Phlebotomine sand fly (Diptera: Psychodidae) fauna, blood meal source, and detection of Leishmania (Kinetoplastida: Trypanosomatidae) DNA in the Gurupi Biological Reserve, Eastern Amazon, Brazil.

This study was conducted in the Gurupi Biological Reserve (REBIO-Gurupi), the largest area of Amazon rainforest in Maranhão State, Brazil. The objectives were to survey the sand fly (Diptera: Psychodidae) fauna of REBIO-Gurupi, identify blood meal sources, and investigate the presence of Leishmania (Ross, 1903) (Kinetoplastida: Trypanosomatidae) DNA. Individuals were collected using Centers for Disease Control (CDC) light traps and black and white Shannon traps in May and Jun 2022 and Jan 2023. DNA was extracted from female sand flies and subjected to amplification and sequencing of cytochrome b molecular marker (CYTB) for identification of blood meal sources and the first internal transcribed spacer (ITS-1) of ribosomal DNA for Leishmania detection. A total of 514 sand flies individuals were sampled, of which 93 were identified at the genus or series level (9 taxa) and 421 were identified at the species level (24 taxa). Psychodopygus davisi (Root, 1934) (41.1%), Nyssomyia antunesi (Coutinho, 1939) (10.3%), and Psychodopygus (Mangabeira, 1941) Chagasi Series Barretto, 1962 (9.7%) were the most frequently collected. Human (Homo sapiens, Primates, Hominidae) and tapir (Tapirus terrestris, Perissodactyla, Tapiridae) DNA was detected in 10 female sand flies. Leishmania (Leishmania) infantum Cunha and Chagas, 1937 DNA was detected in 2 specimens of Ps. davisi. Given the presence of vectors of Leishmania in REBIO-Gurupi, it is imperative to conduct more comprehensive studies on the interactions among sand flies, Leishmania, and pathogen reservoirs in the area.

Mosquito blood-feeding patterns and nesting behavior of American crows, an amplifying host of West Nile virus.

BACKGROUND: Although American crows are a key indicator species for West Nile virus (WNV) and mount among the highest viremias reported for any host, the importance of crows in the WNV transmission cycle has been called into question because of their consistent underrepresentation in studies of Culex blood meal sources. Here, we test the hypothesis that this apparent underrepresentation could be due, in part, to underrepresentation of crow nesting habitat from mosquito sampling designs. Specifically, we examine how the likelihood of a crow blood meal changes with distance to and timing of active crow nests in a Davis, California, population. METHODS: Sixty artificial mosquito resting sites were deployed from May to September 2014 in varying proximity to known crow nesting sites, and Culex blood meal hosts were identified by DNA barcoding. Genotypes from crow blood meals and local crows (72 nestlings from 30 broods and 389 local breeders and helpers) were used to match mosquito blood meals to specific local crows. RESULTS: Among the 297 identified Culex blood meals, 20 (6.7%) were attributable to crows. The mean percentage of blood meals of crow origin was 19% in the nesting period (1 May-18 June 2014), but 0% in the weeks after fledging (19 June-1 September 2014), and the likelihood of a crow blood meal increased with proximity to an active nest: the odds that crows hosted a Culex blood meal were 38.07 times greater within 10 m of an active nest than > 10 m from an active nest. Nine of ten crow blood meals that could be matched to a genotype of a specific crow belonged to either nestlings in these nests or their mothers. Six of the seven genotypes that could not be attributed to sampled birds belonged to females, a sex bias likely due to mosquitoes targeting incubating or brooding females. CONCLUSION: Data herein indicate that breeding crows serve as hosts for Culex in the initial stages of the WNV spring enzootic cycle. Given their high viremia, infected crows could thereby contribute to the re-initiation and early amplification of the virus, increasing its availability as mosquitoes shift to other moderately competent later-breeding avian hosts.

Effect of Dinotefuran, Permethrin, and Pyriproxyfen (Vectra® 3D) on the Foraging and Blood-Feeding Behaviors of Aedes albopictus Using Laboratory Rodent Model.

