Blood-engorged Females Research Articles

Changes in mosquito species and blood meal composition associated with adulticide applications

Although adulticide application is a pillar in the integrated management of nuisance and vector mosquitoes, non-target effects of adulticide applications within ecosystems are a substantial concern. However, the impacts of adulticide applications on non-target organisms are not necessarily detrimental, and in some cases, may provide benefits to certain organisms or wildlife. Here, we hypothesized that adulticide applications have beneficial non-target impacts on vertebrate wildlife through reduced biting pressure. To test this, we collected mosquitoes from ultra-low volume Permanone-treated (intervention) and untreated (reference) areas and assessed mosquito abundance and diversity, and abundance of blood-engorged female mosquitoes. We performed DNA barcoding analysis on mosquito blood meals to identify host species. Our results demonstrated a significant reduction in mosquito abundance by 58.9% in the intervention areas, taking into account the reduction in reference areas. Consequently, this decline led to a 64.5% reduction in the abundance of blood-engorged females. We also found a temporal dynamic of mosquito composition driven by mosquito control actions in which different mosquito species became dominant at treated sites while composition at reference areas remained similar during the same period. The present study suggests that the beneficial effects of mosquito control treatments for humans extend to other vertebrates, which represents an unstudied and rarely recognized non-target impact.

Anopheline diversity in urban and peri-urban malaria foci: comparison between alternative traps and seasonal effects in a city in the Western Brazilian Amazon

BackgroundContinuous vector surveillance and sustainable interventions are mandatory in order to prevent anopheline proliferation (or spread to new areas) and interrupt malaria transmission. Anopheline abundance and richness were evaluated in urban and peri-urban malaria foci at a medium-sized city in the Brazilian Amazon, comparing the protected human landing catch technique (PHLC) and alternative sampling methods over different seasonal periods. Additional information was assessed for female feeding behaviour and faunal composition.MethodsAnophelines were sampled bimonthly in four urban and peri-urban sites in the city of Porto Velho, state of Rondônia, Brazil. The average number of captured mosquitoes was compared between an PHLC (gold standard), a tent trap (Gazetrap), and a barrier screen by means of generalized linear mixed models (GLMM), which also included season and environment (peri-urban/urban) as predictors.ResultsOverall, 2962 Anopheles individuals belonging to 12 species and one complex were caught; Anopheles darlingi represented 86% of the individuals. More mosquitoes were captured in the peri-urban setting, and the urban setting was more diverse. The model estimates that significantly more anophelines were collected by PHLC than by the Screen method, and Gazetrap captured fewer individuals. However, the Screen technique yielded more blood-engorged females. The peak hours of biting activity were from 6 to 7 p.m. in urban areas and from 7 to 8 p.m. in peri-urban areas.ConclusionsAlthough peri-urban settings presented a greater abundance of anophelines, Shannon and Simpson diversities were higher in urban sites. Each technique proved to be useful, depending on the purpose: PHLC was more effective in capturing the highest anopheline densities, Gazetrap caught the greatest number of species, and the barrier screen technique captured more engorged individuals. There was no seasonal effect on Anopheles assemblage structure; however, a more diverse fauna was caught in the transitional season. Biting activity was more intense from 6 to 8 p.m., with a predominance of An. darlingi.

Blood Feeding Patterns of Aedes aegypti Populations in Senegal.

Aedes aegypti plays an important role in the transmission of several arboviruses of medical importance. The availability of information on the blood-feeding preferences of mosquito vectors is a critical step in the understanding of the transmission of human pathogens and implementation of control strategies. In Senegal, no data currently exist on the feeding pattern of Ae. aegypti in urban areas. To fill this gap, Ae. aegypti blood-fed females were collected in five localities by aspiration and using BG Sentinel 2 traps. Collections were carried out monthly between July and November 2019 inside and outside human dwellings. The origin of the blood meal of Ae. aegypti females were identified by an ELISA technique. A total of 1,710 blood-engorged females were examined and showed that Ae. aegypti preferentially fed on human with 78.6% of the identified blood meals. The other blood meals were from animals including dog, cat, horse, cattle, sheep, and rat. This is the first report on the feeding behavior of Ae. aegypti in urban settings in West Africa. It demonstrated that this species is highly anthropophilic.

