Evidence for involvement of a transformer paralogue in sex determination of the wasp Leptopilina clavipes.

Transformer (tra) is the central gear in many insect sex determination pathways and transduces a wide range of primary signals. Mediated by transformer-2 (tra2) it directs sexual development into the female or male mode. Duplications of tra have been detected in numerous Hymenoptera, but a function in sex determination has been confirmed only in Apis mellifera. We identified a tra2 orthologue (Lc-tra2), a tra orthologue (Lc-tra) and a tra paralogue (Lc-traB) in the genome of Leptopilina clavipes (Hymenoptera: Cynipidae). We compared the sequence and structural conservation of these genes between sexual (arrhenotokous) and asexual all-female producing (thelytokous) individuals. Lc-tra is sex-specifically spliced in adults consistent with its orthologous function. The male-specific regions of Lc-tra are conserved in both reproductive modes. The paralogue Lc-traB lacks the genomic region coding for male-specific exons and can only be translated into a full-length TRA-like peptide sequence. Furthermore, unlike LC-TRA, the LC-TRAB interstrain sequence variation is not differentiated into a sexual and an asexual haplotype. The LC-TRAB protein interacts with LC-TRA as well as LC-TRA2. This suggests that Lc-traB functions as a conserved element in sex determination of sexual and asexual individuals.

Maternal provision of non-sex-specific transformer messenger RNA in sex determination of the wasp Asobara tabida.

In many insect species maternal provision of sex-specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer-2 (tra2) orthologues in As. tabida. Of these, At-dsx and At-tra are sex-specifically spliced, indicating a conserved function in sex determination. At-tra and At-tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non-sex-specific splice form of At-tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex-specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms.

Genetics of decayed sexual traits in a parasitoid wasp with endosymbiont-induced asexuality.

Trait decay may occur when selective pressures shift, owing to changes in environment or life style, rendering formerly adaptive traits non-functional or even maladaptive. It remains largely unknown if such decay would stem from multiple mutations with small effects or rather involve few loci with major phenotypic effects. Here, we investigate the decay of female sexual traits, and the genetic causes thereof, in a transition from haplodiploid sexual reproduction to endosymbiont-induced asexual reproduction in the parasitoid wasp Asobara japonica. We take advantage of the fact that asexual females cured of their endosymbionts produce sons instead of daughters, and that these sons can be crossed with sexual females. By combining behavioral experiments with crosses designed to introgress alleles from the asexual into the sexual genome, we found that sexual attractiveness, mating, egg fertilization and plastic adjustment of offspring sex ratio (in response to variation in local mate competition) are decayed in asexual A. japonica females. Furthermore, introgression experiments revealed that the propensity for cured asexual females to produce only sons (because of decayed sexual attractiveness, mating behavior and/or egg fertilization) is likely caused by recessive genetic effects at a single locus. Recessive effects were also found to cause decay of plastic sex-ratio adjustment under variable levels of local mate competition. Our results suggest that few recessive mutations drive decay of female sexual traits, at least in asexual species deriving from haplodiploid sexual ancestors.

Phylogenetic distribution and evolutionary dynamics of the sex determination genes doublesex and transformer in insects.

Sex determination in insects is characterized by a gene cascade that is conserved at the bottom but contains diverse primary signals at the top. The bottom master switch gene doublesex is found in all insects. Its upstream regulator transformer is present in the orders Hymenoptera, Coleoptera and Diptera, but has thus far not been found in Lepidoptera and in the basal lineages of Diptera. transformer is presumed to be ancestral to the holometabolous insects based on its shared domains and conserved features of autoregulation and sex-specific splicing. We interpret that its absence in basal lineages of Diptera and its order-specific conserved domains indicate multiple independent losses or recruitments into the sex determination cascade. Duplications of transformer are found in derived families within the Hymenoptera, characterized by their complementary sex determination mechanism. As duplications are not found in any other insect order, they appear linked to the haplodiploid reproduction of the Hymenoptera. Further phylogenetic analyses combined with functional studies are needed to understand the evolutionary history of the transformer gene among insects.

