Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis.

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host's metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly's resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse's viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse's reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.

Genotyping of whole genome amplified reduced representation libraries reveals a cryptic population of Culicoides brevitarsis in the Northern Territory, Australia.

BACKGROUND: The advent of genotyping by Next Generation Sequencing has enabled rapid discovery of thousands of single nucleotide polymorphism (SNP) markers and high throughput genotyping of large populations at an affordable cost. Genotyping by sequencing (GBS), a reduced representation library sequencing method, allows highly multiplexed sequencing of genomic subsets. This method has limitations for small organisms with low amounts of genomic DNA, such as the bluetongue virus (BTV) vectors, Culicoides midges. RESULTS: This study employed the GBS method to isolate SNP markers de novo from whole genome amplified Culicoides brevitarsis genomic DNA. The individuals were collected from regions representing two different Australian patterns of BTV strain distribution: the Northern Territory (NT) and the east coast. We isolated 8145 SNPs using GBS. Phylogenetic analysis conducted using the filtered 3263 SNPs revealed the presence of a distinct C. brevitarsis sub-population in the NT and this was confirmed by analysis of mitochondrial DNA. Two loci showed a very strong signal for selection and were unique to the NT population. Bayesian analysis with STRUCTURE indicated a possible two-population cluster. CONCLUSIONS: The results suggest that genotyping vectors with high density markers in combination with biological and environmental data is useful. However, more extensive sampling over a wider spatial and temporal range is needed. The presence of sub-structure in populations and loci under natural selection indicates the need for further investigation of the role of vectors in shaping the two Australian systems of BTV transmission. The described workflow is transferable to genotyping of small, non-model organisms, including arthropod vectors of pathogens of economic and medical importance.

Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera: Ceratopogonidae), using multi-locus DNA microsatellites.

Bluetongue virus (BTV) is a major pathogen of ruminants that is transmitted by biting midges (Culicoides spp.). Australian BTV serotypes have origins in Asia and are distributed across the continent into two distinct episystems, one in the north and another in the east. Culicoides brevitarsis is the major vector of BTV in Australia and is distributed across the entire geographic range of the virus. Here, we describe the isolation and use of DNA microsatellites and gauge their ability to determine population genetic connectivity of C. brevitarsis within Australia and with countries to the north. Eleven DNA microsatellite markers were isolated using a novel genomic enrichment method and identified as useful for genetic analyses of sampled populations in Australia, northern Papua New Guinea (PNG) and Timor-Leste. Significant (P < 0.05) population genetic subdivision was observed between all paired regions, though the highest levels of genetic sub-division involved pair-wise tests with PNG (PNG vs. Australia (FST = 0.120) and PNG vs. Timor-Leste (FST = 0.095)). Analysis of multi-locus allelic distributions using STRUCTURE identified a most probable two-cluster population model, which separated PNG specimens from a cluster containing specimens from Timor-Leste and Australia. The source of incursions of this species in Australia is more likely to be Timor-Leste than PNG. Future incursions of BTV positive C. brevitarsis into Australia may be genetically identified to their source populations using these microsatellite loci. The vector's panmictic genetic structure within Australia cannot explain the differential geographic distribution of BTV serotypes.

Aedes albopictus saliva contains a richer microbial community than the midgut.

