White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Molecular tools for studying the major malaria vector Anopheles funestus: improving the utility of the genome using a comparative poly(A) and Ribo-Zero RNAseq analysis.

BACKGROUND: Next-generation sequencing (NGS) offers great opportunities for studying the biology of insect vectors of disease. Prerequisites for successful analyses include high quality annotated genome assemblies and that techniques designed for use with model organisms be tested and optimised for use with these insects. We aimed to test and improve genomic tools for studying the major malaria vector Anopheles funestus. RESULTS: To guide future RNAseq transcriptomic studies of An. funestus, we compared two methods for enrichment of non-ribosomal RNA for analysis: enrichment of polyadenylated RNA and ribosomal RNA depletion using a kit designed to deplete human/rat/mouse rRNA. We found large differences between the two methods in the resulting transcriptomes, some of which is due to differential representation of polyadenylated and non-polyadenylated transcripts. We used the RNAseq data for validation and targeted manual editing of the draft An. funestus genome annotation, validating 62 % of annotated introns, manually improving the annotation of seven gene families involved in the detoxification of xenobiotics and integrated two published transcriptomic datasets with the recently published genome assembly. CONCLUSIONS: The mRNA enrichment method makes a substantial, replicable difference to the transcriptome composition, at least partly due to the representation of non-polyadenylated transcripts in the final transcriptome. Therefore, great care should be taken in comparing gene expression data among studies. Ribosomal RNA depletion of total RNA using a kit designed to deplete human/rat/mouse rRNA works in mosquitoes and, we argue, results in a truer representation of the transcriptome than poly(A) selection. The An. funestus genome annotation can be considerably improved with the help of these new RNAseq data and further guided manual gene editing efforts will be of great benefit to the Anopheles research community for studies of this insect's genome and transcriptome.

Population genomics of the killer whale indicates ecotype evolution in sympatry involving both selection and drift.

The evolution of diversity in the marine ecosystem is poorly understood, given the relatively high potential for connectivity, especially for highly mobile species such as whales and dolphins. The killer whale (Orcinus orca) has a worldwide distribution, and individual social groups travel over a wide geographic range. Even so, regional populations have been shown to be genetically differentiated, including among different foraging specialists (ecotypes) in sympatry. Given the strong matrifocal social structure of this species together with strong resource specializations, understanding the process of differentiation will require an understanding of the relative importance of both genetic drift and local adaptation. Here we provide a high-resolution analysis based on nuclear single-nucleotide polymorphic markers and inference about differentiation at both neutral loci and those potentially under selection. We find that all population comparisons, within or among foraging ecotypes, show significant differentiation, including populations in parapatry and sympatry. Loci putatively under selection show a different pattern of structure compared to neutral loci and are associated with gene ontology terms reflecting physiologically relevant functions (e.g. related to digestion). The pattern of differentiation for one ecotype in the North Pacific suggests local adaptation and shows some fixed differences among sympatric ecotypes. We suggest that differential habitat use and resource specializations have promoted sufficient isolation to allow differential evolution at neutral and functional loci, but that the process is recent and dependent on both selection and drift.

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis.

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

Genome sequence for "Candidatus Mycoplasma haemominutum," a low-pathogenicity hemoplasma species.

We present the genome sequence of "Candidatus Mycoplasma haemominutum" strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis.

Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes.Analysis showed that in hemoplasmas genes involved in carbohydrate metabolism are limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source. The majority of the pentose phosphate pathway enzymes that catalyze the de novo synthesis of ribonucleotides were absent, as were cell division protein FtsZ and chaperonins GroEL/ES. Uncharacterized protein paralogs containing putative surface expression motifs, comprised 62% of M. haemofelis and 19% of Mycoplasma suis genome coverage respectively, the majority of which were present in a small number of unstructured islands. Limited mass spectrometry and immunoblot data matched a number of characterized proteins and uncharacterized paralogs, confirming their expression and immunogenicity in vivo.These data have allowed further characterization of these important pathogens, including their limited metabolic capabilities, which may contribute to their uncultivatable status. A number of immunogenic proteins, and a potential mechanism for host immune system evasion, have been identified.

A comprehensive genetic analysis of candidate genes regulating response to Trypanosoma congolense infection in mice.

BACKGROUND: African trypanosomes are protozoan parasites that cause "sleeping sickness" in humans and a similar disease in livestock. Trypanosomes also infect laboratory mice and three major quantitative trait loci (QTL) that regulate survival time after infection with T. congolense have been identified in two independent crosses between susceptible A/J and BALB/c mice, and the resistant C57BL/6. These were designated Tir1, Tir2 and Tir3 for Trypanosoma infection response, and range in size from 0.9-12 cM. PRINCIPAL FINDINGS: Mapping loci regulating survival time after T. congolense infection in an additional cross revealed that susceptible C3H/HeJ mice have alleles that reduce survival time after infection at Tir1 and Tir3 QTL, but not at Tir2. Next-generation resequencing of a 6.2 Mbp region of mouse chromosome 17, which includes Tir1, identified 1,632 common single nucleotide polymorphisms (SNP) including a probably damaging non-synonymous SNP in Pram1 (PML-RAR alpha-regulated adaptor molecule 1), which was the most plausible candidate QTL gene in Tir1. Genome-wide comparative genomic hybridisation identified 12 loci with copy number variants (CNV) that correlate with differential gene expression, including Cd244 (natural killer cell receptor 2B4), which lies close to the peak of Tir3c and has gene expression that correlates with CNV and phenotype, making it a strong candidate QTL gene at this locus. CONCLUSIONS: By systematically combining next-generation DNA capture and sequencing, array-based comparative genomic hybridisation (aCGH), gene expression data and SNP annotation we have developed a strategy that can generate a short list of polymorphisms in candidate QTL genes that can be functionally tested.

