Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes Highlights d High-density Wolbachia strains found in An. moucheti and An. demeilloni mosquitoes d Infections are visualized in the ovaries, and maternal transmission was observed d Sequencing at depths and coverages comparable to other known Wolbachia strains d Homologs of cytoplasmic incompatibility factor genes are present in both genomes

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Insect-specific flaviviruses (ISFs), are a group of nonpathogenic flaviviruses that only infect insects. ISFs can have a high prevalence in mosquito populations, but their transmission routes are not well understood.

Skin snip evaluation for onchocerciasis has insufficient sensitivity when skin microfilarial (mf) densities are low, such as following ivermectin treatment. Mf density is suitable for assessing microfilaricidal efficacy but only serves as an indirect indicator of macrofilaricidal activity. We assessed circulating nucleic acids from Onchocerca volvulus as an alternative to skin snips. We screened a plasma sample set of infected individuals followed up at four, 12 and 21 months after microfilaricidal (ivermectin, n = four), macrofilaricidal (doxycycline, n = nine), or combination treatment (n = five). Two parasite-derived miRNAs, cel-miR-71-5p and bma-lin-4, and O-150 repeat DNA were assessed. Highly abundant DNA repeat families identified in the O. volvulus genome were also evaluated. miRNAs were detected in two of 72 plasma samples (2.8%) and two of 47 samples (4.3%) with microfilaridermia using RT-qPCR. O-150 DNA was detected in eight (44.4%) baseline samples by qPCR and the number of positives declined post-treatment. One doxycycline-treated individual remained O-150 positive. However, only 11 (23.4%) samples with microfilaridermia were qPCR-positive. Analysis by qPCR showed novel DNA repeat families were comparatively less abundant than the O-150 repeat. Circulating parasite-derived nucleic acids are therefore insufficient as diagnostic tools or as biomarkers of treatment efficacy for O. volvulus. Onchocerciasis, or "river blindness" is a parasitic disease caused by the filarial worm Onchocerca volvulus. Over 100 million people are at risk of infection, of which 99% reside in 31 sub-Saharan African countries endemic for onchocerciasis 1,2. In Africa, the standard elimination strategy consists of annual mass drug administration (MDA) with ivermectin (Mectizan; IVM). IVM kills microfilariae (mf) in the skin and is used in MDA programmes, which aim to reduce disease burden and block transmission to black fly vectors (Simulium spp.) 3. A specific and sensitive diagnostic test is needed for onchocerciasis 'end-game' scenarios, both to verify elimination and to detect cases when endemicity levels no longer justify MDA 4. High sensitivity is required for O. volvulus hypoendemic areas to detect low mf densities, as well as occult and amicrofilaridermic infections, and to monitor infection recrudescence 5. High specificity is also required to discriminate between closely related filarial nematodes with overlapping geographic distributions. This is particularly relevant in areas co-endemic for O. volvulus and the filarial worm Loa loa, such as in "hypoendemic hotspots" 6 , where IVM treatment can cause serious adverse events (SAEs) when L. loa microfilaraemia is high (>30,000 mf/ml) 7. In these areas, alternative strategies with drugs that are safe to use with loiasis will be required to meet elimination targets 8. Alternative 'test and (not) treat' (TaNT) approaches, Loa-first and Oncho-first, can be used to identify and exclude people at risk of SAEs or those not infected with onchocerciasis 9. While the new rapid Loa...

Significance Lymphatic filariasis caused by Wuchereria bancrofti , Brugia malayi , and Brugia timori affects 51 million people, leading to severe physical and mental disabilities. A mutualistic symbiosis between these filarial nematodes and Wolbachia bacteria has been exploited as a new curative treatment. Epidemiological modeling of anti- Wolbachia treatment assumes that transmission persists due to the lag phase before microfilariae (mf) become removed from circulation. Here, we show that Wolbachia -depleted mf cannot develop within the mosquito vector—a phenotype associated with down-regulation of B. malayi mf-specific chitinase, an enzyme essential for exsheathment. Our findings add to the broad range of host biological processes dependent on Wolbachia and suggest that anti- Wolbachia treatment mediates a more accelerated impact on elimination of lymphatic filariasis than currently predicted.

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis , divided into several ‘supergroups’ based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens, including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles gambiae and Anopheles funestus complexes appear at low density, established solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within Anopheles demeilloni and Anopheles moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here, we utilize genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size, with this reduction potentially attributable to degenerated prophage regions. Even with this reduction, we confirmed the presence of cytoplasmic incompatibility (CI) factor genes within both strains, with wAnD maintaining intact copies of these genes while the cifB gene was interrupted in wAnM, so functional analysis is required to determine whether wAnM can induce CI. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, rather than by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes, to our knowledge, that enable us to study the relationship between natural strain Plasmodium malaria parasites and their anopheline hosts.

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis, divided into several ‘supergroups’ based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles (A.) gambiae and A. funestus complexes appear at low density based solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within A. demeilloni and A. moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here we utilise genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size. We further confirm the presence of cytoplasmic incompatibility factor genes, despite noticeably few prophage regions. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, and unlikely to be by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes that enable us to study the relationship between natural strains Plasmodium malaria parasites and their Anopheline hosts.Impact statementWolbachia naturally infects a wide range of arthropod species, including insect vectors of human pathogens, where they may play a role in inhibiting their replication. These bacteria have been commonly found within Aedes (Ae.) albopictus and Culex pipiens mosquitoes but have been noticeably absent in the Anopheles mosquito genera, which includes all species responsible for malaria transmission. Recent PCR-based methods have suggested the potential for natural Wolbachia strains within the A. gambiae species complex, which includes major malaria vector species including A. gambiae s.s., A. coluzzii and A. arabiensis. We recently reported the presence of stable Wolbachia strains naturally occurring within two different Anopheles species (A. demeilloni and A. moucheti). In this study, we perform comparative genomic analysis of these two Wolbachia genomes against each other and published Wolbachia strains. The current assemblies are some of the smallest sequenced Wolbachia strains of insects, although their metabolic pathway repertoire is comparable to other strains. Interestingly, prophage fragments were identified within only one of the two strains. The findings of this study will be of significant interest to researchers investigating Wolbachia as a potential malaria biocontrol strategy, giving greater insight into the evolution and diversity of this obligate intracellular endosymbiont.Data summarySequence data generated and used for this analysis are available in the National Centre for Biotechnology Information Sequence Read Archive (NCBI SRA bioproject number PRJNA642000). The two assembled Wolbachia genomes are available with genome accession numbers GCA_018491735.2 and GCA_018491625.2. Additional Wolbachia genomes used for comparative analysis are described in the supplementary material.The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Additional supplementary data files used to generate several figures can be found at: https://figshare.com/projects/Wolbachia_endosymbionts_in_two_Anopheles_species_indicates_independent_acquisitions_and_lack_of_prophage_elements/126533

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, far more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities are associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique.