Mitochondrial superoxide dismutase overexpression enhanced insect quality and modestly improved the mating success of irradiated transgenic males competing against irradiated WT counterparts in leks. Increased levels of SOD2 in transgenic males led to higher adult emergence rates, a lower proportion of deformities (partially emerged adults and wing damage), and on average 3% more fliers than WT insects, regardless of irradiation treatment. While the improvement in the mating success was modest (22%), irradiated SOD2 5.2 males were more competitive in leks considering that these transgenic insects mated more frequently in sectors with a high frequency of matings than normoxia-irradiated WT males competing with non-irradiated males. Contrary to our hypothesis, SOD2 overexpression did not interact synergistically with short-term low-oxygen conditioning hormesis to improve the mating success of transgenic males. Hypoxic or severe-hypoxic conditioning alone increased the total antioxidant capacity across all treatments relative to control (Nx-0 Gy) and similarly improved mating success in irradiated males from both lines (WT and SOD2 5.2) compared to non-irradiated rivals in leks.
We previously showed that SOD2 overexpression increased mating success of normoxia-irradiated males up to 50% compared to irradiated WT males in small-scale mate choice tests 40. In the sexual competitiveness tests under semi-natural field-cage conditions, the sexual advantage of SOD2 5.2 males over WT rivals observed in the laboratory was not as readily detectable, and the increase in mating performance was only 22% greater and non-significant. In our field cage tests however, normoxia-irradiated SOD2 5.2 males mated more often in competitive lekking places where a higher frequency of mating events occurred than did normoxia-irradiated WT males, indicating that the transgenic insects might be more competitive than wild-type males in conditions where lekking occurs in the wild. We discuss four potential reasons to explain the inability to detect a difference in mating success in field cages when the improvement of SOD2 on mating was evident in small-cage trails40. The four factors are: (1) differences in optimal lek size, (2) context-dependent female mate choice, (3) irradiation dose rate, and (4) study statistical power.
First, although both small-scale and large-scale (field cages) mate choice tests were performed with a 2:1 male: female ratio, the total number of flies differed dramatically between the field cages (50 males, 25 females) and the small cages (2 males, 1 female), possibly diluting the sexual advantage of normoxia-irradiated transgenic males we observed in the small cages40. The larger groups of males and females in the field cages may favor the formation of large leks that, in turn, may have disrupted the ability of SOD2 5.2 males to monopolize both matings and access to females. This argument is reinforced by the results from our high-density condition tests that showed no difference in mating success of non-irradiated and normoxia-irradiated males competing for the same female (100 males: 25 females), despite non-irradiated males mating more often than irradiated males in tests with lower densities (50 males: 25 females). Overall, male aggregation in leks benefits both high-ranking (attractive) and low-ranking (less attractive) males because it facilitates the access to females while reducing the risk of predation41–43. However, differences in optimal lek size between low-ranking and high-ranking males can limit mating success and the benefits of these aggregations. That is, attractive males obtain sexual advantages only in small leks, while unattractive males gain substantially in large leks44,45.
The contrast in optimal lek sizes between attractive and less attractive males drives the overcompensation approach adopted for SIT programs. For C. capitata inundative releases larger than 100 sterile males: 1 wild male are sometimes used in SIT programs to counterbalance the quality losses experienced by mass-reared and irradiated insects27,46. Because this overcompensation approach makes SIT relatively costly, alternative approaches that improve male quality instead of increasing release ratios have gained momentum in recent years47,48. The use of transgenesis to increase enzymatic antioxidant activity can be used as an additional strategy to enhance sterile male quality40. Perhaps males overexpressing SOD2 will show increased tolerance to low-oxygen environments, a factor not evaluated in this study, but critical to ensuring the quality and sexual competitiveness of sterile insects commonly shipped under hypoxic or anoxic environments over long distances30.
Second, the discrepancy between small cages40 and the field cages in this study could be a result of females in small arenas experiencing intense sexual harassment due to limited chances to escape the copulation attempts of insistent males, regardless of the male’s sexual quality. In a field cage, however, females have more opportunities to assess male condition without as much sexual harassment by opportunistic males. Thus, it is expected that mate choice tests in large field cages can provide a better understanding of female sexual preferences because they are more reflective of sexual selection in the field than mate choice tests in small cages in the laboratory. There is evidence that fruit fly females become less sexually selective in environments that favor sexual harassment, such as mass-rearing conditions where flies are kept in cages at high densities49. In addition, female perception of male sexual signals can differ according to the environment50,51. Anastrepha suspensa males rely on chemical and acoustic displays to court females visiting leks52. Hence, it is reasonable to assume that the female perception of acoustic vibration, wing beat frequency, pheromone composition and quality might differ if those signals were displayed in an enclosed small plastic cage rather than in a semi-open field cage.
