Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Diversity of cave Phlebotomines (Diptera: Psychodidae) from a Colombian cave

Sandflies are vector species of Leishmania, among many other pathogens, with a global distribution and a variety of ecological niches. Previous samplings have found that karstic formations (i.e., caves and folds formed by the erosion of limestone) serve as a natural habitat to sandfly species. The majority of samplings of cave sandfly diversity have occurred in Brazil and to date none have studied the species composition in a cave in the Northern Andes. We collected sandflies in the Cave-Los Guacharos-, in the state of Antioquia, Colombia. The sampling was carried out during two consecutive nights in September 2019. CDC-type light traps were installed inside the cavern and in other surrounding karst systems (caves and folds). In total, we identified 18 species of sandfly from the cave and surrounding karst systems, including three new records for Colombia (Bichromomyia olmeca nociva, Brumptomyia brumpti, and Warileya leponti), and provide the first karstic reports for four other species (Lutzomyia gomezi, Lutzomyia hartmanni, Pintomyia ovallesi, and Psychodopygus panamensis). We then used the results of our survey and published literature to test two hypotheses. First, that sandfly diversity in Neotropical caves is richest nearer to the equator and second that there is a phylogenetic signal of karstic habitat use in sandflies. Counter to our predictions, we found no evidence that diversity follows a latitudinal gradient. Further, we find no evidence of a phylogenetic signal of karstic habitat use, instead finding that the use of caves likely evolved multiple times across several genera. Our results highlight the importance of a wide sampling to understand the natural habitat of sandflies and other disease vectors.

Rapid Differentiation of Vector Species: Development of Microsatellite Markers for Population Genetics of Biting Midges and a Potential Tool for Species Identification of Culicoides Sonorensis

Background: Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, a SNP analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance. Methods: The aim of the current study was to develop a reliable and affordable way of differentiating the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers which were also tested for species differentiation.Results: A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recover the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately differentiating the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested. Conclusions: These microsatellites provide an affordable way in which to differentiate the species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America.

Climate Suitable Conditions Index and Autochthonous Dengue Infections: an Early Warning Index

Background: Aedes aegypti is the primary vector of dengue and contributes to most major epidemics of this virus worldwide. Aedes albopictus is also blamed for certain epidemics, as the 2014 Guangdong dengue outbreak. In Guangdong province, Ae. albopictus is thought to be the dominant vector species, with Ae. aegypti absent from most areas. Whether or not primary mosquito vectors are present, optimal climatic conditions for dengue vector species may play a substantial role in epidemics of the virus. We hypothesise that although vector species are required to initiate and establish an outbreak, favourable weather conditions may then grow and perpetuate the outbreak via complex effects on vector sympatry or interactions.Methods: Vector spices-specific suitable conditions index (SCI) and autochthonous dengue case data were fitted to negative binomial (NB) regression models. After accounting for potential confounders, we assessed the relationship between SCI and autochthonous dengue cases. We assumed SCI interaction was a proxy for vector species sympatry and SCI difference a proxy for interspecies competition. Finally, we explored the relationship between these assumed conditions and the autochthonous dengue case. Results: Autochthonous dengue cases are associated negatively with SCI for Ae.albopictus and positively with SCI for Ae.aegypti. According to the NB regression models, autochthonous dengue cases increased 4% (Incident Rate Ratio (IRR): 1.04, 95% CI: 1.02, 1.06) for every unit increase in SCI for Ae. aegypti, but decreased by 3% (IRR: 0.97, 95% CI: 0.96, 0.99) for Ae. albopictus SCI. There was also an interaction between two SCIs and a positive effect of the difference in SCIs on autochthonous dengue cases. These findings support the hypothesis that vector sympatry and interactions may influence the risk of a dengue outbreak.Conclusions: Our results confirm the hypothesis that the dengue virus is more transmissible in regions with warmer weather conditions (high SCI for Ae. aegypti). SCI of Ae. aegypti would be a valuable index to predict dengue transmission even in the absence of Ae. aegypti but with Ae. albopictus present. The results also support that the SCI is beneficial for evaluating dengue outbreak risk in terms of vector sympatry and interactions in the absence of entomology data in future research.

Species complex of mosquitos captured by mechanical traps and an equine host

Field studies were conducted to evaluate the efficacy of commercially available mosquito traps to capture suspected West Nile Virus (WNV) vectors and compare the relative abundance, and species composition of mosquitoes captured by traps. Traps included the Mosquito Magnet (MM) Pro, MM-Liberty, MM-X, the CDC-1012, and the Home and Garden (HGM) prototype trap. In the first study, conducted at the University of Florida Horse Teaching Unit (HTU), Gainesville, FL, from May 17 to September 30, 2002, the CDC-1012 caught significantly more mosquitoes than the other traps. The HGM trap did not catch any mosquitoes and was replaced with a horse on July 17. Percentages of WNV vector species captured in traps and vacuumed from the horse, respectively, were Culex nigripalpus Theobald 85-91% and 27%, and Mansonia titillans (Walker) 2-5% and 40%. In the second study, performed for 5 days in April 2003, the MM-Pro, MM-X, and CDC-1012 traps caught significantly more mosquitoes than did the MM-Liberty trap. All traps captured primarily Cx. salinarius Coquillett, Cx. erraticus (Dyar and Knab), and Anopheles crucians Wiedemann. The horse attracted a greater percentage of Ma. titillans and less Cx. erraticus than did the mechanical traps. The mechanical traps may offer horses some degree of protection from suspected WNV species, however, a large number of Ma. titillans were recovered from the horse but the traps captured very few.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI’s nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

