Detection of microfilariae (Nematoda: Filarioidea) in mosquito vectors trapped at bird and wildlife facilities in Durban, South Africa

Abstract

Mosquitoes belonging to the genera, Aedes, Anopheles, Coquillettidia, Culex and Mansonia are vectors of microfilariae (Superfamily: Filarioidea), the specialised larvae of filarial nematodes that infect the lymphatic system, tissues and body cavities of vertebrates. It was previously established that the microfilariae of Dirofilaria repens and Acanthocheilonema reconditum circulate within the domestic animals in South Africa, however, there is a lack of information relating to the presence and prevalence of microfilariae harboured in mosquito vectors in the region. The objectives of this study were to: i. capture mosquitoes from two animal-dense localities, namely, Centre for the Rehabilitation of Wildlife (CROW) and the Umgeni River Bird Park located in eThekwini (Durban) Metropolitan Municipality, KwaZulu-Natal (KZN) Province in South Africa and identify them using morphological means; ii. determine if microfilariae are present in the mosquito species captured from the two animal-dense sites iii. determine the prevalence of microfilariae in potential mosquito vectors by means of dissection and microscopy and iv. infer molecular identifications of microfilariae in potential mosquito vectors collected from areas with high animal densities in Durban, KZN. To adress the objectives of the study, mosquitoes were captured from the Centre for the Rehabilitation of Wildlife (CROW) and the Umgeni River Bird Park (URBP) in Durban per month from April 2021 to March 2022. All collected mosquitoes were identified morphologically using standard taxonomic keys. The prevalence of microfilariae in wild-caught mosquitoes, was determined by microscopically dissecting the mosquitos’ Malpighian tubules, thoracic muscles, head and mouthparts in search of microfilariae. Of the total number of mosquitoes collected (n= 2886), 1490 moquitoes were manually dissected. More specifically, a minimum of 59 mosquitoes per month at each of the two study localities was calculated to be dissected with 95% confidence level and a 5% margin of error, using Raosoft® (Sample Size Calculator; Raosoft inc.). The sample size was based on estimated 4% prevalence of microfilariae obtained from a study in West Africa (Abogye-Antwi et al., 2015). The remaining 1396 mosquitoes that were collected from the two sites, were pooled into conspecific groups for further molecular screening. Polymerase chain reaction (PCR) was used to detect Filarioidea DNA from the mosquitoes using primer pairs designed to amplify the internal transcribed spacer-2 (ITS2) and the filarial mitochondrial DNA cytochrome oxidase subunit I (COI) gene regions. The amplicons for the COI and ITS2 genes were cloned to determine if multiple infections were present. In addition, to infer potential identity, a BLASTn search and phylogenetic inference using a maximum likelihood approach was employed. A total of 1490 female mosquitoes were dissected during the study period, with 765 mosquitoes collected at from CROW and 725 collected at URBP. Across both sites, twelve mosquito species were caught and divided into pools including, Culex pipiens (s.l) (54.1%), Mansonia africana (32.0%), Coquillettidia microannulata (5.6%), Aedes aegypti (5.2%), Anopheles species (1.2%), with the remaining 1.9% (n=27) comprising seven other species including Eretmapodites quinquevitattus, Culex neavei, Cx. subdentatus, Cx. cinerrellus, Cx. duttoni, Cx. simposoni and Cx. univitattus. The most abundant mosquito species were Culex pipiens (s.l) (54.1%) and Mansonia africana (32.0%), both of which have a high transmission potential for microfilariae. No microfilariae were detected in the dissected mosquitoes. This result demonstrated that prevalence of microfilariae circulating in the most common mosquitoes in Durban was lower than 4%, as used to calculate the sample size of mosquiotes dissected in the study. Amplification of the COI and ITS2 genes of microfilarial DNA determined that one Cx. pipiens pool was found to be positive with Onchocercidae sp, thus supporting Cx. pipiens as a possible vector in the region. Phylogenetic analysis did not yield a genus or species level identification as variation in COI and ITS2 topologies, because molecular libraries available for the Onchocercidae were incomplete, and could not support a definitive identification. The ITS2 provided the most acceptable topology (81% bootstrap support; 99.05% identity), inferring that the positive Onchocercidae sp. is closely related to Mansonella. It is recommended that, to determine true prevalence of microfilariae, the two common mosquito species in Durban, Culex pipiens and Mansonia africana, should be targeted for surveillance. Additionally, sampling should be intensified between November and February (wet season) in which a larger sample size should be collected due to the very low prevalence in the eThekwini Metropolitan Municipality, KZN, South Africa.