Impact of Cypermethrin persistence on stomoxyine abundance and Trypanosoma Vivax infection.

Stomoxyine biting flies play a pivotal role as mechanical vectors of Animal African Trypanosomosis (AAT), inducing painful bites and significant disturbances in cattle, thereby affecting productivity. This study delves into the dynamics of cypermethrin persistence on cattle, a key measure aimed at mitigating stomoxyine populations and the associated transmission of trypanosomes. Conducted within a cattle settlement in Ido, Ibadan, southwest Nigeria, data collection occurred biennially from 2016 to 2022. Cypermethrin application followed a restricted protocol triggered by high fly density. Stomoxyine flies were systematically trapped every three months over a five-day period, employing four Nzi traps per collection cycle. The flies were taxonomically categorised, and their relative abundance assessed. Additionally, engorged flies underwent ITS-PCR analysis to detect T. vivax presence in their mouthparts. The investigation unveiled a noteworthy decline in cypermethrin persistence from 10.7 to 4.9 days over the six-year span, significantly influencing stomoxyine population dynamics and T. vivax infection rates. Despite escalated cypermethrin utilisation, stomoxyine abundance exhibited stability, hinting at potential insecticidal resistance. Notably, cypermethrin persistence was at its highest during July-September, aligning with heightened rainfall and humidity conducive to stomoxyine larval development. The heightened probability of detecting T. vivax DNA in stomoxyine flies as cypermethrin persistence dwindled underscores the intricate interplay between reduced insecticide efficacy and heightened pathogen dissemination. This underscores the imperative for integrated, enhanced control strategies considering the prevailing T. vivax resistance to trypanocides and diminishing cypermethrin efficacy.

Vector competence of Aedes albopictus field populations from Reunion Island exposed to local epidemic dengue viruses.

Dengue virus (DENV) is the most prevalent mosquito-borne Flavivirus that affects humans worldwide. Aedes albopictus, which is naturally infected with the bacteria Wolbachia, is considered to be a secondary vector of DENV. However, it was responsible for a recent DENV outbreak of unprecedented magnitude in Reunion Island, a French island in the South West Indian Ocean. Moreover, the distribution of the cases during this epidemic showed a spatially heterogeneous pattern across the island, leading to questions about the differential vector competence of mosquito populations from different geographic areas. The aim of this study was to gain a better understanding of the vector competence of the Ae. albopictus populations from Reunion Island for local DENV epidemic strains, while considering their infection by Wolbachia. Experimental infections were conducted using ten populations of Ae. albopictus sampled across Reunion Island and exposed to three DENV strains: one strain of DENV serotype 1 (DENV-1) and two strains of DENV serotype 2 (DENV-2). We analyzed three vector competence parameters including infection rate, dissemination efficiency and transmission efficiency, at different days post-exposition (dpe). We also assessed whether there was a correlation between the density of Wolbachia and viral load/vector competence parameters. Our results show that the Ae. albopictus populations tested were not able to transmit the two DENV-2 strains, while transmission efficiencies up to 40.79% were observed for the DENV-1 strain, probably due to difference in viral titres. Statistical analyses showed that the parameters mosquito population, generation, dpe and area of sampling significantly affect the transmission efficiencies of DENV-1. Although the density of Wolbachia varied according to mosquito population, no significant correlation was found between Wolbachia density and either viral load or vector competence parameters for DENV-1. Our results highlight the importance of using natural mosquito populations for a better understanding of transmission patterns of dengue.

Seasonal dynamics, Leishmania diversity, and nanopore-based metabarcoding of blood meal origins in Culicoides spp. in the newly emerging focus of leishmaniasis in Northern Thailand.

