De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

BACKGROUND: Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles malaria vector populations. RESULTS: Anopheles mosquitoes were sampled in malaria endemic forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp., and Anopheles dirus in Cambodia. The most frequent mosquito species sampled at both study sites are human malaria vectors. Small and long RNA sequences were depleted of mosquito host sequences, de novo assembled and clustered to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 115 novel virus sequences, and evidence supports a functional status for at least 86 of the novel viral contigs. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders were found in these Anopheles from Africa and Asia. The remaining non-host RNA assemblies that were unclassified by sequence similarity to known viruses were clustered by small RNA profiles, and 39 high-quality independent contigs strongly matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are entirely undescribed viruses. One thousand five hundred sixty-six additional high-quality unclassified contigs matched a pattern consistent with Piwi-interacting RNAs (piRNAs), suggesting that strand-biased piRNAs are generated from the natural virome in Anopheles. To functionally query piRNA effect, we analyzed piRNA expression in Anopheles coluzzii after infection with O'nyong nyong virus (family Togaviridae), and identified two piRNAs that appear to display specifically altered abundance upon arbovirus infection. CONCLUSIONS: Anopheles vectors of human malaria in Africa and Asia are ubiquitously colonized by RNA viruses, some of which are monophyletic but clearly diverged from other arthropod viruses. The interplay between small RNA pathways, immunity, and the virome may represent part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and could potentially influence vector competence.

Correction to: De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

Following the publication of this article [1], the authors reported that the original shading in columns 3 and 4 of Table 3, which indicated the presence or absence of viruses in each library, had been removed during typesetting.

Wolbachia and Asaia Distribution among Different Mosquito Vectors Is Affected by Tissue Localization and Host Species.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia-Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

Complete genome of Aedes japonicus narnavirus from wild caught mosquitoes collected in Ohio, USA.

Narnaviruses infect several genera of mosquitoes including Culex and Aedes . The narnavirus genome is a positive, single stranded RNA encoding an RNA-dependent RNA polymerase gene. The partial genome of a narnavirus identified in wild Aedes japonicus mosquitoes collected in Wooster, Ohio, USA was obtained using metagenomic analyses. Rapid amplification of 5'-cDNA ends (RACE) and Sanger sequencing were used to obtain the remaining genomic sequence of this strain. The complete genome is composed of 3153 nucleotides and has 98.4% and 99.1% nucleotide sequence identity with Aedes japonicus narnavirus genomes identified in Netherlands and Japan.

Invasive Aedes japonicus Mosquitoes Dominate the Aedes Fauna Collected with Gravid Traps in Wooster, Northeastern Ohio, USA.

Aedes japonicus (Diptera: Culicidae), or the Asian rock pool mosquito, is an invasive mosquito in Europe and America. It was first detected outside of Asia in 1990 in Oceania. It has since expanded to North America and Europe in 1998 and 2000, respectively. Even though it is classified as a secondary vector of pathogens, it is competent to several arboviruses and filarial worms, and it is contributing to the transmission of La Crosse virus (LACV) and West Nile virus (WNV). In this study, CDC light, BG-sentinel, and gravid traps were used to collect mosquitoes between June and October 2021, in Wooster, Northeastern Ohio, USA. Morphological identification or/and Sanger sequencing were performed to identify the collected mosquitoes. Our results revealed that (adult) Ae. japonicus mosquitoes were the most abundant mosquito species collected with gravid traps in Wooster in 2021, confirming its establishment in Ohio. Molecular analyses of Ae. japonicus showed 100% nucleotide similarity with Ae. japonicus collected in Iowa (USA) and Canada, suggesting multiple introductions. Its presence may increase the risk of future arbovirus outbreaks in Wooster, Ohio. This study stresses the importance of actively monitoring the density and distribution of all members of the Ae. japonicus complex.

Effects of Mating on Gene Expression in Female Insects: Unifying the Field.

There is intense interest in controlling insect reproductive output. In many insect species, reproductive output is profoundly influenced by mating, including the receipt of sperm and seminal fluid molecules, through physiological and behavior changes. To understand these changes, many researchers have investigated post-mating gene expression regulation. In this review, we synthesize information from studies both across and within different species about the impact of mating, or components of mating, on female gene expression patterns. We found that genes related to the roles of metabolism, immune-response, and chemosensation are regulated by mating across many different insect species. We highlight the few studies that have taken the important next step of examining the functional consequences of gene expression regulation which is crucial in order to understand the mechanisms underlying the mating-regulated control of female lifespan and reproduction and to make use of such knowledge to propagate or control insect populations. The potential of cross-study comparisons is diminished by different studies using different methods. Thus, we also include a consideration of how future studies could be designed to facilitate cross-study comparisons and a call for collaboration across researchers studying different insect species and different aspects of insect biology.

Insemination in Aedes aegypti and Aedes albopictus.

