Epigenetic inheritance and genome regulation: is DNA methylation linked to ploidy in haplodiploid insects?

Organisms show great variation in ploidy level. For example, chromosome copy number varies among cells, individuals and species. One particularly widespread example of ploidy variation is found in haplodiploid taxa, wherein males are typically haploid and females are typically diploid. Despite the prevalence of haplodiploidy, the regulatory consequences of having separate haploid and diploid genomes are poorly understood. In particular, it remains unknown whether epigenetic mechanisms contribute to regulatory compensation for genome dosage. To gain greater insights into the importance of epigenetic information to ploidy compensation, we examined DNA methylation differences among diploid queen, diploid worker, haploid male and diploid male Solenopsis invicta fire ants. Surprisingly, we found that morphologically dissimilar diploid males, queens and workers were more similar to one another in terms of DNA methylation than were morphologically similar haploid and diploid males. Moreover, methylation level was positively associated with gene expression for genes that were differentially methylated in haploid and diploid castes. These data demonstrate that intragenic DNA methylation levels differ among individuals of distinct ploidy and are positively associated with levels of gene expression. Thus, these results suggest that epigenetic information may be linked to ploidy compensation in haplodiploid insects. Overall, this study suggests that epigenetic mechanisms may be important to maintaining appropriate patterns of gene regulation in biological systems that differ in genome copy number.     

One simple,rapid and economical method for ploidy detection of Trichogramma dendrolimi Matsumura (Hymenoptera Trichogrammatidae)

Ploidy diversity provides valuable scientific information, thus making the detection technique of ploidy important. However, traditional methods of cytological observation and flow cytometry are either laborious or expensive. We here report a simple and rapid, effective and economical quantitative PCR (qPCR) approach to determine the ploidy of a parasitoid species Trichogramma dendrolimi Matsumura, an economically important biocontrol agent. We applied a mitochondrial gene cytochrome oxidase (COI) and a nuclear gene forkhead to evaluate the mitochondrial number per nuclear copy in a thelytokous Wolbachia-infected strain of T. dendrolimi and its bisexual uninfected counterparts. The 2^−ΔCq values calculated from C_q values which resulted from qPCR experiments were significantly larger in haploid males than that in diploid females. Haploid males possessed about 2.69 times mitochondrial number per nuclear copy as diploid females. Not a single significant difference was found between diploid females from thelytokous and bisexual strains. Based on the differences in relative mitochondrial content, we were allowed to distinguish between haploid males and diploid females. Moreover, the number of mitochondria significantly decreased with higher ploidy level but was not affected by Wolbachia-infection. Our study supplied an available tool to investigate the ploidy diversity in sex determination of T. dendrolimi and thelytokous manipulation of Wolbachia, which is the crucial step to further study their underlying mechanisms. This will in turn contribute to the biocontrol efficiency by enhancing the female production and hence the parasitism rate.     

Effects of ploidy and sex-locus genotype on gene expression patterns in the fire ant Solenopsis invicta

Males in many animal species differ greatly from females in morphology, physiology and behaviour. Ants, bees and wasps have a haplodiploid mechanism of sex determination whereby unfertilized eggs become males while fertilized eggs become females. However, many species also have a low frequency of diploid males, which are thought to develop from diploid eggs when individuals are homozygous at one or more sex determination loci. Diploid males are morphologically similar to haploids, though often larger and typically sterile. To determine how ploidy level and sex-locus genotype affect gene expression during development, we compared expression patterns between diploid males, haploid males and females (queens) at three developmental timepoints in Solenopsis invicta. In pupae, gene expression profiles of diploid males were very different from those of haploid males but nearly identical to those of queens. An unexpected shift in expression patterns emerged soon after adult eclosion, with diploid male patterns diverging from those of queens to resemble those of haploid males, a pattern retained in older adults. The finding that ploidy level effects on early gene expression override sex effects (including genes implicated in sperm production and pheromone production/perception) may explain diploid male sterility and lack of worker discrimination against them during development.     