BACKGROUND: Clinical cases of leishmaniasis caused by Leishmania (Mundinia) parasites have been increasingly reported in Southeast Asia, particularly Thailand. Recent evidence has shown that Leishmania (Mundinia) parasites successfully developed into infective metacyclic promastigotes in Culicoides biting midges, strongly supporting their putative role in disease transmission. However, Culicoides diversity, host preference, and Leishmania prevalence in endemic areas remain largely unknown. METHODS: We investigated the seasonal dynamics, infection prevalence, and blood meal identification of Culicoides collected from the emerging focus of visceral leishmaniasis in Lampang Province, Northern Thailand, during 2021-2023. Midge samples were molecularly screened for Leishmania using SSU rRNA-qPCR and ITS1-PCR, followed by Sanger plasmid sequencing, and parasite haplotype diversity was analyzed. Host blood meal origins were comparatively identified using host-specific Cytb-PCRs and a nanopore-based metabarcoding approach. RESULTS: A total of 501 parous and gravid females and 46 blood-engorged ones belonging to at least 17 species of five subgenera (Remmia, Trithecoides, Avaritia, Hoffmania, and Meijerehelea) and two species groups (Shortti and Calvipalpis) were collected with temporal differences in abundance. Leishmania was detected by SSU rRNA-qPCR in 31 samples of at least 11 midge species, consisting of Culicoides oxystoma, C. guttifer, C. orientalis, C. mahasarakhamense, C (Trithecoides) spp., C. innoxius, C. shortti, C. arakawae, C. sumatrae, C. actoni, and C. fulvus, with the overall infection prevalence of 5.7%. The latter six species represent the new records as putative leishmaniasis vectors in Northern Thailand. The ITS1-PCR and plasmid sequencing revealed that Leishmania martiniquensis was predominantly identified in all qPCR-positive species, whereas L. orientalis was identified only in three C. oxystoma samples. The most dominant haplotype of L. martiniquensis in Thailand was genetically intermixed with those from other geographical regions, confirming its globalization. Neutrality test statistics were also significantly negative on regional and country-wide scales, suggesting rapid population expansion or selective sweeps. Nanopore-based blood meal analysis revealed that most Culicoides species are mammalophilic, with peridomestic and wild mammals (cow, pig, deer, and goat-like species) and humans as hosts, while C. guttifer and C. mahasarakhamense fed preferentially on chickens. CONCLUSIONS: This study revealed seasonal dynamics and sympatric circulation of L. martiniquensis and L. orientalis in different species of Culicoides. Evidence of human blood feeding was also demonstrated, implicating Culicoides as putative vectors of human leishmaniasis in endemic areas. Further research is therefore urgently needed to develop vector control strategies and assess the infection status of their reservoir hosts to effectively minimize disease transmission.

Rapid detection of Impatiens necrotic spot virus from thrips vectors using reverse transcription-recombinase polymerase amplification.

The plant virus, Impatiens necrotic spot virus (INSV), is an economically important pathogen of vegetables, fruits, and ornamental crops. INSV is vectored by the western flower thrips, Frankliniella occidentalis, a small insect pest that is globally distributed. In recent years, INSV outbreaks have reached epidemic levels in the Salinas Valley of California-an agriculturally rich region where most of the lettuce (Lactuca sativa) is produced in the United States. Due to the obligate nature in which virus transmission occurs, new tools that could rapidly detect INSV from thrips vectors would enhance our ability to predict where virus outbreaks may occur. Here, we report on the development of a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect INSV from individual thrips. The assay uses crude extraction methods, is performed at a single temperature of 42 °C, can be completed in 25 min, and provides sensitivity levels that are comparable to other available detection methods. When the assay was used on field populations of thrips, INSV was successfully identified and quantified from individual larvae and adults. The work provides a new cost-effective surveillance tool that can rapidly detect INSV from its insect vector and from plants.

Triatomines know no boundaries: Triatoma delpontei Romaña & Abalos, 1947 (Heteroptera: Reduviidae) discovered in Mato Grosso do Sul State, Brazil.

Triatominae are recognized as vectors of Trypanosoma cruzi, a protozoan which is the etiological agent of Chagas disease. A specimen of Triatoma delpontei was found at Porto Murtinho in Mato Grosso do Sul State, Brazil. This is the first report of the occurrence of T. delpontei to the state of Mato Grosso do Sul, Brazil. With the present finding, the total number of triatomines recorded in Mato do Grosso do Sul reaches 17 species, while T. delpontei, previously recorded only from Rio Grande do Sul, is now recorded to a second Brazilian state. Based on the information available in the literature, a meticulous and organized compilation has been crafted, highlighting the cytogenetics differentiations of the species occurring in this state. This work emphasizes the importance of continuous research and surveillance on Triatominae, recognized as vectors of T. cruzi.

Perspicacious insights into plant-virus-vector interactions applying omics.

Transmission of plant viruses by insect vectors is facilitated by unequivocal tri-partite interactions among host plants, viruses, and associated vectors. The advent of next-generation sequencing including whole genome sequencing, RNA/small RNA sequencing, proteomics, and metabolomics aided in elucidating the molecular mechanisms involved in virus transmission by insect vectors and infection in host plants.

Comparative analysis of the microbiota of sand fly vectors of Leishmania major and L. tropica in a mixed focus of cutaneous leishmaniasis in southeast Tunisia; ecotype shapes the bacterial community structure.

