Anh Ly, Nfn Aysha, Nicole Rodriguez
Department of Biology, Rutgers University, Camden NJ 08102
Abstract
Climate change has substantially increased the frequency and intensity of heat waves, leading to heightened temperatures worldwide. This escalation poses a significant threat to insects because they rely on external factors such as sunlight or shade to regulate their internal body temperatures. Insects play a crucial role in our ecosystem such as decomposing organic matter, pollinating crops, maintaining healthy soil, and controlling pests. Maximizing conservation efforts might not be possible without understanding how these organisms respond to heat stress. In this study, Drosophila melanogaster was used as a model for insects, an ectothermic species. We conducted thermal and developmental assays to examine the impact of heat stress on the reproductive and developmental success of D. melanogaster. After 24 hours of mating at a 2:1 female-to-male ratio under control (20°C) and mild heat stress (30°C) conditions, D. melanogaster eggs were collected. Subsequent development of these eggs into pupae and adults was recorded as percentages to measure developmental success. Our results showed a severe reduction in developmental viability in both Wild-type and TrpA1-Knockout genotypes under heat stress. Interestingly, we found that pupae were more susceptible to heat stress than larvae. Due to the low power, we did not see any significant effects of temperature on D. melanogaster’s oviposition rates, or the average number of eggs produced. However, there is a trend that increasing temperatures affect the oviposition rates in wild-type (temperature-sensitive) D. melanogaster but not TrpA1 mutants (temperature-insensitive). Nevertheless, this study provides a framework for understanding the reproductive and developmental responses of Drosophila melanogaster to heat stress.
Introduction
Optimal temperature is essential for many biological processes. In insects, thermal regulation depends heavily on external sources such as sunlight or shade. Thus, thermotaxis, or movement from unfavorable to favorable temperatures in response to stimuli, becomes crucial. It necessitates the ability to detect hazardous temperatures and distinguish between potentially harmful and hospitable environments in animals (Sokabe & Tominaga, 2009; Barbagallo & Garrity, 2015). Under extreme temperatures, Drosophila melanogaster females quickly decide whether to engage in mating or to escape, using a neural pathway activated during the temperature-sensing process. Intriguingly, it was previously found that losing the very mechanism to sense temperature that helps increase survival rates under extreme temperatures helps increase mating activities (Miwa et al, 2018). Yet, the consequential impact of increased mating activities on offspring survival rates remains unknown.
Heat stress has also been known to reduce the reproduction and development of different stages in Drosophila, with increasing temperatures often resulting in decreased survival rates (Huang et al., 2020). Adult D. melanogaster thrive under 25°C and show increased developmental rates at temperatures between 28°C and 30°C. Temperatures exceeding 35°C are likely to cause harmful or even lethal effects. (Simões et al., 2021 & Chen et al., 2013). To detect temperature changes and avoid potential heat threats, D. melanogaster use multiple classes of thermoreceptors: Transient Receptor Potential A1 (TrpA1) for mild heat stress (activated at above 24°C), and Painless for noxious heat (activated at approximately 40°C) (Barbagallo & Garrity, 2015 & Lee & Montell, 2013).
In a previous study, the removal of the TrpA1 channel during thermal stress was known to enhance mating behaviors in Drosophila melanogaster, but the subsequent impact on developmental viability remained unexplored (Miwa et al., 2018). Other research endeavors have assessed the influence of heat stress on the egg-to-adult ratio during development (Kirk Green et al., 2019). However, they did not account for the reduction in mating behaviors under elevated temperatures, as only wild-type Drosophila with normal heat-sensing capacity were used. Therefore, it is imperative to investigate the effects of increased temperature on the development of Drosophila melanogaster that cannot detect heat stress and thus mate at a regular frequency. Mating activities may have a profound impact on fertility by influencing egg production, however, population preservation depends more strongly on offspring viability and development, rather than just mating frequencies.
Therefore, in this study, we aim to investigate the effects of heat stress during development on egg-to-pupa-to-adult viability in TrpA1-knockout (temperature-insensitive) Drosophila melanogaster, which mate at a normal frequency under heat stress. We explore sublethal heat stress at 30°C and hypothesize that offspring development will be negatively affected at high temperatures, despite an increase in mating activities. To test our hypothesis, we use two genotypes: Wild Type as the control and TRPA1 to study the effects of mild heat stress.