Species Diversity of Arthropods Founds in Refuse Dumpsites in Ifite Awka, Anambra State and Its Public Health Risk

A survey of some refuse dumpsites in Ifite, Awka, Anambra, Nigeria State were carried out for 5 weeks to determine the arthropod vectors associated with those refuse dumps and their relative abundance per site and species diversity. Five refuse dumpsites were randomly selected based on their composition and human activities within the environment. The sampled sites are Commissioners Quarters (site A), Second Market (site B), Miracle Junction (site C), Star-Lodge Junction (site D) and Wintess Hotel (site E). Fourteen species of arthropods were collected in eight orders and eleven families namely; Muscidae, Culicidae, Blattidae, Calliphoridae, Formicidae, Elateridae, Xystodesmidae, Polydesmidae, Acrididae, Lygaeidae and Selonopidae. The most abundant vector species encountered in all five study sites was Musca domestica, the dominant species, followed by Aedes. spp., and P. americana which occurred in all study sites except site A. Site A, M. domestica 12 (38.71%), followed by A. aegypti 6 (19.35%), site B, M. domestica 15 (25.42%), followed by A. aegypti 12 (20.34%) and P. Americana 10 (16.95%), site C, M. domestica 17 (28.89%), followed by A. aegypti 7 (15.56%) and P. Americana 5 (11.11%), site D, M. domestica 17 (27.42%), followed by A. aegypti 11 (17.74%) and P. americana 7 (11.29%), site E, M. domestica 30 (32.26%), followed by A. spp.,17 (18.28%) and P. Americana 16 (17.20%).The abundance of these vectors suggests the prevalence of arthropod vector borne disease in Ifite, Awka environment. Proper disposal of refuse dumps and public enlightenment on the dangers of indiscriminate dumping of refuse to the general public is highly recommended to avert health and environmental problems associated with these arthropod vectors.

Transcriptome of the Maize Leafhopper ( Dalbulus maidis ) and Its Transcriptional Response to Maize Rayado Fino Virus (MRFV), Which It Transmits in a Persistent, Propagative Manner

The transcriptome of the corn leafhopper, D. maidis , revealed conserved biochemical pathways for immunity and discovered transcripts responsive to MRFV-infected plants at two time points, providing a basis for functional identification of genes that either limit or promote the virus-vector interaction. Compared to other hopper species and the propagative plant viruses they transmit, D. maidis shared 15 responsive transcripts with S. furcifera (to southern rice black-streaked dwarf virus [SRBSDV]), one with G. nigrifrons (to maize fine streak virus [MFSV]), and one with P. maidis (to maize mosaic virus [MMV]), but no virus-responsive transcripts identified were shared among all four hopper vector species.

Ecology of Powassan Virus in the United States

Zoonotic viruses threaten the lives of millions of people annually, exacerbated by climate change, human encroachment into wildlife habitats, and habitat destruction. The Powassan virus (POWV) is a rare tick-borne virus that can cause severe neurological damage and death, and the incidence of the associated disease (Powassan virus disease) is increasing in the eastern United States. The mechanisms by which POWV is maintained in nature and transmitted to humans are complex and only partly understood. This review provides an overview of what is known about the vector species, vector-host transmission dynamics, and environmental and human-driven factors that may be aiding the spread of both the vector and virus.

Species shifting composition of the Anopheles vector in Wongsorejo district - Banyuwangi, Indonesia

Malaria’s cases have been reported to occur annually in Wongsorejo district of Banyuwangi, East Java since 2002. However, there is a significant decrease of malaria cases during these last years, which might be related to the malaria vector species shifting composition. The objective of this research was to observe some important bionomic characteristics of malaria vector Anopheles in this area from 2015 until 2020 which include species identification, blood feeding behaviour and biting preference of vector. The data collection i.e. determination of Anopheles diversity and behaviour was conducted monthly for 3-6 months annually from 2015 – 2018. In 2019-2020 we sampled irregularly to see the trend. The results showed that there were species shifting of Anopheles vectors in this area. The proportion of Anopheles (An.) sundaicus and An. subpictus, which were previously reported until 2015 as the main Anopheles species in this area, significantly decreased in 2016 - 2018. An. indefinitus & An. vagus was becoming the majority of Anopheles species. During 2019-2020, An. vagus became the main species identified in the sampling area. The predominant species of An. vagus and An. indefinitus has exophagic and zoophilic preference behaviour. Furthermore, An. indefinitus has not previously been identified as a vector for malaria, compared to An. sundaius, and An. subpictus which are well known as an important primary malaria vector on Java Island, Indonesia.