Defining X-ray radiation dose for female sterilization

To determine an optimal X-ray dose for use in the operational tests, we first tested the lowest dose that could completely sterilize Ae. aegypti females and have a minimum impact on male mosquitoes under laboratory conditions. To this end, we subjected 24-h-old pupae (referred to as 0-h of X-ray irradiation in this study, see “Methods” section) to various doses of X-ray irradiation. We use fecundity (number of eggs laid per female) and fertility (egg hatch rate) as two indicators for sterility. As expected, the fecundity of irradiated females exhibited a dose-dependent response (Fig. 1A). Between doses of 10 to 20 Gy, the average number of eggs laid per female significantly decreased from 83.5 eggs/female at 10 Gy to 0.89 eggs/female at 20 Gy, compared to 101.2 eggs/female in those non-irradiated (0 Gy) controls. At doses of 25 Gy or higher, essentially none of the irradiated females produced eggs, except for one female that produced 4 eggs (Fig. 1A), and none of these eggs hatched (Fig. 1A, B). Therefore, a dose of 25 Gy on 24-h-old pupae is sufficient to induce complete sterility in females under laboratory conditions.

Fig. 1

Fecundity and fertility after various X-ray irradiation doses. Effects of various doses of X-ray irradiation on egg production (A) and hatch rate (B) in irradiated females mated with wild-type males. Effects of various doses of X-ray irradiation on egg production (C) and hatch rate (D) in wild-type females mated with irradiated males. The number of samples in each group is shown above the X-axis. Error bars represent the median and interquartile range. Kruskal-Wallis test and Dunn’s multiple comparisons post hoc test shows the difference between control and individual irradiation dose; doses not sharing the same letter are significantly different

We also investigated these X-ray doses on male fertility by crossing irradiated males with wild-type females. Expectedly, no difference in the fecundity of these wild-type females was observed across different experimental groups (Fig. 1C). However, the egg hatch rate exhibited a clear dose-dependent reduction (Fig. 1D), dropping from 28.3 at 10 Gy to 0.98% at 35 Gy, in line with previous reports [35,36,37]. In the subsequent experiments, we chose the X-ray dose of 30 Gy to ensure complete female sterilization after irradiation.

Irradiated mosquitos show no cost to longevity under stress conditions

To evaluate the effect on mosquitoes’ longevity, we examined the lifespan of mosquitoes that emerged from pupae exposed to 30 Gy irradiation. Under optimal laboratory conditions where constant water and sugar were provided, both irradiated females and males showed a significant decrease in lifespan, compared to non-irradiated (0 Gy) controls (Fig. 2A, B). Previous publications estimated that the average lifespan of wAlbB-SG males under the field condition was around 4.5 days [26, 38], significantly shorter than those under laboratory conditions (about 24.5 days). We next investigated the lifespan of those irradiated mosquitoes under stress conditions resembling field conditions by removing sugar or water or both. Under the water-only condition (without sugar supply), the lifespan of irradiated females was comparable to those of control females (Fig. 2C). Under this condition, irradiated males, however, survived better than control males (Fig. 2D). Of note, under the extreme condition of depletion of both sugar and water, there was an increase in lifespan observed in both irradiated males and females, compared to the controls (Fig. 2E, F). These results thus showed that a dose of 30 Gy irradiation does not cause a reduction to mosquitoes’ lifespan under stress conditions.

Fig. 2

Effects of 30 Gy X-ray irradiation on Aedes longevity. Kaplan-Meier survival curves of female (A) and male (B) adults derived from irradiated pupae with constant water and sugar supply. Kaplan-Meier survival curves of female (C) and male (D) adults derived from irradiated pupae with water supply only. Kaplan-Meier survival curves of female (E) and male (F) adults derived from irradiated pupae without water and sugar supply. The number of samples in each figure is shown in the graph key. Pairwise comparisons of survival curves are performed by log-rank (Mantel-Cox) test; *** (p < 0.001), ** (p < 0.01), * (p < 0.05), ns (not significant)

X-ray irradiation disrupts ovariole maturation in female Ae. aegypti

Although irradiation also induces sterility in females [35], the underlying mechanism is not fully understood. Previous publications reported altered gross ovary morphology in irradiated Aedes females, but did not provide a detailed analysis [29, 37]. Therefore, we investigated the underlying cause of sterility in irradiated female mosquitoes.