Evaluating sampling strategies for enzootic Venezuelan equine encephalitis virus vectors in Florida and Panama.

Determining effective sampling methods for mosquitoes are among the first objectives in elucidating transmission cycles of vector-borne zoonotic disease, as the effectiveness of sampling methods can differ based on species, location, and physiological state. The Spissipes section of the subgenus Melanoconion of Culex represents an understudied group of mosquitoes which transmit Venezuelan equine encephalitis viruses (VEEV) in the Western Hemisphere. The objective of this study was to determine effective collection methods that target both blood-engorged and non-engorged females of the Spissipes section of Culex subgenus Melanoconion to test the hypothesis that favorable trapping methods differ between species and by physiological status within a species. Mosquitoes were collected using two commercially available traps, (CDC-light trap and BG-Sentinel trap), two novel passive traps (a novel mosquito drift fence and pop-up resting shelters), and two novel aspirators, (a small-diameter aspirator and a large-diameter aspirator) in Darién, Panama, and Florida, USA. The total number of female mosquitoes collected for each species was compared using rarefaction curves and diversity metrics. We also compared the utility of each trap for collecting total females and blood-engorged females of four Spissipes section mosquito species in Florida and Darién. In Darién, it was found that both blood-engorged and unfed females of Cx. pedroi were most effectively collected using the mosquito drift fence at 57.6% and 61.7% respectively. In contrast, the most unfed Cx. spissipes were collected using the mosquito drift fence (40.7%) while blood-engorged females were collected effectively by pop-up resting shelters (42.3%). In Florida, the best sampling technique for the collection of blood-engorged Cx. panocossa was the large diameter aspirator at 41.9%, while the best trap for collecting Cx. cedecei was the pop-up resting shelter at 45.9%. For unfed female Spissipes section mosquitoes in Florida, the CDC light trap with CO2 collected 84.5% and 98.3% of Cx. cedecei and Cx. panocossa respectively in Florida. Rarefaction analysis, and both the Shannon and Simpsons diversity indices all demonstrated that the mosquito drift fence was capable of collecting the greatest diversity of mosquito species regardless of location. The finding that the proportions of unfed and blood-engorged mosquitoes collected by traps differed both among and between species has implications for how studies of VEEV vectors will be carried out in future investigations. In Florida a combination of pop-up resting shelters and use of a large-diameter aspirator would be optimal for the collection of both VEEV vectors for host-use studies. Results demonstrate that traps can be constructed from common materials to collect mosquitoes for VEEV vector studies and could be assessed for their utilization in vectors of other systems as well. Unfortunately, no single method was effective for capturing all species and physiological states, highlighting a particular need for assessing trap utility for target species of a study.

The Absence of Abdominal Pigmentation in Livestock Associated Culicoides following Artificial Blood Feeding and the Epidemiological Implication for Arbovirus Surveillance.

Culicoides midges (Diptera: Ceratopogonidae), the vectors of economically important arboviruses such as bluetongue virus and African horse sickness virus, are of global importance. In the absence of transovarial transmission, the parity rate of a Culicoides population provides imperative information regarding the risk of virus dispersal. Abdominal pigmentation, which develops after blood feeding and ovipositioning, is used as an indicator of parity in Culicoides. During oral susceptibility trials over the last three decades, a persistent proportion of blood engorged females did not develop pigment after incubation. The present study, combining a number of feeding trials and different artificial feeding methods, reports on this phenomenon, as observed in various South African and Italian Culicoides species and populations. The absence of pigmentation in artificial blood-fed females was found in at least 23 Culicoides species, including important vectors such as C. imicola, C. bolitinos, C. obsoletus, and C. scoticus. Viruses were repeatedly detected in these unpigmented females after incubation. Blood meal size seems to play a role and this phenomenon could be present in the field and requires consideration, especially regarding the detection of virus in apparent “nulliparous” females and the identification of overwintering mechanisms and seasonally free vector zones.

