The influence of developmental temperature on sperm form and function in Callosobruchus maculatus

This thesis examines the effects of the thermal rearing environment on larval development and its influence on the expression of primary reproductive traits in Callosobruchus maculatus. Chapter 1 provided basic outline to the thesis, starting with a general introduction to sexual selection and specifically its role in the evolution of genitalia and primary reproductive traits. These traits appear to exhibit a level of phenotypic plasticity and thus the opening chapter introduces this concept and its role in evolution and diversification. Phenotypic plasticity in the primary reproductive traits of insects and the evolutionary consequences of this plasticity are discussed. Chapter 2 examined the effect of larval rearing temperature on larval development and offspring size. Larvae go through 4 larval instars prior to pupation. Unsurprisingly larvae reared at 17°C took approximately four times longer to develop than those larvae reared at 33°C but those beetles that completed their development at the coolest temperature were the largest, in agreement with Bergmann’s rule. This chapter paved way for the experiments carried out in subsequent chapters. Chapter 3 investigated the effect of developmental temperature on the expression of primary reproductive traits (e.g. sperm length and egg size) and life-history traits (e.g. adult longevity and fecundity). Sperm length exhibited phenotypic plasticity, being shortest when larvae developed at the temperature extremes (17°C & 33°C). Beetles raised at 17°C inseminated the fewest sperm despite the fact that these males had the largest absolute testes size. Developmental temperature affected female fecundity; females that underwent larval development at 17°C laid the fewest eggs. These females along with those reared at 33°C also laid the largest eggs. In general egg size tended to increase with decreasing developmental temperature (in agreement with Bergmann’s rule) although sperm size gives a more mixed response being largest at an intermediate temperature. This is in agreement with the effect of developmental temperature on sperm size in dung flies, but in guppies and a species of snail sperm size increased with decreasing temperature. Chapter 4 used thermal switch experiments to identify when, during larval development, the expression of sperm length was sensitive to temperature. Thermal switching involves an abrupt shift in the thermal environment experienced by the developing larvae, such that development starts at one environment and is completed at either a higher or lower thermal rearing environment. There appeared to be two thermal sensitive periods (TSP) for the expression of sperm length: one during the very stages of ontogeny and one around the time of instar III to instar IV. This is the first demonstration of a TSP in relation to the phenotypic expression of the sperm. The findings of this chapter are discussed in relation to previous shift-once or shift-twice experiments on reptilian embryos and a handful of insects. Chapter 5 studied the consequences of thermal environment during larval development and as adults on copula duration. Both larval rearing temperature and post-eclosion temperature affected copula duration; males reared at the lowest temperature taking longer to complete copulation. This is the first demonstration that larval rearing temperature affects copulatory behaviour. It is argued that because thermal heterogeneity is likely to be common in nature, this under-examined component of male behaviour could account for much variation in copulatory behaviour in nature. Chapter 6 examined the effect of larval rearing temperature on the outcome of sperm competition. Males reared at 17°C were less successful at sperm defence and sperm offence, whilst males reared at 33°C were less successful at sperm offence in comparison to males reared at 27°C. This is the first study to report an effect of developmental temperature on the outcome of postcopulatory sexual selection. The results are discussed in light of the physiological consequences of developmental temperature on male ejaculatory characteristics. Chapter 7 discusses the possible mechanisms and consequences of phenotypic plasticity in reproductive traits as a result of experiencing heterogeneous environments during development, and how these processes might interact with postcopulatory sexual selection. Further work on understanding the role of phenotypic plasticity in the evolution of primary reproductive traits is required

Experimental evidence for stronger impacts of larval but not adult rearing temperature on female fertility and lifespan in a seed beetle

Temperature impacts behaviour, physiology and life-history of many life forms. In many ectotherms, phenotypic plasticity within reproductive traits could act as a buffer allowing adaptation to continued global warming within biological limits. But there could be costs involved, potentially affecting adult reproductive performance and population growth. Empirical data on the expression of reproductive plasticity when different life stages are exposed is still lacking. Plasticity in key components of fitness (e.g., reproduction) can impose life-history trade-offs. Ectotherms are sensitive to temperature variation and the resulting thermal stress is known to impact reproduction. So far, research on reproductive plasticity to temperature variation in this species has focused on males. Here, I explore how rearing temperature impacted female reproduction and lifespan in the bruchid beetle Callosobruchus maculatus by exposing them to four constant temperatures (17 °C, 25 °C, 27 °C and 33 °C) during larval or adult stages. In these experiments, larval rearing cohorts (exposed to 17 °C, 25 °C, 27 °C and 33 °C, from egg to adulthood) were tested in a common garden setting at 27 °C and adult rearing cohorts, after having developed entirely at 27 °C, were exposed to four constant rearing temperatures (17 °C, 25 °C, 27 °C and 33 °C). I found stage-specific plasticity in all the traits measured here: fecundity, egg morphological dimensions (length and width), lifespan and egg hatching success (female fertility). Under different larval rearing conditions, fecundity and fertility was drastically reduced (by 51% and 42%) at 17 °C compared to controls (27 °C). Female lifespan was longest at 17 °C across both larval and adult rearing: by 36% and 55% compared to controls. Collectively, these results indicate that larval rearing temperature had greater reproductive impacts. Integrating both larval and adult rearing effects, I present evidence that female fertility is more sensitive during larval development compared to adult rearing temperature in this system

