Hematophagous Species Research Articles

Emergence patterns and ecological associations of some haematophagous blackfly species along an oligotrophic hydrosystem

Emergence patterns of blackflies (Diptera: Simuliidae) were investigated for 2 years at nine sites in the Plitvice Lakes area of Croatia, using pyramid-type traps. A total of 38 012 specimens representing 10 haematophagous species were collected. The dominant species was Simulium angustipes Edwards, 1915, a vector transmitting avian trypanosomes. Water temperature, alkalinity, conductivity and habitat type represented the factors with the greatest degree of influence on blackfly species composition, structure and distribution along a longitudinal gradient of this oligotrophic karstic hydrosystem. Simulium angustipes was highly associated with lake outlets. The blackfly communities at all sites were dominated by species typical of the rhithral zone, but there was a shift in species composition along a longitudinal gradient from the hypocrenal-epirhithral to the epirhithral-metarhithral zone. Multiple generations per year were detected in S. angustipes, whereas in Simulium costatum Friederichs, 1920, the number of generations differed between sites with constant and those with variable water temperature.

Ecological correlates of body size in gamasid mites parasitic on small mammals: abundance and niche breadth

We studied ecological correlates of body size (abundance and niche breadth) in gamasid mites parasitic on small mammals in 28 regions of the Palearctic. We predicted that smaller species would be characterized by higher abundance than larger species, all else (e.g. host species) being equal. We also predicted that host specificity of mites would decrease (that is, number of host species they use would increase) with an increase in their body size. We focused on mites collected from host bodies that include a) species that feed solely on host’s blood (obligate exclusive haematophages), b) species that feed on both host’s blood and small arthropods (obligate non‐exclusive haematophages), and c) facultative haematophages. We expected that the relationship between body size and abundance and/or host specificity would be more pronounced in obligate exclusively haematophagous mites than for obligate non‐exclusively and facultative haematophagous mites. Across all mite species across regions, mean abundance correlated negatively with body size. The same was true for obligate haematophagous species, but not for facultative haematophages. When mite communities on the same host in a location were considered, the negative body mass–abundance relationship was found in only 3 of 44 communities. Nevertheless, a meta‐analytic (across host species) estimate of the slope of this relationship appeared to be significantly negative. No significant relationship between mite body size and host specificity was found in the analyses across all mite species as well as in obligate exclusive or obligate non‐exclusive haematophages. However, the number of hosts used by facultative haematophagous mites decreased significantly with an increase in their body size. We explain the relationships between morphological (body size) and ecological (abundance and niche breadth) properties of ectoparasites by their interactions with hosts or physical environment.

Antigenic and genetic characterization of rabies virus isolates from Uruguay

After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp.) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149–1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to T. brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp. specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report of rabies virus having been detected in non-hematophagous bats in this country.

First isolation and genotyping of Toxoplasma gondii from bats (Mammalia: Chiroptera)

There are currently no reports on the isolation and molecular examination of Toxoplasma gondii from bats. Here, we report the isolation and genotypic characterisation of two T. gondii isolates from bats. A total of 369 bats from different municipalities in São Paulo state, southeastern Brazil, were captured and euthanised, and collected tissues (heart and pectoral muscle) were processed for each bat or in pools of two or three bats and bioassayed in mice (a total of 283 bioassays). Eleven PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) markers were used to genotype positive samples: SAG1, SAG2 (5′–3′SAG2 and alt. SAG2), SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, CS3 and Apico. The parasite was isolated from two bats from São Paulo city: an insectivorous bat, the velvety free-tailed bat Molossus molossus, and a hematophagous bat, the common vampire bat Desmodus rotundus. Isolates were designated TgBatBr1 and TgBatBr2, respectively. The genotype of the isolate from M. molossus (TgBatBr1) has been previously described in an isolate from a capybara from São Paulo state, and the genotype from the D. rotundus isolate (TgBatBr2) has already been identified in isolates from cats, chickens, capybaras, sheep, a rodent and a common rabbit from different Brazilian states, suggesting that this may be a common T. gondii lineage circulating in some Brazilian regions. Isolation of T. gondii from a hematophagous species is striking. This study reveals that bats can share the same isolates that are found in domesticated and wild terrestrial animals. This is the first report of the isolation and genotyping of T. gondii in chiropterans.