A theory of natural selection incorporating interaction among individuals. IX. Use of groups consisting of a mating pair and sterile diploid caste members

This paper and the next member of the series, deal with genetical mechanisms responsible for the evolution of eusociality (a level of social organization that includes differentiated sterile castes) among the “social” insects. Eusociality has evolved in a number of different species. Two different types of genetic systems are represented among these species: diplodiploidy (both sexes diploid) and haplodiploidy (haploid males and diploid females). The present paper examines the evolution of a sterile caste system in the context of diplodiploidy, and the next paper considers the evolution of eusociality in the context of haplodiploidy.The present study demonstrates that if the sterile diploid caste members are related to the reproductive members of the group, eusociality can evolve. This is true because the caste associate gene effects are included in the function determining gene frequency change (i.e. Δp_i). Also, since the caste gene effects are expressed only through the associate dimension of gene activity, they can cause morphological and behavioral adaptations to occur which are peculiar to the caste members, and need not be expressed in the reproducing members of the group. Thus caste differentiation is possible.     

Wolbachia in the invasive European paper wasp Polistes dominulus

The European paper wasp Polistes dominulus has been expanding its North American range since its introduction in the 1970s. We screened P. dominulus from Italy and the northeastern U.S. for the presence of the intracellular reproductive symbiont Wolbachia. Infection rates among females varied from 16% to 87% among U.S. sites and from 33% to 71% in Italy. We also found infected haploid and diploid males, indicating that this is not a male-killing Wolbachia infection. Our data show that infected individuals from New York, Massachusetts, and Italy carry the same Wolbachia strain, and that some mtDNA haplotypes include both infected and uninfected individuals. We discuss possible implications of Wolbachia infection in this invasive social hymenopteran. Received 28 October 2005; revised 25 January 2006; accepted 17 February 2006.     

A theory of natural selection incorporating interaction among individuate. X. Use of groups consisting of a mating pair together with haploid and diploid caste members

This paper and the previous member of the series, deal with genetical mechanisms responsible for the evolution of eusociality (a level of social organization that includes differentiated sterile castes) among the “social” insects. Eusociality has evolved in a number of different species. Two different types of genetic systems are represented among these species: diplodiploidy (both sexes diploid) and haplodiploidy (haploid males and diploid females). The previous paper examined the evolution of a sterile caste system in the context of diplodiploidy, and the present paper considers the evolution of eusociality in the context of haplodiploidy.The present study demonstrates that selection operating with regard to random groups within the haplodiploid inheritance system cannot result in the evolution of a sterile caste system. Thus haplodiploidy, in itself, is not sufficient for the evolution of eusociality. However, if the sterile caste members are related to the reproductive members of the group, the appropriate caste associate gene effects are included in the function determining gene frequency change (i.e. Δp_i), and therefore, eusociality can evolve. This is true for both haploid and diploid castes.In comparing the two modes of inheritance, it is demonstrated that haplodiploidy provides up to 37·5% increased selection efficiency relative to diplodiploidy in evolving a social caste system in the absence of inbreeding.     

Reproductivity of early males of the temperate paper wasp Polistes rothneyi iwatai

In Polistes paper wasps, haploid early males can mate with early emerging females and leave viable offspring. In contrast, diploid early males are eventually sterile because they contribute triploid offspring via diploid sperm. Clarifying the ploidy of early males is important for determining whether early male production is a reproductive strategy for the species. We examined the mating behavior and the ploidy of early males in the Japanese paper wasp, Polistes rothneyi iwatai van der Vecht. Thirteen early males from four colonies were all diploid. Two of the nine early males (22.2%) attempted to mate with females, but only one individual (11.1%) was successful (the female's spermatheca contained spermatozoa). These results suggest that although most early males of P. rothneyi iwatai do not produce offspring, their mating may be linked to the occasional production of triploid females.     