The formation of Aedes ovariole, similar to that of Drosophila melanogaster, begins in the late larval stage [39]. In control pupae, the pre-ovarioles (defined as the immature form of ovariole) formed and resided on the surface of the ovary (Fig. 3A for ovary and 3I for ovariole). The ovary grew in size after female emergence and matured by day 3 (Fig. 3B, C). A closer examination revealed that in 24-h-old pupae (0-h of irradiation), the pre-ovarioles in Aedes mosquitoes were elongated in shape, with several germline cells situated within the tubular structure formed by surrounding somatic cells (Fig. 3I). The future oocyte of the first germline cyst was evident by its condensed chromosome and located at the posterior end of the germ cell cluster (Fig. 3I, white arrow). During maturation, the germ cells proliferated, and the somatic cells divided and bisected the ovariole to form two segments, an anterior portion filled with undifferentiated germ cells (covered by a layer of inner germarial sheath cells, IGS cells) and the posterior part with a developing germline cyst covered by a single layer of follicular cells, which eventually budded out of the germarium to become the primary follicle (Fig. 3J). After female emergence, the primary follicle continued to grow, expanded its volume, and reached the mature (previtellogenic arresting) stage by day 3, while the secondary follicle formed in the germarium (Fig. 3K).

Fig. 3

X-ray irradiation disrupts the development of ovary. Representative confocal microscopy images of ovaries (A–H) and ovarioles (I–P) with Vasa (Green), Phalloidin (Red), and DNA (Grey) staining at day 0 (A and I), day 1 (B and J), and day 3 (C and K) in control females, and day 0 (D and L), day 1 (E and M), day 3 (F and N), day 7 (G and O), and day 14 (H and P) in irradiated females after irradiation. A–H Red arrows indicate one ovariole (green marked by Vasa staining) in each sample. I,J,L and M White arrows indicate the future oocyte as indicated by the condensed chromosome. Yellow arrows in J and K indicate follicular cells bisecting the germarium into two segments, and the yellow arrow in M shows that no follicular cells bisect the germarial region of the ovariole from irradiated females. K, N-P follicular cells are present in control follicles (yellow arrowhead in K) but absent in ovarioles from irradiated females (yellow arrowheands in N–P). Scale bars in A (for A–H) and I (for I–P) are 50 µm

To understand the effects of X-ray irradiation, we traced the development of the ovary and ovariole from day 0 to day 14 post-irradiation. As expected, in pupae right after irradiation (day 0, about one hour after the irradiation), the ovaries of irradiated females were morphologically similar to those of control ovaries (Fig. 3D, compared to 3A). By day 1 (24 h post-irradiation), no obvious overall morphological difference was observed in comparison to the control (Fig. 3E, compared to 3B). However, while control ovaries grew and matured by day 3, the ovaries of irradiated females did not grow and were similar in size to those of day 1 ovaries (Fig. 3F, compared to 3C). These ovaries remained underdeveloped even by day 14 (Fig. 3G, H, compared to 3C). A close-up examination, however, showed that ovariole segmentation of irradiated females was blocked at day 1 (Fig. 3M) compared to the ovarioles of non-irradiated controls (Fig. 3J). While control ovarioles contained two segments (Fig. 3J), no segmentation was observed for ovarioles of irradiated females (Fig. 3M). Of note, ovarioles of irradiated females contained fewer visible somatic cells than those of the controls, and in general, lacked the layer of follicular cells covering developing germ cell cyst (Fig. 3M, compared to 3 J). Interestingly, the development of the germline cyst in the ovarioles of irradiated females was not affected, as the oocyte of the first germline cyst was specified and located at the posterior position (Fig. 3M, white arrow), similar to that of the control ovarioles (Fig. 3J). Although the germline cyst was able to grow, as evidenced by the enlarged nurse cell nuclei, it failed to bud out of and separated from the germarium and eventually fused with the anterior germarium (Fig. 3N). Furthermore, while the germ cells in the primary follicle of the control ovarioles were surrounded by a single layer of follicular cells (Fig. 3K), in irradiated female mosquitoes, the germ cells of the primary follicle were not covered by a layer of follicular cells and were arrested in development (Fig. 3N–P). Meanwhile, the anterior part of the germarium shrank and did not produce the secondary follicle. A reduction of IGS cells was observed in the anterior segment of the ovarioles from irradiated females (Fig. 3N, compared to 3 K). In summary, these results show that the ovariole maturation was blocked by irradiation, likely due to the lack of somatic cells (including IGS and follicular cells), which consequently disrupts oogenesis, resulting in sterility.

