Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40–62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Isolation and characterization of a novel Wolbachia bacteriophage from Allonemobius socius crickets in Missouri

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOKue of the Mediterranean flour moth Ephestia kuehniella (94.18% identity) and WOLig of the coronet moth, Craniophora ligustri (96.86% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40-62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Sorted gene genealogies and species-specific nonsynonymous substitutions point to putative postmating prezygotic isolation genes inAllonemobiuscrickets

In theAllonemobius sociuscomplex of crickets, reproductive isolation is primarily accomplished via postmating prezygotic barriers. We tested seven protein-coding genes expressed in the male ejaculate for patterns of evolution consistent with a putative role as postmating prezygotic isolation genes.Our recently diverged species generally lacked sequence variation. As a result,ω-based tests were only mildly successful. Some of our genes showed evidence of elevatedωvalues on the internal branches of gene trees. In a couple genes these internal branches coincided with both species branching events of the species tree, betweenA. fasciatusand the other two species, and betweenA. sociusandA. sp. nov.Tex. In comparison, more successful approaches were those that took advantage of the varying degrees of lineage sorting and allele sharing among our young species. These approaches were particularly powerful within the contact zone. Among the genes we tested we found genes with genealogies that indicated relatively advanced degrees of lineage sorting across both allopatric and contact zone alleles. Within a contact zone between two members of the species complex, only a subset of genes maintained allelic segregation despite evidence of ongoing gene flow in other genes. The overlap in these analyses wasarginine kinase(AK) andapolipoprotein A-1 binding protein(APBP). These genes represent two of the first examples of sperm maturation, capacitation, and motility proteins with fixed non-synonymous substitutions between species-specific alleles that may lead to postmating prezygotic isolation. Both genes express ejaculate proteins transferred to females during copulation and were previously identified through comparative proteomics. We discuss the potential function of these genes in the context of the specific postmating prezygotic isolation phenotype among our species, namely conspecific sperm precedence and the superior ability of conspecific males to induce oviposition in females.

The influence of male age and simulated pathogenic infection on producing a dishonest sexual signal

In recent years, studies have shown that reproductive effort decelerates in response to pathogenic infection. If infection substantially reduces a host's residual reproductive value (RRV), however, then an acceleration of effort may instead occur (e.g. terminal investment). Reproductive acceleration would theoretically allow hosts to maintain or exaggerate their sexual signal upon infection. This would create a deceptive message from the perspective of the chooser, who may unwittingly copulate with an infected mate to their detriment. Using the cricket Allonemobius socius , we assessed the potential for reduced RRV to accelerate male reproductive effort and create a dishonest signal. RRV was manipulated through male age and simulated pathogenic insult. Reproductive effort was measured as calling song energetics, mating success, latency to mate and nuptial gift size. We show that males adopted either an accelerated or decelerated reproductive strategy upon infection, and that this decision was probably mediated by RRV. Moreover, males who accelerated their effort produced a dishonest signal by increasing their song energetics while providing fewer paternal resources (i.e. smaller gifts). Our study is one of the few to document the existence of dishonest signals and relate dishonesty to a potential reduction in female fitness, underscoring the conflict inherent in sexual reproduction.