No Need to Discriminate? Reproductive Diploid Males in a Parasitoid with Complementary Sex Determination

Diploid males in hymenopterans are generally either inviable or sterile, thus imposing a severe genetic load on populations. In species with the widespread single locus complementary sex determination (sl-CSD), sex depends on the genotype at one single locus with multiple alleles. Haploid (hemizygous) individuals are always males. Diploid individuals develop into females when heterozygous and into males when homozygous at the sex determining locus. Our comparison of the mating and reproductive success of haploid and diploid males revealed that diploid males of the braconid parasitoid Cotesia glomerata sire viable and fertile diploid daughters. Females mated to diploid males, however, produced fewer daughters than females mated to haploid males. Nevertheless, females did not discriminate against diploid males as mating partners. Diploid males initiated courtship display sooner than haploid males and were larger in body size. Although in most species so far examined diploid males were recognized as genetic dead ends, we present a second example of a species with sl-CSD and commonly occurring functionally reproductive diploid males. Our study suggests that functionally reproductive diploid males might not be as rare as hitherto assumed. We argue that the frequent occurrence of inbreeding in combination with imperfect behavioural adaptations towards its avoidance promote the evolution of diploid male fertility.     

Evolution and maintenance of haploid–diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus

The evolutionary stability of haploid–diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long‐standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year‐round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid–diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.     

Diploid males and their triploid offspring in the paper wasp Polistes dominulus

Although the hymenopteran sex-determining mechanism generally results in haploid males and diploid females, diploid males can be produced via homozygosity at the sex-determining locus. Diploid males have low fitness because they are effectively sterile or produce presumably sterile triploid offspring. Previously, triploid females were observed in three species of North American Polistes paper wasps, and this was interpreted as indirect evidence of diploid males. Here we report what is, to our knowledge, the first direct evidence: four of five early male-producing Polistes dominulus nests from three populations contained diploid males. Because haploid males were also found, however, the adaptive value of early males cannot be ignored. Using genetic and morphological data from triploid females, we also present evidence that both diploid males and triploid females remain undetected throughout the colony cycle. Consequently, diploid male production may result in a delayed fitness cost for two generations. This phenomenon is particularly relevant for introduced populations with few alleles at the sex-determining locus, but cannot be ignored in native populations without supporting genetic data. Future research using paper wasp populations to test theories of social evolution should explicitly consider the potential impacts of diploid males.     

The role of male disease susceptibility in the evolution of haplodiploid insect societies

Heterozygosity at loci affecting resistance against parasites can benefit host fitness. We predict that, in haplodiploid species, haploid males will suffer decreased parasite resistance relative to diploid females. We suggest that elevated susceptibility in haploid males has shaped the evolution of social behaviour in haplodiploid species. Male susceptibility will select for behavioural adaptations that limit males' exposure to pathogens and that limit male transmission of pathogens within and between colonies. The relatedness-asymmetry hypothesis that has been advanced to explain female-only workers does not make these predictions. We review the relevant evidence for genetic effects on parasite resistance in insects and summarize empirical evidence that relates to the haploid-susceptibility hypothesis.     

Life‐history traits of the Whiting polyploid line of the parasitoid Nasonia vitripennis

In hymenopterans, males are normally haploid (1n) and females diploid (2n), but individuals with divergent ploidy levels are frequently found. In species with ‘complementary sex determination’ (CSD), increasing numbers of diploid males that are often infertile or unviable arise from inbreeding, presenting a major impediment to biocontrol breeding. Non‐CSD species, which are common in some parasitoid wasp taxa, do not produce polyploids through inbreeding. Nevertheless, polyploidy also occurs in non‐CSD Hymenoptera. As a first survey on the impacts of inbreeding and polyploidy of non‐CSD species, we investigate life‐history traits of a long‐term laboratory line of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (‘Whiting polyploid line’) in which polyploids of both sexes (diploid males, triploid females) are viable and fertile. Diploid males produce diploid sperm and virgin triploid females produce haploid and diploid eggs. We found that diploid males did not differ from haploid males with respect to body size, progeny size, mate competition, or lifespan. When diploid males were mated to many females (without accounting for mating order), the females produced a relatively high proportion of male offspring, possibly indicating that these males produce less sperm and/or have reduced sperm functionality. In triploid females, parasitization rate and fecundity were reduced and body size was slightly increased, but there was no effect on lifespan. After one generation of outbreeding, lifespan as well as parasitization rate were increased, and a body size difference was no longer apparent. This suggests that outbreeding has an effect on traits observed in an inbred polyploidy background. Overall, these results indicate some phenotypic detriments of non‐CSD polyploids that must be taken into account in breeding.     