Implication of haematophagous arthropod salivary proteins in host-vector interactions.

The saliva of haematophagous arthropods contains an array of anti-haemostatic, anti-inflammatory and immunomodulatory molecules that contribute to the success of the blood meal. The saliva of haematophagous arthropods is also involved in the transmission and the establishment of pathogens in the host and in allergic responses. This survey provides a comprehensive overview of the pharmacological activity and immunogenic properties of the main salivary proteins characterised in various haematophagous arthropod species. The potential biological and epidemiological applications of these immunogenic salivary molecules will be discussed with an emphasis on their use as biomarkers of exposure to haematophagous arthropod bites or vaccine candidates that are liable to improve host protection against vector-borne diseases.

A multimodal bait for trapping blood-sucking arthropods

Artificial baits constitute important tools for the detection and sampling of blood-sucking arthropods, in particular those that are vectors of parasites affecting human health. At present, many different devices have been proposed to attract blood-sucking arthropods, mostly based on the attractiveness of particular chemicals or blends. However, most of them revealed themselves as unpractical (e.g. they require an electrical supply), expensive (e.g. gas bottles) or not efficient enough. On the other hand, the use of living baits is as effective but it has practical constraints and/or raises ethical questions. We present here a multimodal lure to attract blood-sucking arthropods designed taking into account both practical constraints and costs. The main characteristics of our bait are: (1) artificiality (no living-host); (2) multimodality (it associates heat, carbon dioxide and chemical attractants); (3) independency from any energy source; (4) no need for gas bottles; (5) easy to prepare and use in the field; (6) low cost. We tested the ability of the bait to attract blood-sucking arthropods in the laboratory and in the field, using capture sticky-traps. Our bait evinced to be almost as efficient as live hosts (mice) for the capture of Chagas disease and Borrelia vectors in Bolivia. The multimodal lure here presented is a generalist bait, i.e. effective for attracting different haematophagous species.

Bullous Leg Lesions Caused by Culicoides Midges after Travel in the Amazon Basin

A 36-year-old man presented with 1 day of blistering lesions on the lower extremities. Two days before presentation, he had traveled to several rural communities in the Peruvian Amazon. During that trip, he had hiked through several areas of damp soil, decaying plants, and dense grass reaching up to 50 cm in height. He awoke the next morning with numerous confluent, erythematous, pruritic, non-blanching papules that were circumferential around both ankles. Over the next day, multiple bullae up to 4.5 cm in diameter developed with surrounding edema (Figure 1). The patient was treated with prednisone (50 mg daily) by mouth for 3 days, followed by routine wound care with topical antimicrobials, dressings, and topical hydrocortisone for pruritus. The lesions stabilized in size by the third day and had largely resolved by 14 days with minimal scarring. Figure 1. View of the patient's right ankle 1 day after travel, with prominent bullae and numerous confluent papules. Soft-tissue edema is obscuring the medial malleolus. This figure appears in color at www.ajtmh.org. This case was caused by midge bites, likely from the hematophagous species Culicoides paraensis and insinuatus (Figure 2). These small insects are widespread in the Peruvian Amazon, where they favor wet areas, grass, and decaying vegetation such as the remains of banana (platano) trees.1 Approximately 1 mm in size and noiseless, they are difficult to see and may be overlooked in vector-avoidance strategies despite being vectors for Oropouche virus and other pathogens. Bites to humans may manifest as an immediate-type hypersensitivity reaction with urticaria or as presumably delayed-type reactions with papular, ulcerating, or bullous lesions that can require weeks to resolve.2 Travelers to rural regions in the tropics should adhere carefully to vector precautions, including the use of N,N-diethyl-meta-toluamide (DEET), permethrin, and physical barriers such as heavy boots and tucked-in trousers to prevent bites and their potential consequences. Figure 2. Dissecting microscope images (40×) of a female C. paraensis collected near Iquitos, Peru in the vicinity of where the subject traveled. (A) Thorax and legs. (B) Head. (C) Abdomen. (D) Wing. This figure appears in color at www.ajtmh.org.

