Female fruit flies cannot protect stored sperm from high temperature damage

Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. Here, we test whether females from two fruit fly species can protect stored sperm from a high temperature stress. We find that sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is destroying stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to destroy mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change.

Plastic responses of survival and fertility following heat stress in pupal and adult Drosophila virilis

The impact of rising global temperatures on survival and reproduction is putting many species at risk of extinction. In particular, it has recently been shown that thermal effects on reproduction, especially limits to male fertility, can underpin species distributions in insects. However, the physiological factors influencing fertility at high temperatures are poorly understood. Key factors that affect somatic thermal tolerance such as hardening, the ability to phenotypically increase thermal tolerance after a mild heat shock, and the differential impact of temperature on different life stages, are largely unexplored for thermal fertility tolerance. Here, we examine the impact of high temperatures on male fertility in the cosmopolitan fruit fly Drosophila virilis. We first determined whether temperature stress at either the pupal or adult life-history stage impacts fertility. We then tested the capacity for heat-hardening to mitigate heat-induced sterility. We found that thermal stress reduces fertility in different ways in pupae and adults. Pupal heat stress delays sexual maturity, whereas males heated as adults can reproduce initially following heat stress, but lose the ability to produce offspring. We also found evidence that while heat-hardening in D. virilis can improve high temperature survival, there is no significant protective impact of this same hardening treatment on fertility. These results suggest that males may be unable to prevent the costs of high temperature stress on fertility through heat-hardening which limits a species’ ability to quickly and effectively reduce fertility loss in the face of short-term high temperature events.

Multiple recent sex chromosome fusions in Drosophila virilis associated with elevated satellite DNA abundance

Repetitive satellite DNA is highly variable both within and between species, and is often located near centromeres. However, the abundance or array length of satellite DNA may be constrained or have maximum limits. Drosophila virilis contains among the highest relative satellite abundances, with almost half its genome composed of three related 7 bp satellites. We discovered a strain of D. virilis that has 15% more pericentromeric satellite DNA compared to other strains, and also underwent two independent centromere-to-centromere sex chromosome fusion events. These fusions are presumably caused by DNA breakage near the pericentromeric satellites followed by repair using similar repetitive regions of nonhomologous chromosomes. We hypothesized that excess satellite DNA might increase the risk of DNA breaks and genome instability when stressed, which would be consistent with the apparent high rate of fusions we found in this strain. To directly quantify DNA breakage levels between strains with different satellite DNA abundances, we performed the comet assay after feeding flies gemcitabine and administering low-dose gamma radiation. We found a positive correlation between the rate of DNA breakage and satellite DNA abundance. This was further supported by a significant decrease in DNA breakage in an otherwise genetically identical substrain that lost the chromosome fusion and several megabases of satellite DNA. We find that the centromere-to-centromere fusions resulted in up to a 21% nondisjunction rate between the X and Y chromosomes in males, adding a fitness cost. Finally, we propose a model consistent with our data that implicates genome instability as a critical evolutionary constraint to satellite abundance.

Gametic specialization of centromeric histone paralogs in Drosophila virilis

In most eukaryotes, centromeric histone (CenH3) proteins mediate mitosis and meiosis and ensure epigenetic inheritance of centromere identity. We hypothesized that disparate chromatin environments in soma versus germline might impose divergent functional requirements on single CenH3 genes, which could be ameliorated by gene duplications and subsequent specialization. Here, we analyzed the cytological localization of two recently identified CenH3 paralogs, Cid1 and Cid5, in Drosophila virilis using specific antibodies and epitope-tagged transgenic strains. We find that only ancestral Cid1 is present in somatic cells, whereas both Cid1 and Cid5 are expressed in testes and ovaries. However, Cid1 is lost in male meiosis but retained throughout oogenesis, whereas Cid5 is lost during female meiosis but retained in mature sperm. Following fertilization, only Cid1 is detectable in the early embryo, suggesting that maternally deposited Cid1 is rapidly loaded onto paternal centromeres during the protamine-to-histone transition. Our studies reveal mutually exclusive gametic specialization of divergent CenH3 paralogs. Duplication and divergence might allow essential centromeric genes to resolve an intralocus conflict between maternal and paternal centromeric requirements in many animal species.

Evolutionary Dynamics of the Pericentromeric Heterochromatin in Drosophila virilis and Related Species

Pericentromeric heterochromatin in Drosophila generally consists of repetitive DNA, forming the environment associated with gene silencing. Despite the expanding knowledge of the impact of transposable elements (TEs) on the host genome, little is known about the evolution of pericentromeric heterochromatin, its structural composition, and age. During the evolution of the Drosophilidae, hundreds of genes have become embedded within pericentromeric regions yet retained activity. We investigated a pericentromeric heterochromatin fragment found in D. virilis and related species, describing the evolution of genes in this region and the age of TE invasion. Regardless of the heterochromatic environment, the amino acid composition of the genes is under purifying selection. However, the selective pressure affects parts of genes in varying degrees, resulting in expansion of gene introns due to TEs invasion. According to the divergence of TEs, the pericentromeric heterochromatin of the species of virilis group began to form more than 20 million years ago by invasions of retroelements, miniature inverted repeat transposable elements (MITEs), and Helitrons. Importantly, invasions into the heterochromatin continue to occur by TEs that fall under the scope of piRNA silencing. Thus, the pericentromeric heterochromatin, in spite of its ability to induce silencing, has the means for being dynamic, incorporating the regions of active transcription.

The effects of the sex chromosomes on the inheritance of species-specific traits of the copulatory organ shape in Drosophila virilis and Drosophila lummei

The shape of the male genitalia in many taxa is the most rapidly evolving morphological structure, often driving reproductive isolation, and is therefore widely used in systematics as a key character to distinguish between sibling species. However, only a few studies have used the genital arch of the male copulatory organ as a model to study the genetic basis of species-specific differences in the Drosophila copulatory system. Moreover, almost nothing is known about the effects of the sex chromosomes on the shape of the male mating organ. In our study, we used a set of crosses between D. virilis and D. lummei and applied the methods of quantitative genetics to assess the variability of the shape of the male copulatory organ and the effects of the sex chromosomes and autosomes on its variance. Our results showed that the male genital shape depends on the species composition of the sex chromosomes and autosomes. Epistatic interactions of the sex chromosomes with autosomes and the species origin of the Y-chromosome in a male in interspecific crosses also influenced the expression of species-specific traits in the shape of the male copulatory system. Overall, the effects of sex chromosomes were comparable to the effects of autosomes despite the great differences in gene numbers between them. It may be reasonably considered that sexual selection for specific genes associated with the shape of the male mating organ prevents the demasculinization of the X chromosome.