Determination of the feeding behavior of Phlebotomus sergenti using multiplex PCR and tent-baited traps in a new focus of Anthroponotic Cutaneous Leishmaniasis in the southeast of Iran.

OBJECTIVES: This study aimed to determine feeding behaviors of Phlebotomus sergenti Parrot, in a new focus of Anthroponotic Cutaneous Leishmaniasis in Bam County, southeast Iran. METHODS: Two methods were used to determine the feeding behavior of Phlebotomus sergenti. In the first method, blood-fed sand flies were captured using a mouth aspirator in human and animal dwellings and consequently, blood meal identification was made using Multiplex PCR. The results were used for calculating Host Feeding Index (HFI) and Forage Ratio (FR) parameters. In the second method, human (Homo sapiens), goat (Capra aegagrus), cattle (Bos taurus), chicken (Gallus gallus) and dog (Canis lupus) were used as baits in tent-baited traps to determine the feeding behavior of Phlebotomus sergenti. RESULTS: Multiplex PCR analysis revealed that the most frequent blood in the stomack of sand flies' were from chicken, but the calculation of the FR revealed that this species prefers canine and poultary blood as meal. Human and animal tent-baited traps revealed that most Phlebotomus sergenti were attracted to chicken rather than the other hosts. CONCLUSIONS: Sand flies are attracted to animals for various reasons such as eating blood, mating on their bodies and laying eggs on their feces. Molecular methods are effective and accurate methods to determine the type of host that sandfly fed on, but they do not show host preferences. The results of the molecular analysis, along with the calculation of HFI and FR, can determine the preferred host of sand flies. The current study revealed that dogs, the secondary reservoir of ACL in Iran, is the first preferred host of Phlebotomus sergenti.

Limited Capacity of Deer To Serve as Zooprophylactic Hosts for Borrelia burgdorferi in the Northeastern United States.

Because deer are considered to be incompetent reservoirs of the agent of Lyme disease (Borrelia burgdorferi sensu stricto) in the northeastern United States, they may serve as zooprophylactic or "dilution" hosts if larvae of the deer tick vector (Ixodes dammini, "northern" clade of Ixodes scapularis) frequently feed on them. To determine whether host-seeking nymphal deer ticks commonly feed on deer as larvae, we used a real-time PCR host bloodmeal remnant identification assay to identify the host on which these ticks had fed. Nymphal lone star ticks (Amblyomma americanum) were collected simultaneously in our sites and provided an index of the availability of deer in these sites. At 3 of the 4 sites, Ixodes nymphs had fed as larvae on a variety of hosts, including mice, birds, and shrews, but rarely on deer (<6% for all sites); in contrast, lone star tick nymphs had commonly fed on deer (31 to 78%). Deer were common larval hosts for Ixodes ticks (39% of bloodmeals) in only one site. The prevalence of B. burgdorferi in host-seeking nymphal deer ticks was associated with mouse-fed ticks (P = 0.007), but there was no association with deer-fed ticks (P = 0.5). The diversity and prevalence of hosts that were identified differed between deer ticks and lone star ticks that were collected simultaneously, demonstrating that there is no confounding of host bloodmeal identification by contaminating environmental DNA (eDNA). We conclude that deer were not common hosts for larval deer ticks, thus limiting their zooprophylactic role in our sites. IMPORTANCE Because deer are incompetent reservoirs for B. burgdorferi, their presence may modulate the force of enzootic transmission by serving as zooprophylactic or "dilution" hosts. Such an effect would depend on the extent to which subadult deer ticks feed on other hosts. We used bloodmeal analysis on nymphal deer ticks to identify the host upon which larvae had fed. We found that lone star ticks collected at the same time as deer ticks commonly fed on deer, but deer ticks did not. We conclude that deer are not a preferred host for larval deer ticks and, thus, are not necessarily zooprophylactic.

Host Contributions to the Force of Borrelia burgdorferi and Babesia microti Transmission Differ at Edges of and within a Small Habitat Patch.