Materials & Methods
Insect rearing and genotypic groups
Wild-type Oregon-R (Stock #5) Drosophila melanogaster flies used in this study were obtained from Bloomington Drosophila Stock Center. The flies were reared on a standard diet (purified water, agar, potassium sodium, calcium chloride, sucrose, dextrose, deactivated yeast, and cornmeal) and were left at room temperature at approximately 20°C. Wild type (Oregon-R) served as the control, temperature-sensitive genotype and TrpA1-Knockout (Stock #26504) served as the experimental, temperature-insensitive animals.
Fly food and oviposition substrate formula
Virgin females of both genotypes were collected prior to the experiment by emptying the original stock of flies into new vials containing standard diets. The food vial with the remaining eggs and pupa was then observed for any new emerging flies. The virgin females were then separated into new food vials. The flies were flipped every 6 hours to prevent the emerged flies from mating (approximately 16 virgin females were collected for each genotype). We prepared grape juice agar plates for collecting eggs (Rockwell et al, 2019). To prepare grape juice agar, 75.2% distilled water, 25.2% grape juice (purchased from convenience store), 3% agar (VWR CAS# 9002-18-0) and 1.2% sucrose (VWR CAS# 57-50-1) were mixed in a 600 mL beaker and heated at around 300°C on a hot plate until all solids completely dissolved and gave one uniform layer. The solution was cooled down for 5 minutes before 2% ethanol (Fisher CAS# 64-17-5) and 1% of acetic acid (VWR CAS# 64-19-7) was added.
To prevent the solutions from solidifying while transferring to 55mm Petri dishes (SKU# 502014-07), the beaker was kept on low heat on the hot plate during the process. Approximately 5 mL of this solution was pipetted to each petri dish. A total of 50 petri dishes were made (approximately 24 petri dishes were used for the experiment) with leftovers. Yeast paste was prepared by mixing 7g activated yeast powder (Carolina CAS# 68 876-77-7) with 9mL distilled water (Featherstone et al, 2009). One drop of thick and fresh yeast paste was added to the center of the grape juice agar petri dish. The agar surface was scraped with a plastic knife to encourage egg-laying (Rockwell et al, 2019).
Experimental Design
In this experiment, the Oregon-R Wild Type and TrpA1 Knockout D. melanogaster received treatments at the following temperatures: 1) Room temperature (20°C) 2) Mild hot temperature (30°C). The mild hot (30°C) treatment groups were kept in the incubators with 70% relative humidity and a photoperiod of 14:10 (L:D). Each experimental group received embryo collection cages with each cage containing two virgin females and one male (Rockwell et al, 2019). A 55mm petri dish containing 5 mL of grape juice agar was used as a food source and oviposition substrate at the bottom of each embryo collection cage. Each trio was kept at their respective temperatures for 24 h to allow mating to occur. The ambient temperature groups across all genotypes were used as the control.
Data Collection
After 24h of mating, the breeding flies were transferred to new food vials. Subsequently, the petri dishes were examined, and the eggs were quantified using a microscope. After the egg counting process, the agar along with the eggs, was inverted and transferred into a fresh food vial containing the standard diet before being placed back to their respective temperature (Rockwell et al, 2019). The number of pupae formation was recorded approximately 10 days after the counting of eggs, and the adult emergence was recorded approximately 5-7 days after pupation.
Statistical Analysis
Data for adult and pupal counts were presented in percent pupation ([number of pupae/number of eggs] × 100) and percent adult eclosion ([number of adults/number of eggs] × 100). To determine if there were any significant differences between the temperature treatment groups, a two-way ANOVA was performed using GraphPad Prism version 8.0.0 for Windows. Egg count data that fell outside of two standard deviations from the mean was considered and removed as outliers. Subsequently, pupa and adult measurements from these eggs were also excluded.
Results
D. melanogaster were allowed to mate for 24 hours in embryo collection cages at their respective temperatures. The average number of eggs for the Wild-Type (Oregon-R, control group) under room temperature 20°C (control temperature), and 30°C (experimental temperature) was not significantly different (Two-way ANOVA, p = 0.29). There were also no statistical differences found between the temperature treatments group in TrpA1ins genotype (Two-way ANOVA, p = 0.27) (Figure 1a).