Contrasted ecological repartition of the Northern Fowl Mite Ornithonyssus sylviarum (Mesostigmata : Macronyssidae) and the Chicken Red Mite Dermanyssus gallinae (Mesostigmata: Dermanyssidae)

The two hematophagous mite species, the Northern Fowl Mite Ornithonyssus sylviarum (Macronyssidae) and the Chicken Red Mite Dermanyssus gallinae (Dermanyssidae), are considered serious pests in fowl farms in the USA and in Europe respectively. However, neither of them seems to be restricted to either of these two continents according to sparse records in the literature. The aim here was to explore the respective ecological repartitions of these two species in France and to compare them with data from the USA. We thus analyzed hematophagous mesostigmatid mites collected in France in natura, in orchards agroecosystems, in pet bird and fowl farms and compared DNA sequences from some North American and French mites in both species. It is remarkable that O. sylviarum has been recurrently encountered in bird nests from France and yet been absent from French layer farms, whereas it is a serious pest in layer farms in the USA. On the other hand, O. sylviarum has been isolated from ornamental bird farms in France (canary, pheasant). This suggests either a strong impact of different farming practices between continents or a colonization in process in Europe. It remains to be explored whether the opposite applies to D. gallinae in the USA. Lastly, mites belonging to the special lineage D. gallinae L1 have been isolated from North American pigeons providing mt and nDNA sequences very close to French L1 isolates. This confirms the specific status of this cryptic entity.

Persistent Wolbachia and Cultivable Bacteria Infection in the Reproductive and Somatic Tissues of the Mosquito Vector Aedes albopictus

BackgroundCommensal and symbiotic microbes have a considerable impact on the behavior of many arthropod hosts, including hematophagous species that transmit pathogens causing infectious diseases to human and animals. Little is known about the bacteria associated with mosquitoes other than the vectorized pathogens. This study investigated Wolbachia and cultivable bacteria that persist through generations in Ae. albopictus organs known to host transmitted arboviruses, such as dengue and chikungunya.Methodology/Principal FindingsWe used culturing, diagnostic and quantitative PCR, as well as in situ hybridization, to detect and locate bacteria in whole individual mosquitoes and in dissected tissues. Wolbachia, cultivable bacteria of the genera Acinetobacter, Comamonas, Delftia and Pseudomonas co-occurred and persisted in the bodies of both males and females of Ae. albopictus initially collected in La Réunion during the chikungunya outbreak, and maintained as colonies in insectaries. In dissected tissues, Wolbachia and the cultivable Acinetobacter can be detected in the salivary glands. The other bacteria are commonly found in the gut. Quantitative PCR estimates suggest that Wolbachia densities are highest in ovaries, lower than those of Acinetobacter in the gut, and approximately equal to those of Acinetobacter in the salivary glands. Hybridization using specific fluorescent probes successfully localized Wolbachia in all germ cells, including the oocytes, and in the salivary glands, whereas the Acinetobacter hybridizing signal was mostly located in the foregut and in the anterior midgut.Conclusions/SignificanceOur results show that Proteobacteria are distributed in the somatic and reproductive tissues of mosquito where transmissible pathogens reside and replicate. This location may portend the coexistence of symbionts and pathogens, and thus the possibility that competition or cooperation phenomena may occur in the mosquito vector Ae. albopictus. Improved understanding of the vectorial system, including the role of bacteria in the vector's biology and competence, could have major implications for understanding viral emergences and for disease control.