In the northeastern United States, the emergence of Lyme disease has been associated, in part, with the increase of small forest patches. Such disturbed habitat is exploited by generalist species, such as white-footed mice, which are considered the host with the greatest reservoir capacity for the agents of Lyme disease (Borrelia burgdorferi sensu stricto) and human babesiosis (Babesia microti). Spatial risk analyses have identified edge habitat as particularly risky. Using a retrotransposon-based quantitative PCR assay for host bloodmeal remnant identification, we directly measured whether the hosts upon which vector ticks fed differed at the edge or within the contiguous small habitat patch. Questing nymphal deer ticks, Ixodes dammini, the northern clade of Ixodes scapularis, were collected from either the edge or within a thicket on Nantucket Island over 3 transmission seasons and tested for evidence of infection as well as bloodmeal hosts. Tick bloodmeal hosts significantly differed by site as well as by year. Mice and deer were identified most often (49.9%), but shrews, rabbits, and birds were also common. Ticks from the edge fed on a greater diversity of hosts than those from the thicket. Surprisingly, mice were not strongly associated with either infection at either sampling site (odds ratio [OR] < 2 for all). Although shrews were not the most common host utilized by ticks, they were highly associated with both infections at both sites (OR = 4.5 and 11.0 for B. burgdorferi and 7.9 and 19.0 for B. microti at the edge and thicket, respectively). We conclude that reservoir hosts may differ in their contributions to infecting ticks between edge and contiguous vegetated patches. IMPORTANCE Habitat fragmentation is thought to be a main factor in the emergence of Lyme disease and other deer tick-transmitted infections. The patchwork of forest and edges promotes altered biodiversity, favoring the abundance of generalist rodents, such as white footed mice, heretofore considered a key tick and reservoir host in the northeastern United States. We used tick bloodmeal analyses to directly identify the hosts from which nymphal deer ticks became infected. We demonstrate that there is considerable microfocality in host contributions to the cohort of infected ticks and that shrews, although they fed fewer ticks than mice, disproportionately influenced the force of pathogen transmission in our site. The venue of transmission of certain deer tick-transmitted agents may comprise a habitat scale of 10 m or fewer and depend on alternative small mammal hosts such as shrews.

Shotgun and TMT-Labeled Proteomic Analysis of the Ovarian Proteins of an Insect Vector, Aedes aegypti (Diptera: Culicidae).

Aedes aegypti [Linnaeus in Hasselquist; yellow fever mosquito] transmits several viruses that infect millions of people each year, including Zika, dengue, yellow fever, chikungunya, and West Nile. Pathogen transmission occurs during blood feeding. Only the females blood feed as they require a bloodmeal for oogenesis; in the bloodmeal, holo-transferrin and hemoglobin provide the females with a high iron load. We are interested in the effects of the bloodmeal on the expression of iron-associated proteins in oogenesis. Previous data showed that following digestion of a bloodmeal, ovarian iron concentrations doubles by 72 hr. We have used shotgun proteomics to identify proteins expressed in Ae. aegypti ovaries at two oogenesis developmental stages following blood feeding, and tandem mass tag-labeling proteomics to quantify proteins expressed at one stage following feeding of a controlled iron diet. Our findings provide the first report of mosquito ovarian protein expression in early and late oogenesis. We identify proteins differentially expressed in the two oogenesis development stages. We establish that metal-associated proteins play an important role in Ae. aegypti oogenesis and we identify new candidate proteins that might be involved in mosquito iron metabolism. Finally, this work identified a unique second ferritin light chain subunit, the first reported in any species. The shotgun proteomic data are available via ProteomeXchange with identifier PXD005893, while the tandem mass tag-labeled proteomic data are available with identifier PXD028242.

Impact of cattle on the abundance of indoor and outdoor resting malaria vectors in southern Malawi.