Fertility and mortality impacts of thermal stress from experimental heatwaves on different life stages and their recovery in a model insect

With climate change creating a more volatile atmosphere, heatwaves that create thermal stress for living systems will become stronger and more frequent. Using the flour beetle Tribolium castaneum, we measure the impacts of thermal stress from experimental heatwaves in the laboratory on reproduction and survival across different insect life stages, and the extent and pace of any recovery. We exposed larvae, pupae, juvenile and mature adult male beetles to 5-day periods of heat stress where temperatures were maintained at either 40°C or 42°C, a few degrees above the 35°C optimum for this species' population productivity, and then measured survival and reproduction compared with controls at 30°C. Mortality due to thermal stress was greatest among juvenile life stages. Male reproductive function was specifically damaged by high temperatures, especially if experienced through pupal or immature life stages when complete sterility was shown at reproductive maturity; larval exposure did not damage adult male fertility. High temperatures impaired testis development and the production of viable sperm, with damage being strongest when experienced during pupal or juvenile adult stages. Despite this disruption, males recovered from heat stress and, depending on the stage of exposure, testis size, sperm production and fertility returned to normal 15–28 days after exposure. Our experiments reveal how thermal stress from heatwave conditions could impact on insect survival and reproduction across different life stages, and the potential and timescales of recovery

Adaptive thermal plasticity enhances sperm and egg performance in a model insect

Rising and more variable global temperatures pose a challenge for biodiversity, with reproduction and fertility being especially sensitive to heat. Here, we assessed the potential for thermal adaptation in sperm and egg function using Tribolium flour beetles, a warm-temperate-tropical insect model. Following temperature increases through adult development, we found opposing gamete responses, with males producing shorter sperm and females laying larger eggs. Importantly, this gamete phenotypic plasticity was adaptive: thermal translocation experiments showed that both sperm and eggs produced in warmer conditions had superior reproductive performance in warmer environments, and vice versa for cooler production conditions and reproductive environments. In warmer environments, gamete plasticity enabled males to double their reproductive success, and females could increase offspring production by one-third. Our results reveal exciting potential for sensitive but vital traits within reproduction to handle increasing and more variable thermal regimes in the natural environment

Age‐specific sensitivity of sperm length and testes size to developmental temperature in the bruchid beetle

In an era of global warming, the negative effects of temperature stress on fertility could intensify predicted losses of biodiversity. Male fertility is particularly sensitive to temperature stress, yet we have an incomplete understanding of when, during reproductive ontogeny, spermatogenesis is most affected. Here, we used a temperature‐switch protocol to identify when during development temperature affects the expression of sperm length and testes size in the bruchid beetle Callosobruchus maculatus. Egg‐to‐adult development took place at 17°C, 27°C or 33°C for either the full duration of pre‐imago development or it was switched at different stages during development. Full development at 17°C or 33°C resulted in significantly shorter sperm than at 27°C. However, when developing larvae were switched to higher or lower temperatures, we observed switch‐specific phenotypic expression of sperm length and testes size. Our key finding was that sperm length was sensitive to high‐temperature stress during the early stages of ontogeny and to low‐temperature stress during the latter stages of ontogeny. Such age‐specific developmental sensitivity suggests that infertility resulting from transient heat stress (i.e. heatwaves) could be mitigated by age‐related developmental heterogeneity within populations. Those individuals able to avoid severe effects of heat stress on fertility, through serendipitously being at less‐sensitive stages of development, could potentially compensate for the loss of fertility experienced by those individuals at more sensitive stages of development

Polyandry provides reproductive and genetic benefits in colonising populations

Polyandry, when females mate with more than one male, is theorised to play an important role in successful colonisation of new habitats. In addition to possible benefits from sexual selection, even mild polyandry could facilitate colonisation by protecting against inbreeding and reducing the costs of mating with incompatible or infertile males. Here, we measure the importance of mild polyandry for population viability and reproductive fitness following experimental founder events into a higher‐temperature regime. Using colonisation experiments with the model beetle Tribolium castaneum, in which females can produce offspring for up to 140 days following a single mating, we founded more than 100 replicate populations using single females that had been given the opportunity to mate with either one or two males and then tracked their subsequent population dynamics. Following population viability and fitness across 10 generations, we found that extinction rates were significantly lower in populations founded by females given polyandrous opportunities to mate with two males (9%) compared to populations founded by monogamous females (34%). In addition, populations founded by females that had been provided with opportunities to store sperm from two different males showed double the median productivity following colonisation compared to monogamous‐founded populations. Notably, we identified short‐term and longer‐term benefits to post‐colonisation populations from double‐mating, with results suggesting that polyandry acts to both protect against mating with incompatible males through the founder event, and reduce inbreeding depression as the colonisation proceeds for 10 generations. Our results therefore show that even mild polyandry provides both reproductive and genetic benefits for colonising populations

