Exploring potentialities of avian genomic research in Nepalese Himalayas.

Nepal, a small landlocked country in South Asia, holds about 800 km of Himalayan Mountain range including the Earth's highest mountain. Within such a mountain range in the north and plain lowlands in the south, Nepal provides a habitat for about 9% of global avian fauna. However, this diversity is underrated because of the lack of enough studies, especially using molecular tools to quantify and understand the distribution patterns of diversity. In this study, we reviewed the studies in the last two decades (2000‒2019) that used molecular methods to study the biodiversity in Nepal to examine the ongoing research trend and focus. Although Nepalese Himalaya has many opportunities for cutting-edge molecular research, our results indicated that the rate of genetic/genomic studies is much slower compared to the regional trends. We found that genetic research in Nepal heavily relies on resources from international institutes and that too is mostly limited to research on species monitoring, distribution, and taxonomic validations. Local infrastructures to carry out cutting-edge genomic research in Nepal are still in their infancy and there is a strong need for support from national/international scientists, universities, and governmental agencies to expand such genomic infrastructures in Nepal. We particularly highlight avian fauna as a potential future study system in this region that can be an excellent resource to explore key biological questions such as understanding eco-physiology and molecular basis of organismal persistence to changing environment, evolutionary processes underlying divergence and speciation, or mechanisms of endemism and restrictive distribution of species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40657-021-00290-5.

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida, USA.

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two-step PCR assay to detect Wolbachia in wild-collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop-mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia-positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.

Experimental Evaluation of the Role of Ecologically-Relevant Hosts and Vectors in Japanese Encephalitis Virus Genotype Displacement.

Japanese encephalitis virus (JEV) is a flavivirus that is maintained via transmission between Culex spp. mosquitoes and water birds across a large swath of southern Asia and northern Australia. Currently JEV is the leading cause of vaccine-preventable encephalitis in humans in Asia. Five genotypes of JEV (G-I⁻G-V) have been responsible for historical and current outbreaks in endemic regions, and G-I and G-III co-circulate throughout Southern Asia. While G-III has historically been the dominant genotype worldwide, G-I has gradually but steadily displaced G-III. The objective of this study was to better understand the phenomenon of genotype displacement for JEV by evaluating both avian host and mosquito vector susceptibilities to infection with representatives from both G-I and G-III. Since ducks and Culex quinquefasciatus mosquitoes are prevalent avian hosts and vectors perpetuating JEV transmission in JE endemic areas, experimental evaluation of virus replication in these species was considered to approximate the natural conditions necessary for studying the role of host, vectors and viral fitness in the JEV genotype displacement context. We evaluated viremia in ducklings infected with G-I and G-III, and did not detect differences in magnitude or duration of viremia. Testing the same viruses in mosquitoes revealed that the rates of infection, dissemination and transmission were higher in virus strains belonging to G-I than G-III, and that the extrinsic incubation period was shorter for the G-I strains. These data suggest that the characteristics of JEV infection of mosquitoes but not of ducklings, may have play a role in genotype displacement.

Colonized Sabethes cyaneus, a Sylvatic New World Mosquito Species, Shows a Low Vector Competence for Zika Virus Relative to Aedes aegypti.

The introduction of Zika virus (ZIKV) to the Americas raised concern that the virus would spill back from human transmission, perpetuated by Aedes aegypti, into a sylvatic cycle maintained in wildlife and forest-living mosquitoes. In the Americas, Sabethes species are vectors of sylvatic yellow fever virus (YFV) and are therefore candidate vectors of a sylvatic ZIKV cycle. To test the potential of Sabethes cyaneus to transmit ZIKV, Sa. cyaneus and Ae. aegypti were fed on A129 mice one or two days post-infection (dpi) with a ZIKV isolate from Mexico. Sa. cyaneus were sampled at 3, 4, 5, 7, 14, and 21 days post-feeding (dpf) and Ae. aegypti were sampled at 14 and 21 dpf. ZIKV was quantified in mosquito bodies, legs, and saliva to measure infection, dissemination, and potential transmission, respectively. Of 69 Sa. cyaneus that fed, ZIKV was detected in only one, in all body compartments, at 21 dpf. In contrast, at 14 dpf 100% of 20 Ae. aegypti that fed on mice at 2 dpi were infected and 70% had virus in saliva. These data demonstrate that Sa. cyaneus is a competent vector for ZIKV, albeit much less competent than Ae. aegypti.

Mosquitoes as a Potential Vector of Ranavirus Transmission in Terrestrial Turtles.

Ranaviruses are significant pathogens of amphibians, reptiles, and fishes, contributing to mass mortality events worldwide. Despite an increasing focus on ranavirus ecology, our understanding of ranavirus transmission, especially among reptilian hosts, remains limited. For example, experimental evidence for oral transmission of the virus in chelonians is mixed. Consequently, vector-borne transmission has been hypothesized in terrestrial turtle species. To test this hypothesis, mosquitoes captured during a 2012/2013 ranavirus outbreak in box turtles from southwestern Indiana were pooled by genus and tested for ranavirus DNA using qPCR. Two of 30 pools tested positive for ranavirus. Additionally, an individual Aedes sp. mosquito observed engorging on a box turtle also tested positive for ranavirus. Although our approach does not rule out the possibility that the sequenced ranavirus was simply from virus in bloodmeal, it does suggests that mosquitoes may be involved in virus transmission as a mechanical or biological vector among ectothermic vertebrates. While additional studies are needed to elucidate the exact role of mosquitoes in ranavirus ecology, our study suggests that a greater focus on vector-borne transmission may be necessary to fully understand ranaviral disease dynamics in herpetofauna.