Parasite-host relationships of water mites (Acari: Hydrachnidia) and black flies (Diptera: Simuliidae) in southeastern Spain.

BACKGROUND: Documentation on water mites in Spain is scarce, as is information on the parasite-host relationship between certain water mite species and representatives of the dipteran family Simuliidae. The discomfort caused to humans and animals by black flies seems to be increasing in recent years. In this context, an investigation of parasitic water mites is of great importance, not only from the point of view of biodiversity, but also in terms of their potential to control black fly populations. METHODS: Rivers across a wide region of eastern Spain were sampled to determine the specific richness of simuliid dipterans and to investigate their possible parasites, such as water mites, mermithid nematodes and microsporidia (fungal microbes). Data on environmental variables, abundance, prevalence and intensity of parasitism on the collected specimens were analyzed. RESULTS: In 10 streams, 15,396 simuliid pupae were collected and checked for the presence of water mite larvae; 426 pupae in seven streams were found to be associated with water mite larvae. Of the 21 simuliid species identified based on morphological characters, eight were found to be associated with water mite larvae. Water mite infection was not equally distributed among black fly species. Also, the prevalence of parasitism was low and differed among simuliid species, ranging from one to 13 water mites per black fly pupa. Variation at the intra- and interspecific levels was detected in terms of the number of water mites inside the black fly cocoons. Free-living deutonymphal and adult water mites representing 15 different species of six genera and five families were morphologically identified. The taxonomic identity of the parasitic mite larvae is unclear at present. Morphologically, they fit descriptions of larval Sperchon (Hispidosperchon) algeriensis Lundblad, 1942, but the possibility cannot be excluded that they represent Sperchon algeriensis, the most abundant species at the adult stage in this study and unknown at the larval stage, or even another species of the genus. A molecular analysis produced for the first time cytochrome oxidase I gene sequences for S. algeriensis. CONCLUSIONS: Our results contribute to current knowledge on Spanish Hydrachnidia and their relationships with simuliids as hosts. However, further research is needed to evaluate the diversity, distribution, bioecology and prevalence of this parasitism.

Onchocerciasis in the Ntui Health District of Cameroon: epidemiological, entomological and parasitological findings in relation to elimination prospects.

BACKGROUND: Despite decades of community-directed treatment with ivermectin (CDTI), onchocerciasis transmission persists in Cameroon and has been associated with increased risk for epilepsy in endemic communities. We investigated the onchocerciasis situation in the Ntui Health District (a known onchocerciasis focus in Cameroon where the Sanaga River constitutes the main source of black fly vectors) using parasitological, entomological and serological parameters. METHODS: In July 2021, community-based surveys were conducted in four villages (Essougli, Nachtigal, Ndjame and Ndowe). Onchocerciasis was diagnosed via microscopic examination of skin snips. Using rapid diagnostic tests, we screened children aged 3-6 years for Ov16 antibodies as a proxy for recent onchocerciasis transmission. Monthly black fly biting rates were obtained from the two riverside villages (Nachtigal and Essougli) for 12 consecutive months (July 2021 to June 2022) using the human landing catch technique. Some black flies were dissected each month to check for infection. RESULTS: Overall, 460 participants were recruited; mean age was 32.1 (range: 3-85) years with 248 (53.9%) being males. Among skin snipped participants (n = 425), onchocerciasis prevalence was 14.6%. Participants with epilepsy (n = 25) were more often skin snip positive (45.8% vs 12.7%; P < 0.001) and had higher microfilarial loads (9.2 ± 22.0 vs 0.7 ± 3.5 microfilariae/skin snip; P < 0.001) compared to their peers without epilepsy. Eight (6.5%) of the 123 tested children were Ov16 seropositive. The breeding sites we investigated along the Sanaga River during the current study harbored fewer vectors (annual biting rates reaching 530,322 vs 606,370 in the Mbam River) and exhibited lower black fly infection rates (annual transmission potentials reaching 1479 vs 4488 in the Mbam River) when compared to recent entomological reports in Cameroon. CONCLUSION: Despite substantial biting rates, black fly infection rates (by microscopy) in the Ntui Health District were rather low resulting in overall low transmission potentials in study villages. Thanks to CDTI, O. volvulus infection in both humans and insects is on the decrease. However, there is evidence that O. volvulus is still endemic in these communities. Reducing the vector population will further accelerate onchocerciasis elimination prospects.