Tropical rat mites (Ornithonyssus bacoti) – serious ectoparasites

In Germany there is limited information available about the distribution of the tropical rat mite (Ornithonyssus bacoti) in rodents. A few case reports show that this hematophagous mite species may also cause dermatitis in man. Having close body contact to small rodents is an important question for patients with pruritic dermatoses. The definitive diagnosis of this ectoparasitosis requires the detection of the parasite, which is more likely to be found in the environment of its host (in the cages, in the litter or in corners or cracks of the living area) than on the hosts' skin itself. A case of infestation with tropical rat mites in a family is reported here. Three mice that had been removed from the home two months before were the reservoir. The mites were detected in a room where the cage with the mice had been placed months ago. Treatment requires the eradication of the parasites on its hosts (by a veterinarian) and in the environment (by an exterminator) with adequate acaricides such as permethrin.

Antibody responses of domestic animals to salivary antigens of Triatomainfestans as biomarkers for low-level infestation of triatomines

Hematophagous arthropods such as Triatoma infestans, the vector of Trypanosoma cruzi, elicit host-immune responses during feeding. Characterization of antibody responses to salivary antigens offers the potential to develop immunologically based monitoring techniques for exposure to re-emergent triatomine bug populations in peridomestic animals. IgG-antibody responses to the salivary antigens of T. infestans have been detected in chickens as soon as 2 days after the first exposure to five adult bugs. Chickens and guinea pigs regularly exposed to this number of triatomines showed a significantly lower anti-saliva antibody titre than animals exposed to 25 adults and fifth instars of four different T. infestans strains originating from Bolivia and from Northern Chile. Highly immunogenic salivary antigens of 14 and 21 kDa were recognised by all chicken sera and of 79 kDa by all guinea pig sera. Cross-reactivity studies using saliva or salivary gland extracts from different hematophagous species, e.g. different triatomines, bed bugs, mosquitoes, sand flies and ticks, as well as chicken sera exposed to triatomines and mosquitoes, demonstrated that the 14 and 21 kDa salivary antigens were only found in triatomines. Sera from peridomestic chickens and guinea pigs in sites of known T. infestans challenge in Bolivia also recognised the 14 and 21 kDa antigens. These represent promising epidemiological markers for the detection of small numbers of feeding bugs and hence may be a new tool for vector surveillance in Chagas disease control programs.

Exploration of the susceptibility of AChE from the poultry red mite Dermanyssus gallinae (Acari: Mesostigmata) to organophosphates in field isolates from France

The red fowl mite Dermanyssus gallinae (De Geer, 1778) is a hematophagous mite species, which is very commonly found in layer facilities in Europe. The economic and animal health impact of this parasite is quite important. In laying hen houses, organophosphates are almost the only legally usable chemicals. Detecting a target resistance can be useful in order to limit the emergence of resistant populations. The acetylcholinesterase (AChE) activity and the enzyme sensitivity to paraoxon was investigated in 39 field samples and compared to a susceptible reference strain (SSK). Insensitivity factor values (expressed as IC50 ratio) obtained from field isolates compared to SSK revealed some polymorphism but not exceeding a 6-fold difference. The kinetic characteristics of AChE from some field samples showed some difference in KM values for acetylthiocholine and inhibition kinetics performed with diethyl paraoxon exhibited a 5.5-fold difference in the bimolecular rate constant in one field isolate. Taken together, these data suggested that differences in AChE susceptibility to organophosphates may exist in D. gallinae but no resistant population was found.

Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus nymph

Studies were carried out to evaluate the efficacy of the growth regulator, triflumuron (TFM) (Starycide sc 480 Bayer), for disrupting the development of Rhodnius prolixus fifth-instar nymph by oral, topical or continuous treatment. All treatments were able to induce high levels of mortality, delay development and molt inhibition. Oral treatment induced molt inhibition in all insects that survived at doses of 0.25, 0.50 and 5.0 mg/mL of a blood meal. The highest levels of both mortality in 24 h and molt inhibition were always observed after topical treatment. The lowest doses needed to obtain considerable biological effects were always observed after continuous treatment. In this way, the highest levels of mortality within 30 days were detected after continuous treatment, which also induced an extended inter-molting period, a lower number of over-aged nymphs and the highest level of molting in nymphs that survived. Moreover, the effects of TFM on insects were often displayed in a dose response manner. These results indicate that TFM acts as a potent growth inhibitor of R. prolixus nymphs and has the potential to be used in integrated vector control programs against hematophagous triatomine species.