Dinotefuran-Permethrin-Pyriproxyfen (DPP) is used to kill and repel mosquitoes from dogs. However, the influence of the product on the host-seeking behavior of mosquitoes remains unknown. The interference of DPP with the host selection of unfed female Aedes albopictus was investigated. A total of 18 animals (9 mice and 9 rats) were divided into three groups of six animals each. DU: DPP treated rats (n = 3) with untreated mice (n = 3), UD: DPP treated mice (n = 3) with untreated rats (n = 3) and control UU: untreated mice (n = 3) and untreated rats (n = 3). In each group, the rats and mice were placed 30 cm apart. After sedation, the animals in each group were exposed twice (Day 1 and Day 7 post-treatment) for one hour to 71 ± 3 female mosquitoes. Mosquitoes were categorized after the 2-h post-exposure period as dead or alive. Blood-meal origin was determined from mosquitoes using a newly customized duplex qPCR. The highest values of forage ratio (1.36 ≥ wi ≤ 1.88) and selection index (0.63 ≥ Bi ≤ 0.94) for rat hosts indicates a preference of mosquitoes for this species as compared to mice when co-housed during the exposure. The mosquitoes only seldom fed on mice, even in the untreated group. The anti-feeding effect of DPP was therefore only assessed on rat's hosts. The results showed that DPP, when directly applied on rats, provided a direct protection of 82% and 61% on Day 1 and Day 7, respectively, while when applied on mice hosts (UD), the DPP provided an indirect protection of 21% and 10% on Day 1 and Day 7, respectively. The results showed also that DPP, when applied on rats, provided a direct protection against Ae. albopictus bites. This effect did not result in increased exposure of the untreated host placed in the same cage at a distance of 30 cm.

An Improved Multiplex Polymerase Chain Reaction (PCR) Assay for the Identification of Mosquito (Diptera: Culicidae) Blood Meals.

The analysis of vertebrate blood meals serves as an integral component of vector incrimination studies where feeding preferences and host associations influence vector-borne disease transmission. Diagnostic polymerase chain reaction (PCR)-based techniques have been widely used to determine host associations, yet applications for Culex (Diptera: Culicidae), which feed primarily on bird populations, have been limited by multistep PCR techniques that approach each potential host species singly. As a result, we have developed a multiplexed primer set targeting mitochondrial cytochrome b sequences that can distinguish human, bird, and mammalian host blood meals in a single PCR reaction, an improvement over previous analyses relying on single primers or other multiplex primer approaches through the inclusion of avian primers. To validate this new methodology, we demonstrate its application on blood samples as well as field-collected Culex samples. Although designed for applications with mosquito vectors, this multiplex PCR assay is not mosquito-specific, and should serve as a valuable tool for identifying the blood meals of other blood-feeding arthropods, contributing greatly to the study of vector-borne disease.

Evaluation of resting traps to examine the behaviour and ecology of mosquito vectors in an area of rapidly changing land use in Sabah, Malaysian Borneo.

BACKGROUND: Widespread deforestation occurring in the tropics is hypothesized to impact the transmission of vector-borne diseases (VBD). Predicting how environmental changes will impact VBD transmission is dependent on understanding the ecology and behaviour of potential vector species outside of domestic settings. However there are few reliable sampling tools for measuring the habitat preference and host choice of mosquito vectors; with almost none suitable for sampling recently blood-fed, resting mosquitoes. This study evaluated the use of two mosquito traps: the resting bucket (RB) and sticky resting bucket (SRB) traps relative to CDC backpack aspiration (CDC) for sampling mosquitoes resting in a range of habitats representing a gradient of deforestation. Eight habitats were selected for sampling around two villages in Kudat District, Malaysian Borneo, to reflect the range of habitats available to mosquitoes in and around human dwellings, and nearby forest habitats where reservoir hosts are present: secondary forest (edge, interior and canopy); plantations (palm and rubber); and human settlements (inside, under and around houses). RESULTS: Over 31 days, 2243 mosquitoes were collected in 5748 discrete collections. Nine mosquito genera were sampled with Aedes and Culex species being present in all habitats and most abundant. RB and CDC backpack aspiration were most efficient for sampling Culex whereas CDC backpack aspiration and SRB were most efficient for Aedes. Most Aedes identified to species level were Ae. albopictus (91%), with their abundance being highest in forest edge habitats. In contrast, Culex were most abundant under houses. Most blood-fed mosquitoes (76%) were found in human settlements; with humans and chickens being the only blood source. CONCLUSIONS: RB and SRB traps proved capable of sampling mosquitoes resting in all sampled habitats. However, sampling efficiency was generally low (c.0.1 per trap per day), necessitating traps to be deployed in high numbers for mosquito detection. None of the traps were effective for sampling zoonotic malaria vectors; however, SRB collected relatively higher numbers of the dengue vector Ae. albopictus. The higher abundance of mosquitoes in forest edge habitats indicates the potential value of these traps for investigating sylvatic dengue transmission. This study has demonstrated the merits in application of simple resting traps for characterising mosquito vector resting behaviour outside of the home.