The interruption of transmission of onchocerciasis in Kaduna, Kebbi and Zamfara states, Nigeria: another milestone achievement.

BACKGROUND: More than 40 million people live in onchocerciasis-endemic areas in Nigeria. For at least 19 y, mass drug administration (MDA) with ivermectin was implemented with at least 65% total population coverage in Kaduna, Kebbi and Zamfara states. Impact surveys done using skin biopsies yielded no infections. Serological and entomological assessments were undertaken to determine if onchocerciasis transmission had been interrupted and MDA could be stopped. METHODS: The presence of onchocerciasis-specific immunoglobulin G4 antibody was measured by enzyme=linked immunosorbent assay conducted on dried blood spots collected from 5- to 9-year-old children resident in each state. O-150 polymerase chain reaction testing of Simulium damnosum s.l. heads for Onchocerca volvulus DNA was done on black flies collected by human landing capture and Esperanza window traps. RESULTS: A total of 9078 children were surveyed across the three states. A total of 6139 vectors were collected from Kaduna state, 129 from Kebbi state and 2 from Zamfara state; all were negative. Kebbi and Zamfara states did thousands of hours of black fly catching and intensive river prospecting. The resulting low fly catch was due to a low fly population incapable of sustaining transmission. CONCLUSION: Onchocerciasis transmission has been interrupted and the three states meet World Health Organization thresholds: seropositivity in children <0.1% and <1/2000 infective black flies with 95% confidence. The 2.2 million people in Kaduna state and 4 million in Kebbi and Zamfara states no longer need ivermectin for onchocerciasis.

Survey and Phylogenetic Analysis of Leucocytozoon (Apicomplexa: Haemosporida) Parasites in Mississippi Black Flies (Diptera: Simuliidae).

For several years, there have been continuous reports of black flies in Mississippi with evidence of transmission of Leucocytozoon spp. This study was conducted to determine the presence and diversity of Leucocytozoon spp. in black flies captured throughout Mississippi. Thirty-three collections, consisting of 346 specimens, were made during the 2-yr period (2015-2016) at 10 locations around the state. In addition to these systematic biweekly collections, 46 specimens were taken in 10 collections at four additional sites from 2009 to 2014, as well as 388 specimens taken in 14 collections during a severe black fly outbreak during March and April of 2018. Out of 186 pooled samples, 21 samples were positive for haemosporidian DNA. Eighteen of those samples were identified as Leucocytozoon spp. A phylogenetic tree was constructed using the novel sequences along with existing sequences from the MalAvi database. Results showed several of the sequences constructed in this study had high divergence from the existing sequences from the database.

A Survey on Native and Invasive Mosquitoes and Other Biting Dipterans in Northern Spain.