BACKGROUND: Past findings demonstrate that arthropods can egest midgut microbiota into the host skin leading to dual colonization of the vertebrate host with pathogens and saliva microbiome. A knowledge gap exists on how the saliva microbiome interacts with the pathogen in the saliva. To fill this gap, we need to first define the microbial composition of mosquito saliva. METHODS: The current study aimed at analyzing and comparing the microbial profile of Aedes albopictus saliva and midgut as well as assessing the impact of Zika virus (ZIKV) infection on the midgut and saliva microbial composition. Colony-reared Ae. albopictus strains were either exposed to ZIKV infectious or noninfectious bloodmeal. At 14 ays postinfection, the 16S V3-V4 hypervariable rRNA region was amplified from midgut and saliva samples and sequenced on an Illumina MiSeq platform. The relative abundance and diversity of midgut and saliva microbial taxa were assessed. RESULTS: We observed a richer microbial community in the saliva compared with the midgut, yet some of the microbial taxa were common in the midgut and saliva. ZIKV infection did not impact the microbial diversity of midgut or saliva. Further, we identified Elizabethkingia spp. in the Ae. albopictus saliva. CONCLUSIONS: This study provides insights into the microbial community of the Ae. albopictus saliva as well as the influence of ZIKV infection on the microbial composition of its midgut and saliva. The identification of Elizabethkingia spp., an emerging pathogen of global health significance, in Ae. albopictus saliva is of medical importance. Future studies to assess the interactions between Ae. albopictus saliva microbiome and ZIKV could lead to novel strategies for developing transmission barrier tools.

Viral metagenomics demonstrates that domestic pigs are a potential reservoir for Ndumu virus.

BACKGROUND: The rising demand for pork has resulted in a massive expansion of pig production in Uganda. This has resulted in increased contact between humans and pigs. Pigs can act as reservoirs for emerging infectious diseases. Therefore identification of potential zoonotic pathogens is important for public health surveillance. In this study, during a routine general surveillance for African swine fever, domestic pigs from Uganda were screened for the presence of RNA and DNA viruses using a high-throughput pyrosequencing method. FINDINGS: Serum samples from 16 domestic pigs were collected from five regions in Uganda and pooled accordingly. Genomic DNA and RNA were extracted and sequenced on the 454 GS-FLX platform. Among the sequences assigned to a taxon, 53% mapped to the domestic pig (Sus scrofa). African swine fever virus, Torque teno viruses (TTVs), and porcine endogenous retroviruses were identified. Interestingly, two pools (B and C) of RNA origin had sequences that showed 98% sequence identity to Ndumu virus (NDUV). None of the reads had identity to the class Insecta indicating that these sequences were unlikely to result from contamination with mosquito nucleic acids. CONCLUSIONS: This is the first report of the domestic pig as a vertebrate host for Ndumu virus. NDUV had been previously isolated only from culicine mosquitoes. NDUV therefore represents a potential zoonotic pathogen, particularly given the increasing risk of human-livestock-mosquito contact.

Zika virus and temperature modulate Elizabethkingia anophelis in Aedes albopictus.

BACKGROUND: Vector-borne pathogens must survive and replicate in the hostile environment of an insect's midgut before successful dissemination. Midgut microbiota interfere with pathogen infection by activating the basal immunity of the mosquito and by synthesizing pathogen-inhibitory metabolites. METHODS: The goal of this study was to assess the influence of Zika virus (ZIKV) infection and increased temperature on Aedes albopictus midgut microbiota. Aedes albopictus were reared at diurnal temperatures of day 28 °C/night 24 °C (L) or day 30 °C/night 26 °C (M). The mosquitoes were given infectious blood meals with 2.0 × 108 PFU/ml ZIKV, and 16S rRNA sequencing was performed on midguts at 7 days post-infectious blood meal exposure. RESULTS: Our findings demonstrate that Elizabethkingia anophelis albopictus was associated with Ae. albopictus midguts exposed to ZIKV infectious blood meal. We observed a negative correlation between ZIKV and E. anophelis albopictus in the midguts of Ae. albopictus. Supplemental feeding of Ae. albopictus with E. anophelis aegypti and ZIKV resulted in reduced ZIKV infection rates. Reduced viral loads were detected in Vero cells that were sequentially infected with E. anophelis aegypti and ZIKV, dengue virus (DENV), or chikungunya virus (CHIKV). CONCLUSIONS: Our findings demonstrate the influence of ZIKV infection and temperature on the Ae. albopictus microbiome along with a negative correlation between ZIKV and E. anophelis albopictus. Our results have important implications for controlling vector-borne pathogens.