Delimiting species boundaries within Dermanyssus Dugès, 1834 (Acari:Dermanyssidae) using a total evidence approach

The genus Dermanyssus is currently composed of 24 hematophagous mite species and includes the Poultry Red Mite, Dermanyssus gallinae, a serious pest in poultry houses. Morphologically, Dermanyssus species fall into two groups corresponding to Moss’ gallinae-group and to hirsutus-group + Microdermanyssus. Species of the gallinae-group exhibit high levels of morphological variability, and are nearly impossible to distinguish. Species of the second group display consistent characters and host associations and are easily distinguishable. Species of the gallinae-group tend to be the major problems in poultry houses and it is unknown whether D. gallinae is the only pest, or if there are numerous cryptic species present in the system. Twenty species of Dermanyssus were tested phylogenetically based on 46 morphological characters. A subset of species, mainly of the gallinae-group, represented each by several populations, was sequenced for two mitochondrial and one nuclear gene regions. This allowed testing their specific status and their interrelationships based and on morphological and molecular characters. The molecular data was analysed separately and in combination with morphological characters. As expected, morphology did a poor job resolving relationships. Molecular data proved more informative. The resulting phylogenetic hypotheses brought some information about interrelationships among species of the gallinae-group showing a split into two main clades. The invasion of human managed environments seems to occur only in taxa within one of the two clades. The host spectrum seems to get enlarged in more derived taxa in the same clade. A delineation of six species within the gallinae-group is provided. Additionally, a key for morphological identification of these species is provided. D. gallinae appears to be the only pest in poultry houses, but is composed of several different and more or less strongly isolated lineages. A new species found from the black swift is described.

Detection of Host Virus-Reactive Antibodies in Blood Meals of Naturally Engorged Mosquitoes

Although serosurvey in human or animals is a useful and straightforward strategy routinely used for public health, it often faces different types of impediments: ethics, beliefs, limitation by animal owners, hazard of access to wild animals. To survey virus circulation, we applied the enzyme-linked immunosorbent assay (ELISA) technique to detect Dengue and Japanese encephalitis (JE) virus-reactive antibodies in blood meals collected from mosquitoes without regard to the potential of mosquito species to be a virus vector. ELISA was performed on mosquito colonies and wild specimens collected from farms and urban areas. Blood meals from Aedes aegypti freshly fed on naturally infected volunteers showed the same levels of dengue immunoglobulin (Ig)G and IgM as the sera directly collected from volunteers. A significant clearance of antibodies during the digestion process started from 13 hours after blood meal, and a negative baseline was reached after 30 hours. The ELISA test performed on wild mosquitoes showed that 37% of Culex quinquefasciatus mosquitoes that engorged on humans in a dengue urban endemic area tested positive for dengue IgG, and in a JE virus-endemic area, 88% of Culex tritaeniorhynchus mosquitoes that engorged on pigs from a large pig farm tested positive for JE virus antibodies versus 11% in a small farm. The main limitation of the ELISA method is the antibody cross-reactivity among flaviviruses; also, sampling strategy should be adjusted to take into account that the actual host from which the blood meal was taken may not be determined. Nevertheless, ELISA performed on recently (1-2 days) engorged mosquito, or any other hematophagous arthropod species, could potentially be used as a "wild phlebotomist" to monitor the prevalence or emergence of a variety of pathogens, with less of the practical, ethical, or risk limitations due to direct blood collection from humans and wild or domestic animals.