Artificial resting sites: An alternative sampling method for adult mosquitoes.

Mosquito collections are commonly conducted with baited traps predominantly attracting host-seeking females. In contrast, resting sites are generally colonized by a broader range of the mosquito population, including a higher proportion of males and blood-engorged females. This study evaluates the sampling success of different artificial resting sites, attached to a deciduous or coniferous tree at different heights. As standard sampling method, carbon dioxide-baited Biogents Sentinel traps (BG traps) were operated in parallel. BG traps caught a higher number of specimens compared to the resting sites. However, the proportion of blood-engorged females and males was higher in resting sites. More Culiseta spp. specimens were collected in resting sites compared to BG traps, but less Aedes spp. specimens. In general, fewer specimens and species were recorded in small resting sites and at top height level compared to medium or large resting sites at medium or ground level. The proportion of males was highest at the ground, while the proportion of engorged females was highest at medium and top level. Due to the higher proportion of blood-engorged females, artificial resting sites are especially useful for studies of host-feeding patterns or xenosurveillance. Low costs and efforts allow a cost-effective increase of the number of resting sites per sampling site to collect more mosquitoes.

Microbiome analysis of the midguts of different developmental stages of Argas persicus in China

Argas persicus is an ectoparasite of poultry. The bacterial community structure and the pathogenic bacteria associated with different developmental stages of A. persicus have implications for control. Argas persicus were collected from chickens in the city of Jiuquan in Gansu, China. Bacterial DNA was extracted from the midgut contents of blood engorged larvae, nymphs and adult females. The V3-V4 hypervariable regions of 16S rRNA genes were sequenced using the IonS5™XL platform. Identification of Rickettsia spp. and detection of Coxiella burnetii were performed using PCR on target genes. The bacterial diversity within larvae was the highest and the bacterial diversity within nymphs was greater than that of adults. At different classification levels, seven bacterial phyla were common phyla, 27 genera were common genera, and 18 species were common species in the three samples. At the phylum level, Proteobacteria showed a marked predominance in all samples. Rickettsia, Stenotrophomonas, Spiroplasma, and Coxiella were the dominant bacteria at the genus level. The Rickettsia species in A. persicus was identified as Rickettsia hoogstraalii and the Coxiella species was identified as a Coxiella-like endosymbiont. Additionally, some bacterial species such as Pseudomonas geniculata, Sphingomonas koreensis, and Acinetobacter haemolyticus were reported here for the first time in A. persicus.

Examining the Relationship of Clinical and Laboratory Parameters With Infectiousness to Phlebotomus perniciosus and Its Potential Infectivity in Dogs With Overt Clinical Leishmaniasis.

Infected dogs are considered the main domestic animal reservoirs for Leishmania infantum parasite. Infectiousness to competent phlebotomine vectors has been associated with many factors, the main being the severity of the disease exhibited by infected dogs. This study examines the relationship between different clinical parameters and the infectiousness to colonized Phlebotomus perniciosus sand flies having a blood meal on dogs. Data obtained in the present study come from an untreated group of Leishmania sick dogs submitted to xenodiagnosis for the evaluation of a spot on insecticide solution. Seventeen dogs were diagnosed as affected by leishmaniasis through clinical examination, immunofluorescence antibody test (IFAT) serology, and loop-mediated isothermal amplification (LAMP). The disease severity (clinical score) was staged by using a numeric value derived from eight clinical and parasitological parameters. Xenodiagnosis was performed on caged dogs exposed for 1.5 h to sand-fly bites. The following parameters related to sand flies were examined: blood feeding (% of blood engorged females), promastigote detection (% of promastigote-positive sand flies), promastigote burden, and the promastigote stage maturation (potential transmissibility rate). Statistical relationship between the clinical score and entomological parameters was investigated, as well as the possible correlation between each clinical and laboratory parameters and sand fly infection/infectivity. The severity of clinical score may influence the blood feeding by, and the probability of promastigote detection in, sand flies; skin lesions seem to be the main factor that influences the rate of blood feeding. Promastigote burden is related to IFAT titer, skin lesions, and clinical score. All entomological parameters are strongly related among them. This study confirms that both P. perniciosus infection and infectivity are influenced by a dog's clinical condition.

