Cryopreservation of Anopheles stephensi embryos

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

Molecular Analysis Reveals a High Diversity of Anopheline Mosquitoes in Yanomami Lands and the Pantanal Region of Brazil

Identifying the species of the subfamily Anophelinae that are Plasmodium vectors is important to vector and malaria control. Despite the increase in cases, vector mosquitoes remain poorly known in Brazilian indigenous communities. This study explores Anophelinae mosquito diversity in the following areas: (1) a Yanomami reserve in the northwestern Amazon Brazil biome and (2) the Pantanal biome in southwestern Brazil. This is carried out by analyzing cytochrome c oxidase (COI) gene data using Refined Single Linkage (RESL), Assemble Species by Automatic Partitioning (ASAP), and tree-based multi-rate Poisson tree processes (mPTP) as species delimitation approaches. A total of 216 specimens collected from the Yanomami and Pantanal regions were sequenced and combined with 547 reference sequences for species delimitation analyses. The mPTP analysis for all sequences resulted in the delimitation of 45 species groups, while the ASAP analysis provided the partition of 48 groups. RESL analysis resulted in 63 operational taxonomic units (OTUs). This study expands our scant knowledge of anopheline species in the Yanomami and Pantanal regions. At least 18 species of Anophelinae mosquitoes were found in these study areas. Additional studies are now required to determine the species that transmit Plasmodium spp. in these regions.

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.

Detection of Insect-Specific Flaviviruses in Mosquitoes (Diptera: Culicidae) in Northeastern Regions of South Africa

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

The effect of irrigation on malaria vector bionomics and transmission intensity in western Ethiopia

Background Irrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia. Methods Comparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package. Results A total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [t(3) = 5.14, P = 0.0143] and CDC LT catches [t(271.97) = 7.446, P < 0.0001). The overall sporozoite rate of anopheline species from the nearby clusters was 10-fold higher compared to the faraway clusters. Conclusions Significantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area. Graphical abstract

Updated distribution of anopheline mosquitoes in Hokkaido, Japan, and the first evidence of Anopheles belenrae in Japan

Background In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. Methods A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2. Results A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. Conclusion The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan. Graphical abstract

Prevalence and distribution of anopheline mosquitoes in malaria endemic areas of Asir Region, Saudi Arabia

To study the prevalence of anopheline mosquitoes, over 180 sites were sampled in malaria-endemic areas of Asir region, Saudi Arabia, during June 1999-April 2001. A total of 7085 larval and 754 adult female Anopheles spp. specimens were collected. Seven species were identified: An. dthali, An. rupicolus, An. sergentii, An. arabiensis, An. multicolor, An. turkhudi and An. pretoriensis. Both An. arabiensis and An. sergentii are known vectors of malaria in the region. An. dthali occurred in all sites and was the most abundant species. An. turkhudi was collected in low numbers as larvae only. An. multicolor and An. pretoriensis were recorded for the first time in Asir region. An. sergentii is a species of the northern areas of the region, whereas An. arabiensis was more prevalent in the south

Malaria vector dynamics and utilization of insecticide-treated nets in low-transmission setting in Southwest Ethiopia: implications for residual transmission

Background Understanding the behaviour of local malaria vectors is essential as effectiveness of the commonly used vector-targeted malaria control tools heavily relies on behaviour of the major malaria vectors. This study was conducted to determine species composition, biting behaviour, host preference and infectivity of anopheline mosquitoes, and assess utilization of insecticide-treated nets (ITNs) in a low transmission setting in Southwest Ethiopia. Methods Adult anopheline mosquitoes were collected using human landing catches (HLCs), Centers for Disease Control and Prevention (CDC) light traps (LTs) and Pyrethrum Spray Catches (PSCs) from June 2016 to May 2018 in Kishe, Jimma Zone, Southwest Ethiopia. The anopheline mosquitoes were morphologically identified. Moreover, sub-sample of An. gambiae s.l. was identified to species using polymerase chain reaction (PCR). Circum-sporozoite proteins (CSPs) and blood meal sources of the anopheline mosquitoes were tested using enzyme-linked immunosorbent assay (ELISA). In addition, a cross-sectional survey was conducted to assess ITN utilization by the inhabitants. Results A total of 3659 anopheline mosquitoes comprising An. coustani complex (84.4%), An. gambiae s.l. (11.3%), and An. pharoensis and An. squamosus comprising less than 5% were collected. The anopheline mosquitoes showed marked outdoor (67%) and early evening (63%) biting behaviour. An. coustani complex and An. gambiae s.l. were predominantly zoophilic and anthropophilic, respectively. None of the sampled anopheline were CSP-positive. Most of the households (97.8%) owned at least one ITN, with modest usage by the inhabitants (73.4%). ITN usage was significantly higher among under-five children (AOR = 7.9, 95% CI: 4.41–14.03), household heads and spouses (AOR = 4.8, 95% CI: 3.0–7.59), those with sufficient access to ITNs (AOR = 1.8, 95% CI: 1.39–2.35), and who were not utilizing alternative mosquito repellents (AOR = 2.2, 95% CI: 1.58–2.99). Conclusion The anopheline mosquito species exhibited predominantly outdoor and early evening biting activity. Household ITN coverage was high with slight gap in usage. Vector control interventions should target outdoor and early biting vectors to further suppress the local mosquito population. Moreover, sensitization of the community on consistent use of ITNs is required.

Updated Distribution of Anopheline Mosquitoes (Diptera: Culicidae) in Hokkaido, Japan, and the First Evidence of Anopheles (Anopheles) Belenrae in Japan

Background: In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015.Methods: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a CDC miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to PCR-based sequence analysis of the ITS2 region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method. Results: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae Rueda (2005), was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido.Conclusion: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.