Nucleolus size varies with sex,ploidy and gene dosage in insects

The nucleolus constitutes a cytologically visible phenotype for ribosomal DNA (rDNA). Nucleolar size, as determined by silver staining, is a good indicator of cell proliferation rate and biosynthetic activity. Nevertheless, the relationship between rDNA content and sexual dimorphism for nucleolar size is not well documented. In the present study, the impact of sex and ploidy level on nucleolar size is investigated in three haplo/diploid and three diplo/diploid species of insect. Nucleolar sizes are found to be proportional to ploidy level in the haplo/diploid hymenopterans Trypoxylon albitarse and Nasonia vitripennis. Conversely, in the ant Messor barbarus, nucleolar sizes are larger in haploid males (winged) than diploid females (apterous). Among the diplo/diploid species, evidence for gene dosage compensation on nucleolar activity is suggested by the absence of sex differences in Drosophila simulans, a species in which rDNA is limited to the X chromosome. By contrast, in the grasshopper Stenobothrus festivus, another species with rRNA genes restricted to the X chromosome, the size of the nucleolus is significantly larger in females than in males. Additionally, in the grasshopper Chorthippus parallelus, where rDNA is distributed evenly on several autosomes of males and females, the females also show larger nucleoli than males. In both grasshopper species, the magnitude of the female/male ratio for nucleolus area is very similar to the body size ratio, suggesting that body size, as well as sex, ploidy, gene dosage and physiological activity, may be an important determinant of nucleolus area.     

Complementary Sex Determination in the Parasitic Wasp Diachasmimorpha longicaudata

We studied the sex determination in Diachasmimorpha longicaudata, a parasitoid braconid wasp widely used as biological control agent of fruit pest tephritid flies. We tested the complementary sex determination hypothesis (CSD) known in at least 60 species of Hymenoptera. According to CSD, male or female development depends on the allelic composition of one sex locus (single-locus CSD) or multiple sex loci (multiple-locus CSD). Hemizygote individuals are normal haploid males, and heterozygotes for at least one sex locus are normal diploid females, but homozygotes for all the sex loci are diploid males. In order to force the occurrence of diploid males in D. longicaudata, we established highly inbred lines and examined their offspring using chromosome counting, flow cytometry, and sex ratio analysis. We found that when mother-son crosses were studied, this wasp produced about 20% of diploid males out of the total male progeny. Our results suggest that this parasitoid may represent the second genus with multiple-locus CSD in Hymenoptera. Knowledge about the sex determination system in D. longicaudata is relevant for the improvement of mass rearing protocols of this species. This information also provides the necessary background for further investigations on the underlying molecular mechanisms of sex determination in this species, and a better insight into the evolution of this pathway in Hymenoptera in particular and insects in general.     

Further Studies on the Role of Polyploidy in the Evolution of Meloidogyne

Two tetraploid isolates of Meloidogyne hapla, 86P and E289P, with haploid chromosome numbers of 34 and 28, respectively, were studied cytogenetically and biologically in relation to the diploid populations, 86-Va (n = 17) and E289-Taiwan (n = 14), from which they had been originally isolated. Both isolates were quite stable, converting to diploidy at the low rate of about 2.5%. The tetraploid isolate 86P maintained itself in competition with its diploid counterpart in mixed cultures, although an initial frequency of 50% polyploidy was reduced to about 9% at the end of the sixth generation. Both tetraploid isolates could maintain themselves in greenhouse cultures without artificial selection for at least 2 years. Crosses between diploid females and tetraploid males resulted in a few triploid females that produced mostly nonviable eggs, suggesting partial reproductive isolation between the two ploidy forms. Ten generations of propagation of only polyploid females of isolate 86P that were associated with males failed to yield an obligatorily amphimictic isolate that would not convert at all to diploidy. If one accepts a previous assumption that the present day amphimictic root-knot nematodes are tetraploids derived from diploid ancestors, results of the present study are not inconsistent with an evolutionary trend toward an even higher level of ploidy in Meloidogyne, presumably octaploidy.     

