Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface

Abstract

The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya's Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies.

Fig 1. Map of mosquito sampling areas near Lakes Victoria and Baringo in Kenya.

Fig 2. HRM profiles of selected mosquito bloodmeal sources using cyt b (A) and 16S rRNA (B).

Vertebrate species in the legend are ordered from their lowest to highest 16S rRNA melting temperatures.

Fig 3. Melt rates of serially diluted pure and mixed blood for calibration of identifications and sensitivity validations using cyt b (A) and 16S rRNA (B).

Vertebrate species in the legend are ordered from their lowest to highest melting temperatures.

Fig 4. Melt rates of field collected mixed mosquito blood meals using cyt b (A) and 16S rRNA (B).

Vertebrate species in the legend are ordered from their lowest to highest melting temperatures.

Fig 5. Melt rates of mosquito cyt b amplicons alongside selected vertebrate bloodmeal amplicons.

Species in the legend are ordered from their lowest to highest melting temperatures.

Fig 6. Melt rate profiles of Bunyamwera S segment amplicons detected in blood-fed mosquitoes.