Project description:ObjectiveIn insects, closely related species are often difficult or impossible to distinguish solely by morphological traits. Mitochondrial DNA (mtDNA) markers are often useful and reliable for distinguishing closely related species. However, useful mtDNA markers can be unavailable, particularly when such species pairs experienced hybrid introgression in the past. Although polymorphic nuclear DNA markers would be necessary to distinguish such species pairs, recombination, multiple copies, and slower mutation rates of the nuclear DNA compared with those of mtDNA often make it challenging. The objective of this study was to develop a multiplex polymerase chain reaction that can reliably amplify and distinguish the Tpi sequences of Eurema mandarina and Eurema hecabe.ResultsWe successfully analyzed the nucleotide sequences of the Z chromosome-linked triose phosphate isomerase (Tpi) gene to develop a multiplex polymerase chain reaction (PCR) that amplified ca. 120-bp products for E. mandarina and ca. 375-bp products for E. hecabe. We suggest that multiplex PCR using Tpi with appropriately designed primers can be used to accurately and reliably distinguish between other closely related Lepidoptera species.
Project description:Theory predicts unified sex ratios for most organisms, yet biases may be engendered by selfish genetic elements such as endosymbionts that kill or feminize individuals with male genotypes. Although rare, feminization is established for Wolbachia-infected Eurema butterflies. This paradigm is presently confined to islands in the southern Japanese archipelago, where feminized phenotypes produce viable all-daughter broods. Here, we characterize sex bias for E. hecabe in continental Australia. Starting with 186 wild-caught females, we reared >6000 F1-F3 progeny in pedigree designs that incorporated selective antibiotic treatments. F1 generations expressed a consistent bias across 2 years and populations that was driven by an ~5% incidence of broods comprising ⩾80% daughters. Females from biased lineages continued to overproduce daughters over two generations of outcrossing to wild males. Treatment with antibiotics of differential strength influenced sex ratio only in biased lineages by inducing an equivalent incomplete degree of son overproduction. Brood sex ratios were nevertheless highly variable within lineages and across generations. Intriguingly, the cytogenetic signature of female karyotype was uniformly absent, even among phenotypic females in unbiased lineages. Molecular evidence supported the existence of a single Wolbachia strain at high prevalence, yet this was not clearly linked to brood sex bias. In sum, we establish an inherited, experimentally reversible tendency for incomplete offspring bias. Key features of our findings clearly depart from the Japanese feminization paradigm and highlight the potential for more subtle degrees of sex distortion in arthropods.
Project description:Arthropod sex ratios can be manipulated by a diverse range of selfish genetic elements, including maternally inherited Wolbachia bacteria. Feminization by Wolbachia is rare but has been described for Eurema mandarina butterflies. In this species, some phenotypic and functional females, thought to be ZZ genetic males, are infected with a feminizing Wolbachia strain, wFem. Meanwhile, heterogametic WZ females are not infected with wFem. Here, we establish a quantitative PCR assay allowing reliable sexing in three Eurema species. Against expectation, all E. mandarina females, including wFem females, had only one Z chromosome that was paternally inherited. Observation of somatic interphase nuclei confirmed that W chromatin was absent in wFem females, but present in females without wFem. We conclude that the sex bias in wFem lines is due to meiotic drive (MD) that excludes the maternal Z and thus prevents formation of ZZ males. Furthermore, wFem lines may have lost the W chromosome or harbour a dysfunctional version, yet rely on wFem for female development; removal of wFem results in all-male offspring. This is the first study that demonstrates an interaction between MD and Wolbachia feminization, and it highlights endosymbionts as potentially confounding factors in MD of sex chromosomes.