Manipulation of arthropod sex determination by endosymbionts: diversity and molecular mechanisms.

Arthropods exhibit a large variety of sex determination systems both at the chromosomal and molecular level. Male heterogamety, female heterogamety, and haplodiploidy occur frequently, but partially different genes are involved. Endosymbionts, such as Wolbachia, Cardinium,Rickettsia, and Spiroplasma, can manipulate host reproduction and sex determination. Four major reproductive manipulation types are distinguished: cytoplasmic incompatibility, thelytokous parthenogenesis, male killing, and feminization. In this review, the effects of these manipulation types and how they interfere with arthropod sex determination in terms of host developmental timing, alteration of sex determination, and modification of sexual differentiation pathways are summarized. Transitions between different manipulation types occur frequently which suggests that they are based on similar molecular processes. It is also discussed how mechanisms of reproductive manipulation and host sex determination can be informative on each other, with a special focus on haplodiploidy. Future directions on how the study of endosymbiotic manipulation of host reproduction can be key to further studies of arthropod sex determination are shown.

Cuticular hydrocarbon divergence in the jewel wasp Nasonia: evolutionary shifts in chemical communication channels?

The evolution and maintenance of intraspecific communication channels constitute a key feature of chemical signalling and sexual communication. However, how divergent chemical communication channels evolve while maintaining their integrity for both sender and receiver is poorly understood. In this study, we compare male and female cuticular hydrocarbon (CHC) profiles in the jewel wasp genus Nasonia, analyse their chemical divergence and investigate their role as species-specific sexual signalling cues. Males and females of all four Nasonia species showed unique, nonoverlapping CHC profiles unambiguously separating them. Surprisingly, male and female phylogenies based on the chemical distances between their CHC profiles differed dramatically, where only male CHC divergence parallels the molecular phylogeny of Nasonia. In particular, N. giraulti female CHC profiles were the most divergent from all other species and very different from its most closely related sibling species N. oneida. Furthermore, although our behavioural assays indicate that female CHC profiles can generally be perceived as sexual cues attracting males in Nasonia, this function has apparently been lost in the highly divergent female N. giraulti CHC profiles. Curiously, N. giraulti males are still attracted to heterospecific, but not to conspecific female CHC profiles. We suggest that this striking discrepancy has been caused by an extensive evolutionary shift in female N. giraulti CHC profiles, which are no longer used as conspecific recognition cues. Our study constitutes the first report of an apparent abandonment of a sexual recognition cue that the receiver did not adapt to.

Polyploidy in animals: effects of gene expression on sex determination, evolution and ecology.

Polyploidy is rarer in animals than in plants. Why? Since Muller's observation in 1925, many hypotheses have been proposed and tested, but none were able to completely explain this intriguing fact. New genomic technologies enable the study of whole genomes to explain the constraints on or consequences of polyploidization, rather than focusing on specific genes or life history characteristics. Here, we review a selection of old and recent literature on polyploidy in animals, with emphasis on the consequences of polyploidization for gene expression patterns and genomic network interactions. We propose a conceptual model to contrast various scenarios for changes in genomic networks, which may serve as a framework to explain the different evolutionary dynamics of polyploidy in animals and plants. We also present new insights of genetic sex determination in animals and our emerging understanding of how animal sex determination systems may hamper or enable polyploidization, including some recent data on haplodiploids. We discuss the role of polyploidy in evolution and ecology, using a gene regulation perspective, and conclude with a synopsis regarding the effects of whole genome duplications on the balance of genomic networks. See also the sister articles focusing on plants by Ashman et al. and Madlung and Wendel in this themed issue.

Adaptive latitudinal cline of photoperiodic diapause induction in the parasitoid Nasonia vitripennis in Europe.