X-ray irradiation blocks proliferation and causes apoptosis during ovariole maturation

The lack of somatic cells (IGS and follicular cells) in the ovarioles of irradiated females could be caused by a defective proliferation and/or apoptosis induced by irradiation. To understand the cause of these irradiation-induced ovariole maturation defects, we used anti-Phospho-Histone H3 (pSer10), PH3, and anti-cleaved Caspase3, CC3, antibodies to detect the proliferative or apoptotic cells, respectively, in these ovaries. In the control ovaries, active proliferation was observed in the somatic cells residing in the middle of the germarium during the process of germarium segmentation (Fig. 4A, B, and K), indicating that both cell proliferation and migration are required to bisect the germarium into the anterior section with undifferentiated germ cells and the posterior compartment containing a developing germline cyst (the primary follicle). Subsequently, during the growth stage of the primary follicle, extensive proliferation of the follicular cells was observed (Fig. 4C, K). After reaching the mature (previtellogenic arresting) stage, no active proliferation was detected in the primary follicle, and only limited proliferation of the follicular cells was observed in the germarium (Fig. 4D, E, and K). On the contrary, a significant reduction of proliferating follicular cells was detected in the irradiated samples. The reduced proliferation was observed as early as day 0 (several hours after irradiation), although the difference is not statistically significant (Fig. 4F, K). However, a significant reduction of proliferating follicular cells was detected during the ovariole maturation process (Fig. 4G–K).

Fig. 4

X-ray irradiation blocks ovariole maturation. A–J Representative confocal microscopy images of ovarioles with Vasa (Green), PH3 (Red), and DNA (Grey) staining at day 0 (A), day 1 (B), day 3 (C), day 7 (D), and day 14 (E) in controls, and at day 0 (F), day 1 (G), day 3 (H), day 7 (I), and day 14 (J) in irradiated females after irradiation. Yellow arrows indicate PH3-positive cells. K Quantification of the average PH3-positive K. L–U Representative confocal microscopy images of ovarioles with Vasa (Green), cleaved-Caspase3 (Red), and DNA (Grey) staining at day 0 (L), day 1 (M), day 3 (N), day 7 (O), and day 14 (P) in controls, and at day 0 (Q), day 1 (R), day 3 (S), day 7 (T), and day 14 (U) in irradiated females after irradiation. Arrows point to somatic cells. The arrowhead points to the germ cell. The scale bar in A (for all image panels) is 50 µm. V Quantification of the cleave-Caspase3-positive somatic cells per ovariole. Error bars in K and V represent mean ± SEM. Comparisons of control and irradiated samples are performed by t-test; *** (P < 0.001), ** (P < 0.01), * (P < 0.05), ns (not significant). The number of samples in each group is shown above the X-axis

In addition to the reduced proliferation, apoptosis resulting from irradiation might also contribute to the deformative ovarioles. In controls, no apoptotic cells, including germ cells and somatic cells (IGS and follicular cells), were observed at day 0 or day 1 ovarioles, and very few apoptotic follicular cells were detected in the ovarioles during the ovariole maturation process (Fig. 4L–P, and V). However, low levels of the apoptotic signal were occasionally detected as early as a few hours after irradiation, but a significant increase in apoptotic cells was observed in day 1, day 3, and day 7 ovarioles of irradiated females (Fig. 4R–V). On day 1, apoptotic cells were somatic cells and mainly detected in the middle germarial region where active proliferation was previously observed during germarium segmentation (Fig. 4B, R), in line with the notion that the mitotic cells are more vulnerable to irradiation [40]. On day 3, ovarioles contained few somatic cells, and apoptosis was frequently detected in these remaining follicle cells (Fig. 4S). It is difficult to distinguish the somatic cell type (IGS or follicular cells) in these ovarioles due to the deformed morphology and lack of cell type markers (Fig. 4S). Of note, apoptosis was also detected in germ cells at this time point (Fig. 4S, yellow arrowhead), presumably as the consequence of a lack of support from these somatic cells and consistent with the observed shrinkage of the germarium. From day 7 onwards, the number of apoptotic cells reduced (likely due to the significant reduction of somatic cells by this time point), and most of them were follicular cells (Fig. 4T–V). Collectively, X-ray irradiation caused a reduction of somatic cell proliferation and an elevation of apoptosis in both somatic cells and germ cells, which led to the blockage of ovariole maturation and consequently resulted in the sterility of irradiated females.

