https://orcid.org/0000-0002-4234-1239
Figures
Abstract
Sterile Insect Technique (SIT), Incompatible Insect Technique (IIT) or a combination of the two has become alternative promising vector control approaches. In order to apply these approaches, the targeted mosquitoes need to be sterilized and released. So far, the irradiation of mosquitoes has been conducted at the pupae or adult stages. In this study, we investigated the possibility of applying X-ray irradiation at the egg stage and also assessed the effect on the development and sterility of both wild-type and Wolbachia trans-infected Aedes aegypti mosquito vectors. The eggs of both wild-type and Wolbachia trans-infected lines were irradiated using X-ray at the doses of 1, 3, 5 and 7 Gy. Development of immature stages was observed. For wild-type Ae. aegypti, X-ray irradiation at the doses from 3 Gy decreased the development of the first-instar larvae and increased the development of the third-instar larvae but there was no effect on pupae. For Wolbachia trans-infected ones, a irradiation dose as low as 1 Gy could increase the development of the forth-instar larvae while an irradiation dose of 7 Gy induced significantly high mortality to the pupae (p < 0.05). To assess sterility, males and females that emerged from irradiated eggs were mated with the non-irradiated ones. Our results showed that an irradiation dose of 7 Gy significantly caused more than 90% sterility in both wild-type males and females (p < 0.05). However, this irradiation dose could be reduced to 5 Gy to sterilize both males and females infected with Wolbachia. Our findings revealed, for the first time, that applying a low-dose X-ray irradiation at the egg stage could sterilize both wild-type and Wolbachia trans-infected Ae. aegypti when they become adults. Egg irradiation could make the implementation of SIT, IIT or combined SIT/IIT for vector control much more feasible as the sterile eggs are easier to distribute and operate when compared to other developmental stages of mosquitoes.
Citation: Kittayapong P, Ninphanomchai S, Thayanukul P, Limohpasmanee W (2025) Effect of egg irradiation on development and sterility of wild-type and Wolbachia trans-infected Aedes aegypti mosquito vectors. PLoS One 20(9): e0333297. https://doi.org/10.1371/journal.pone.0333297
Editor: Jiang-Shiou Hwang, National Taiwan Ocean University, TAIWAN
Received: December 2, 2024; Accepted: September 11, 2025; Published: September 25, 2025
Copyright: © 2025 Kittayapong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This investigation received financial support through PK from the International Atomic Energy Agency (IAEA/CRP/D44004/23918 and IAEA/TC/RAS/5082) and Mahidol University. The funders play no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Aedes aegypti is a widespread and invasive mosquito species that is highly abundant in tropical countries [1]. Significant public health diseases, such as dengue, chikungunya and Zika, are transmitted by Ae. aegypti mosquitoes [2,3]. Currently, there are no highly efficient vaccines available for protection of dengue, chikungunya, and Zika diseases but several vaccine candidates are undergoing evaluation [4]. Regular immature surveillance and implementation of appropriate control measures have been recommended by WHO for vector control of Aedes mosquitoes [5]. However, challenges in insecticide resistance and treating small larval breeding sites have been highlighted [4]. Over the past decade, several technologies have been developed to strengthen the conventional vector control methods [6], i.e., the release of genetically modified mosquitoes (GMM) or transgenic males to reduce the target populations [6–9], the exploit of cytoplasmic incompatibility induction properties of Wolbachia by the incompatible insect technique (IIT) to enable the production of non-viable eggs [6,10,11], and the release of insects sterilized by radiation, known as the sterile insect technique (SIT), in order to reduce the reproduction of natural populations of the same species [6,12]. Because of social concerns, cultural acceptance, and regulatory approval, the use of some methods, such as GMM and IIT, for vector control has encountered a number of challenges, therefore, the radiation-based approach has been considered as the most feasible and safest solution [6].