BACKGROUND: Understanding the blood feeding preferences and resting habits of malaria vectors is important for assessing and designing effective malaria vector control tools. The presence of livestock, such as cattle, which are used as blood meal hosts by some malaria vectors, may impact malaria parasite transmission dynamics. The presence of livestock may provide sufficient blood meals for the vectors, thereby reducing the frequency of vectors biting humans. Alternatively, the presence of cattle may enhance the availability of blood meals such that infectious mosquitoes may survive longer, thereby increasing the risk of malaria transmission. This study assessed the effect of household-level cattle presence and distribution on the abundance of indoor and outdoor resting malaria vectors. METHODS: Houses with and without cattle were selected in Chikwawa district, southern Malawi for sampling resting malaria vectors. Prokopack aspirators and clay pots were used for indoor and outdoor sampling, respectively. Each house was sampled over two consecutive days. For houses with cattle nearby, the number of cattle and the distances from the house to where the cattle were corralled the previous night were recorded. All data were analysed using generalized linear models fitted with Poisson distribution. RESULTS: The malaria vectors caught resting indoors were Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis and Anopheles funestus s.s. Outdoor collections consisted primarily of An. arabiensis. The catch sizes of indoor resting An. gambiae sensu lato (s.l.) were not different in houses with and without cattle (P = 0.34). The presence of cattle near a house was associated with a reduction in the abundance of indoor resting An. funestus s.l. (P = 0.04). This effect was strongest when cattle were kept overnight ≤ 15 m away from the houses (P = 0.03). The blood meal hosts varied across the species. CONCLUSION: These results highlight differences between malaria vector species and their interactions with potential blood meal hosts, which may have implications for malaria risk. Whereas An. arabiensis remained unaffected, the reduction of An. funestus s.s. in houses near cattle suggests a potential protective effect of cattle. However, the low abundance of mosquitoes reduced the power of some analyses and limited the generalizability of the results to other settings. Therefore, further studies incorporating the vectors' host-seeking behaviour/human biting rates are recommended to fully support the primary finding.

Transcriptome of the Aedes aegypti Mosquito in Response to Human Complement Proteins.

Aedes aegypti is the primary mosquito vector of several human arboviruses, including the dengue virus (DENV). Vector control is the principal intervention to decrease the transmission of these viruses. The characterization of molecules involved in the mosquito physiological responses to blood-feeding may help identify novel targets useful in designing effective control strategies. In this study, we evaluated the in vivo effect of feeding adult female mosquitoes with human red blood cells reconstituted with either heat-inactivated (IB) or normal plasma (NB). The RNA-seq based transcript expression of IB and NB mosquitoes was compared against sugar-fed (SF) mosquitoes. In in vitro experiments, we treated Aag2 cells with a recombinant version of complement proteins (hC3 or hC5a) and compared transcript expression to untreated control cells after 24 h. The transcript expression analysis revealed that human complement proteins modulate approximately 2300 transcripts involved in multiple biological functions, including immunity. We also found 161 upregulated and 168 downregulated transcripts differentially expressed when human complement protein C3 (hC3) and human complement protein C5a (hC5a) treated cells were compared to the control untreated cells. We conclude that active human complement induces significant changes to the transcriptome of Ae. aegypti mosquitoes, which may influence the physiology of these arthropods.

Mosquito bloodmeal preferences in two zoological gardens in Germany.

Because they provide a high density and diversity of vertebrate species, small water pools and shaded environments, zoological gardens offer ideal living conditions for numerous mosquito species. Depending on their host preferences and vector competencies, these species may be able to transmit pathogens between native and non-adapted exotic blood host species, thereby causing morbidity and mortality among valuable zoo animals. To determine the extent to which native mosquito species feed on captive and wild animals, as well as on humans, in two German zoological gardens, mosquitoes were collected over two seasons by trapping and aspirating. A total of 405 blood-fed specimens belonging to 16 mosquito taxa were collected. Genetic bloodmeal analysis revealed 56 host species, mainly representing mammals of the zoo animal population, including exotic species previously not known as blood hosts of the mosquito species collected. These results indicate opportunistic feeding patterns with low host-specificity in the analysed mosquitoes, although these could be grouped, according to their bloodmeals, into 'amphibian-', 'non-human mammal-' and 'non-human mammal and human-' feeding species. As the blood-feeding preferences of vector-competent mosquito species are major determinants of vector capacity, information on the blood-feeding behaviour of mosquitoes in zoos is crucial to the success of targeted vector management.