X-ray irradiation does not interrupt the development of Aedes aegypti testes

Given the strong reduction of fertility in irradiated males at 30 Gy, we intended to understand the reason behind this sterility. Ae. aegypti testis development, similar to that of D. melanogaster, occurs earlier than ovary development ([41] and data not shown). By 24-h-old pupal stage (or day 0 of irradiation), the testes already contain germ cells at different differentiating stages (Fig. 5A), arranged in a gradient from the anterior tip (germline stem cells) to the posterior end (elongating spermatids). Testes continued to grow and by day 1, mature sperms were present at the posterior end (white arrow in Fig. 5C). From day 3 to day 14, testes progressively reduced their size (Fig. 5E, G, and I), presumably due to the usage of sperms during the mating process. Surprisingly, no obvious morphological change was observed in the day 0 to day 3 testes of irradiated males, compared to those of the control testes (Fig. 5A–F). Only by day 7 and day 14, some testes of irradiated males exhibited some abnormal patches of Vasa-positive germ cell clusters at ectopic positions in the middle of the testes (Fig. 5G–J, red arrows, and Additional file 1: Fig. S1A-J). A reduction of Vasa-positive early-stage germ cells was also observed in day 14 testes of irradiated males. Hence, unlike its strong effect on the ovariole morphology, X-ray irradiation did not have a strong impact on the overall structure of the testes during its mating competitive stage. It is worth noting that testes from irradiated males exhibited a reduction in germ cell proliferation measured by PH3, compared to the controls, although it is not statistically significant between day 0 to day 7 (Fig. 5A–K), and only by day 14, testes of irradiated males showed significantly reduced germ cell proliferation (Fig. 5K). Furthermore, more testes from irradiated males contained some apoptotic cells when compared to control testes from day 3 onwards (Additional file 1: Fig. S1K). Collectively, X-ray irradiation on 24-h-old pupae has a limited effect on the overall morphology of testes during its early stage.

Fig. 5

X-ray irradiation does not interrupt the development of testis. A–J Representative confocal microscopy images of testes with Vasa (green), PH3 (red), and DNA (grey) staining at day 0 (A), day 1 (B), day 3 (C), day 7 (D), and day 14 (E) in controls, and at day 0 (F), day 1 (G), day 3 (H), day 7 (I), and day 14 (J) in irradiated males after irradiation. White arrows in C and D indicate mature sperm. Red arrows in H and J indicate Vasa-positive germ cells located at ectopic position (compared to G and I). The scale bar in A (for all image panels) is 50 µm. K Percentage of PH3-positive testes. Comparisons of control and irradiated samples are performed by Fisher’s exact test; **** (P < 0.0001), ns (not significant). The number of samples in each group is shown above each column

X-ray irradiation causes chromosomal damage to sperm

Next, we investigated whether X-ray irradiation affected sperm quantity and quality, which could have led to the observed male sterility. We first checked whether irradiation might affect the production of sperm, although there was no overall abnormality in the testis morphology. While each control male contained about 10,000 sperm, the irradiated male on average harboured significantly fewer sperm in its testes and seminal vesicles (Fig. 6A). Males, in general, produce an excess number of sperm via continuous spermatogenesis. Thus, the 50% reduction in sperm in irradiated males might not explain the high sterility observed. To address this, we examined the number of inseminated sperm in spermathecae of females mated with control or irradiated males. Interestingly, there was no significant difference in terms of sperm quantity in spermathecae of females mated with control or irradiated males (Fig. 6B). These results suggest that irradiation indeed affects spermatogenesis (by reducing sperm production) but does not affect mating and insemination between wild-type females and irradiated males. It further indicates that reduced sperm production in irradiated males is not the underlying cause of the strong sterility observed. We moved on to examine the portion of dead/damaged sperm using a live/dead sperm viability assay. Notably, the sperm samples of irradiated males showed a higher portion of dead or damaged sperm than those of control males (Fig. 6C), and the sperm survival rate dropped from 71.7% in controls to 47.0% in irradiated samples (Fig. 6D), indicating that quality of sperm is strongly affected by irradiation. To further address DNA damage in these irradiated samples, we examined two additional double-strand break (DSB) markers, p-γ-H2Av and TUNEL staining. γ-H2Av is a conserved histone H2A variant of human γ-H2Ax and its phosphorylation serves as an indicator for DNA DSB. Ae. aegypti genome harbours one functional ortholog of the human H2A variant (AAEL012499) [42, 43]. As expected, in the control testes, p-γ-H2Av was only detected in meiotic spermatocytes but not in other germ cells (Fig. 6E). In the irradiated samples, ectopic p-γ-H2Av was detected in most germ cells, including GSCs, spermatogonia, and early differentiating spermatids (Fig. 6F). In the control testes, TUNEL staining did not detect consistent signals above the background (Fig. 6G), while its signal decorated many differentiating spermatids in the irradiated samples (Fig. 6H), showing DNA damage in these spermatids. These results together demonstrate that X-ray irradiation induces chromosomal damage to male germ cells. Hence, the observed male sterility after pupal irradiation is likely due to a decline in sperm quality.