Radio-sensitivity varies with age or age within the life stage [13] and thus different developmental stages among Aedes mosquitoes are not equally affected by radiation [14]. However, due to great variations in life cycle, life span, and exposure pathway, interaction between radiation and a wide range of mosquito species is not very well understood [15,16]. On the other hand, production of sterile males for release through application of the SIT approach has been mainly obtained by irradiating pupae [17–19]. To date, only one study has been conducted to compare the sensitivity of immature stages of Ae. aegypti with radiation, but that study focused on gamma radiation [20]. The use of gamma radiation has become problematic because of government regulation, supply, usage and disposal of radioactive isotopes [19,21]; as a result, an adequate alternative technology without the security risk, such as X-ray technology, has been urgently needed [21]. X-ray is among the ionizing radiation sources that have been utilized in sterile insect releasing program [22] and X-ray based sterilization has been studied for a variety of insect pests including mosquitoes [21,23–26]. However, only X-ray irradiation of pupae [23–25,27] or adults [26] has been studied. To date, no X-ray studies have been conducted on eggs or larvae of Ae. aegypti for comparison. Radiation has an effect on each developmental life cycle of Ae. aegypti [20], and the impact on each life history trait of the species varies with dosage [15].
In this study, we aimed to investigate the effect of X-ray irradiation at the egg stage on the development and sterility of Wolbachia trans-infected and uninfected Aedes aegypti mosquito vectors. Our findings should provide useful information on optimal dosage for radiation-induced sterility of Ae. aegypti at the egg stage which is most appropriate and feasible for SIT, IIT or combined SIT/IIT implementation for vector control.
Materials and methods
Ethics consideration
The experiments were reviewed and approved by the Faculty of Science, Mahidol University-Institutional Animal Care and Use Committee (MUSC-IACUC) (MUSC64-044-593).
Mosquito colony and rearing
Both wild-type Wolbachia-uninfected and wAlbB Wolbachia trans-infected Aedes aegypti mosquito colonies were used in the present study. A wild-type Wolbachia-uninfected Ae. aegypti mosquito colony was originally established from mosquito eggs, which were collected by using ovitraps placed in several households in communities in Chatuchak District, Bangkok. A wAlbB Wolbachia trans-infected Ae. aegypti colony was obtained by direct microinjecting wAlbB Wolbachia into wild-type Ae. aegypti female mosquitoes from the Chatuchak colony described above. The wAlbB Wolbachia strain was extracted from the Ae. albopictus colony originating from Plaeng Yao District, Chachoengsao Province. An establishment of wAlbB Wolbachia trans-infected Ae. aegypti mosquitoes was done using the method described by Ruang-areerate and Kittayapong [28]. The characteristics of the wAlbB Wolbachia trans-infected Ae. aegypti mosquitoes were demonstrated in Kittayapong et al. [29].
In the experiments, mosquitoes were reared in an aluminum mosquito rearing cage sized 30 cm x 30 cm x 30 cm. in a screened climatic control insectary at the Center of Excellence for Vectors and Vector-Borne Diseases (CVVD), Faculty of Science, Mahidol University at Salaya, Nakhon Pathom, Thailand, with 75 ± 2% relative humidity, 27 ± 2°C, and a photoperiod of L12:D12, and were fed with 10% sucrose solution. Males and females were allowed to mate for 2–3 days; then the females were fed with pig blood by using the Hemotek membrane feeding system (Hemotek Ltd., UK) for 3–4 consecutive days after mating. The blood, obtained from a qualified slaughterhouse, was treated with 10% of EDTA (SCHARLAU, Spain) as an anticoagulant. Egg papers were placed in the containers half-filled with water inside the cage 1–3 days following blood-feeding. After 3–4 days, the egg papers were then collected, dried for 1–3 days at room temperature, and placed on a plastic tray prior to the irradiation process.
Irradiation and sex separation procedure
In this experiment, a total of 2,000 Wolbachia uninfected Ae. aegypti eggs, stored not more than one month, were counted and separated into 5 portions of 400 eggs. Each portion of eggs was placed on a paper and then transferred into a plastic container volume 1,038.69 cm3 (diameter 11.5 cm) covered with a screened lid. One portion of eggs was used as a control. Four portions of eggs were transported by an air-condition car from the Center of Excellence for Vectors and Vector-Borne Diseases (CVVD), Faculty of Science, Mahidol University at Salaya, Nakhon Pathom Province to the Thailand Institute of Nuclear Technology (TINT) (Public Organization), Ministry of Higher Education, Science, Research and Innovation in Nakhon Nayok Province for irradiation. The distance from CVVD, where the rearing facility was located, to TINT was about 100 km or 3 hours by car for a round-trip. Irradiation was conducted by the experienced and trained staff of TINT.