Seasonal Distribution, Blood-Feeding Habits, and Viruses of Mosquitoes in an Open-Faced Quarry in Connecticut, 2010 and 2011.

Seasonal abundance of mosquitoes, their viruses, and blood-feeding habits were determined at an open-faced quarry in North Branford, CT, in 2010 and 2011. This unique habitat had not previously been sampled for mosquitoes and mosquito-borne viruses. Thirty species of mosquitoes were identified from 41,719 specimens collected. Coquillettidia perturbans, Aedes trivittatus, and Ae. vexans were the most abundant species and represented 34.5%, 17.7%, and 14.8% of the totals, respectively. Jamestown Canyon virus was isolated from 6 species of mosquitoes collected from mid-June through July: Cq. perturbans (3 pools), Ae. cantator (3), Ae. trivittatus (2), Ae. aurifer (1), Ae. excrucians (1), and Culex pipiens (1). West Nile virus was cultured from 8 pools of Cx. pipiens and from 1 pool of Culiseta melanura collected from mid-August through late September. Cache Valley virus was isolated from 4 species of mosquitoes in 3 genera from about mid-August through late September 2011: Cq. perturbans (5 pools), Ae. trivittatus (2), Anopheles punctipennis (1), and An. quadrimaculatus (1). Nine different mammalian hosts were identified as sources of blood for 13 species of mosquitoes. White-tailed deer, Odocoileus virginianus, were the most common mammalian hosts (90.8%), followed by raccoon, Procyon lotor (3.1%), coyote, Canis latrans (2.4%), and human, Homo sapiens (1.2%). Exclusive mammalian blood-feeding mosquitoes included: Ae. canadensis, Ae. cantator, Ae. excrucians, Ae. japonicus, Ae. vexans, An. punctipennis, and Cx. salinarius. Fourteen species of birds, mostly Passeriformes, were identified as sources of blood from 6 mosquito species. Five species that fed on mammals (Ae. thibaulti, Ae. trivittatus, Ae. cinereus, Cq. perturbans, and Cx. pipiens) also fed on birds.

Blood Meal Source Characterization Using Illumina Sequencing in the Chagas Disease Vector Rhodnius pallescens (Hemiptera: Reduviidae) in Panamá.

Accurate blood meal identification is critical to understand hematophagous vector-host relationships. This study describes a customizable Next-Generation Sequencing (NGS) approach to identify blood meals from Rhodnius pallescens (Hemiptera: Reduviidae) triatomines using multiple barcoded primers and existing software to pick operational taxonomic units and match sequences for blood meal identification. We precisely identified all positive control samples using this method and further examined 74 wild-caught R. pallescens samples. With this novel blood meal identification method, we detected 13 vertebrate species in the blood meals, as well as single and multiple blood meals in individual bugs. Our results demonstrate the reliability and descriptive uses of our method.

Molecular detection of Leishmania parasites and host blood meal identification in wild sand flies from a new endemic rural region, south of Iran.

Zoonotic Cutaneous Leishmaniosis (ZCL) remains the most crucial vector-borne public health disease particularly in endemic rural parts of Iran. The main aim of this study is to identify wild sand flies (Diptera: Psychodidae), determine their infection rate, and differentiate their host blood meal sources using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Sand fly populations were caught with sticky paper traps from 10 different villages in the county of Darab, Fars province, southern Iran. Following their species identification, they were used in one step PCR to determine their infection with Leishmania spp. parasites. They were then subjected to PCR-RFLP protocol to identify and differentiate their blood meal sources. Two genera of Phlebotomus and Sergentomyia comprising 13 species of sand flies were identified in this region. From a total of 150 parous female sand flies, encompassing 4 different medically important species, 7 specimens (4.7%) including 6 Phlebotomus papatasi and 1 Phlebotomus bergeroti were infected with Leishmania major. Molecular data indicated that about 32% of female sand flies fed on man, while nearly 43% fed on rodent and canine hosts. Molecular detection is an efficient way of differentiating the source of blood meals in female sand flies feeding on different vertebrate hosts. It is suggested that P. papatasi is not highly anthropophagic and appears to be an opportunistic feeder on man. This species is, however, the primary vector of ZCL in this region.

Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding.

BACKGROUND: Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. METHODS: We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. RESULTS: Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. CONCLUSIONS: Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided.

Identification of sandflies (Diptera: Psychodidae: Phlebotominae) blood meals in an endemic Leishmaniasis area in Brazil / Identificação do repasto sanguíneo de flebotomíneos (Diptera: Psychodidae: Phlebotominae) provenientes de área endêmica de Leishmaniose no Brasil

SUMMARYThe aim of this study was to identify blood meals of female sandflies captured in the municipality of Governador Valadares, an endemic area of visceral and cutaneous leishmaniasis, in the State of Minas Gerais, Brazil. From May 2011 to January 2012, captures were performed using HP light traps in four districts. There were 2,614 specimens (2,090 males and 524 females) captured; 97 engorged females were identified belonging to the species Lutzomyia longipalpis(82.1%) and Lutzomyia cortelezzii(17.9%). Considering simple and mixed feeding, the enzyme-linked immunosorbent assay revealed a predominance of chicken blood (43.6%) in Lutzomyia longipalpis, showing the important role that chickens exert around the residential areas of Governador Valadares. This finding increases the chances of sandflies contact with other vertebrates and consequently the risk of leishmaniasis transmission.

RESUMOO objetivo deste estudo foi identificar o repasto sanguíneo de fêmeas de flebotomíneos capturadas no município de Governador Valadares, área endêmica de leishmaniose visceral e tegumentar no Estado de Minas Gerais, Brasil. Entre maio de 2011 e janeiro 2012 foram realizadas capturas com armadilhas luminosas HP em quatro bairros. Foram capturados 2.614 exemplares (2.090 machos e 524 fêmeas). Noventa e sete fêmeas ingurgitadas foram identificadas como pertencentes às espécies Lutzomyia longipalpis(82,1%) e Lutzomyia cortelezzii(17,9%). Considerando a alimentação simples e a mista, o ensaio imunoenzimático revelou em Lutzomyia longipalpisuma predominância de sangue de galinhas (43,6%), mostrando o importante papel que galinhas podem exercer no peridomicílio, aumentando a chance de contato dos flebotomíneos com outros vertebrados e, consequentemente, o risco de transmissão da leishmaniose.

Blood meal analysis and virus detection in blood-fed mosquitoes collected during the 2006-2007 Rift Valley fever outbreak in Kenya.

BACKGROUND: Rift Valley fever (RVF) is a zoonosis of domestic ruminants in Africa. Blood-fed mosquitoes collected during the 2006-2007 RVF outbreak in Kenya were analyzed to determine the virus infection status and animal source of the blood meals. MATERIALS AND METHODS: Blood meals from individual mosquito abdomens were screened for viruses using Vero cells and RT-PCR. DNA was also extracted and the cytochrome c oxidase 1 (CO1) and cytochrome b (cytb) genes amplified by PCR. Purified amplicons were sequenced and queried in GenBank and Barcode of Life Database (BOLD) to identify the putative blood meal sources. RESULTS: The predominant species in Garissa were Aedes ochraceus, (n=561, 76%) and Ae. mcintoshi, (n=176, 24%), and Mansonia uniformis, (n=24, 72.7%) in Baringo. Ae. ochraceus fed on goats (37.6%), cattle (16.4%), donkeys (10.7%), sheep (5.9%), and humans (5.3%). Ae. mcintoshi fed on the same animals in almost equal proportions. RVFV was isolated from Ae. ochraceus that had fed on sheep (4), goats (3), human (1), cattle (1), and unidentified host (1), with infection and dissemination rates of 1.8% (10/561) and 50% (5/10), respectively, and 0.56% (1/176) and 100% (1/1) in Ae. mcintoshi. In Baringo, Ma. uniformis fed on sheep (38%), frogs (13%), duikers (8%), cattle (4%), goats (4%), and unidentified hosts (29%), with infection and dissemination rates of 25% (6/24) and 83.3% (5/6), respectively. Ndumu virus (NDUV) was also isolated from Ae. ochraceus with infection and dissemination rates of 2.3% (13/561) and 76.9% (10/13), and Ae. mcintoshi, 2.8% (5/176) and 80% (4/5), respectively. Ten of the infected Ae. ochraceus had fed on goats, sheep (1), and unidentified hosts (2), and Ae. mcintoshi on goats (3), camel (1), and donkey (1). CONCLUSION: This study has demonstrated that RVFV and NDUV were concurrently circulating during the outbreak, and sheep and goats were the main amplifiers of these viruses respectively.