Molecular identification of bloodmeal sources and trypanosomes in Glossina spp., Tabanus spp. and Stomoxys spp. trapped on cattle farm settlements in southwest Nigeria.

The interactions of host, vector and parasite in bovine trypanosomiasis transmission cycles in southwest Nigeria are not yet well understood. Trypanosoma (Trypanosomatida: Trypanosomatidae) species infection prevalences and bloodmeal sources were determined in transmitting vectors of the genera Glossina (Diptera: Glossinidae), Tabanus (Diptera: Tabanidae) and Stomoxys (Diptera: Muscidae) collected using Nzi traps in cattle settlements in southwest Nigeria. Sequenced cytochrome B mitochondrial DNA segments obtained from vector digestive tracts identified bloodmeal sources from eight host species, namely human, cattle, hippopotamus, giraffe, gazelle, spotted hyena, long-tailed rat and one unidentified species. Overall, 71.1% [95% confidence interval (CI) 63.0-78.1], 33.3% (95% CI 21.9-47.0) and 22.2% (95% CI 16.2-29.9), respectively, of Glossina, Tabanus and Stomoxys flies were positive for trypanosomes. The observed trypanosome species were Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma evansi, Trypanosoma simiae and Trypanosoma godfreyi. Trypanosome DNA was more prevalent in tsetse (34.8% Tr. vivax, 51.1% Tr. b. brucei, 5.2% Tr. congolense, 4.4% Tr. simiae and 24.4% mixed infections) than in other flies and the main determinants in all flies were seasonal factors and host availability. To the best of the present group's knowledge, this is the first report of Trypanosoma species in Tabanus and Stomoxys flies in Nigeria. It indicates that vector control programmes should always consider biting flies along with tsetse flies in the control of human and animal trypanosomiasis.

Host use patterns of Culicoides spp. biting midges at a big game preserve in Florida, U.S.A., and implications for the transmission of orbiviruses.

Culicoides spp. biting midges (Diptera: Ceratopogonidae) are vectors of pathogens that have a significant economic impact on the livestock industry. White-tailed deer (Odocoileus virginianus), a farmed species in the U.S.A., are susceptible to two Culicoides spp. borne orbiviruses: bluetongue virus and epizootic haemorrhagic disease virus. Elucidating host-vector interactions is an integral step in studying disease transmission. This study investigated the host range of Culicoides spp. present on a big game preserve in Florida on which a variety of Cervidae and Bovidae freely roam. Culicoides were captured with Centers for Disease Control and Prevention (CDC) miniature light traps run twice weekly on the preserve for 18 consecutive months (July 2015-December 2016). Host preference was quantified through forage ratios, based upon PCR-based bloodmeal analysis of Culicoides spp. and overall animal relative abundance on the preserve. Culicoides stellifer preferentially fed on Cervus spp. and fallow deer (Dama dama) and displayed a relative avoidance of Bovidae and white-tailed deer. Culicoides debilipalpis preferred white-tailed deer and avoided all Bovidae. Culicoides pallidicornis and Culicoides biguttatus showed preferences for white-tailed deer and Père David's deer (Elaphurus davidianus), respectively. These results add to current knowledge of preferred hosts of Florida Culicoides spp. and have implications for the spread of orbiviruses. Copyright © 2018 John Wiley & Sons, Ltd.

Molecular bloodmeal analyses reveal that Trypanosoma cruzi-infected, native triatomine bugs often feed on humans in houses in central Brazil.