Four portions of 400 eggs were irradiated with X-ray irradiator model RS 2400 (Rad Source Technologies, Inc., USA) at the irradiation doses of 1 Gy, 3 Gy, 5 Gy and 7 Gy respectively. For the RS 2400 setting, the irradiator basically auto-generated the irradiation time based on parameters that were formerly established by the manufacturer. In general, the irradiator was operated under precise settings: power of 145 Kev, current of 37.5 mA, and a dose rate of 0.24 Gy per second or 14.63 Gy per minute. Therefore, in this study, the irradiation time for 1 Gy, 3 Gy, 5 Gy, and 7 Gy was auto-generated as 4.17 seconds, 12.50 seconds, 20.83 seconds, and 29.17 seconds respectively.
Mosquito development process
After irradiation, eggs were transported from TINT back to CVVD. Then they were hatched in deionized water and newly emerged larvae were transferred into a plastic tray sized 32 cm x 42 cm x 5 cm with a total of 100 larvae per tray. Larval diets were provided at an amount between 0.5–2.0 g per day according to the developmental stage. Dead larvae were removed by using a dropper and the number of dead larvae was counted and recorded daily. When larvae developed into pupae, male and female pupae were sex separated using the local pupal sex separator modified from the larval-pupal sex separator (John Hock Co., Ltd., USA). Then they were separately transferred into a mosquito cage sized 20 cm x 20 cm x 20 cm. The number of non-emerged male and female pupae was counted and recorded. After pupae became adults, 10% of sucrose solution was provided inside the mosquito cage.
Sterility test
After emergence, Wolbachia uninfected Ae. aegypti aged 2–3 days were cross-mated according to the following mating pairs: 1) males emerged from irradiated eggs and non-irradiated females (IR Non-WolB M x Non-IR Non-WolB F); 2) females emerged from irradiated eggs and non-irradiated males (IR Non-WolB F x Non-IR Non-WolB M); and 3) non-irradiated males and non-irradiated females (Non-IR Non-WolB M x Non-IR Non-WolB F). Mosquitoes were allowed to mate for 2–3 days and then blood feeding was provided. Blood-fed females were individually transferred into a small plastic cup and the condition for oviposition was provided. Females were allowed to lay eggs for 3–5 days and the number of eggs laid per female was counted and recorded. Eggs were hatched as previously described and the number of hatched eggs was counted and recorded for assessment of sterility. The induce sterility (IS) was assessed in order to evaluate the effect of sterility, and it was calculated as 100% minus the residual fertility value, which was calculated as IS = 100%–(Ho/Hn) where Ho was the mean egg hatch rate of treatment cages, and Hn was the mean egg hatch rate of fertile control cages [24]. Four replicates were conducted for each experiment.
The same experiments were conducted on wAlbB Wolbachia trans-infected Ae. aegypti with three cross-mating pairs as follows: 1) males emerged from irradiated eggs and non-irradiated females (IR WolB M x Non-IR WolB F); 2) females emerged from irradiated eggs and non-irradiated males (IR WolB F x Non-IR WolB M); and 3) non-irradiated males and non-irradiated females (Non-IR WolB M x Non-IR WolB F).
Statistical analysis
Data was entered and cleaned using Microsoft Office Excel 2016 and statistical analysis was performed using SPSS 18.0 (Mahidol University License, SPSS Inc., Chicago, USA). Numbers of eggs, larvae, pupae, and egg hatch rate were analyzed by using paired-sample t-test; and induced sterility (IS) was analyzed and compared with a theoretical value of 100 (IS = 100 when the male mosquito is completely sterile) [29] by using one sample t-test. P-values of less or equal to 0.05 were considered significant.
Results
Development of wild-type Aedes aegypti after being irradiated at the egg stage
Results showed that X-ray irradiation of the wild-type Wolbachia uninfected Ae. aegypti eggs with the irradiation doses from 3 Gy to 5 Gy significantly decreased the development of the first instar larvae, whereas the irradiation doses from 3 Gy to 7 Gy significantly increased the development of the third instar larvae when compared to those of the controls (Fig 1, Table 1). When larvae developed into pupae, late development or high mortality of pupae was observed at the irradiation dose of 7 Gy when compared to other irradiation doses or the controls; but this difference was not statistically significant (p < 0.05).