Living in seasonally changing environments requires adaptation to seasonal cycles. Many insects use the change in day length as a reliable cue for upcoming winter and respond to shortened photoperiod through diapause. In this study, we report the clinal variation in photoperiodic diapause induction in populations of the parasitoid wasp Nasonia vitripennis collected along a latitudinal gradient in Europe. In this species, diapause occurs in the larval stage and is maternally induced. Adult Nasonia females were exposed to different photoperiodic cycles and lifetime production of diapausing offspring was scored. Females switched to the production of diapausing offspring after exposure to a threshold number of photoperiodic cycles. A latitudinal cline was found in the proportion of diapausing offspring, the switch point for diapause induction measured as the maternal age at which the female starts to produce diapausing larvae, and the critical photoperiod for diapause induction. Populations at northern latitudes show an earlier switch point, higher proportions of diapausing individuals and longer critical photoperiods. Since the photoperiodic response was measured under the same laboratory conditions, the observed differences between populations most likely reflect genetic differences in sensitivity to photoperiodic cues, resulting from local adaptation to environmental cycles. The observed variability in diapause response combined with the availability of genomic tools for N. vitripennis represent a good opportunity to further investigate the genetic basis of this adaptive trait.

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.

Temperature stress increases hybrid incompatibilities in the parasitic wasp genus Nasonia.

Hybrid incompatibilities, measured as mortality and sterility, are caused by the disruption of gene interactions. They are important post-zygotic isolation barriers to species hybridization, and much effort is put into the discovery of the genes underlying these incompatibilities. In hybridization studies of the haplodiploid parasitic wasp genus Nasonia, genic incompatibilities have been shown to affect mortality and sterility. The genomic regions associated with mortality have been found to depend on the cytotype of the hybrids and thus suggest cytonuclear incompatibilities. As environmental conditions can affect gene expression and gene interaction, we here investigate the effect of developmental temperature on sterility and mortality in Nasonia hybrids. Results show that extreme temperatures strongly affect both hybrid sterility (mainly spermatogenic failure) and mortality. Molecular mapping revealed that extreme temperatures increase transmission ratio distortion of parental alleles at incompatible loci, and thus, cryptic incompatible loci surface under temperature stress that remain undiscovered under standard temperatures. Our results underline the sensitivity of hybrid incompatibilities to environmental factors and the effects of unstable epistasis.

DNA methylation plays a crucial role during early Nasonia development.

Although the role of DNA methylation in insect development is still poorly understood, the number and role of DNA methyltransferases in insects vary strongly between species. DNA methylation appears to be widely present among the social hymenoptera and functional studies in Apis have suggested a crucial role for de novo methylation in a wide variety of developmental processes. The sequencing of three parasitoid Nasonia genomes revealed the presence of three Dnmt1 (Dnmt1a, Dnmt1b and Dnmt1c) genes and one Dnmt2 and Dnmt3 gene, suggesting a role of DNA methylation in Nasonia development. In the present study we show that in Nasonia vitripennis all Dnmt1 messenger RNAs (mRNAs) and Dnmt3 mRNA are maternally provided to the embryo and, of these, Dnmt1a is essential during early embryogenesis. Lowering of maternal Dnmt1a mRNA results in embryonic lethality during the onset of gastrulation. This dependence on maternal Dnmt1a during embryogenesis in an organismal group outside the vertebrates, suggests evolutionary conservation of the function of Dnmt1 during embryogenesis.

Haldane's rule in the 21st century.

Haldane's Rule (HR), which states that 'when in the offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous (heterogametic) sex', is one of the most general patterns in speciation biology. We review the literature of the past 15 years and find that among the ∼85 new studies, many consider taxa that traditionally have not been the focus for HR investigations. The new studies increased to nine, the number of 'phylogenetically independent' groups that comply with HR. They continue to support the dominance and faster-male theories as explanations for HR, although due to increased reliance on indirect data (from, for example, differential introgression of cytoplasmic versus chromosomal loci in natural hybrid zones) unambiguous novel results are rare. We further highlight how research on organisms with sex determination systems different from those traditionally considered may lead to more insight in the underlying causes of HR. In particular, haplodiploid organisms provide opportunities for testing specific predictions of the dominance and faster X chromosome theory, and we present new data that show that the faster-male component of HR is supported in hermaphrodites, suggesting that genes involved in male function may evolve faster than those expressed in the female function.