The Vector - Host - Pathogen Interface: The Next Frontier in the Battle Against Mosquito-Borne Viral Diseases?

An unprecedented spread of mosquito-borne viruses and increasing populations of mosquito vectors has led to an increase in the frequency of mosquito-borne virus disease outbreaks. Recent outbreaks of Zika virus (ZIKV) and yellow fever virus (YFV), among others have led to a concerted effort to understand the biology of mosquito-borne viruses and their interaction with their vector mosquito and vertebrate hosts. Recent studies have aimed to understand the vector-host-pathogen interface and how it influences infection, tropism and disease severity in the vertebrate host. The initial replication of the pathogen at the skin bite site is crucial in determining the progression of the infection in the vertebrate host. Delineating the role of the commensal microbes in the mosquito saliva as well as how they interact with the vertebrate host keratinocytes will improve our understanding of disease immunopathology and may lead to new therapeutics.

Zika Virus Infection Results in Biochemical Changes Associated With RNA Editing, Inflammatory and Antiviral Responses in Aedes albopictus.

Rapid and significant range expansion of both the Zika virus (ZIKV) and its Aedes vector species has resulted in the declaration of ZIKV as a global health threat. Successful transmission of ZIKV by its vector requires a complex series of interactions between these entities including the establishment, replication and dissemination of the virus within the mosquito. The metabolic conditions within the mosquito tissues play a critical role in mediating the crucial processes of viral infection and replication and represent targets for prevention of virus transmission. In this study, we carried out a comprehensive metabolomic phenotyping of ZIKV infected and uninfected Ae. albopictus by untargeted analysis of primary metabolites, lipids and biogenic amines. We performed a comparative metabolomic study of infection state with the aim of understanding the biochemical changes resulting from the interaction between the ZIKV and its vector. We have demonstrated that ZIKV infection results in changes to the cellular metabolic environment including a significant enrichment of inosine and pseudo-uridine (Ψ) levels which may be associated with RNA editing activity. In addition, infected mosquitoes demonstrate a hypoglycemic phenotype and show significant increases in the abundance of metabolites such as prostaglandin H2, leukotriene D4 and protoporphyrinogen IX which are associated with antiviral activity. These provide a basis for understanding the biochemical response to ZIKV infection and pathology in the vector. Future mechanistic studies targeting these ZIKV infection responsive metabolites and their associated biosynthetic pathways can provide inroads to identification of mosquito antiviral responses with infection blocking potential.

Corrigendum: Zika Virus Infection Results in Biochemical Changes Associated With RNA Editing, Inflammatory and Antiviral Responses in Aedes albopictus.

[This corrects the article DOI: 10.3389/fmicb.2020.559035.].

Increased temperatures reduce the vectorial capacity of Aedes mosquitoes for Zika virus.

Rapid and significant range expansion of both Zika virus (ZIKV) and its Aedes vector species has resulted in ZIKV being declared a global health threat. Mean temperatures are projected to increase globally, likely resulting in alterations of the transmission potential of mosquito-borne pathogens. To understand the effect of diurnal temperature range on the vectorial capacity of Ae. aegypti and Ae. albopictus for ZIKV, longevity, blood-feeding and vector competence were assessed at two temperature regimes following feeding on infectious blood meals. Higher temperatures resulted in decreased longevity of Ae. aegypti [Log-rank test, χ2, df 35.66, 5, P < 0.001] and a decrease in blood-feeding rates of Ae. albopictus [Fisher's exact test, P < 0.001]. Temperature had a population and species-specific impact on ZIKV infection rates. Overall, Ae. albopictus reared at the lowest temperature regime demonstrated the highest vectorial capacity (0.53) and the highest transmission efficiency (57%). Increased temperature decreased vectorial capacity across groups yet more significant effects were measured with Ae. aegypti relative to Ae. albopictus. The results of this study suggest that future increases in temperature in the Americas could significantly impact vector competence, blood-feeding and longevity, and potentially decrease the overall vectorial capacity of Aedes mosquitoes in the Americas.

