Single-locus DNA barcoding and species delimitation of the sandfly subgenus Evandromyia (Aldamyia).

Sandfly specimens from the subgenus Evandromyia (Aldamyia) Galati, 2003 (Diptera: Psychodidae: Phlebotominae) were collected between 2012 and 2019 from nine localities in seven Brazilian states, morphologically-identified, and then DNA barcoded by sequencing the mitochondrial cytochrome c oxidase subunit I (coi) gene. Forty-four new barcode sequences generated from 10 morphospecies were combined with 49 previously published sequences from the same subgenus and analysed using sequence-similarity methods (best-match criteria) to assess their ability at specimen identification, while four different species delimitation methods (ABGD, GMYC, PTP and TCS) were used to infer molecular operational taxonomic units (MOTUs). Overall, seven of the 11 morphospecies analysed were congruent with both the well-supported clades identified by phylogenetic analysis and the MOTUs inferred by species delimitation, while the remaining four morphospecies - E. carmelinoi, E. evandroi, E. lenti and E. piperiformis - were merged into a single well-supported clade/MOTU. Although E. carmelinoi, E. evandroi and E. lenti were indistinguishable using coi DNA barcodes, E. piperiformis did form a distinct phylogenetic cluster and could be correctly identified using best-match criteria. Despite their apparent morphological differences, we propose on the basis of the molecular similarity of their DNA barcodes that these latter four morphospecies should be considered members of a recently-diverged species complex.

The transcriptome of the mosquito Aedes fluviatilis (Diptera: Culicidae), and transcriptional changes associated with its native Wolbachia infection.

BACKGROUND: Wolbachia is a bacterial endosymbiont that naturally infects a wide range of insect species, and causes drastic changes to host biology. Stable infections of Wolbachia in mosquitoes can inhibit infection with medically important pathogens such as dengue virus and malaria-causing Plasmodium parasites. However, some native Wolbachia strains can enhance infection with certain pathogens, as is the case for the mosquito Aedes fluviatilis, where infection with Plasmodium gallinaceum is enhanced by the native wFlu Wolbachia strain. To better understand the biological interactions between mosquitoes and native Wolbachia infections, and to investigate the process of pathogen enhancement, we used RNA-Seq to generate the transcriptome of Ae. fluviatilis with and without Wolbachia infection. RESULTS: In total, we generated 22,280,160 Illumina paired-end reads from Wolbachia-infected and uninfected mosquitoes, and used these to make a de novo transcriptome assembly, resulting in 58,013 contigs with a median sequence length of 443 bp and an N50 of 2454 bp. Contigs were annotated through local alignments using BlastX, and associated with both gene ontology and KEGG orthology terms. Through baySeq, we identified 159 contigs that were significantly upregulated due to Wolbachia infection, and 98 that were downregulated. Critically, we saw no changes to Toll or IMD immune gene transcription, but did see evidence that wFlu infection altered the expression of several bacterial recognition genes, and immune-related genes that could influence Plasmodium infection. wFlu infection also had a widespread effect on a number of host physiological processes including protein, carbohydrate and lipid metabolism, and oxidative stress. We then compared our data set with transcriptomic data for other Wolbachia infections in Aedes aegypti, and identified a core set of 15 gene groups associated with Wolbachia infection in mosquitoes. CONCLUSIONS: While the scale of transcriptional changes associated with wFlu infection might be small, the scope is rather large, which confirms that native Wolbachia infections maintain intricate molecular relationships with their mosquito hosts even after lengthy periods of co-evolution. We have also identified several potential means through which wFlu infection might influence Plasmodium infection in Ae. fluviatilis, and these genes should form the basis of future investigation into the enhancement of Plasmodium by Wolbachia.

Broader prevalence of Wolbachia in insects including potential human disease vectors.

Wolbachia are intracellular, maternally transmitted bacteria considered the most abundant endosymbionts found in arthropods. They reproductively manipulate their host in order to increase their chances of being transmitted to the offspring, and currently are being used as a tool to control vector-borne diseases. Studies on distribution of Wolbachia among its arthropod hosts are important both for better understanding why this bacterium is so common, as well as for its potential use as a biological control agent. Here, we studied the incidence of Wolbachia in a broad range of insect species, collected from different regions of Brazil, using three genetic markers (16S rRNA, wsp and ftsZ), which varied in terms of their sensitivity to detect this bacterium. The overall incidence of Wolbachia among species belonging to 58 families and 14 orders was 61.9%. The most common positive insect orders were Coleoptera, Diptera, Hemiptera and Hymenoptera, with Diptera and Hemiptera having the highest numbers of Wolbachia-positive families. They included potential human disease vectors whose infection status has never been reported before. Our study further shows the importance of using quantitative polymerase chain reaction for high-throughput and sensitive Wolbachia screening.

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion.

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.

Plasmodium falciparum ookinete invasion of the midgut epithelium of Anopheles stephensi is consistent with the Time Bomb model.

Plasmodium falciparum gametocytes grown in vitro were fed through membrane feeders to laboratory-reared Anopheles stephensi mosquitoes. Intact midguts, including entire bloodmeal contents, were removed between 24 and 48 h post-bloodfeeding. Giemsa-stained histological sections were prepared from the midguts and examined by light microscopy. Contrary to previous reports, ookinetes were clearly visible within midgut epithelial cells, demonstrating intracellular migration across the midgut wall. Ookinetes entered epithelial cells through the lateral apical membrane at sites where 3 adjacent cells converged. There was no evidence for the existence of a morphologically distinct group of epithelial cells preferentially invaded by ookinetes. However, ookinete penetration was associated with significant morphological changes to invaded cells, including differential staining, condensation and fragmentation of the nucleus, vacuolization, loss of microvilli and various degrees of extrusion into the midgut lumen. Epithelial cells completely separated from the midgut wall were found within the midgut lumen. These cells were associated with invading parasites suggesting that ookinete penetration resulted in complete ejection of invaded cells from the midgut wall. Small clusters of morphologically altered midgut cells and invading parasites spanning the membranes of adjacent abnormal epithelial cells were observed, consistent with intracellular movement of ookinetes between neighbouring midgut cells. Extruded epithelial cells were also observed rarely in uninfected midguts. Epithelial cell extrusion, therefore, may be a general mechanism of tissue repair through which damaged cells are removed from the midgut wall rather than a parasite-specific response. These observations demonstrate that human malaria parasite infection of mosquitoes is consistent with, and provides further support for, the Time Bomb model of ookinete invasion of the mosquito midgut epithelium previously proposed for rodent malaria parasites.