Sterility of wild-type Aedes aegypti after being irradiated at the egg stage
In terms of the sterility of males emerging from irradiated eggs, it was found that the irradiation doses starting from 3 Gy onward significantly reduced the total number of eggs and the total number of hatched eggs of the cross-mating pairs: males emerged from irradiated eggs and non-irradiated females (IR Non-WolB M x Non-IR Non-WolB F), with a significant reduction of egg hatch rates at the irradiation doses starting from 1 Gy onward (0.59 ± 0.14 vs 0.70 ± 0.09, df = 29, t = 3.808, p = 0.001) (Fig 2, Table 2). However, males became nearly complete sterile at the irradiation dose of 7 Gy (IS = 99.92; 0.08 ± 0.18 vs 0.70 ± 0.09, df = 16.693, t = 29, p = 0.000) (Fig 2, Table 2).
For sterility of females emerged from irradiated eggs, it was found that the irradiation dose of 7 Gy significantly reduced the total number of eggs of the cross-mating pairs: females emerged from irradiated eggs and non-irradiated males (IR Non-WolB F x Non-IR Non-WolB M), and a significant reduction of egg hatch rates was observed at the irradiation doses starting from 3 Gy onward (0.58 ± 0.10 vs 0.64 ± 1.01, df = 29, t = 2.376, p = 0.024) (Fig 3, Table 3); and females became nearly complete sterile at the irradiation dose of 7 Gy (IS = 99.95; 0.05 ± 0.14 vs 0.64 ± 1.01, df = 29, t = 17.124, p = 0.000).
To summarize, when wild-type Wolbachia uninfected eggs of Ae. aegypti were irradiated with X-ray, the irradiation dose of 7 Gy significantly induced more than 90% sterility of both males and females. Therefore, an irradiation dose of 7 Gy could be the optimum dose for irradiation of wild-type Wolbachia uninfected Ae. aegypti eggs.
Development of Wolbachia trans-infected Aedes aegypti after being irradiated at the egg stage
When Wolbachia trans-infected Ae. aegypti eggs were irradiated, it was found that the irradiation dose from 1 Gy onward significantly increased the development of the fourth-instar larvae when compared to those of the controls (Fig 4, Table 4). When larvae developed into pupae, significantly high mortality of both male and female pupae was observed at the irradiation dose of 7 Gy when compared to those of the controls.
Sterility of Wolbachia trans-infected Aedes aegypti after being irradiated at the egg stage
In terms of the sterility of males emerged from irradiated eggs, it was found that the irradiation dose of 1 Gy significantly reduced the total number of eggs and the total number of hatched eggs of the cross-mating pairs: males emerged from irradiated eggs and non-irradiated females (IR WolB M x Non-IR WolB F); and a significant reduction of the egg hatch rates was observed when the irradiation dose was at 3 Gy (0.60 ± 0.13 vs 0.70 ± 0.11, df = 29, t = 3.113, p = 0.004) (Fig 5, Table 5). Dramatically significant reduction of the egg hatch rates was observed when the irradiation doses were at 5 Gy (0.14 ± 0.29 vs 0.70 ± 0.11, df = 29, t = 10.691, p = 0.000) and at 7 Gy (0.05 ± 0.19 vs 0.70 ± 0.11, df = 29, t = 16.824, p = 0.000) (Fig 5, Table 5).
For sterility of females emerged from irradiated eggs, it was found that the irradiation dose of 1 Gy significantly reduced the total number of eggs and the total number of hatched eggs of the cross-mating pairs: females emerged from irradiated eggs and non-irradiated males (IR WolB F x Non-IR WolB M). A significant reduction of the egg hatch rates was observed when the irradiation doses were at 1 Gy (0.45 ± 0.22 vs 0.63 ± 0.11, df = 29, t = 3.940, p = 0.000) and at 3 Gy (0.16 ± 0.26 vs 0.63 ± 0.11, df = 29, t = 10.020, p = 0.000) (Fig 6, Table 6). Moreover, dramatically significant reduction of the egg hatch rates was observed or females became nearly complete sterile when the irradiation dose was at 5 Gy (IS = 99.91, 0.09 ± 0.18 vs 0.63 ± 0.11, df = 29, t = 14.707, p = 0.000) (Fig 6, Table 6).
In summary, the X-ray irradiation dose of 7 Gy significantly induced male sterility, whereas the irradiation dose of only 5 Gy significantly induced female sterility. Therefore, an X-ray irradiation dose of 5 Gy could be the optimum dose for irradiation of Wolbachia trans-infected Ae. aegypti eggs as it could induce high level of sterility in males and nearly complete sterility in females.