Temperatures did not have a significant effect on percent pupation in TrpA1ins (ANOVA, p = 0.18), but it did have a significant impact on the WT strain (ANOVA, p = 0.03) (Figure 1b). At higher temperatures, the percentage of pupation dropped significantly, leading to an almost threefold decrease from 20°C (mean ± SEM; 56.7% ± 2.7%) to 30°C (21.3% ± 10.1%) in WT. A similar trend was also observed in TrpA1ins from 20°C (39.2% ± 13.9%) to 30°C (13.6% ± 5.1%).Significant differences were observed in the percentage of adult eclosion for both the WT (ANOVA, p = 0.0004) and the TrpA1ins group (ANOVA, p = 0.0052) (Figure 1c). Higher temperatures significantly reduced egg-to-adult developmental viability to nearly zero in both genotypes. At 30°C, percent eclosion in the WT strain dropped from 60.0% to 4.8%, and in TrpA1ins from 48.9% to 0.81%.
Figure 1. Effects of temperatures on Developmental Metrics in Wild Type (Oregon-R) and TrpA1ins mutant D. melanogaster.a) The average number of eggs (Mean ± SEM), b) percent pupation against the side of the vial & on the food surface (percent of eggs that successfully pupated)(Mean ± SEM), c) percent adult eclosion (percent of eggs that successfully emerged as adults) (Mean ± SEM) for each temperature treatment across two genotypes: Wild-type (control group; n = 4 at 20°C, n = 6 at 30°C) and TrpA1ins (experimental group; n = 5 for both temperatures).
Discussion
Our study aimed to understand the impact of heat stress on the developmental success of Drosophila melanogaster, focusing on both temperature-sensitive and temperature-insensitive genotypes. We hypothesized that increased temperature would decrease developmental success in both genotypes. To test this hypothesis, we used embryo collection cages and allowed D. melanogaster to mate at a 2:1 female-to-male ratio for 24 hours under 20°C (control) and 30°C (experimental) conditions. We recorded the total number of eggs and monitored their subsequent development into pupae and adults to measure developmental viability. Our result indicated that temperature did not affect oviposition rates in either genotypes. However, a trend suggested that increased temperatures decreased egg-laying in the WT strain. Increasing the number of replicates may unveil more significant differences. Among the laid eggs, increased temperature reduced percent pupation in only the WT strain. However, there was a trend indicating that higher temperatures also negatively affected the TrpA1 mutant. The egg-to-adult success rate severely decreased as temperature increased in both genotypes, suggesting a general negative impact on developmental success regardless of temperature sensitivity.
Our data confirmed the findings of previous studies, which indicated that increased temperatures negatively impact egg-to-pupa development in wild-type Drosophila (Evans et al., 2018). Additionally, we observed a severe reduction in developmental success in TrpA1-knockout (temperature-insensitive) animals. This indicates that eggs and larvae are highly susceptible to temperature fluctuations because early embryonic stages lack the ability to produce heat shock proteins (Evans et al., 2018). In addition, larvae can move freely and use thermotaxis to avoid extreme temperatures, thereby making it unnecessary to invest in additional heat protective mechanisms. However, since temperature remains constant in the food vials used in this study, thermotaxis becomes ineffective, posing a significant risk to larvae as it is their primary means of heat protection.
Percent pupation, however, had some limitations. During our experiment, only visible pupae were counted, typically found on the food surface or along the vial walls. It is likely that additional pupae were present beneath the food surface but were not counted, which may have impacted the accuracy of the percent pupation data. Despite this limitation, our results still suggest that most of the eggs and larvae were highly vulnerable to heat stress.
In Drosophila, the pupal stage is generally known to be the most resistant to heat stress, while the larval stage is the least (Evans et al., 2018, Moghadam et al., 2019). However, in our study, we observed more eggs developing to pupae than pupae emerging as adults, in both wild-type and TrpA1-knockout animals. Nearly all pupae failed to develop into adults, suggesting that pupae might be less resistant to heat stress compared to eggs or larvae. This contradicts the findings of other studies. One possible explanation is that the duration of heat exposure was too long for the developing offspring to withstand. According to a previous study, variations in temperature and exposure duration (even in hours) resulted in decreased emergence rates (Kirk Green et al., 2019). Given the setup of our experiment, by the time the eggs reached the pupal stage, they had endured heat stress for approximately 7-10 days. Therefore, it’s not surprising that most did not successfully transition into adults.