Experimental evolution reveals that sperm competition intensity selects for longer, more costly sperm

It is the differences between sperm and eggs that fundamentally underpin the differences between the sexes within reproduction. For males, it is theorized that widespread sperm competition leads to selection for investment in sperm numbers, achieved by minimizing sperm size within limited resources for spermatogenesis in the testis. Here, we empirically examine how sperm competition shapes sperm size, after more than 77 generations of experimental selection of replicate lines under either high or low sperm competition intensities in the promiscuous flour beetle Tribolium castaneum. After this experimental evolution, populations had diverged significantly in their sperm competitiveness, with sperm in ejaculates from males evolving under high sperm competition intensities gaining 20% greater paternity than sperm in ejaculates from males that had evolved under low sperm competition intensity. Males did not change their relative investment into sperm production following this experimental evolution, showing no difference in testis sizes between high and low intensity regimes. However, the more competitive males from high sperm competition intensity regimes had evolved significantly longer sperm and, across six independently selected lines, there was a significant association between the degree of divergence in sperm length and average sperm competitiveness. To determine whether such sperm elongation is costly, we used dietary restriction experiments, and revealed that protein-restricted males produced significantly shorter sperm. Our findings therefore demonstrate that sperm competition intensity can exert positive directional selection on sperm size, despite this being a costly reproductive trait

Facultative polyandry protects females from compromised male fertility caused by heatwave conditions

Why is polyandry such a common mating behaviour when it exposes females to a range of significant fitness costs? Here, we investigated whether polyandry protects females against reduced male fertility caused by thermal stress from heatwave conditions. Sperm production and function are vulnerable to heat, and heatwave conditions are forecast to increase as our climate warms, so we examined these effects on female reproduction and mating behaviour in the flour beetle, Tribolium castaneum, a promiscuous ectotherm model in which fertility is damaged by environmental warming. We tested whether polyandrous matings, or polyandrous sperm stores, protect females against reduced male fertility caused by heatwave conditions, and whether females flexibly adjust their remating behaviour to enable fertility rescue. We found that polyandry protected females against reduced male fertility: monogamous matings with males exposed to heatwave conditions halved female offspring production, but opportunities to mate with five of these males allowed normal female reproductive output. By contrast with this fertility improvement following polyandrous mating opportunities, there was no protective benefit for females already carrying sperm stores from multiple males, which suffered similar heatwave damage within the female tract as monogamous sperm stores. Importantly, female polyandry was flexible to male condition, with females showing greater motivation to remate with new males if their previous mate had been damaged by heatwave exposure, enabling a rapid reproductive rescue. Our results reveal that flexible polyandry enables females to rescue their fertility when male reproductive function is compromised by heatwave conditions, a phenomenon that may become more prevalent under climate change

Experimental evolution with an insect model reveals that male homosexual behaviour occurs due to inaccurate mate choice

The existence of widespread male same-sex sexual behaviour (SSB) is puzzling: why does evolution allow costly homosexual activity to exist, when reproductive fitness is primarily achieved through heterosexual matings? Here, we used experimental evolution to understand why SSB occurs in the flour beetle Tribolium castaneum. By varying the adult operational sex ratio across 82–106 generations, we created divergent evolutionary regimes that selected for or against SSB depending upon its function. Male-biased (90:10 M:F) regimes generated strong selection on males from intrasexual competition, and demanded improved ability to locate and identify female mates. By contrast, Female-biased regimes (10:90 M:F) generated weak male–male competition, and relaxed selection on mate-searching abilities in males. If male SSB functions through sexually selected male–male competition, it should be more evident within Male-biased regimes, where reproductive competition is nine times greater, than in the Female-biased regimes. By contrast, if SSB exists due to inaccurate mate choice, it should be reduced in Male-biased regimes, where males experience stronger selection for improved mate finding and discrimination abilities than in the Female-biased regime, where most potential mating targets are female. Following these divergent evolutionary regimes, we measured male engagement in SSB through choice experiments simultaneously presenting female and male mating targets. Males from both regimes showed similar overall levels of mating activity. However, there were significant differences in levels of SSB between the two regimes: males that evolved through male-biased operational sex ratios located, mounted and mated more frequently with the female targets. By contrast, males from female-biased selection histories mated less frequently with females, exhibiting almost random choice between male and female targets in their first mating attempt. Following experimental evolution, we therefore conclude that SSB does not function through sexually selected male–male competition, but instead occurs because males fail to perfectly discriminate females as mates