Aedes mosquitoes are the vectors of several arboviruses that cause human disease. A better understanding of their reproduction helps to improve their management and contributes insights into the fundamental biology of mosquitoes. During mating, inseminated mosquito females receive seminal fluids and sperm from males that they then store in the spermathecae. In Aedes aegypti and Aedes albopictus, most mated females become resistant to further insemination within 2 h of initial insemination. Although the male seminal fluids are known to be involved in initiating the resistance of inseminated females to further insemination, the mechanism underlying this resistance is not well-understood. The determination of insemination status is a key step in investigating the behavioral and molecular interactions between males and females and for exploring the proximate influences and evolutionary implications of interspecific copulations. Several methods exist for determining insemination status, as discussed here. The choice of method depends on the research question and the availability of resources.

Determining the Insemination Status of Aedes Mosquitoes.

Within the genus Aedes, the reproductive biology of two species has been most thoroughly studied: Aedes aegypti and Aedes albopictus In these species, females tend to copulate with one or more males once sexually mature. Within a few hours after an initial insemination, most females become refractory to insemination for the rest of their lives. Aedes females store sperm in three sclerotized spherical structures called spermathecae, where they can remain viable for >3 mo after copulation. This protocol outlines a quick (∼3-5 min per female once you are practiced in dissection) and effective method for dissecting the female spermathecae from Ae. aegypti and Ae. albopictus mosquitoes and scoring them for the presence or absence of sperm in cases in which the researcher does not need the female for further studies.

Factors enhancing the transmission of mosquito-borne arboviruses in Africa.

Arthropod-borne viruses (Arboviruses) replicate in vertebrates and invertebrates and are mainly transmitted by mosquitoes. Between 2000 and 2021, several arbovirus outbreaks were recorded in African countries, including dengue, yellow fever, Chikungunya, Zika, and O'nyong nyong. Most often, the causes and factors involved in these outbreaks are unknown. We aimed to understand current knowledge regarding factors responsible for the persistent transmission and emergence of mosquito-borne arboviruses in Africa and to identify critical research gaps important for preventing future outbreaks. We used a systematic literature review between 2020 and 2021, to show that the main identified factors favoring the arbovirus outbreak in Africa are low vaccination coverage, high density and diversity of competent mosquitoes, insecticide resistance of mosquito vectors, and a scarcity of data on arboviruses. Further studies on arboviruses may include studies of competence to viral strains and the susceptibility of mosquito vectors to insecticides. Because of the detrimental effects of insecticides on human health and the environment, viral paratransgenesis and other biological control methods should be explored as alternatives or as supplements to insecticides. Graphical abstract: Illustration of factors identified for promoting the transmission of arbovirus in Africa. The main factors are the lack of drugs and vaccines, low coverage of vaccination when a vaccine exists, competence of mosquitoes to viruses, diversity and high density of vectors. Climate change, urbanization, deforestation and agricultural practices, lead to a richness and high density of vectors.

Interaction of RNA viruses of the natural virome with the African malaria vector, Anopheles coluzzii.

Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. Here, we describe biological interactions between these two viruses and An. coluzzii mosquitoes. Viral abundance varies reproducibly during mosquito development. DNA forms of these viruses were not detected, and thus viral persistence is likely based on vertical transmission of RNA genomes. At least Anopheles C virus is vertically transmitted by an intraembryonic route. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. One possible mechanism for this could be interactions with host immunity, and functional genomic analysis indicated differential influence of at least the Toll and JAK/STAT immune signaling pathways upon the viruses. The nonrandom distributions and interactions with host immunity suggest that these and other members of the natural virome may constitute a source of unrecognized heterogeneity in mosquito vector populations.

A Systematic Review of the Natural Virome of Anopheles Mosquitoes.

Anopheles mosquitoes are vectors of human malaria, but they also harbor viruses, collectively termed the virome. The Anopheles virome is relatively poorly studied, and the number and function of viruses are unknown. Only the o'nyong-nyong arbovirus (ONNV) is known to be consistently transmitted to vertebrates by Anopheles mosquitoes. A systematic literature review searched four databases: PubMed, Web of Science, Scopus, and Lissa. In addition, online and print resources were searched manually. The searches yielded 259 records. After screening for eligibility criteria, we found at least 51 viruses reported in Anopheles, including viruses with potential to cause febrile disease if transmitted to humans or other vertebrates. Studies to date have not provided evidence that Anopheles consistently transmit and maintain arboviruses other than ONNV. However, anthropophilic Anopheles vectors of malaria are constantly exposed to arboviruses in human bloodmeals. It is possible that in malaria-endemic zones, febrile symptoms may be commonly misdiagnosed. It is also possible that anophelines may be inherently less competent arbovirus vectors than culicines, but if true, the biological basis would warrant further study. This systematic review contributes a context to characterize the biology, knowledge gaps, and potential public health risk of Anopheles viruses.