Identification of three novel superantigen-encoding genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP.

The acquisition of superantigen-encoding genes by Streptococcus pyogenes has been associated with increased morbidity and mortality in humans, and the gain of four superantigens by Streptococcus equi is linked to the evolution of this host-restricted pathogen from an ancestral strain of the opportunistic pathogen Streptococcus equi subsp. zooepidemicus. A recent study determined that the culture supernatants of several S. equi subsp. zooepidemicus strains possessed mitogenic activity but lacked known superantigen-encoding genes. Here, we report the identification and activities of three novel superantigen-encoding genes. The products of szeF, szeN, and szeP share 59%, 49%, and 34% amino acid sequence identity with SPEH, SPEM, and SPEL, respectively. Recombinant SzeF, SzeN, and SzeP stimulated the proliferation of equine peripheral blood mononuclear cells, and tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) production, in vitro. Although none of these superantigen genes were encoded within functional prophage elements, szeN and szeP were located next to a prophage remnant, suggesting that they were acquired by horizontal transfer. Eighty-one of 165 diverse S. equi subsp. zooepidemicus strains screened, including 7 out of 15 isolates from cases of disease in humans, contained at least one of these new superantigen-encoding genes. The presence of szeN or szeP, but not szeF, was significantly associated with mitogenic activity in the S. equi subsp. zooepidemicus population (P < 0.000001, P < 0.000001, and P = 0.104, respectively). We conclude that horizontal transfer of these novel superantigens from and within the diverse S. equi subsp. zooepidemicus population is likely to have implications for veterinary and human disease.

Single gene differentiation by DNA-modified carbon electrodes using an AC impedimetric approach.

A simple and novel electrochemical biosensor is described for differentiating between differing gene sequences on the basis of DNA hybridization events. Polyethylenimine-modified screen-printed carbon electrodes were used to immobilize single-stranded PCR fragments from plasmid DNA from the gene for pyruvate kinase. AC impedimetric measurements were first performed on these systems in buffer and then upon exposure to single-stranded DNA. When the electrode and solution DNA were complementary, a large drop in impedance was measured. Complementary DNA could be clearly detected at concentrations down to 1 fg/mL. Higher concentrations gave faster hybridization with saturation occurring at levels above 1 ng/mL. Responses were much lower upon exposure to noncomplementary DNA, even at higher concentrations, with the sensor showing a high degree of selectivity. This sensor format offers great promise for many DNA hybridization applications and lends itself to mass fabrication.

Seasonally hibernating phenotype assessed through transcript screening.

Hibernation is a seasonally entrained and profound phenotypic transition to conserve energy in winter. It involves significant biochemical reprogramming, although our understanding of the underpinning molecular events is fragmentary and selective. We have conducted a large-scale gene expression screen of the golden-mantled ground squirrel, Spermophilus lateralis, to identify transcriptional responses associated specifically with the summer-winter transition and the torpid-arousal transition in winter. We used 112 cDNA microarrays comprising 12,288 probes that cover at least 5,109 genes. In liver, the profiles of torpid and active states in the winter were almost identical, although we identified 102 cDNAs that were differentially expressed between winter and summer, 90% of which were downregulated in the winter states. By contrast, in cardiac tissue, 59 and 115 cDNAs were elevated in interbout arousal and torpor, respectively, relative to the summer active condition, but only 7 were common to both winter states, and during arousal none was downregulated. In brain, 78 cDNAs were found to change in winter, 44 of which were upregulated. Thus transcriptional changes associated with hibernation are qualitatively modest and, since these changes are generally less than twofold, also quantitatively modest. Unbiased Gene Ontology profiling of the transcripts suggests a winter switch to beta-oxidation of lipids in liver and heart, a reduction in metabolism of toxic compounds and the urea cycle in liver, and downregulated electron transport in the brain. We identified just one strongly winter-induced transcript common to all tissues, namely an RNA-binding protein, RBM3. This analysis clearly differentiates responses of the principal tissues, identifies a large number of new genes undergoing regulation, and broadens our understanding of affected cellular processes that, in part, account for the winter-adaptive hibernating phenotype.

Evidence of oxidative stress in chronic venous ulcers.

Reactive oxygen species have been implicated in the impaired healing of chronic leg ulcers but little direct evidence is available. We have observed a significant (p < 0.01) elevation of the allantoin : uric acid percentage ratio, a marker of oxidative stress, in wound fluid from chronic leg ulcers (median 17, range 8-860) compared to both paired plasma (median 2, range 1-8) and acute surgical wound fluid (median 4, range 3-7). However, the allantoin : uric acid percentage ratio did not differ significantly between chronic wounds that healed and those that failed to heal. Neutrophil elastase was elevated 30- to 1300-fold in chronic wound fluid compared to plasma and there was a correlation (r(2) = 0.742) between wound fluid elastase and the allantoin : uric acid percentage ratio. Total antioxidant capacity of wound fluid, as measured with a chemiluminescence assay, did not show a correlation (r(2) = 0.03) with the observed oxidative stress. These observations suggest that conditions of localized oxidative stress, possibly related to neutrophil-associated production of reactive oxygen species, are present in chronic leg ulcers. It is possible that future therapeutic strategies aimed at reducing oxidative stress, in addition to good standard care, could improve healing rates of chronic wounds.