Revisiting the paradigm of anhematophagy in male mosquitoes.

Female mosquitoes are reproductively obligate bloodfeeders which feed on vertebrate blood to obtain nutrients required for egg production (driving transmission of vector-borne pathogens in the process), and which rely on plant sugars for their non-reproductive energy requirements. Male mosquitoes, on the other hand, are thought to rely exclusively on plant sugars for their energetic needs; indeed, this dichotomy is one of the central tenets of medical entomology. Here, we show that male Culex tarsalis and Aedes aegypti mosquitoes will readily take blood from a membrane feeder when reared under dehydration conditions with no toxic effects. Mosquitoes with impaired humidity detection do not increase their bloodfeeding rates when dehydrated compared to wild-type controls. While conventionally reared males ignore a human host, dehydrated males are attracted to and attempt to probe, with some success, although they cannot access host capillaries. However, they will take blood from a vertebrate host wound. When fed a blood meal containing West Nile virus, male mosquitoes can become infected with and orally transmit the pathogen at rates and titers equivalent to females. These data suggest that under some circumstances male mosquitoes may be able to probe and/or ingest blood and transmit pathogens to vertebrate hosts, and that their role in maintaining pathogen transmission cycles should be re-examined.

Eilat virus (EILV) causes superinfection exclusion against West Nile virus (WNV) in a strain-specific manner in Culex tarsalis mosquitoes.

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the USA. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vector Culex tarsalis is also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells and C. tarsalis mosquitoes. The titres of both WNV strains - WN02-1956 and NY99 - were suppressed by EILV in C6/36 cells as early as 48-72 h post-superinfection at both m.o.i. values tested in our study. The titres of WN02-1956 at both m.o.i. values remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titres. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In C. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titres at 3 days post-superinfection, but this effect disappeared at 7 days post-superinfection. In contrast, WN02-1956 infection titres were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, in C. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

Eilat virus (EILV) causes superinfection exclusion against West NILE virus (WNV) in a strain specific manner in Culex tarsalis mosquitoes.

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the United States. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vector Culex tarsalis is also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells and Culex tarsalis mosquitoes. The titers of both WNV strains-WN02-1956 and NY99-were suppressed by EILV in C6/36 cells as early as 48-72 h post superinfection at both multiplicity of infections (MOIs) tested in our study. The titers of WN02-1956 at both MOIs remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titers. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In Cx. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titers at 3 days post superinfection, but this effect disappeared at 7 days post superinfection. In contrast, WN02-1956 infection titers were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, in Cx. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains.

Immune-related transcripts, microbiota and vector competence differ in dengue-2 virus-infected geographically distinct Aedes aegypti populations.

BACKGROUND: Vector competence in Aedes aegypti is influenced by various factors. Crucial new control methods can be developed by recognizing which factors affect virus and mosquito interactions. METHODS: In the present study we used three geographically distinct Ae. aegypti populations and compared their susceptibility to infection by dengue virus serotype 2 (DENV-2). To identify any differences among the three mosquito populations, we evaluated expression levels of immune-related genes and assessed the presence of microbiota that might contribute to the uniqueness in their vector competence. RESULTS: Based on the results from the DENV-2 competence study, we categorized the three geographically distinct Ae. aegypti populations into a refractory population (Vilas do Atlântico), a susceptible population (Vero) and a susceptible but low transmission population (California). The immune-related transcripts were highly expressed in the California population but not in the refractory population. However, the Rel-1 gene was upregulated in the Vilas do Atlântico population following ingestion of a non-infectious blood meal, suggesting the gene's involvement in non-viral responses, such as response to microbiota. Screening of the bacteria, fungi and flaviviruses revealed differences between populations, and any of these could be one of the factors that interfere with the vector competence. CONCLUSIONS: The results reveal potential factors that might impact the virus and mosquito interaction, as well as influence the Ae. aegypti refractory phenotype.

Intrinsic variation in the vertically transmitted core virome of the mosquito Aedes aegypti.

Virome studies among metazoans have revealed the ubiquity of RNA viruses in animals, contributing to a fundamental rethinking of the relationships between organisms and their microbiota. Mosquito viromes, often scrutinized due to their public health relevance, may also provide insight into broadly applicable concepts, such as a "core virome," a set of viruses consistently associated with a host species or population that may fundamentally impact its basic biology. A subset of mosquito-associated viruses (MAVs) could comprise such a core, and MAVs can be categorized as (i) arboviruses, which alternate between mosquito and vertebrate hosts, (ii) insect-specific viruses, which cannot replicate in vertebrate cells, and (iii) viruses with unknown specificity. MAVs have been widely characterized in the disease vector Aedes aegypti, and the occurrence of a core virome in this species has been proposed but remains unclear. Using a wild population previously surveyed for MAVs and a common laboratory strain, we investigated viromes in reproductive tissue via metagenomic RNA sequencing. Virome composition varied across samples, but four groups comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a toti-like virus (Totiviridae), unclassified Riboviria, and four orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the partiti-like virus, toti-like virus, and one orthomyxo-like virus were assembled and analysed phylogenetically. Multigenerational maintenance of these MAVs was confirmed by RT-PCR, indicating vertical transmission as a mechanism for persistence. This study provides fundamental information regarding MAV ecology and variability in A. aegypti and the potential for vertically maintained core viromes at the population level.