PURPOSE: Haematophagous Diptera, such as mosquitoes (Culicidae), biting midges (Ceratopogonidae), and black flies (Simuliidae), are important insects for public and animal health due to their capacity to bite and transmit pathogens. Outdoor recreation areas are usually affected by biting species and provide suitable habitats to both adult and immature stages. This study aimed to determine the species diversity and larval sites of these Diptera groups in two golf courses. METHODS: A multi-method collection approach using ultraviolet-CDC traps, human landing catches, collection in breeding sites, and ovitraps was implemented during summer 2020 in northern Spain. Insects were determined by morphological features accompanied by DNA barcoding. RESULTS: A total of ten native mosquito species were recorded either as adults or as larval stages. The invasive species Aedes japonicus was collected only at egg or pupa stage in ovitraps. Culex pipiens s.l. and Culex torrentium were both common mosquito species accounting for 47.9% of the total larval site collections and their larvae might be found in a wide range of natural and artificial sites. Culiseta longiareolata specimens were also prominent (30.1% of the total) and occurred exclusively in man-made water-filled containers. A total of 13 Culicoides species were identified, 10 of which were captured by ultraviolet-CDC traps, particularly members of the Obsoletus complex (Culicoides obsoletus/Culicoides scoticus, 74.9%) and seven species by emergence traps, being the two most abundant C. kibunensis (44.8%) and C. festivipennis (34.9%). Simulium cryophilum was also collected hovering around the operator under field sampling. CONCLUSION: A comprehensive representation of the blood-sucking Diptera fauna and their larval sites was obtained by the multi-method approach in two Spanish golf courses.

Descriptions of two new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Taiwan and Japan, and a revised description of S. (S.) quinquestriatum (Shiraki).

Species in the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) in Taiwan and Japan are morphologically reevaluated. Simulium (S.) yanpingense sp. nov. is described as the third member in this species-group from Taiwan and a revised description of S. (S.) quinquestriatum (Shiraki) is provided. The species previously regarded as S. (S.) quinquestriatum in Japan is described as a new species, S. (S.) tanegashimaense. Simulium (S.) yanpingense sp. nov. is similar to S. (S.) pingtungense Huang & Takaoka from Taiwan, in many characteristics including the haired basal portion of the female radius, dark leg color, and male scutum with brassy hairs, but it is distinguished from the latter species by the number of male upper-eye (large) facets, which are in 17 (rarely 18) vertical columns and 17 or 18 horizontal rows in this new species but in 19 vertical columns and 20 horizontal rows in S. (S.) pingtungense. Simulium (S.) tanegashimaense sp. nov. is similar to S. (S.) quinquestriatum originally described from Taiwan by having the male scutum with yellow fine hairs, but is distinguished from the latter species by the number of male upper-eye (large) facets, which are in 17 (rarely 16) vertical columns and 17 horizontal rows in this new species but in 19 or 20 vertical columns and 19 or 20 horizontal rows in S. (S.) quinquestriatum. The morphological differences between these two new species and their related species from India, Malaysia, Thailand and Vietnam are noted.

Chromosomal and Molecular Diversity in the Simulium ornatum Group (Diptera: Simuliidae) in the Western Tian Shan Range of Central Asia.

By any measure, such as abundance, species diversity or geographic range, the Simulium ornatum species group is one of the most successful Palearctic taxa of black flies. To explore potential diversity in this group in the Tian Shan range of Central Asia, we focused on Kyrgyzstan, in which three nominal morphospecies have been recorded. Among our samples, we morphologically identified S. mesasiaticum Rubtsov and a second possible species tentatively identified as S. ferganicum Rubtsov. By analyzing banding patterns of the larval polytene chromosomes, we discovered two fixed inversions, two sex-linked rearrangements, and 19 autosomal rearrangements, including supernumerary B chromosomes. The chromosomal data indicate minimal diversity of only one or two species across the surveyed area of nearly 50,000 km2. Mitochondrial DNA (CO1) sequences fell into three distinct clusters, possibly representing separate species. The chromosomal, molecular, and morphological data indicate that Kyrgyz populations are unique within the S. ornatum group, but the data sets are not entirely congruent. Thus, reconciling data sets and assigning existing names is tentative. Simulium mesasiaticum is linked with undifferentiated sex chromosomes, one of the three CO1 clades, and higher elevations, whereas S. ferganicum is tenuously associated with differentiated sex chromosomes, a separate CO1 clade, and lower elevations. These associations leave one Kyrgyz larva, which is in a third CO1 clade, unlinked to a formal species name. Our analyses also indicate that S. ornatum Meigen sensu stricto, contrary to previous reports, does not occur in Kyrgyzstan and should be deleted from the country's faunal list.