A microsatellite marker linkage map of the housefly, Musca domestica: evidence for male recombination.

We present the first molecular marker linkage map for Musca domestica containing 35 microsatellite plus six visible markers. We report the development of 33 new microsatellite markers of which 19 are included in the linkage map. Two hundred and thirty-six F2 individuals were genotyped from three crosses yielding a linkage map consisting of five linkage groups that represent the five autosomes of the housefly. The map covers a total of 229.6 cM with an average marker spacing of 4.4 cM spanning approximately 80.2% of the genome. We found up to 29% recombination in male houseflies in contrast to most previous studies. The linkage map will add to genetic studies of the housefly.

A comparison of recombination frequencies in intraspecific versus interspecific mapping populations of Nasonia.

We present the first intraspecific linkage map for Nasonia vitripennis based on molecular markers. The map consists of 36 new microsatellite markers, extracted from the Nasonia genome sequence, and spans 515 cM. The five inferred linkage groups correspond to the five chromosomes of Nasonia. Comparison of recombination frequencies of the marker intervals spread over the whole genome (N=33 marker intervals) between the intraspecific N. vitripennis map and an interspecific N. vitripennis x N. giraulti map revealed a slightly higher (1.8%) recombination frequency in the intraspecific cross. We further considered an N. vitripennis x N. longicornis map with 29 microsatellite markers spanning 430 cM. Recombination frequencies in the two interspecific crosses differed neither between reciprocal crosses nor between mapping populations of embryos and adults. No major chromosomal rearrangements were found for the analyzed genomic segments. The observed differential F(2) hybrid male mortality has no significant effect on the genome-wide recombination frequency in Nasonia. We conclude that interspecific crosses between the different Nasonia species, a hallmark of Nasonia genetics, are generally suitable for mapping quantitative and qualitative trait loci for species differences.

Phylogeography of Nasonia vitripennis (Hymenoptera) indicates a mitochondrial-Wolbachia sweep in North America.

Here we report evidence of a mitochondrial-Wolbachia sweep in North American populations of the parasitoid wasp Nasonia vitripennis, a cosmopolitan species and emerging model organism for evolutionary and genetic studies. Analysis of the genetic variation of 89 N. vitripennis specimens from Europe and North America was performed using four types of genetic markers: a portion of the mitochondrial cytochrome oxidase I gene, nine polymorphic nuclear microsatellites, sequences from 11 single-copy nuclear markers and six Wolbachia genes. The results show that the European populations have a sevenfold higher mitochondrial sequence variation than North American populations, but similar levels of microsatellite and nuclear gene sequence variation. Variation in the North American mitochondria is extremely low (pi=0.31%), despite a highly elevated mutation rate (approximately 35-40 times higher than the nuclear genes) in the mitochondria of Nasonia. The data are indicative of a mitochondrial sweep in the North American population, possibly due to Wolbachia infections that are maternally co-inherited with the mitochondria. Owing to similar levels of nuclear variation, the data could not resolve whether N. vitripennis originated in the New or the Old World.

Segregation distortion and the evolution of sex-determining mechanisms.

Segregation distorters are alleles that distort normal segregation in their own favour. Sex chromosomal distorters lead to biased sex ratios, and the presence of such distorters, therefore, may induce selection for a change in the mechanism of sex determination. The evolutionary dynamics of distorter-induced changes in sex determination has only been studied in some specific systems. Here, we present a generic model for this process. We consider three scenarios: a driving X chromosome, a driving Y chromosome and a driving autosome with a male-determining factor. We investigate how the invasion prospects of a new sex-determining factor are affected by the strength of distortion and the fitness effect of the distorting allele. Our models show that in many cases, segregation distortion does create selection pressure, allowing novel sex-determining alleles to spread. When distortion leads to female-biased sex ratios, a new masculinizing gene can invade, leading to a new male heterogametic system. When distortion leads to male-biased sex ratios, a feminizing factor can invade and cause a switch to female heterogamety. In many cases, the distorter-induced change in the sex-determining system eventually leads to loss of the distorter from the population. Hence, the presence of sex chromosomal distorters will often only be transient, and the distorters may remain unnoticed. The role of segregation distortion in the evolution of sex determination may, therefore, be underestimated.

