperature, and duration of the treatment (log-rank test, p < 0.05) (Table S1). In addition, longevity at densities of between 40 and 80 males/2 mL with lower temperatures (7 and 14 • C) and the same duration showed no significant difference, except for durations of 12 h and 48 h at temperatures of 7 and 14 • C, respectively. was significantly reduced with increasing density, temperature, and duration of the treatment (log-rank test, p < 0.05) (Table S1). In addition, longevity at densities of between 40 and 80 males/2 mL with lower temperatures (7 and 14 °C) and the same duration showed no significant difference, except for durations of 12 h and 48 h at temperatures of 7 and 14 °C, respectively.

Discussion
Handling, packaging, and transporting male insects are critical steps in SIT programs. Considering the fragility of mosquitoes, it is challenging to maintain the quality of the sterile male specimens during handling, packaging, and transport. The main objective of packaging and transportation development is to construct appropriate methods associated with easy handling, space efficiency, and suitable conditions that can maintain the quality of the sterile male mosquitoes in long-term transportation. The authors of the present study investigated key factors that potentially influence the packaging and transporting of gamma-sterilized male Ae. aegypti, i.e., compaction, temperature, and duration, at the laboratory scale. In addition, we simulated packaging and transportation; subsequently, we observed the sensitivity of sterile male Ae. aegypti to the packaging and transportation factors of treatments i.e., mortality, flight ability, longevity, and induced sterility.
In the present study, the mortality rate of gamma-sterilized male Ae. aegypti in all treatments ranged from 5.0 ± 2.6% to 87.5 ± 6.29% and showed a tendency to significantly increase with increases in density, temperature, and duration of the packaging and simulated transportation. However, no difference was found between the densities of 40 and 80 males/2 mL (Table 2), so the density of 80 males/2 mL is better from a performance point Insects 2022, 13, 847 9 of 13 of view. We found that a temperature of 7 • C and a density of 80 males/2 mL comprised the optimal setup with the smallest effect on the mortality of the gamma-sterilized male Ae. aegypti, which was less than 20% at all durations (Table 2). These findings are close to those of previous studies, which showed that temperatures ranging from 7 to 10 • C during transportation resulted in maintaining the survival of Ae. aegypti [41,43]. Regarding species, previous studies showed that Ae. albopictus also have a similar tolerance to temperatures from 7 to 10 • C for packing and transportation [43,45]. In addition, An. arabiensis could be immobilized at a temperature ranging from 4 to 10 • C for durations of up to 24 h without any significant adverse impacts [42]. Our findings are also close to those resulting from the temperature treatments used to maintain Mediterranean fruit flies [46] and tsetse fly Glossina palpalis gambiensis [47] for long-distance transportation (around 10 • C).
Flight ability is one of the essential quality attributes of sterile male insects in SIT programs. This parameter is associated with the ability of sterile males to disperse, survive, find food, and seek and mate wild-type females in the field, which determines the success of SIT implementation [48]. In this study, the flight ability of gamma-sterilized male Ae. aegypti was significantly affected by all treatment factors, i.e., density, temperature, and duration. In general, flight ability was considerably reduced with increases in density, temperature, and duration. However, no difference was found between densities of 40 and 80 males/2 mL. We found that a temperature of 7 • C and a density of 80 males/2 mL with a duration of no more than 24 h comprised the optimal setup which could maintain the flight ability from 78.13 ± 1.49% to 100.00 ± 0.0% (Table 3). A similar result was reported by Mastronikolos et al. [45], who transported irradiated male Ae. albopictus from Italy to Greece by compacting them at temperatures of 8 to 14 • C. They found that this temperature treatment during transportation could maintain the flight ability by more than 60%. In addition, the chilled temperature treatment during packaging and transportation was expected to lead to an immobilization state, reducing interaction and friction among individual gamma-sterilized male mosquitoes inside the compaction packing device. Such friction may lead the physical damage, including missing scales, head damage, abdomen damage, wing damage, antenna damage, and leg damage that potentially reduce flight ability [41].
Regarding induced sterility, we found that the factors during packaging and simulated transportation treatments, i.e., density, temperature, and duration, did not affect male Ae. aegypti's sterility. Induced sterility ranged from 95.34 ± 2.27% to 99.52 ± 0.24%; however, there was no statistical difference (Table 4). In the present study, based on our previous study, the gonad cells of male Ae. aegypti were exposed to gamma irradiation at a dose of 70 Gy (Cobalt-60), therefore causing dominant lethal mutation, to induce sterility [40]. In the SIT, sterility can be defined as any complete or partial structural or functional failure to produce gametes or viable zygotes that can be induced by ionizing energy such as gamma irradiation [49,50]. Based on the data presented here, density, temperature, and duration probably only affect somatic cells, thus affecting the physical and fitness of gamma-sterilized male Ae. aegypti, but not affecting gonad cells to induce sterility. Our findings are similar to those of a previous study conducted by Sasmita et al. [51], who reported that packing and transportation treatment did not affect the induced sterility of gamma-sterilized male Ae. aegypti.