DNA barcoding of tropical black flies (Diptera: Simuliidae) in Thailand: One decade of progress.

Black flies (Diptera: Simuliidae) are important blood sucking insects because they are the vectors of disease agents transmitted to human and other animals. Rapid and correct species identifications are necessary for all aspects of the study of black flies. DNA barcodes based on mitochondrial cytochrome c oxidase I (COI) have been effectively used for the determination of black fly species. However, the success of this method requires a large and reliable COI sequence library. In this study, 171 DNA barcoding sequences from 17 black fly species were added to NCBI GenBank database, six of these species were reported for the first time. Efficacy of DNA barcodes for species identification was examined using 1,286 sequences representing 89 nominal species of black flies in Thailand. A considerable level of success (90%) was achieved but efficiency of COI sequences for species identification was very low in the following species-groups; Simulium asakoae, S. feuerborni, S. multistriatum and S. striatum. Incomplete lineage sorting or inadequate variation of this genetic marker for differentiation of recently diverged species are the more likely explanations, and thus, more variable genetic markers are needed. Other reasons for unsuccessful DNA barcoding are imperfect taxonomy and the misidentification of sources of reference sequences. Because many new black fly species in Thailand were described recently, a reassessment of the COI sequences reported previously is necessary.

Community Structure, Biodiversity and Spatiotemporal Distribution of the Black Flies (Diptera: Simuliidae) Using Malaise Traps on the Highest Mountain in Thailand.

Black flies form a group of small blood-sucking insects of medical and veterinary importance. This study aimed to investigate the community structure, biodiversity and spatial and temporal distribution of adult black flies in tropical rain forests, by using malaise traps in Doi Inthanon National Park, northern Thailand. Malaise traps were placed along six elevational gradients (400 m to 2500 m, above sea level) at Doi Inthanon National Park, Chiang Mai province, from December 2013 to November 2014. A total of 9406 adult female black flies belonging to five subgenera-Daviesellum (2%), Gomphostilbia (23%), Montisimulium (11%), Nevermannia (16%) and Simulium (48%)-were collected. Among 44 taxa found, S. tenebrosum complex had the highest relative abundance (11.1%), followed by the S. asakoae species-group (9.6%), the S. striatum species-group (7.7%), S. inthanonense (6.6%), S. doipuiense complex (6.4%), S. chomthongense complex (5.3%), S. chumpornense (5.1%) and S. nigrogilvum (4.1%). Two human-biting species-S. nigrogilvum and species in the S. asakoae species-group-were found in all of the collection sites with 100% species occurrence. Species richness was highest at mid elevation (1400 m), which is represented by 19 black fly species. The peak and lowest seasonal abundance was observed in the rainy and hot season, respectively. Seasonal species richness was highest in the cold season, except for that from elevation sites at 700 m, 1700 m and 2500 m. This study revealed that the malaise trap is effective in providing important data for further monitoring of the effects of environmental changes and conservation planning on the biodiversity of black flies in Doi Inthanon National Park.

Effects of Bacillus thuringiensis var. israelensis on the Black Fly Communities (Diptera, Simuliidae) in Tropical Streams.

Bacillus thuringiensis var. israelensis (Bti) Berliner, 1911 is widely used in the biological control of black fly and mosquito populations. The objective of this study was to evaluate the effects of Bti on the black fly communities in streams in the Atlantic Forest domain. The study was carried out in eight streams of Serra do Mar, in the municipality of Ubatuba, São Paulo. Some parts of the streams in this locality have received applications of Bti fortnightly for more than 25 years by the sanitary agency of the region. In each stream, two sections were sampled, with and without application of Bti (June 2015 and 2016). Black flies were collected and identified to the species level in the laboratory with 1382 larvae being identified, distributed in six species. Of this total, 73% of the individuals were found in sections where Bti was not applied. There was a difference in the abundance of simulids between the sections with and without Bti application, being more abundant in the latter. We measured the head capsule of individuals of the most abundant species, Simulium pertinax Kollar 1832, using a stereoscopic microscope with millimeter lens. These measurements demonstrated that there was a difference between the sections regarding the age structure of Simuliidae populations. In the sections without Bti application, there was a higher proportion of larvae in the last instar, while in the Bti-treated sections, smaller instars were predominant, possibly due to constant recolonization processes.