Spatio-temporal distribution of Spiroplasma infections in the tsetse fly (Glossina fuscipes fuscipes) in northern Uganda.

Tsetse flies (Glossina spp.) are vectors of parasitic trypanosomes, which cause human (HAT) and animal African trypanosomiasis (AAT) in sub-Saharan Africa. In Uganda, Glossina fuscipes fuscipes (Gff) is the main vector of HAT, where it transmits Gambiense disease in the northwest and Rhodesiense disease in central, southeast and western regions. Endosymbionts can influence transmission efficiency of parasites through their insect vectors via conferring a protective effect against the parasite. It is known that the bacterium Spiroplasma is capable of protecting its Drosophila host from infection with a parasitic nematode. This endosymbiont can also impact its host's population structure via altering host reproductive traits. Here, we used field collections across 26 different Gff sampling sites in northern and western Uganda to investigate the association of Spiroplasma with geographic origin, seasonal conditions, Gff genetic background and sex, and trypanosome infection status. We also investigated the influence of Spiroplasma on Gff vector competence to trypanosome infections under laboratory conditions. Generalized linear models (GLM) showed that Spiroplasma probability was correlated with the geographic origin of Gff host and with the season of collection, with higher prevalence found in flies within the Albert Nile (0.42 vs 0.16) and Achwa River (0.36 vs 0.08) watersheds and with higher prevalence detected in flies collected in the intermediate than wet season. In contrast, there was no significant correlation of Spiroplasma prevalence with Gff host genetic background or sex once geographic origin was accounted for in generalized linear models. Additionally, we found a potential negative correlation of Spiroplasma with trypanosome infection, with only 2% of Spiroplasma infected flies harboring trypanosome co-infections. We also found that in a laboratory line of Gff, parasitic trypanosomes are less likely to colonize the midgut in individuals that harbor Spiroplasma infection. These results indicate that Spiroplasma infections in tsetse may be maintained by not only maternal but also via horizontal transmission routes, and Spiroplasma infections may also have important effects on trypanosome transmission efficiency of the host tsetse. Potential functional effects of Spiroplasma infection in Gff could have impacts on vector control approaches to reduce trypanosome infections.

Delineation of the population genetic structure of Culicoides imicola in East and South Africa.

BACKGROUND: Culicoides imicola Kieffer, 1913 is the main vector of bluetongue virus (BTV) and African horse sickness virus (AHSV) in Sub-Saharan Africa. Understanding the population genetic structure of this midge and the nature of barriers to gene flow will lead to a deeper understanding of bluetongue epidemiology and more effective vector control in this region. METHODS: A panel of 12 DNA microsatellite markers isolated de novo and mitochondrial DNA were utilized in a study of C. imicola populations from Africa and an outlier population from the Balearic Islands. The DNA microsatellite markers and mitochondrial DNA were also used to examine a population of closely related C. bolitinos Meiswinkel midges. RESULTS: The microsatellite data suggest gene flow between Kenya and south-west Indian Ocean Islands exist while a restricted gene flow between Kenya and South Africa C. imicola populations occurs. Genetic distance correlated with geographic distance by Mantel test. The mitochondrial DNA analysis results imply that the C. imicola populations from Kenya and south-west Indian Ocean Islands (Madagascar and Mauritius) shared haplotypes while C. imicola population from South Africa possessed private haplotypes and the highest nucleotide diversity among the African populations. The Bayesian skyline plot suggested a population growth. CONCLUSIONS: The gene flow demonstrated by this study indicates a potential risk of introduction of new BTV serotypes by wind-borne infected Culicoides into the Islands. Genetic similarity between Mauritius and South Africa may be due to translocation as a result of human-induced activities; this could impact negatively on the livestock industry. The microsatellite markers isolated in this study may be utilised to study C. bolitinos, an important vector of BTV and AHSV in Africa and identify sources of future incursions.