Aedes Vector Surveillance in the Southeastern United States Reveals Growing Threat of Aedes japonicus japonicus (Diptera: Culicidae) and Aedes albopictus.

Mosquito-borne diseases are on the rise globally, and have the potential to thrive along the Gulf Coast of the United States, where subtropical conditions may facilitate the introduction or movement of mosquito vectors. Despite surveillance efforts, Aedes aegypti (L.) had not been detected in the Gulf state of Alabama for nearly three decades. The detection of Ae. aegypti in Alabama may suggest remnant or reemergent populations of this vector. We conducted adult sampling between May and August of 2018 to capture mosquitoes during a time frame when all species should be active. This was to ensure no species were missed due to overwintering and to identify the distributions of Aedes mosquitoes of medical importance. No Ae. aegypti were detected in Alabama over the period of this study. We detected Aedes albopictus (Skuse) in 65 counties and the recently invasive Aedes japonicus japonicus (Theobald) in 30 counties across the state. These results indicate that while Ae. aegypti was recently reported from parts of Alabama, the state is not experiencing a major resurgence of the species, whereas Ae. albopictus remains ubiquitous. Further, results indicate that a third wave of Aedes invasion may be occurring, that of Ae. japonicus japonicus. All three of these species are medically important vectors and may pose threats to the public health of the Gulf Coast of the United States.

Killer yeasts exert anti-plasmodial activities against the malaria parasite Plasmodium berghei in the vector mosquito Anopheles stephensi and in mice.

BACKGROUND: Wickerhamomyces anomalus is a yeast associated with different insects including mosquitoes, where it is proposed to be involved in symbiotic relationships with hosts. Different symbiotic strains of W. anomalus display a killer phenotype mediated by protein toxins with broad-spectrum antimicrobial activities. In particular, a killer toxin purified from a W. anomalus strain (WaF17.12), previously isolated from the malaria vector mosquito Anopheles stephensi, has shown strong in vitro anti-plasmodial activity against early sporogonic stages of the murine malaria parasite Plasmodium berghei. RESULTS: Here, we provide evidence that WaF17.12 cultures, properly stimulated to induce the expression of the killer toxin, can directly affect in vitro P. berghei early sporogonic stages, causing membrane damage and parasite death. Moreover, we demonstrated by in vivo studies that mosquito dietary supplementation with activated WaF17.12 cells interfere with ookinete development in the midgut of An. stephensi. Besides the anti-sporogonic action of WaF17.12, an inhibitory effect of purified WaF17.12-killer toxin was observed on erythrocytic stages of P. berghei, with a consequent reduction of parasitaemia in mice. The preliminary safety tests on murine cell lines showed no side effects. CONCLUSIONS: Our findings demonstrate the anti-plasmodial activity of WaF17.12 against different developmental stages of P. berghei. New studies on P. falciparum are needed to evaluate the use of killer yeasts as innovative tools in the symbiotic control of malaria.

Denaturing Gradient Gel Electrophoresis Analysis of Bacteria in Italian Ticks and First Detection of Streptococcus equi in Rhipicephalus bursa from the Lazio Region.

Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two Rhipicephalus sanguineus ticks from dogs (Lombardia), two Rhipicephalus bursa ticks from bovines (Lazio), and three Ixodes ricinus ticks from humans (Marche). The major result was the first report of the zoonotic agent Streptococcus equi in ticks. S. equi is a species complex of highly contagious pathogens. Subsequent to S. equi detection in a R. bursa tick removed from a bovine of Lazio in 2012, we studied 95 R. bursa samples collected from 3 bovines, 3 ponies, and 1 sheep grazing in the same area in 2012 and from 6 ponies grazing there in 2017. The results of a specific PCR assay indicated a not sporadic occurrence of S. equi in ticks. This finding provides a basis for assessing the potential of ticks to harbor and disperse S. equi.

The Genomes of Four Meyerozyma caribbica Isolates and Novel Insights into the Meyerozyma guilliermondii Species Complex.