Insect hypersensitivity beyond bee and wasp venom allergy.

The bites of blood-feeding insects regularly induce sensitization to salivary proteins and cause local hypersensitivity reactions in over 90% of the population, representing either an IgE-mediated immediate wheal and flare reaction or a T cell-driven delayed papule. Long-lasting large local reactions and bullous reactions may cause significant discomfort and reduction in quality-of-life. Anaphylaxis is rarely reported though proven for several insects, above all mosquitoes, horse flies, and kissing bugs. Recently, salivary gland proteins have been thoroughly studied in some blood-feeding insect species, and several allergens have been identified. Interestingly, many of them belong to the same protein families as the well-known honeybee and wasp venom allergens (phospholipases, hyaluronidases, antigens 5, serine proteases) though sequence identities are mostly low. There is still insufficient evidence for the proposed cross-reactivity between salivary proteins from blood-feeding insects and Hymenoptera venom allergens.

Management Strategies for Reducing the Risk of Equines Contracting Vesicular Stomatitis Virus (VSV) in the Western United States.

Vesicular stomatitis viruses (VSVs) cause a condition known as vesicular stomatitis (VS), which results in painful lesions in equines, cattle, swine, and camelids, and when transmitted to humans, can cause flu-like symptoms. When animal premises are affected by VS, they are subject to a quarantine. The equine industry more broadly may incur economic losses due to interruptions of animal trade and transportation to shows, competitions, and other events. Equine owners, barn managers, and veterinarians can take proactive measures to reduce the risk of equines contracting VS. To identify appropriate risk management strategies, it helps to understand which biting insects are capable of transmitting the virus to animals, and to identify these insect vectors' preferred habitats and behaviors. We make this area of science more accessible to equine owners, barn managers, and veterinarians, by (1) translating the most relevant scientific information about biting insect vectors of VSV and (2) identifying practical management strategies that might reduce the risk of equines contracting VSV from infectious biting insects or from other equines already infected with VSV. We address transmission risk at four different spatial scales-the animal, the barn/shelter, the barnyard/premises, and the surrounding environment/neighborhood-noting that a multiscale and spatially collaborative strategy may be needed to reduce the risk of VS.

Using Digital Image Analysis to Quantify Small Arthropod Vectors.

Quantifying arthropod vectors can be a time-consuming process. Here, we describe a technique to count large samples of small arthropods using ImageJ. ImageJ is an open source image processing software, produced by the National Institutes of Health, with a straightforward interface that has proven useful in quantifying small organisms (i.e., cells, pollen, eggs). In 2017, we deployed CDC light traps baited with carbon dioxide among seven sites to capture black flies (Diptera: Simuliidae). Samples of the captured specimens were photographed, and then quantified manually and automatically, using ImageJ. We compared the accuracy of three types of automated counts to manual counts of black flies using an information-theoretic approach. We found that changing the particle size produced counts closest to those obtained by manual counts. Even over a large range of values, from tens to thousands of flies, our automated counts were often identical to and almost always within 5% of the manual counts. When different, automated counts were usually slightly less than manual counts, and thus conservative estimates. This automated technique is simple, repeatable, requires minimal training, and can reduce the time needed to quantify small arthropods such as black flies.

Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium.