Complementary sex determination in the genus Diadegma (Hymenoptera: Ichneumonidae)

In the evolution of sexual reproduction we would expect to see a close association between mating systems and sex determination mechanisms. Such associations are especially evident in the insect order Hymenoptera which shows great diversity with respect to both of these characteristics. The ancestral sex determination mechanism in this order is thought to be single‐locus complementary sex determination (sl‐CSD), which is inbreeding sensitive, and where inbreeding results in the production of sterile diploid males rather than daughters. Presently, however, there is insufficient data to give strong support to the hypothesis that sl‐CSD is truly the ancestral condition in the Hymenoptera, principally because of the difficulty of reliably determining the degree of male ploidy. Here we show that six ichneumonid parasitoids from the polyphyletic genus Diadegma are subject to sl‐CSD, using neuronal cell DNA flow cytometry to distinguish ploidy levels. The presence of sl‐CSD in these six species, together with earlier evidence from the authors for D. chrysostictos, provides considerable support for the notion that sl‐CSD was ancestral in the Aculeata/Ichneumonoidea clade, which contains all eusocial Hymenoptera. Moreover, because flow cytometry discriminates reliably between haploid and diploid males, and is independent of the maternal sex allocation or the need for genetic markers, it has considerable potential for the determination of ploidy more generally.     

Hybrid incompatibilities in the parasitic wasp genus Nasonia: negative effects of hemizygosity and the identification of transmission ratio distortion loci

The occurrence of hybrid incompatibilities forms an important stage during the evolution of reproductive isolation. In early stages of speciation, males and females often respond differently to hybridization. Haldane's rule states that the heterogametic sex suffers more from hybridization than the homogametic sex. Although haplodiploid reproduction (haploid males, diploid females) does not involve sex chromosomes, sex-specific incompatibilities are predicted to be prevalent in haplodiploid species. Here, we evaluate the effect of sex/ploidy level on hybrid incompatibilities and locate genomic regions that cause increased mortality rates in hybrid males of the haplodiploid wasps Nasonia vitripennis and Nasonia longicornis. Our data show that diploid F(1) hybrid females suffer less from hybridization than haploid F(2) hybrid males. The latter not only suffer from an increased mortality rate, but also from behavioural and spermatogenic sterility. Genetic mapping in recombinant F(2) male hybrids revealed that the observed hybrid mortality is most likely due to a disruption of cytonuclear interactions. As these sex-specific hybrid incompatibilities follow predictions based on Haldane's rule, our data accentuate the need to broaden the view of Haldane's rule to include species with haplodiploid sex determination, consistent with Haldane's original definition.     

Sex-Specific Differences in Pathogen Susceptibility in Honey Bees (Apis mellifera)

Sex-related differences in susceptibility to pathogens are a common phenomenon in animals. In the eusocial Hymenoptera the two female castes, workers and queens, are diploid and males are haploid. The haploid susceptibility hypothesis predicts that haploid males are more susceptible to pathogen infections compared to females. Here we test this hypothesis using adult male (drone) and female (worker) honey bees (Apis mellifera), inoculated with the gut endoparasite Nosema ceranae and/or black queen cell virus (BQCV). These pathogens were chosen due to previously reported synergistic interactions between Nosema apis and BQCV. Our data do not support synergistic interactions between N. ceranae and BQCV and also suggest that BQCV has limited effect on both drone and worker health, regardless of the infection level. However, the data clearly show that, despite lower levels of N. ceranae spores in drones than in workers, Nosema-infected drones had both a higher mortality and a lower body mass than non-infected drones, across all treatment groups, while the mortality and body mass of worker bees were largely unaffected by N. ceranae infection, suggesting that drones are more susceptible to this pathogen than workers. In conclusion, the data reveal considerable sex-specific differences in pathogen susceptibility in honey bees and highlight the importance of ultimate measures for determining susceptibility, such as mortality and body quality, rather than mere infection levels.