Fig. 6

X-ray irradiation affects sperm viability. A Quantification of sperm number in testes and seminal vesicle of wild-type and 30 Gy irradiated males. B Quantification of sperm number in spermathecae of wild-type females mated with wild-type or 30 Gy irradiated males. C Representative microscopy images of live sperm (Green) or dead sperm (Red) of wild-type and 30 Gy irradiated males. D Percentage of live sperm of wild-type or 30 Gy irradiated males. E-F Anti-H2Av staining only labels meiotic spermatocytes (red arrow) in wild-type testis. H2Av signals are also detected in GSCs, spermatogonia (white arrow), and differentiating spermatids (yellow arrow) in irradiated males (F). G–H TUNEL staining does not detect consistent signals in wild-type testes (G), but is elevated (decorating spermatid DNA, white arrow) in irradiated males (H). G’ and H’ are close-up views for the regions indicated in G and H. Comparisons of controls and irradiated samples are performed by paired t-test; *** (P < 0.001), ns (not significant). Error bars represent mean ± SEM. The number of samples in each group is shown above the X-axis

Wolbachia-infected Ae. aegypti strain is more sensitive to X-ray irradiation

So far, we have tested the working dose of X-ray irradiation during the pupal stage on wild-type Ae. aegypti (NEA-EHI strain) and investigated the cause of sterility in both female and male mosquitoes. Building upon these findings, we moved on to test the dose of X-ray irradiation on wAlbB-SG, the Wolbachia-infected Ae. aegypti (NEA-EHI strain) used in the IIT-SIT operational test under laboratory conditions. Our results showed that both egg number and hatch rate of irradiated wAlbB-SG females reduced significantly in a dose-dependent manner (Fig. 7A, B). wAlbB-SG females subjected to 25 or 30 Gy irradiation did not produce any eggs, only 4 out of 132 females subjected to a dose of 20 Gy irradiation laid few eggs and none of these eggs hatched (Fig. 7A, B). Thus, the minimal dose to induce complete sterility in females of wAlbB-SG strain is 20 to 25 Gy, lower than that of the wild-type strain (25–30 Gy), indicating that the Wolbachia-infected strain is likely more sensitive to X-ray irradiation than its parental Ae. aegypti (NEA-EHI strain). Similarly, it was reported that Wolbachia-infected Ae. aegypti lines (Brazil and Mexican strains) are more radiosensitive to irradiation than the uninfected ones [44]. As expected, wAlbB-SG females mated with irradiated wAlbB-SG males and produced a comparable number of eggs as wAlbB-SG females mated with control males (Fig. 7C). However, the hatch rate of these eggs showed a dose-dependent reduction, compared to the controls (Fig. 7D), which is consistent with the previous report [26]. Collectively, wAlbB-SG mosquitoes are likely more sensitive to X-ray irradiation, compared to the uninfected controls, and a lower dose of X-ray (20 to 25 Gy) is able to achieve complete female sterilization.

Fig. 7

Effect of irradiation dose on the fecundity and hatch rate in wAlbB-SG. Effects of various doses of X-ray irradiation on egg production (A) and hatch rate (B) for irradiated wAlbB-SG females mated with wild-type wAlbB-SG males. Effects of various doses of X-ray irradiation on egg production (C) and hatch rate (D) for wild-type wAlbB-SG females mated with irradiated wAlbB-SG males. The number of samples in each group is shown above the X-axis. Error bars represent median and interquartile range. Kruskal-Wallis test and Dunn’s multiple comparisons post hoc test shows the difference between control and individual irradiation dose; doses not sharing the same letter are significantly different