Host acceptance and sex allocation of Nasonia wasps in response to conspecifics and heterospecifics.

Species recognition is an important aspect of an organism's biology. Here, we consider how parasitoid wasps vary their reproductive decisions when their offspring face intra- and interspecific competition for resources and mates. We use host acceptance and sex ratio behaviour to test whether female Nasonia vitripennis and Nasonia longicornis discriminate between conspecifics and heterospecifics when ovipositing. We tested pairs of conspecific or heterospecific females ovipositing either simultaneously or sequentially on a single host, using strains varying in their recent history of sympatry. Both N. vitripennis and N. longicornis rejected parasitized hosts more often than unparasitized hosts, although females were more likely to superparasitize their own species in the sequential treatment. However, sex ratio behaviour did not vary, suggesting similar responses towards conspecifics and heterospecifics. This contrasts with theory predicting that heterospecifics should not influence sex ratios as their offspring do not influence local mate competition, where conspecifics would. These non-adaptive sex ratios reinforce the lack of adaptive kin discrimination in N. vitripennis and suggest a behavioural constraint. Discrimination between closely related species is therefore context dependent in Nasonia. We suggest that isolating mechanisms associated with the speciation process have influenced behaviour to a greater extent than selection on sex ratios.

Identification and characterization of the doublesex gene of Nasonia.

The doublesex (dsx) gene of the parasitic wasp Nasonia vitripennis is described and characterized. Differential splicing of dsx transcripts has been shown to induce somatic sexual differentiation in Diptera and Lepidoptera, but not yet in other insect orders. Two spliceforms of Nasonia dsx mRNA are differentially expressed in males and females. In addition, in a gynandromorphic line that produces haploids (normally males) with full female phenotypes, these individuals show the female spliceform, providing the first demonstration of a direct association of dsx with somatic sex differentiation in Hymenoptera. Finally, the DNA binding (DM) domain of Nasonia dsx clusters phylogenetically with dsx from other insects, and Nasonia dsx shows microsynteny with dsx of Apis, further supporting identification of the dsx orthologue in Nasonia.

Transcriptome and proteome analysis of ovaries of arrhenotokous and thelytokous Venturia canescens.

Under arrhenotoky, unfertilized haploid eggs develop as males but under thelytoky they develop into diploid females after they have undergone diploidy restoration. In the parasitoid wasp Venturia canescens both reproductive modes occur. Thelytoky is genetically determined but the underlying genetics of diploidy restoration remain unknown. In this study we aim to identify the genes and/or proteins that control thelytoky. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of total ovarian RNA and two-dimensional protein electrophoresis in combination with mass spectrometry revealed putative transcripts and proteins involved in arrhenotokous and thelytokous development. The detected tubulin and actin protein differences are most likely functionally related to the two types of reproduction.

Reproductive strategies under multiparasitism in natural populations of the parasitoid wasp Nasonia (Hymenoptera).

Parasitoid Nasonia wasps adjust their progeny sex ratio to the presence of conspecifics to optimize their fitness. Another trait under female control is the induction of offspring diapause. We analysed progeny sex ratios and the proportion of diapausing offspring of individual Nasonia females in host patches parasitized by two species, Nasonia vitripennis and Nasonia giraulti, in North American field populations using microsatellite fingerprinting. Both Nasonia species produced similar sex ratios on hosts that were co-parasitized by their own species as by the other species, indicating that females do not distinguish between con- and heterospecific clutches. The sex ratios of the diapause and adult fractions of mixed broods from single females were not correlated. We found further indications that N. vitripennis females take the emergence time of the offspring into account in their sex allocation. The reproductive strategies of Nasonia under multiparasitism are largely adaptive, but also partially constrained by information.