Genetic variation, DNA barcoding and blood meal identification of Culicoides Latreille biting midges (Diptera: Ceratopogonidae) in Thailand

Biting midges of the genus Culicoides Latreille are blood sucking insects of medical and veterinary importance. Many species are vectors of disease agents transmitted to humans and other animals. Therefore, rapid and accurate species identification is essential for appreciation of all aspects of these insects. In this study, DNA barcode efficacy and molecular identification of host blood sources were examined in biting midges from Thailand. A total of 203 barcoding sequences were obtained from 16 Culicoides taxa. Intraspecific genetic divergence varied from 0.28% to 9.90% for specimens collected in Thailand. Despite this high level of genetic variation, DNA barcode identifications in the Barcoding of Life Data System had a considerable success rate (90%). Phylogenetic analyses and distance-based species delimitation methods indicated the possibility of cryptic species in four taxa, namely, Culicoides actoni Smit, C. arakawae Arakawa, C. huffi Causey and C. jacobsoni Macfie. Further investigations will be required to examine the species status of these lineages. Host blood meal identifications from 42 blood engorged females of 10 Culicoides taxa revealed three animal hosts: chicken, cattle and buffalo. Most of this information agrees with previous knowledge but this is the first report of C. actoni, C. fulvus and C. huffi feeding on chicken.

Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as leucocytozoon vectors

Identification of the host blood meal of hematophagous insects can provide significant information regarding host preferences and the possibility of the transmission of disease agents. Currently, this knowledge is limited for black flies in the Oriental region. In this study, we used cytochrome b gene sequences for identification of blood meal sources of two black fly taxa, the Simulium asakoae complex and S. chumpornense Takaoka & Kuvangkadilok in Thailand. A total of 4,260 wild adult females were visually screened revealing 24 blood-engorged females from which cytochrome b was successfully amplified in 19 individuals. Comparisons in GenBank database revealed that all are closest to chicken (Gallus gallus) with sequence similarity of >98%. Therefore, these black fly species are feeding on chickens. We also molecularly investigated the hemosporidian blood protozoa genus Leucocytozoon in black flies and found 13 of 19 blood-engorged females positive for this protozoon. Sequence analysis revealed that this Leucocytozoon DNA could be assigned into two previously recognized groups, one with a Leucocytozoon reported from domestic chickens and black flies in Thailand, and another close to the L. schoutedeni. Our results indicate a high possibility that the S. asakoae complex and S. chumpornense are natural vectors of Leucocytozoon.

Host Associations Of Biting Midges (Diptera: Ceratopogonidae: Culicoides) Near Sentinel Chicken Surveillance Locations In Florida, USA.

Quantifying host use is important for understanding transmission of vector-borne pathogens. Despite the importance of biting midges (Diptera: Ceratopogonidae) in pathogen transmission, the vector-host relationships of most Culicoides species are poorly documented, even in locations where active arbovirus surveillance is conducted. Polymerase chain reaction-based blood-meal analysis was performed on 663 blood-engorged Culicoides females collected by 7 Florida mosquito control districts at 24 sentinel chicken arbovirus surveillance sites in 2017. A total of 638 blood meals were successfully analyzed to determine host species source, representing 11 Culicoides species. The most commonly bitten host was domestic chicken (Gallus gallus) (presumably sentinel chickens), constituting 565 of 638 (88.6%) the total blood meals. Other common hosts included humans (5.8%), white-tailed deer (Odoocoileus virginianus) (2.5%), and brown basilisk (Basiliscus vittatus) (1.6%). Significant differences in distribution of mammal and avian blood meals were found for a number of Culicoides species, and these patterns did not vary across locations. These results indicate that sentinel chickens are exposed to bites by Culicoides, potentially exposing them to Culicoides-borne pathogens. The findings that vertebrate host use was consistent across locations suggests that each Culicoides species has affinity for one or more specific animal groups, and does not feed randomly upon available animals.