Third, it is possible that the dissimilarities between this study and our previous work40 were due to differences in dose rates from different gamma irradiation sources used for each experiment. Insects in our previous small-scale mate choice tests were irradiated at a dose rate of ~ 8 Gy/min40, while males tested in field cages were irradiated at a dose rate of ~ 97 Gy/min, approximately 12 times faster. If radiation-induced oxidative damage is proportional to dose rate, then insects irradiated at lower dose rates will accumulate less oxidative damage in their cells. A few studies have assessed the effect of irradiation dose rate on fruit flies’ sterility and performance53,54. For instance, dose rates ranging from 5 Gy to 80 Gy per minute affected neither fly quality (emergence and flight ability) nor sterility of Bactrocera tryoni54. However, B. tryoni individuals irradiated at high dose rates showed increased mortality under starvation conditions compared to those non-irradiated or irradiated at low dose rates54. Nonetheless, the effect of irradiation dose rates on sexual performance or oxidative stress has not been determined in tephritid fruit flies.
Last, our field-cage tests comparing the sexual competitiveness of normoxia-irradiated SOD2 and WT males were based on small sample size (n = 9). Therefore, this study had less statistical power to detect a modest difference of ~ 10% in mating success between the WT and SOD2 lines than our previous study where we had many more replicates of small cages (~ 50). Post-hoc power analysis indicated that a sample size of 776 field cages would have 80% power to detect a small biological effect size consistent with a 10% difference in the mating success of normoxia-irradiated SOD2 5.2 males relative to their normoxia-irradiated WT counterparts. Thus, we recognize the limitations of inferences we can make due to our small sample size, potentially leading to inaccurate conclusions if we accept our null hypothesis of equal sexual competitiveness of transgenic and WT males irradiated in normoxia (the null hypothesis of no difference between treatments is equivalent to an RSI = 0.50).
The enzymatic antioxidative protection offered by enhanced expression of SOD2 to irradiated transgenic males reinforces SOD2’s primary role in protecting somatic cells against radiation-generated oxidative stress. Both SOD2 5.2 and WT males had similar total antioxidant activity, but only insects overexpressing SOD2 showed partial sexual enhancement and significant improvements in quality control parameters. Our findings corroborate the idea that endogenous antioxidant enzymes can play critical roles in the mechanisms of sexual selection in the face of severe oxidative stress conditions, such as gamma irradiation37. Even though SOD2 activity was not measured in this study, our previous work showed that the mitochondrial superoxide dismutase activity of SOD2 5.2 males was 50% greater than WT males40. Superoxide dismutase (SOD) is a critical component of the antioxidant defenses of aerobic organisms, particularly its mitochondrial version (SOD2) that is directly linked to protection against mitochondrial ROS generated as inevitable by-products of cellular energy generation55,56. Many vertebrates and invertebrates use antioxidant enzymes like SOD as a first line of protection against reactive oxygen species (ROS) and reactive nitrogen species (RNS) within cells16,57,58. Additionally, non-enzymatic antioxidants, such as glutathione and carotenoid pigments, can also play important direct and indirect roles in organismal antioxidative defense systems and should not be ignored14,59. More work on small-molecule antioxidants is needed in the context of sexual selection and male mating competitiveness in SIT.
The increase in total antioxidant activity we observed in response to anoxic conditioning in A. suspensa is not exclusive to our study. Previous correlative studies have shown that 1 hour of anoxic conditioning of pharate adults (two days before emergence) prior to irradiation (70 Gy) resulted in greater antioxidant capacity, specifically much higher SOD2 activity, less oxidative damage, better insect quality, and greater mating success than unconditioned irradiated males37,38. While we also observed an increase in sexual performance following low-oxygen conditioning, the conditioning treatment resulted in a reduction in insect quality, particularly emergence and percentage of fliers, contrary to what is reported in C. capitata under similar treatment conditions60. Usually, short term exposure (~ 1h) of fruit fly species used in operational SIT programs does not result in detrimental effects29.
Overall, increased antioxidant capacity during hypoxic or anoxic events has been observed for numerous organisms ranging from arthropods to diving seals and turtles, and increased antioxidant capacity is recognized as a common mechanism used to protect the organism from the stress of reoxygenation after hypoxia or anoxia exposure61. This study extends our earlier work on antioxidant protection in A. suspensa37 by directly comparing severe hypoxic (0.4 ± 0.1 kPa of O2, 0.8 ± 0.2 kPa of CO2) atmosphere to short-term hypoxic (7.3 ± 1.2 kPa of O2, 4.5 ± 0.8 kPa of CO2) conditioning treatments. Here males irradiated in both nitrogen and in a hypoxic atmosphere composed mainly of nitrogen with low levels of O2 and CO2 exhibited a similar protective effect to short-term anoxic conditioning. This finding is especially valuable, considering that SIT programs worldwide rely on natural oxygen depletion to safeguard sterile insect quality from the oxidative damage generated during irradiation, handling, and shipment procedures30.
In conclusion, we corroborate the previous findings of López-Martínez and Hahn37,38 and extend the same hormetic mechanism, first described for anoxic-conditioning, to less oxygen depleted hypoxic treatments. We also extend our earlier work on the benefits of SOD2 overexpression40 and show that while elevated SOD2 is beneficial, the benefits are not as dramatic in realistic field-cage settings. Alternative treatments focusing on the vast range of possibilities offered by enzymatic and non-enzymatic antioxidant protection to safeguard sterile insect quality and sexual competence should be extensively explored in future studies that seek to improve overall quality of irradiated insects used in SIT programs.