The identification of bloodmeal sources in triatomine bugs (Hemiptera: Reduviidae) is important in understanding vector-host associations and in measuring the risk for Chagas' disease transmission. The bloodmeal sources of triatomines infected with Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) caught in houses in central Brazil (Goiás State and the Federal District) were investigated during 2012-2014. Mitochondrial cytochrome b amplicons were used to identify bloodmeals through high-resolution melting and DNA sequencing. Most bugs were found to have fed on either humans (45.7%) or chickens (43.1%). Human blood was detected in Triatoma sordida (n = 22/50 bugs), Triatoma pseudomaculata (n = 7/11 bugs), Panstrongylus megistus (n = 10/24 bugs), Panstrongylus geniculatus (n = 1/3 bugs) and Rhodnius neglectus (n = 18/28 bugs) (all: Hemiptera: Reduviidae). Sequencing identified Necromys (Rodentia: Cricetidae) mouse blood in P. geniculatus and Tropidurus (Squamata: Tropiduridae) lizard blood in T. pseudomaculata and T. sordida. These findings reveal new vector-host associations. The present results suggest frequent contact between humans and T. cruzi-infected triatomines in central Brazil and indicate that Chagas' disease transmission by native vectors is an ongoing threat.

iDNA screening: Disease vectors as vertebrate samplers.

In the current context of global change and human-induced biodiversity decline, there is an urgent need for developing sampling approaches able to accurately describe the state of biodiversity. Traditional surveys of vertebrate fauna involve time-consuming and skill-demanding field methods. Recently, the use of DNA derived from invertebrate parasites (leeches and blowflies) was suggested as a new tool for vertebrate diversity assessment. Bloodmeal analyses of arthropod disease vectors have long been performed to describe their feeding behaviour, for epidemiological purposes. On the other hand, this existing expertise has not yet been applied to investigate vertebrate fauna per se. Here, we evaluate the usefulness of hematophagous dipterans as vertebrate samplers. Blood-fed sand flies and mosquitoes were collected in Amazonian forest sites and analysed using high-throughput sequencing of short mitochondrial markers. Bloodmeal identifications highlighted contrasting ecological features and feeding behaviour among dipteran species, which allowed unveiling arboreal and terrestrial mammals of various body size, as well as birds, lizards and amphibians. Additionally, lower vertebrate diversity was found in sites undergoing higher levels of human-induced perturbation. These results suggest that, in addition to providing precious information on disease vector host use, dipteran bloodmeal analyses may represent a useful tool in the study of vertebrate communities. Although further effort is required to validate the approach and consider its application to large-scale studies, this first work opens up promising perspectives for biodiversity monitoring and eco-epidemiology.

Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito, Aedes aegypti.

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya.

BACKGROUND: Small islands serve as potential malaria reservoirs through which new infections might come to the mainland and may be important targets in malaria elimination efforts. This study investigated malaria vector species diversity, blood-meal hosts, Plasmodium infection rates, and long-lasting insecticidal net (LLIN) coverage on Mageta, Magare and Ngodhe Islands of Lake Victoria in western Kenya, a region where extensive vector control is implemented on the mainland. RESULTS: From trapping for six consecutive nights per month (November 2012 to March 2015) using CDC light traps, pyrethrum spray catches and backpack aspiration, 1868 Anopheles mosquitoes were collected. Based on their cytochrome oxidase I (COI) and intergenic spacer region PCR and sequencing, Anopheles gambiae s.l. (68.52%), Anopheles coustani (19.81%) and Anopheles funestus s.l. (11.67%) mosquitoes were differentiated. The mean abundance of Anopheles mosquitoes per building per trap was significantly higher (p < 0.001) in Mageta than in Magare and Ngodhe. Mageta was also the most populated island (n = 6487) with low LLIN coverage of 62.35% compared to Ngodhe (n = 484; 88.31%) and Magare (n = 250; 98.59%). Overall, 416 (22.27%) engorged Anopheles mosquitoes were analysed, of which 41 tested positive for Plasmodium falciparum infection by high-resolution melting (HRM) analysis of 18S rRNA and cytochrome b PCR products. Plasmodium falciparum infection rates were 10.00, 11.76, 0, and 18.75% among blood-fed An. gambiae s.s. (n = 320), Anopheles arabiensis (n = 51), An. funestus s.s. (n = 29), and An. coustani (n = 16), respectively. Based on HRM analysis of vertebrate cytochrome b, 16S rRNA and COI PCR products, humans (72.36%) were the prominent blood-meal hosts of malaria vectors, but 20.91% of blood-meals were from non-human vertebrate hosts. CONCLUSIONS: These findings demonstrate high Plasmodium infection rates among the primary malaria vectors An. gambiae s.s. and An. arabiensis, as well as in An. coustani for the first time in the region, and that non-human blood-meal sources play an important role in their ecology. Further, the higher Anopheles mosquito abundances on the only low LLIN coverage island of Mageta suggests that high LLIN coverage has been effective in reducing malaria vector populations on Magare and Ngodhe Islands.

Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey.

Leishmaniasis is an arthropod-borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Aydin province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS-1 real-time PCR. The detection of Leishmania DNA in wild-caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Aydin province.

Molecular Confirmation of Frogs (Anura) as Hosts of Corethrellidae (Diptera) in the Southeastern United States.

Flies in the family Corethrellidae Edwards 1932 (Diptera) are known to be attracted to the mating calls of male frogs. For the first time, the hosts of corethrellids were identified to species by analyzing bloodmeals taken from resting female flies. A portion of the cytochrome b gene was amplified and sequenced from blood-engorged flies using vertebrate-specific primers. The flies were collected over 6 yr at two locations in the southeastern United States from resting boxes and natural resting sites (rodent burrows). Potential host abundance focused on frog surveillance, and estimation relied on visual encounters, passive trapping (artificial refugia), and call surveys. This study confirms that corethrellids take blood from tree frogs (Hylidae); however, it was found that true frogs (Lithobates Fitzinger 1843 (Ranidae: Anura) sp.) were the principal host selected by Corethrella brakeleyi (Coquillett 1902) (~73% of identified bloodmeals). These preliminary data suggest that host selection of Corethrella Freeman 1962 sp. is not necessarily correlated with host calling abundance.

Detecting Burrowing Owl Bloodmeals in Pulex irritans (Siphonaptera: Pulicidae).

Pulex irritans L. is a cosmopolitan flea species that infests a wide variety of hosts. In North America it generally parasitizes large wild mammals, but in the Pacific Northwest an association has emerged between P. irritans and the western burrowing owl (Athene cunicularia hypugaea). While investigators have recognized this association for decades, it has not been clear if P. irritans feeds on burrowing owls, or if the owls serve exclusively as phoretic hosts. Here we describe using a real-time assay that was originally developed to identify bloodmeals in Ugandan cat fleas (Ctenocephalides felis Bouché) to detect burrowing owl DNA in P. irritans collected from burrowing owls in southern Idaho. Of 50 fleas tested, 12 had no detectable vertebrate bloodmeal. The remaining 38 (76%) contained burrowing owl DNA. The assay did not detect vertebrate DNA in unfed fleas exposed to owl or mouse pelts and is therefore unlikely to detect DNA in fleas from vertebrates that have served exclusively as phoretic hosts. We conclude that P. irritans feeds on burrowing owls. We discuss the potential implications of this finding for burrowing owl conservation and enzootic plague dynamics.

Development and evaluation of real-time PCR assays for bloodmeal identification in Culicoides midges.

Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host-feeding preferences, is limited. Identification of host-feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi-quantitative real-time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan-reactive species group and seven species-specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood-fed midges of various species from several geographic locations in Australia.

Comparison of DNA and Carbon and Nitrogen Stable Isotope-based Techniques for Identification of Prior Vertebrate Hosts of Ticks.