Bloodmeal host identification with inferences to feeding habits of a fish-fed mosquito, Aedes baisasi

The mosquito, Aedes baisasi, which inhabits brackish mangrove swamps, is known to feed on fish. However, its host assemblage has not been investigated at the species level. We amplified and sequenced the cytochrome oxidase subunit I barcoding regions as well as some other regions from blood-fed females to identify host assemblages in the natural populations from four islands in the Ryukyu Archipelago. Hosts were identified from 230 females. We identified 15 host fish species belonging to eight families and four orders. Contrary to expectations from previous observations, mudskippers were detected from only 3% of blood-engorged females. The dominant host was a four-eyed sleeper, Bostrychus sinensis (Butidae, Gobiiformes), in Iriomote-jima Island (61%), while it was a snake eel, Pisodonophis boro (Ophichthidae, Anguilliformes), in Amami-oshima and Okinawa-jima islands (78% and 79%, respectively). Most of the identified hosts were known as air-breathing or amphibious fishes that inhabit mangroves or lagoons. Our results suggest that A. baisasi females locate the bloodmeal hosts within the mangrove forests and sometimes in the adjacent lagoons and land on the surface of available amphibious or other air-breathing fishes exposed in the air to feed on their blood.

Potential of Aedes albopictus as a bridge vector for enzootic pathogens at the urban-forest interface in Brazil

The invasive species Aedes albopictus is present in 60% of Brazilian municipalities, including at the interfaces between urban settings and forests that are zoonotic arbovirus hotspots. We investigated Ae. albopictus colonization, adult dispersal and host feeding patterns in the anthropic-natural interface of three forested sites covering three biomes in Brazil in 2016. To evaluate whether an ecological overlap exists between Ae. albopictus and sylvatic yellow fever virus (YFV) in forests, we performed similar investigations in seven additional urban-forest interfaces where YFV circulated in 2017. We found Ae. albopictus in all forested sites. We detected eggs and adults up to 300 and 500 m into the forest, respectively, demonstrating that Ae. albopictus forest colonization and dispersal decrease with distance from the forest edge. Analysis of the host identity in blood-engorged females indicated that they fed mainly on humans and domestic mammals, suggesting rare contact with wildlife at the forest edge. Our results show that Ae. albopictus frequency declines as it penetrates into the forest and highlight its potential role as a bridge vector of zoonotic diseases at the edge of the Brazilian forests studied.

Vector competence of pre-alpine Culicoides (Diptera: Ceratopogonidae) for bluetongue virus serotypes 1, 4 and 8