A New Glossiphoniid Leech from Catemaco Lake, Veracruz, México

Haementeria acuecueyetzin n. sp. from Catemaco Lake, Veracruz, Mexico, is described based on the examination of 6 specimens. This new hematophagous leech species resembles other members of the genus in the number and position of the eyespots, number of compact salivary glands, and in the presence of 2 pairs of spheroidal mycetomes, but it is distinguished from the other species by having 6 rows of longitudinal smooth white papillae in the dorsal surface and numerous tubercles in dorsal and ventral surfaces. This new species represents the third species of Haementeria in the Northern Hemisphere of the Americas.

Occurrence of a house-infesting Tropical rat mite ( Ornithonyssus bacoti) on murides and human beings

In Germany there is little information available about the distribution of the Tropical rat mite (Ornithonyssus bacoti) in rodents. A few case reports show that this haematophagous mite species may also cause dermatitis in man. All developmental stages are exclusively bloodfeeder. Three children (4, 11 and 15 years old) of a family and a 23-year-old medical student were attacked by the Tropical rat mite. Prior to the consultation of our institution, the patients' conditions had been diagnosed as allergic dermatitis of unclear origin and treated by several antiphlogistic agents, however without success. The conclusive diagnosis, Tropical rat mite dermatitis, was based on the identification of the arthropod Ornithonyssus bacoti in the flats of the patients (husbandry of gerbils, etc.). The diagnosis of a Rat mite dermatitis requires the detection of the parasite, which is more likely to be found in the environment of its host than on the hosts' skin itself.

PERMANENT GENETIC RESOURCES: Development of polymorphic microsatellite loci for the common vampire bat, Desmodus rotundus (Chiroptera: Phylostomidae)

The common vampire bat (Desmodus rotundus) is one of three haematophagous species of bats and the only species in this genus. These New World bats prey on mammals and create significant economic impacts through transmission of rabies in areas where livestock are prevalent. Furthermore, in some portions of their range, it is not uncommon for them to prey upon humans. It is critical to the management of this species and for understanding the spread of bat rabies that detailed studies of D. rotundus population structure be conducted. To further such studies, we have characterized 12 microsatellite loci for this species.

Rhythmic expression of the cycle gene in a hematophagous insect vector

BackgroundA large number of organisms have internal circadian clocks that enable them to adapt to the cyclic changes of the external environment. In the model organism Drosophila melanogaster, feedback loops of transcription and translation are believed to be crucial for the maintenance of the central pacemaker. In this mechanism the cycle (or bmal1) gene, which is constitutively expressed, plays a critical role activating the expression of genes that will later inhibit their own activity, thereby closing the loop. Unlike Drosophila, the molecular clock of insect vectors is poorly understood, despite the importance of circadian behavior in the dynamic of disease transmission.ResultsHere we describe the sequence, genomic organization and circadian expression of cycle in the crepuscular/nocturnal hematophagous sandfly Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Deduced amino acid sequence revealed that sandfly cycle has a C-terminal transactivation domain highly conserved among eukaryotes but absent in D. melanogaster. Moreover, an alternative form of the transcript was also identified. Interestingly, while cycle expression in Drosophila and other Diptera is constitutive, in sandflies it is rhythmic in males and female heads but constitutive in the female body. Blood-feeding, which causes down-regulation of period and timeless in this species, does not affect cycle expression.ConclusionSequence and expression analysis of cycle in L. longipalpis show interesting differences compared to Drosophila suggesting that hematophagous vector species might present interesting new models to study the molecular control of insect circadian clocks.

An alternative method of blood-feeding Culicoides imicola and other haematophagous Culicoides species for vector competence studies

The use of cotton wool pads saturated with blood/virus mixture for oral infection attempts was compared to membrane feeding for the assessment of vector competence in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera, Ceratopogonidae). Although lower infection rates were obtained using pad feeding, it was possible to clearly distinguish the levels of competence between species as well as differences in virus infection rates for various serotypes of bluetongue virus. Reduced infection rates with cotton pad feeding was partly due to a smaller volume of blood meals taken up. However, the method described is likely to be useful in situations where membrane feeding is not viable to separate populations with significant differences in vector competence.