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A human genotyping trial to estimate the post-feeding time from mosquito blood meals.

ABSTRACT: Mosquitoes occur almost worldwide, and females of some species feed on blood from humans and other animals to support ovum maturation. In warm and hot seasons, such as the summer in Japan, fed mosquitoes are often observed at crime scenes. The current study attempted to estimate the time that elapsed since feeding from the degree of human DNA digestion in mosquito blood meals and also to identify the individual human sources of the DNA using genotyping in two species of mosquito: Culex pipiens pallens and Aedes albopictus. After stereomicroscopic observation, the extracted DNA samples were quantified using a human DNA quantification and quality control kit and were genotyped for 15 short tandem repeats using a commercial multiplexing kit. It took about 3 days for the complete digestion of a blood meal, and genotyping was possible until 2 days post-feeding. The relative peak heights of the 15 STRs and DNA concentrations were useful for estimating the post-feeding time to approximately half a day between 0 and 2 days. Furthermore, the quantitative ratios derived from STR peak heights and the quality control kit (Q129/Q41, Q305/Q41, and Q305/Q129) were reasonably effective for estimating the approximate post-feeding time after 2-3 days. We suggest that this study may be very useful for estimating the time since a mosquito fed from blood meal DNA, although further refinements are necessary to estimate the times more accurately.

SUBMITTER: Hiroshige Y

PROVIDER: S-EPMC5472291 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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A human genotyping trial to estimate the post-feeding time from mosquito blood meals.

Hiroshige Yuuji Y Hara Masaaki M Nagai Atsushi A Hikitsuchi Tomoyuki T Umeda Mitsuo M Kawajiri Yumi Y Nakayama Koji K Suzuki Koichi K Takada Aya A Ishii Akira A Yamamoto Toshimichi T

PloS one 20170615 6

Mosquitoes occur almost worldwide, and females of some species feed on blood from humans and other animals to support ovum maturation. In warm and hot seasons, such as the summer in Japan, fed mosquitoes are often observed at crime scenes. The current study attempted to estimate the time that elapsed since feeding from the degree of human DNA digestion in mosquito blood meals and also to identify the individual human sources of the DNA using genotyping in two species of mosquito: Culex pipiens p ...[more]

PMID: 28617865

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Project description:BackgroundControl of malaria parasite transmission can be enhanced by understanding which human demographic groups serve as the infectious reservoirs. Because vector biting can be heterogeneous, some infected individuals may contribute more to human-to-mosquito transmission than others. Infection prevalence peaks in school-age children, but it is not known how often they are fed upon. Genotypic profiling of human blood permits identification of individual humans who were bitten. The present investigation used this method to estimate which human demographic groups were most responsible for transmitting malaria parasites to Anopheles mosquitoes. It was hypothesized that school-age children contribute more than other demographic groups to human-to-mosquito malaria transmission.MethodsIn a region of moderate-to-high malaria incidence in southeastern Malawi, randomly selected households were surveyed to collect human demographic information and blood samples. Blood-fed, female Anopheles mosquitoes were sampled indoors from the same houses. Genomic DNA from human blood samples and mosquito blood meals of human origin was genotyped using 24 microsatellite loci. The resultant genotypes were matched to identify which individual humans were sources of blood meals. In addition, Plasmodium falciparum DNA in mosquito abdomens was detected with polymerase chain reaction. The combined results were used to identify which humans were most frequently bitten, and the P. falciparum infection prevalence in mosquitoes that resulted from these blood meals.ResultsAnopheles females selected human hosts non-randomly and fed on more than one human in 9% of the blood meals. Few humans contributed most of the blood meals to the Anopheles vector population. Children ≤ 5 years old were under-represented in mosquito blood meals while older males (31-75 years old) were over-represented. However, the largest number of malaria-infected blood meals was from school age children (6-15 years old).ConclusionsThe results support the hypothesis that humans aged 6-15 years are the most important demographic group contributing to the transmission of P. falciparum to the Anopheles mosquito vectors. This conclusion suggests that malaria control and prevention programmes should enhance efforts targeting school-age children and males.

Project description:To understand local transmission of vector-borne diseases, it is important to identify potential vectors, characterize their host feeding patterns, and determine if vector-borne pathogens are circulating within the region. This study simultaneously investigated these aspects of disease transmission by collecting engorged mosquitoes within two rural study sites in the central Red River Valley of North Dakota. Mosquitoes were identified, midguts were excised, and the blood was expelled from the midguts. DNA was extracted from blood meals and subjected to PCR and direct sequencing to identify the vertebrate origin of the blood. Using different primer sets, PCR was used to screen for two types of vector-borne pathogens, filarioid nematodes and hemosporidian parasites. White-tailed deer were the primary source of blood meals for the eight aedine mosquito species collected. None of the 288 deer-derived blood meals contained filarioid or hemosporidian DNA. In contrast, 18 of 32 Culex tarsalis and three of three Cx. pipiens blood meals contained avian blood, representing eight different species of birds. Of 24 avian-derived blood meals examined, 12 contained Plasmodium DNA, three of which also contained Leucocytozoon DNA (i.e., dual infection). Potential confounding effects resulting from parasite acquisition and development from previous blood meals (e.g., oocysts) were eliminated because host blood had been removed from the midguts prior to DNA extraction. Thus, specific parasite lineages/species could be unequivocally linked to specific vertebrate species. By combining mosquito identification with molecular techniques for identifying blood meal source and pathogens, a relatively small sample of engorged mosquitoes yielded important new information about mosquito feeding patterns and hemosporidia infections in birds. Thorough analyses of wild-caught engorged mosquitoes and other arthropods represent a powerful tool in understanding the local transmission of vector-borne and zoonotic diseases.

Project description:Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal.Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 ?l) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species.The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal.The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.

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A human genotyping trial to estimate the post-feeding time from mosquito blood meals.

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A human genotyping trial to estimate the post-feeding time from mosquito blood meals.

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