BACKGROUND & OBJECTIVES: The black fly genus Simulium Latreille is one of the most important medical insect group of the family Simuliidae (Diptera) and many species of this genus are important pests of human and animals, while some of them also represent vectors of pathogens. Correct species identification is essential to the implementation of control measures for species of medical or agricultural importance. METHODS: In this study, the usefulness of DNA barcoding was discussed in distinguishing species of Simulium. RESULTS: Analysis showed hidden biodiversity, usually referred to in Simuliidae as cryptic species, which was detected in 15 species. Firstly, intraspecific divergences of eleven species was unexpectedly high and the maximum distances of them ranged from 5.1-16.8%. Based on the differential of K2P (Kimura 2-Parameter) distances, sequences were subdivided into two or three groups, respectively. Secondly, extremely low interspecific divergences were detected in eight groups of species, and shared haplotypes were also found among them. Furthermore, the subdivision within species and shared haplotypes among some species were all supported by the NJ (Neighbour-Joining) analysis. INTERPRETATION & CONCLUSION: Our results confirmed that DNA barcoding was a powerful tool for revealing hidden species diversity of black flies. Further work is needed to reveal ambiguous species delimitation in some problematic species groups.

Revision of Simulium rufibasis (Diptera: Simuliidae) in Japan and Korea: Chromosomes, DNA, and Morphology.

The widespread nominal black fly Simulium (Simulium) rufibasis Brunetti was reexamined morphologically, chromosomally, and molecularly to determine the status of populations in Japan and Korea with respect to S. rufibasis from the type locality in India and to all other known species in the S. (S.) tuberosum species-group. Morphological comparisons established that the species previously known as S. rufibasis in Japan and Korea is distinct from all other species. Consequently, it was described and illustrated as a new species, Simulium (S.) yamatoense. Simulium yokotense Shiraki, formerly a synonym of S. rufibasis, was morphologically reevaluated and considered a species unplaced to species-group in the subgenus Simulium. Chromosomal analyses of S. yamatoense sp. nov. demonstrated that it is unique among all cytologically known species of the S. tuberosum group and is the sister species of the Taiwanese species tentatively known as S. (S.) arisanum Shiraki. Populations of S. yamatoense sp. nov. included two cytoforms, based on the sex chromosomes. Cytoform A, including topotypical representatives, was found in Kyushu, Japan, whereas cytoform B was found in Korea and Honshu, Japan. Molecular analysis based on the COI mitochondrial gene generally corroborated morphological and chromosomal data that S. yamatoense sp. nov. is a distinct species and, like the chromosomal data, indicate that it is most closely related to S. arisanum, with interspecific genetic distance of 2.92-4.63%.

Austrocnephia, new genus, for five species of 'Paracnephia' (Diptera: Simuliidae), with a key to Australian black fly genera.

A segregate of the so-called Australian 'Paracnephia' (Diptera: Simuliidae) is assigned to a new genus, Austrocnephia. The taxon is fully diagnosed and a key to constituent species presented. Two species-groups are recognized: the aurantiaca species-group, comprised of A. aurantiaca (Tonnoir 1925) and A. strenua (Mackerras Mackerras 1950), and the tonnoiri species-group, comprised of A. fuscoflava (Mackerras Mackerras 1948), A. orientalis (Mackerras Mackerras 1950) and A. tonnoiri (Drummond 1931). Both species-groups are diagnosed and the included species fully redescribed. Detailed locality data is given, as is information about biology, when known. Brief comments are offered about the historical biogeography of Austrocnephia. A key to Australian simuliid genera is also provided.

Anti-Wolbachia therapy for onchocerciasis & lymphatic filariasis: Current perspectives.

Onchocerciasis and lymphatic filariasis (LF) are human filarial diseases belonging to the group of neglected tropical diseases, leading to permanent and long-term disability in infected individuals in the endemic countries such as Africa and India. Microfilaricidal drugs such as ivermectin and albendazole have been used as the standard therapy in filariasis, although their efficacy in eliminating the diseases is not fully established. Anti-Wolbachia therapy employs antibiotics and is a promising approach showing potent macrofilaricidal activity and also prevents embryogenesis. This has translated to clinical benefits resulting in successful eradication of microfilarial burden, thus averting the risk of adverse events from target species as well as those due to co-infection with loiasis. Doxycycline shows potential as an anti-Wolbachia treatment, leading to the death of adult parasitic worms. It is readily available, cheap and safe to use in adult non-pregnant patients. Besides doxycycline, several other potential antibiotics are also being investigated for the treatment of LF and onchocerciasis. This review aims to discuss and summarise recent developments in the use of anti-Wolbachia drugs to treat onchocerciasis and LF.