BackgroundBluetongue disease, caused by bluetongue virus serotype 8 (BTV-8), appeared for the first time in the northern part of Europe in 2006, and subsequently rapidly spread causing severe economic losses to the farming industry. The implicated vectors of BTV in Europe are Culicoides species within the subgenus Avaritia (C. chiopterus, C. dewulfi, C. obsoletus and C. scoticus). Epidemiological data from Switzerland have shown that BTV, whose spread was eliminated at an early stage by vaccination campaigns, had not been circulating among livestock at higher altitudes where other species dominate the Culicoides fauna. In this study, we investigated the extent that Culicoides spp. prevailing at higher altitudes (mainly C. grisescens) can act as vectors for BTV.MethodsCulicoides were collected at farms in the pre-alpine region (two sites at 1550 m above sea level, masl, referred to as pre-alpine I; one site at 2030 masl, pre-alpine II) and, for comparative purposes, from the Swiss Plateau (one site, 650 masl). They were fed on bovine blood/BTV suspensions (BTV-1, 4 or 8) and incubated for eight days under a fluctuating temperature regime (13–25 °C, mean 19 °C), reflecting a mid-summer warm spell in the pre-alpine region. Susceptibility to BTV transmission was assessed from head homogenates by RT-qPCR and virus isolation.ResultsOverall, 9196 female Culicoides were exposed to the three BTV strains through an artificial membrane, with feeding rates of 14–27%. Survival rates of blood-engorged Culicoides females at eight days post-infection depended on both virus serotype and altitude of origin. Virus dissemination (Cq ≤ the cut-off value as determined by serial virus dilutions) was confirmed only for BTV-1 in C. scoticus (dissemination efficiency 22.5%; 9/40) and C. obsoletus (5.6%; 1/18) from the Swiss Plateau area. There was no strong evidence of susceptibility to infection for Culicoides from the pre-alpine area when fed with all BTV strains (BTV-1, 4 and 8).ConclusionsThis study confirms the susceptibility of C. scoticus and C. obsoletus to BTV-1 infection, including under cooler temperatures. Culicoides grisescens, which is highly abundant at higher altitudes, cannot be considered a potential vector under these temperature conditions.

Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain.

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host-feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood-engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.

Some observations on overwintering sites of adult Culex quinquefasciatus (Diptera: Culicidae) and strategies followed under natural and seminatural conditions.

Field population of adult Culex quinquefasciatus Say, landed in December, congregated and overwintered in indoor artificial sites in Delhi. Repeated sampling strategy by individually collecting the adults was adopted to study their overwintering strategies for 4 years (December to April). They remained vagile and readily repopulated the resting sites after the removal of samples. A large percentage of females was fertile, unfed and nulliparous indicating that reproduction ceased in them. Adult survival was significantly prolonged to a maximum of 3 months under natural conditions. Gonotrophic cycle also got prolonged. Close to quitting, they became gravid and left in April without oviposition. No adults were observed on the sites for the rest of the year. Oviposition was induced in the blood-engorged females when provided with food, water and outdoor conditions. Oviposition might have been induced directly by water and food provided them energy under seminatural conditions. Eggs were laid singly or in the form of rafts, and the number in both the cases was low. Singly laid eggs did not hatch, and in rafts, hatching was ~80 %. Winter conditions seemed to strongly impact fertility, blood feeding, fecundity, oviposition behaviour, egg hatchability and longevity. Use of the overwintering sites as biological tool, as a part of environmental control in IPM, is suggested for organising antivector measures during winter. There is a need of exploring and creating more sites of this kind.

Blood meal analysis of culicoides (Diptera: ceratopogonidae) in central Tunisia.

To evaluate the host preferences of Culicoides species (Diptera: Ceratopogonidae) in Central Tunisia, we identified the source of blood meals of field collected specimens by sequencing of the cytochrome b (cyt b) mitochondrial locus and Prepronociceptine single copy nuclear gene. The study includes the most common and abundant livestock associated species of biting midges in Tunisia: C. imicola, C. jumineri, C. newsteadi, C. paolae, C. cataneii, C. circumscriptus, C. kingi, C. pseudojumineri, C. submaritimus, C. langeroni, C. jumineri var and some unidentified C. species. Analysis of cyt b PCR products from 182 field collected blood-engorged females’ midges revealed that 92% of them fed solely on mammalian species, 1.6% on birds, 2.4% on insects and 0.8% on reptiles. The blast results identified the blood origin of biting midges to the species level with exact or nearly exact matches (≥98%). The results confirm the presence of several Culicoides species, including proven vectors in Central Tunisia. Blood meal analyses show that these species will indeed feed on bigger mammals, thereby highlighting the risk that these viruses will be able to spread in Tunisia.