Another factor that could have contributed to the low egg-to-pupa and pupa-to-adult developmental viability is egg quality. Heat-stressed breeding Drosophila must cope with the heat by generating heat shock proteins to mitigate the effects of high temperatures by repairing damaged proteins and preventing further denaturation (Krebs and Loeschcke, 1994, Richter et al., 2010). However, producing heat shock proteins is energetically costly, potentially leading to trade-offs in adult Drosophila, resulting in lower-quality eggs as suggested in a previous study (Krebs and Loeschcke, 1994). This reduction in egg quality was also assessed in another study, where offspring from heat-shocked parental Drosophila suffered high mortality rates and low reproductive performance (Kirk Green et al., 2019). Another possible explanation is that high temperatures (30°C) may have reduced the ovary size in breeding females, leading to a decrease in the overall efficiency of their reproductive system. There was also a significant reduction in sperm quality and quantity in the testis tubules of Drosophila (Kirk Green et al., 2019). These changes in reproductive organs might have negatively affected the fertilization process, therefore leading to lower egg quality.
Even when temperatures affected every other developmental stage in Drosophila, it did not have much significant impact on oviposition rates. However, there is a trend that temperatures impacted Wild-type Drosophila, but not TrpA1 mutants. By increasing the replicates/power, we can detect more subtle effects. If there was a significant difference between the temperature treatments in only wild-type Drosophila eggs, it may, for the first time, provide evidence that mating behavior was the only factor affecting oviposition rates.
Potential sources of errors in our study include the inconsistent age and health of the selected mating trios. In future studies, ensuring Drosophila are of the same age and health status could mitigate variations in their mating capacity, thereby reducing significant fluctuations in egg counts. In this study, we identified and removed these fluctuations as outliers (defined as values more than two standard deviations from the mean).
Another limitation might be the variation in the amount and consistency of yeast paste. The quantity of yeast paste can influence Drosophila’s attraction index (Palanca et al., 2013), but it’s still unclear whether it affects the quality and quantity of eggs laid. Other limitations could stem from differences in the diameter of the embryo collection cages and the developing food vials. This made it challenging to accurately and consistently transfer the agar with the eggs (which needed to be done upside down), causing different moisture levels among transferred eggs. If eggs were not transferred correctly, they were at a higher risk of drying out or dying (Rockwell et al., 2019).
For future research building on this study, it is essential to increase the number of replicates for egg count data to detect significant differences in response to temperature across both genotypes. It is also crucial to account for unfertilized eggs to gain better insight into whether the fertilization process was disrupted. This can be achieved by allowing the eggs to develop and then counting the number of larvae, which represents the number of fertilized eggs.
Climate change is increasing global temperatures by increasing the intensity and frequency of heat waves (Marx et al., 2021), which poses a significant risk to insects as they rely on environmental temperatures to regulate their body temperature. Many researchers have studied the effects of heat stress on the development and fertility of Drosophila, but this is the first study to consider the reduced mating frequency in Wild-type Drosophila under heat stress and explore the egg-to-pupa-to-adult developmental viability in genetically modified Drosophila melanogaster that lacks temperature-sensing capabilities. The findings of this study may inform conservation strategies that despite the increase in mating activities and oviposition rates in temperature-insensitive Drosophila under heat stress, subsequent developmental phases of the offspring are still negatively affected. Further research focusing on the fertilization process might shed light on factors that are influencing oviposition rates and can expand our knowledge of how ectothermic species respond to heat stress. This knowledge can enhance conservation efforts for hundreds of endangered insect populations, as well as other ectothermic species facing similar threats due to climate change.
Acknowledgements
We would like to convey our gratitude to Dr. Nathan Fried for his continued mentorship and support. We would also like to thank the Rutgers Camden Department of Biology for providing us with the funding and resources for this project.
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