Discussion
In this study, we found that as X-ray irradiation dose increased, nearly complete sterility was progressively achieved in both wild-type and Wolbachia trans-infected Ae. aegypti. Lower dose of X-ray irradiation was sufficient to induce complete sterility in both wild-type and Wolbachia trans-infected Ae. aegypti when irradiation was done at the egg stage. An X-ray irradiation dose of 5 Gy and 7 Gy could be the optimum doses to induce nearly complete sterility in Wolbachia trans-infected and wild-type Ae. aegypti respectively. Irradiation-induced dominant lethal mutations in the germ cells were the main cause of male sterilization in the SIT approach [30]. However, a higher dose of radiation exposure could decrease male quality if the males were overdosed [31]. Our study showed that an increased irradiation dose could reduce male quality in both wild-type and Wolbachia trans-infected Ae. aegypti, since we observed that less eggs were laid by females that were previously mated with wild-type and Wolbachia trans-infected Ae. aegypti males when those two were exposed to higher doses of radiation. Therefore, optimizing the irradiation step to achieve maximum sterility while minimizing the somatic damage could significantly improve the quality of sterile Ae. aegypti mosquitoes [32].
Irradiation of different mosquito species at various stages of the life cycle showed increased or declined effects on adult life span including its subsequent generations [18,33]. Age had been shown to have an effect on radio-sensitivity, and the older the life stage, or the aging within the life stages, the more radio-resistant the insects became [13]. Treatment of early life stages of mosquitoes, together with environmental alterations, greatly influenced the lifespan of adult mosquitoes [15], so in order to reduce somatic damage, irradiation of insects at or near to the completion of their development, i.e., the late pupal and adult stages of mosquitoes, had been emphasized [14,15,19,23]. However, the optimum developmental stage for irradiation depended on many factors including ease of handling on a mass-production scale, logistics of the irradiation process (e.g., the need to irradiate large numbers of insects), competitiveness of the insects, release methodology, and costs [19]. In this study, we investigated the effect of irradiation at the egg stage on sterility of Wolbachia trans-infected and uninfected Ae. aegypti, in order to find an appropriate and more convenient method for mass production of sterile males prior to the release of sterile males for vector control.
So far, not much information has been available on the irradiation of eggs, especially for mosquitoes. Due to inefficient results in controlling Aedes-transmitted arboviral diseases, research on irradiation of the mosquito stages, beyond adults and immatures, was needed [34]. In this study, we observed a late pupation and high mortality in pupae when Ae. aegypti eggs were irradiated with X-ray at an irradiation dose of 7 Gy. Our results were supported by Tantawy et al. [35] who showed high mortality when eggs of the Anopheles pharoensis were irradiated with gamma radiation. Moreover, our studies showed that an irradiation dose of 7 Gy significantly induced more than 90% sterility in the wild-type Wolbachia uninfected Ae. aegypti males and females after being irradiated at the egg stage. In terms of Wolbachia trans-infected Ae. aegypti, the same irradiation dose of 7 Gy also induced sterility in males, and a lower dose of 5 Gy was sufficient to induce complete sterility in females. Our results were supported by the study of Akter and Khan [20] who found that low dose radiation from 1–10 Gy had significant effects on pupation and adult emergence when they were irradiated at the egg stage. In addition, our results coincided with the study of Furaki et al. [36] who observed a reduction in egg hatching, as well as in adult emergence, among flour beetles and almond moths when their eggs were exposed to UV irradiation. The same study concluded that damage to the surface tissues of the eggs by radiation could be fatal, especially at advanced stages of development [36].
Although pupae is the preferred stages to sterilize during SIT operations, due to limited pupation time, the distances for their distribution are limited to closer localities where the irradiation source is located [34]. Eggs can be distributed to logistically remote field sites where they can hatch, develop into adults, and fly out to compete with wild mosquitoes [37]. The cost-benefit of using the egg stage in field distribution is very promising in low-income endemic countries. Hence, the expansion of sterilized insect programs could be facilitated by using either irradiated eggs or Wolbachia vertical infection [34]. Our results showed the effect of low-dose X-ray irradiation on sterility of both male and female wild-type and Wolbachia trans-infected Ae. aegypti when eggs were irradiated, and how this approach could be applied for SIT operations for vector control in the field.
Acknowledgments
The authors would like to thank Ms. Natchaya Klinpikul and Mr. Kuang Chalongpak for mosquito rearing, Ms. Kanyarat Yimpramote and Ms. Kamolrat Tharaporn for lab assistance, Mr. Thodsapon Thannarin for irradiation process, and Mr. David A. Blyler for English editing.
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