Isolation of haemolytic bacilli from field-collected Culicoides oxystoma and Culicoides peregrinus: potential vectors of bluetongue virus in West Bengal, India.

Two haemolytic bacterial strains of Bacillus pumilus (CU1A, CU1B) and one blood-utilizing strain of Bacillus licheniformis (CU2B) were isolated from relatively low numbers of field-collected females of Culicoides oxystoma and Culicoides peregrinus (Diptera: Ceratopogonidae). A total of 36 females, including 18 of each of C. oxystoma and C. peregrinus (consisting of one and a pool of eight blood-engorged specimens, and one and a pool of eight non-engorged specimens for each species), were tested. In C. oxystoma, all three strains of bacteria were isolated from the one non-engorged, the pool of non-engorged and the pool of blood-engorged females tested, but CU1A and CU2B were not found in the one blood-engorged female tested. In C. peregrinus, all three strains were present in the pool of blood-engorged females. However, the strain CU2B was not found in the pool of non-engorged females. In the one blood-engorged and one non-engorged female tested, CU1A and CU2B were detected. The bacterial strains were identified based on Gram staining, enzyme activity (amylase and protease) and alignment of the 16S rRNA partial gene sequence to that available in the National Center for Biotechnology Information (NCBI) database GenBank. The functional role and significance of these haemolytic and blood-digesting bacteria within the genus Culicoides remain to be determined.

Resting environments of some Costa Rican mosquitoes

The resting sites of tropical American mosquitoes are poorly documented, and the few reports that do exist are largely from opportunistic collections. Since blood-engorged females (used in determining host associations) are more efficiently collected from resting sites than attractive traps, information on resting site utilization has practical value. To investigate differences in the resting sites utilized by tropical mosquitoes, we collected and identified female mosquitoes from one man-made (resting shelter) and three natural (buttress tree roots, hollow trees, and understory vegetation) resting environments at a tropical dry forest location in western Costa Rica. All of the most common species collected demonstrated associations with one or more resting environments. Females of five species (blood-engorged Anopheles albimanus, Uranotaenia apicalis, Uranotaenia lowii, Uranotaenia orthodoxa, and blood-engorged Mansonia titillans) were collected in significantly greater numbers from understory vegetation than other resting environments. Culex erraticus and other members of the subgenus Melanoconion were encountered more often in resting shelters, hollow trees, and buttress roots, while Culex restrictor (blood-engorged) females were associated with hollow trees. Similarity indices indicate that buttress tree roots, hollow trees, and resting shelters are similar with respect to the mosquito communities that utilize them as resting sites, while understory vegetation has a resting fauna that is different than the other environments surveyed here. These results add to the body of information regarding resting sites utilized by tropical American mosquitoes.

Molecular identification of bloodmeals and species composition in Culicoides biting midges

Investigations of host preferences in haematophagous insects, including Culicoides biting midges (Diptera: Ceratopogonidae), are critical in order to assess transmission routes of vector-borne diseases. In this study, we collected and morphologically identified 164 blood-engorged Culicoides females caught in both light traps and permanent 12-m high suction traps during 2008-2010 in Sweden. Molecular analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the biting midges was performed to verify species classification, discern phylogenetic relationships and uncover possible cryptic species. Bloodmeal analysis using universal vertebrate cytochrome b primers revealed a clear distinction in host selection between mammalophilic and ornithophilic Culicoides species. Host sequences found matches in horse (n = 59), sheep (n = 39), cattle (n = 26), Eurasian elk (n = 1) and 10 different bird species (n = 18). We identified 15 Culicoides species previously recorded in Scandinavia and four additional species haplotypes that were distinctly different from the described species. All ornithophilic individuals (n = 23) were caught exclusively in the suction traps, as were, interestingly, almost all mammalophilic species (n = 41), indicating that many biting midge species may be able to cover long distances after completing a bloodmeal. These results add new information on the composition of Culicoides species and their host preferences and their potential long-distance dispersal while blood-engorged.