The longevity of sterile male mosquitoes post-packaging and simulated transportation treatments is correlated to survivability and mating performance and is important for the success of SIT programs [52]. In the present study, the longevity of gamma-sterilized male Ae. aegypti was affected by packaging and transportation treatments and showed a significant reduction with increasing density, temperature, and duration; however, there was no difference between the densities of 40 and 80 males/2 mL. Based on our results, temperatures of 7 and 14 • C and a density of 80 males/2 mL may comprise the optimal conditions for transporting gamma-sterilized male Ae. aegypti, as these conditions maintained a mean longevity ranging from 4.24 ± 0.29 to 5.89 ± 0.42 days (Table S1). Lower temperatures immobilized male Ae. aegypti and minimized physical damage, resulting in the maintenance of longevity post-transportation. Our findings are consistent with those of previous studies showing that lower temperatures during transportation could maintain the survival and longevity of mosquitoes, including Ae. aegypti at temperatures ranging from 7 to 14 • C [41,43], Ae. albopictus at temperatures ranging from 8 to 14 • C [45], and An. arabiensis at temperatures ranging from 4 to 10 • C [42]. Regarding a nonchilled temperature setup, a previous study by Sasmita et al. [51] found that an eight hours of land transportation (temperatures ranging from 22 to 26 • C) could maintain the longevity of gamma-sterilized male Ae. aegypti from 5.4 to 9.1 days on average. However, longer durations must be investigated for practical purposes.
We were able to measure the fitness and quality of the males using induced sterility and other factors reflecting physical ability and survival, i.e., flight ability, longevity, and mortality, despite the mating competitiveness index not being present in our data set due to the number of dependent variables and their combinations. Our experimental data revealed that lower temperature setups (7 and 14 • C) during packaging and simulated transportation could maintain the quality of gamma-sterilized male Ae. aegypti, as assessed through mortality, flight ability, induced sterility, and longevity. We advise that a temperature of 7 • C be used to immobilize and maintain sterile male Ae. aegypti for short-term transportation periods (up to 24 h). As a rule, insects are poikilothermic. Hence, their body temperature is influenced by environmental temperature. A chilling temperature treatment during packaging and transportation causes a quiescence condition and a reduced metabolism rate, consequently reducing the growth and developmental rate of gamma-sterilized male Ae. aegypti [53]. In addition, this chilling temperature was shown to cause immobilization and to prevent lost energy reserve due to movement inside the packaging device. Moreover, it was shown to prevent friction, which potentially causes physical damage [51]. The knockout time related to the temperature changes can be immediate. A study on An. arabiensis showed that an immediate change of temperature from room temperature to between 2 and 11 • C only took from 12 ± 3.6 to 25 ± 2.5 s for complete immobilization [42].
Compaction treatment could be a breakthrough, considering the space efficiency issue in developing packaging and transportation methods for mosquitoes in SIT programs. From a compaction perspective, the key point is finding an optimal density for packaging and transportation that can maintain the quality of sterile male mosquitoes. In the present study, gamma-sterilized male Ae. aegypti were compacted into Falcon tubes with densities of 40, 80, and 120 males/2 mL. The densities in our recent study were not realistic for operational SIT programs. Nevertheless, the results were adequate to prove that the increasing density during transportation was detrimental to the quality of gamma-sterilized male Ae. aegypti, except for the induced sterility parameter. Increasing density resulted in higher mortality and reduced flight ability and longevity. However, no differences were found between the densities of 40 and 80 males/2 mL. In combination with temperature and duration factors, we recommend that 80 males/2 mL be selected as the optimal density for compaction treatment during the short-term transportation of sterile male Ae. aegypti. Higher densities cause sterile male mosquitoes to the crowd, potentially causing physical damage and consequently reducing quality parameters [41] and mating competitiveness [54]. Meanwhile, lower densities are postulated to create space inside the packaging device, potentially causing physical interactions and friction due to vibration during transportation and consequently inducing physical damage and reducing the quality of gamma-sterilized male Ae. aegypti. Our findings are similar to those reported by Chung et al. [41], in that a density of 40 males/cm 3 maintained the survival of more than 80% packed male Ae. aegypti using Falcon tubes at a temperature of 7 • C.
In this study, compaction, temperature, and duration factors were combined in the assessment of the packaging and transportation treatment of gamma-sterilized male Ae. aegypti. Based on the data presented here, we found that compaction with a density of 80 males/2 mL, a temperature of 7 • C, and short-term transportation (no longer than 24 h), comprising an appropriate setup for packaging, could maintain the quality of gammasterilized male Ae. aegypti. Immobilization at chilling temperatures and compaction treatments are beneficial in maintaining the quality and space efficiency of sterile male Ae. aegypti in packaging and transportation methods in operational SIT programs.

Conclusions
The results of experiments of the present study revealed that compaction density, temperature, and duration factors in packaging and simulated transportation treatments significantly affected the quality of gamma-sterilized male Ae. aegypti regarding mortality, flight ability, and longevity. We found that a temperature of 7 • C and a compaction density of 80 males/2 mL could maintain the longevity and quality of gamma-sterilized male Ae. aegypti, with a mortality rate of less than 20%, a flight ability of at least 70%, and an induced sterility of around 98%. Consequently, based on our results, we recommend a temperature of 7 • C, a compaction density of 80 males/2 mL, and short-term transportation (no more than 24 h) as an appropriate treatment for the packaging of sterile male Ae. aegypti in SIT programs. However, this study has not been able to answer whether the vibration factor during transportation affects the quality of mosquitoes. Therefore, further study on the effect of vibration during transportation is needed.