Blood meal identification and feeding habits of uranotaenia species collected in the ryukyu archipelago.

To know the blood meal in the stomach of Uranotaenia species, blood-fed mosquitoes were collected by 4 methods at different sites in the mountain forest of 3 islands, Amamioshima, Okinawajima, and Iriomotejima in the Ryukyu Archipelago, Japan from 2005 to 2012. One hundred twenty-four blood-fed Uranotaenia mosquitoes of 7 species (Ur. jacksoni, nivipleura, ohamai, yaeyamana, annandalei, lateralis, and macfarlanei) were collected. The collection rates are 0.26, 0.6, 0.31, and 0.66 by black light trap, black light blue with dry ice trap, frog call trap, and sweeping net, respectively. The blood meals of 107 females (86.3%) were successfully identified by a polymerase chain reaction-based method. All Uranotaenia species fed on cold-blooded animals, especially amphibians (99.1%), and notably on frogs. They would feed readily on available frogs in a given region having no close connection with the breeding (calling) season of each frog. They also fed on reptiles (0.9%), but not on warm-blooded animals.

Identification of host blood from engorged mosquitoes collected in western Uganda using cytochrome oxidase I gene sequences.

Emerging infectious disease events are frequently caused by arthropod-borne viruses (arboviruses) that are maintained in a zoonotic cycle between arthropod vectors and vertebrate wildlife species, with spillover to humans in areas where human and wildlife populations interface. The greater Congo basin region, including Uganda, has historically been a hot spot for emergence of known and novel arboviruses. Surveillance of arthropod vectors is a critical activity in monitoring and predicting outbreaks of arboviral disease, and identification of blood meals in engorged arthropods collected during surveillance efforts provides insight into the ecology of arboviruses and their vectors. As part of an ongoing arbovirus surveillance project we analyzed blood meals from engorged mosquitoes collected at five sites in western Uganda November 2008-June 2010. We extracted DNA from the dissected and triturated abdomens of engorged mosquito specimens. Mitochondrial cytochrome c oxidase I gene sequence was amplified by PCR and sequenced to identify the source of the mosquito host blood. Blood meals were analyzed from 533 engorged mosquito specimens; 440 of these blood meals were successfully identified from 33 mosquito species. Species identifications were made for 285 of the 440 identified specimens with the remainder identified to genus, family, or order. When combined with published arbovirus isolation and serologic survey data, our results suggest possible vector-reservoir relationships for several arboviruses, including Rift Valley fever virus and West Nile virus.

Establishment of a molecular tool for blood meal identification in Malaysia.

OBJECTIVE: To establish a polymerase chain reaction (PCR) technique based on cytochrome b (cytb) gene of mitochondria DNA (mtDNA) for blood meal identification. METHODS: The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in GenBank. PCR was next performed to compile gene sequences of different species of wild rodents. The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA. A total of 100 blood samples, both from laboratory animals and wild rodents were collected and analyzed. The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species. RESULTS: Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus (rattus) tanezumi, Rattus tiomanicus, Leopoldamys sabanus, Tupaia glis, Tupaia minor, Niviventor cremoriventor, Rhinosciurus laticaudatus, Callosciurus caniseps, Sundamys muelleri, Rattus rajah and Maxomys whiteheadi. The BLAST results confirmed the host with exact or nearly exact matches (>89% identity). Ten new gene sequences have been deposited in GenBank database since September 2010. CONCLUSIONS: This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.