Sympatric speciation in the Simulium arcticum s. l. complex (Diptera: Simuliidae): The Rothfels model updated.

ABSTRACT: We tested the Rothfels sympatric speciation model for black flies by comparing all available data for sex-chromosome diversity with the geographic locations of larval collection sites within the Simulium arcticum complex of black flies (Diptera: Simuliidae). Five separate data sets equaling about 20,000 larvae were included from throughout the geographic range of this complex. We record a total of 31 taxa having unique sex chromosomes, all of which demonstrate linkage disequilibrium with most taxa sharing autosomal polymorphisms. All siblings share portions of their distributions with S. negativum, the presumed oldest member of the complex. Twenty-one of 22 cytotypes have distributions within the ranges of siblings thus supporting the sympatric speciation model of Rothfels. Chromosomally diverse sites may require analysis of as many as 200 larvae to be properly described. There is no effect of any inversions influencing the occurrence of other inversions. Finally, we report a new cytotype, Simulium arcticum IIL-6, which we originally discovered in Alaska. Aspects of future genomic research are discussed as they relate to the main chromosomal structural/functional tenants of the model. OPEN RESEARCH BADGE: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data are available at https://doi.org/10.6084/m9.figshare.7719398.

Proteomes of the female salivary glands of Simulium nigrogilvum and Simulium nodosum, the main human-biting black flies in Thailand.

Although several studies have reported pharmacological and immunological activity, as well as the role of black flies in transmitting pathogens to vertebrate hosts through salivary glands (SG) during blood feeding, SG proteomes of the anthropophilic black flies in Thailand have never been reported. Therefore, this study determined the SG proteomes of female S. nigrogilvum and S. nodosum. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2-DE) gels containing separated SG proteins of individual species were subjected to liquid chromatography-tandem mass spectrometry (LCMS/MS) and an orthologous protein search from eukaryotic organism, nematocera and simuliidae databases for total protein identification. SDS-PAGE and protein staining revealed at least 13 and 9 major protein bands in the SGs of female S. nigrogilvum and S. nodosum, respectively, as well as several minor ones. The 2-DE demonstrated a total of 56 and 41 protein spots for S. nigrogilvum and S. nodosum, respectively. Most of the proteins obtained in both species were enzymes involved in blood feeding, including proteases, apyrases, hyaluronidases, aminopeptidase and elastase. The results obtained in this study provided a new body of knowledge for a better understanding on the role of salivary gland proteins in these black fly species in Thailand.

Evaluation of Larval Medium in the Controlled Current Toxicity Test.

The Controlled Current Toxicity Test (CCTT) is a protocol used by the University of Georgia Black Fly Rearing and Bioassay Laboratory to evaluate the efficacy of larvicides based on Bacillus thuringiensis subsp. israelensis (Bti) against Simulium vittatum larvae. A standard CCTT provides a larval medium with suspended organic nutrients, a sustained current, and a clean surface for black fly larval attachment in each exposure vessel. In an effort to streamline the CCTT and eliminate a potential source of variability, 3 types of larval medium were evaluated: deionized water, distilled water, and moderately hard water, which were compared to the currently used protocol. A statistical significance in mean lethal concentration (LC50) rates was observed between the CCTT (deionized water with suspended organic nutrients) medium and the other 3 experimental media. The presence of suspended organic nutrients in the CCTT significantly improved the efficacy of the Bti-based larvicide. The interaction of the suspended organic nutrient particles and the Bti-based particles in the larvicide appears to produce a mixture that is more efficiently captured and ingested by the black fly larvae than the larvicide formulation particles alone.