Identification of the vertebrate hosts upon which hematophagous arthropods feed provides key information for understanding the ecology and transmission of vector-borne diseases. Bloodmeal analysis of ticks presents unique challenges relative to other vectors, given the long interval between bloodmeal acquisition and host-seeking, during which DNA degradation occurs. This study evaluates DNA-based and stable isotope-based bloodmeal analysis methodologies for the lone star tick, Amblyomma americanum (Linneaus, 1758), in an experimental study with chicken as the known host. We subjected ticks of different ages and environmental rearing conditions to three DNA-based approaches and a stable isotopic analysis, which relies on the natural variation of nitrogen ((15)N/(14)N) and carbon ((13)C/(12)C) isotopes. While all three DNA-based approaches were successful in identifying the bloodmeal host of the engorged nymphs, only the probe-based RT-PCR was able to detect host DNA in aged ticks, the success of which was low and inconsistent across age and rearing treatments. In contrast, the stable isotope analysis showed utility in determining the host across all ages of ticks when isotopic values of ticks were compared with a panel of candidate vertebrate species. There was a positive shift in both δ(13)C and δ(15)N in adult A. americanum until 34 wk postnymphal bloodmeal. Through analyzing the isotopic signatures of eight potential vertebrate host species, we determined that the magnitude of this isotopic shift that occurred with tick age was minor compared with the heterogeneity in the δ(15)N and δ(13)C signatures among species. These results suggest that stable isotopes are a useful tool for understanding tick-host interactions.

Host preferences of ornithophilic biting midges of the genus Culicoides in the Eastern Balkans.

Many biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of a diverse number of pathogens. The identification of their feeding behaviour and of vector-host associations is essential for understanding their transmission capacity. By applying two different nested polymerase chain reaction (PCR) assays, of which one targeted the avian cyt b gene and the other targeted the COI gene of a wide range of vertebrates, we identified the blood hosts of six biting midge species including Culicoides circumscriptus, Culicoides festivipennis, Culicoides punctatus, Culicoides pictipennis, Culicoides alazanicus and Culicoides cf. griseidorsum, the latter two of which are reported in Bulgaria for the first time. Bird DNA was found in 50.6% of 95 investigated bloodmeals, whereas mammalian DNA was identified in 13.7%. Two Culicoides species were found to feed on both birds and mammals. There was remarkable diversity in the range of avian hosts: 23 species from four orders were identified in the abdomens of four Culicoides species. The most common bird species identified was the magpie, Pica pica (n = 7), which was registered in all four ornithophilic biting midge species. Six bloodmeals from the great tit, Parus major, were recorded only in C. alazanicus. None of the studied species of Culicoides appeared to be restricted to a single avian host.

Influence of Bloodmeal Source on Reproductive Output of the Potential West Nile Vector, Culex theileri (Diptera: Culicidae).

Culex theileri Theobald (Diptera: Culicidae) has a wide Afrotropical, southern Palaearctic, northern Oriental, and European distribution. It is mainly considered as a mammophilic mosquito and also feeds on birds and serves as a vector for various zoonotic diseases including West Nile virus. Despite its broad distribution and evidence indicating that Cx. theileri is a competent vector of human and domestic animal pathogens, basic biological and ecological features of this species have not been well investigated. We evaluated the impact of bloodmeal source (human, chicken, cow, and a double bloodmeal such as human and cow or chicken and cow and mixed bloodmeals [cow, chicken, and human] via artificial feeding) on fecundity, hatching rates, developmental times, and viability from egg to adult for laboratory colonized Cx. theileri. Fecundity in mosquitoes that took a chicken bloodmeal, a double bloodmeal and mixed bloodmeals was significantly higher than in females fed on a single cow or single human blood. This is the first study about the bloodmeal sources effect on laboratory-reared Cx. theileri populations and these findings contribute to our understanding of the impact of bloodmeal source on reproduction in Cx. theileri. As it is known that Cx. theileri is a vector for West Nile virus, the potential impacts of bloodmeal source on virus transmission are discussed.