Yeasts of the Meyerozyma guilliermondii species complex are widespread in nature and can be isolated from a variety of sources, from the environment to arthropods to hospital patients. To date, the species complex comprises the thoroughly studied and versatile M. guilliermondii, the hard to distinguish M. caribbica, and Candida carpophila Here we report the whole genome sequencing and de novo assembly of four M. caribbica isolates, identified with the most recent molecular techniques, derived from four Diptera species. The four novel assemblies present reduced fragmentation and comparable metrics (genome size, gene content) to the available genomes belonging to the species complex. We performed a phylogenomic analysis comprising all known members of the species complex, to investigate evolutionary relationships within this clade. Our results show a compact phylogenetic structure for the complex and indicate the presence of a sizable core set of genes. Furthermore, M. caribbica, despite a broad literature on the difficulties of discerning it from M. guilliermondii, seems to be more closely related to C. carpophila Finally, we believe that there is evidence for considering these four genomes to be the first published for the species M. caribbica Raw reads and assembled contigs have been made public to further the study of these organisms.

Mosquitoes can harbour yeasts of clinical significance and contribute to their environmental dissemination.

There is still a lack of studies on fungal microbiota in mosquitoes, compared with the number available on bacterial microbiota. This study reports the identification of yeasts of clinical significance in laboratory mosquito species: Anopheles gambiae, Anopheles stephensi, Culex quinquefasciatus, Aedes albopictus and Aedes aegypti. Among the yeasts isolated, they focused on the opportunistic pathogen Candida parapsilosis, since there is a need to better understand breakthrough candidaemia with resistance to the usual antifungals, which requires careful consideration in the broad-spectrum therapy, as documented in many clinical reports. C. parapsilosis occurs widely and has been isolated from diverse sources, including insects, which may contribute to its dissemination. In this study, it was isolated from the gut of An. gambiae and its presence in developmental stages and organs of different mosquito species was studied. Our results indicated that there was a stable association between C. parapsilosis and reared mosquitoes during the entire life cycle, and in adult male and female gut and gonads. A wide occurrence of C. parapsilosis was also documented in several populations of wild mosquitoes. Based on these findings, it can be said that mosquitoes might participate in the spreading of this opportunistic pathogen, not only as a carrier.

A yeast strain associated to Anopheles mosquitoes produces a toxin able to kill malaria parasites.

BACKGROUND: Malaria control strategies are focusing on new approaches, such as the symbiotic control, which consists in the use of microbial symbionts to prevent parasite development in the mosquito gut and to block the transmission of the infection to humans. Several microbes, bacteria and fungi, have been proposed for malaria or other mosquito-borne diseases control strategies. Among these, the yeast Wickerhamomyces anomalus has been recently isolated from the gut of Anopheles mosquitoes, where it releases a natural antimicrobial toxin. Interestingly, many environmental strains of W. anomalus exert a wide anti-bacterial/fungal activity and some of these 'killer' yeasts are already used in industrial applications as food and feed bio-preservation agents. Since a few studies showed that W. anomalus killer strains have antimicrobial effects also against protozoan parasites, the possible anti-plasmodial activity of the yeast was investigated. METHODS: A yeast killer toxin (KT), purified through combined chromatographic techniques from a W. anomalus strain isolated from the malaria vector Anopheles stephensi, was tested as an effector molecule to target the sporogonic stages of the rodent malaria parasite Plasmodium berghei, in vitro. Giemsa staining was used to detect morphological damages in zygotes/ookinetes after treatment with the KT. Furthermore, the possible mechanism of action of the KT was investigated pre-incubating the protein with castanospermine, an inhibitor of ß-glucanase activity. RESULTS: A strong anti-plasmodial effect was observed when the P. berghei sporogonic stages were treated with KT, obtaining an inhibition percentage up to around 90%. Microscopy analysis revealed several ookinete alterations at morphological and structural level, suggesting the direct implication of the KT-enzymatic activity. Moreover, evidences of the reduction of KT activity upon treatment with castanospermine propose a ß-glucanase-mediated activity. CONCLUSION: The results showed the in vitro killing efficacy of a protein produced by a mosquito strain of W. anomalus against malaria parasites. Further studies are required to test the KT activity against the sporogonic stages in vivo, nevertheless this work opens new perspectives for the possible use of killer strains in innovative strategies to impede the development of the malaria parasite in mosquito vectors by the means of microbial symbionts.

A rapid qPCR method to investigate the circulation of the yeast Wickerhamomyces anomalus in humans.

The yeast Wickerhamomyces anomalus has been proposed for many biotechnological applications in the food industry. However, a number of opportunistic pathogenic strains have been reported as causative agents of nosocomial fungemia. Recognition of potentially pathogenic isolates is an important challenge for the future commercialization of this yeast. The isolation of W. anomalus from different matrices and, recently, from mosquitoes, requires further investigations into its circulation in humans. Here we present a qPCR protocol for the detection of W. anomalus in human blood samples and the results of a screening of 525 donors, including different classes of patients and healthy people.