Phlebotomine sand flies are vectors of the protozoan parasite Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the impact of host species and environment on the gut microbiome. To address this issue, a comparative analysis of the microbiota of sand fly vector populations of Leishmania major and L. tropica in a mixed focus of cutaneous leishmaniasis in Tunisia was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize and compare the overall bacterial and fungal composition of field-collected sand flies: Phlebotomus papatasi, Ph. perniciosus, Ph. riouxi, and Ph. sergenti. Thirty-eight bacterial genera belonging to five phyla were identified in 117 female specimens. The similarities and differences between the microbiome data from different samples collected from three collections were determined using principal coordinate analysis (PCoA). Substantial variations in the bacterial composition were found between geographically distinct populations of the same sand fly species, but not between different species at the same location, suggesting that the microbiota content was structured according to environmental factors rather than host species. These findings suggest that host phylogeny may play a minor role in determining the insect gut microbiota, and its potential to affect the transmission of the Leishmania parasite appear to be very low. These results highlight the need for further studies to decode sand fly Leishmania-microbiota interactions, as even the same bacterial species, such as Enterococcus faecalis, can exert completely opposite effects when confronted with different pathogens within various host insects and vice versa.

Triatomine (Hemiptera: Reduviidae) populations and Trypanosoma cruzi genotyping in peridomestic and sylvatic environments in the semiarid region of Sergipe, Northeastern, Brazil.

We assessed the diversity of triatomines, the rates of natural infection, and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them in two municipalities in the state of Sergipe, Brazil. Active searches for triatomines were conducted in the peridomicily and wild enviroments of 10 villages within the two municipalities. Triatomines were taxonomically identified and their feces were extracted using the abdominal compression method. Parasite detection was performed using optical microscopy. For Trypanosoma cruzi genotyping via PCR-FFLB, 151 samples of the subspecies Triatoma brasiliensis macromelasoma and Triatoma brasiliensis were isolated from both municipalities. In total, 505 triatomines were collected, with Triatoma brasiliensis macromelasoma being the most frequent species (58.81 %). Triatoma b. brasiliensis was the only species in both peridomestic and wild environments. Regarding the other species, T. pseudomaculata was found only in the peridomestic environment; and T. b. macromelasoma and Psammolestes tertius were found in the wild environment. Three Discrete Typing Units were identified: TcI (87.51 %) detected in T. b. brasiliensis and T. b. macromelasoma, TcI+TcIII (10.41 %) in T. b. macromelasoma, and TcI+Trypanosoma rangeli (2.08 %) in T. b. macromelasoma. It is concluded that T. b. macromelasoma is the species collected most frequently in the studied region and the one that presents the highest rates of natural infection, highlighting its epidemiological importance for the vectorial transmission of Chagas disease in Sergipe.

Ongoing transmission of onchocerciasis in the Pru District of Ghana after two decades of mass drug administration with ivermectin and comparative identification of members of the Simulium damnosum complex using cytological and morphological techniques.

BACKGROUND: Human onchocerciasis remains a public health problem in Ghana. Mass drug administration (MDA) with ivermectin (IVM) has reduced disease morbidity and prevalence, but the transmission of onchocerciasis remains ongoing in several endemic foci. We investigated parasite transmission in some endemic communities in Ghana that had received > 18 rounds of annual MDA with IVM and determined the species composition of black fly (Simulium damnosum) vectors in these areas. METHODS: Adult female black flies were collected using human landing catches and identified as either forest or savanna species using morpho-taxonomic keys. The adult flies underwent dissection to determine their parity and detect any O. volvulus larvae, followed by the calculation of entomological indices. Simulium damnosum s.l. larvae were collected and preserved in freshly prepared Carnoy's fixative and were later used for cytotaxonomic studies. RESULTS: A total of 9,983 adult flies were caught: 6,569 and 3,414 in the rainy and dry seasons respectively. Black fly biting activities over the study period showed bimodal or trimodal patterns. The highest monthly biting rate (MBR) of 10,578.75 bites/person/month was recorded in July in Beposo, while the highest monthly transmission potential of 100.69 infective bites/person/month was recorded in Asubende in August. Morphological analysis of 2,032 flies showed that 99.8% (2,028) of the flies were savanna species, with only 4 (0.2%) adult flies being of the forest species. Cytogenetic studies on 114 black fly larvae revealed three cytospecies (Simulium damnosum s.s., S. sirbanum and S. sanctipauli) in the study area. CONCLUSIONS: The present studies confirmed an ongoing transmission of onchocerciasis in the study communities except Abua-1. It also provides further information on biting behaviors and onchocerciasis transmission indices in the study communities. Further, our data confirmed the savanna species (S. damnosum s.s. and S. sirbanum) of the S. damnosum s.l. to be the major vectors of onchocerciasis in the study areas, with only an occasional influx of forest cytotypes.

Abundance and Leishmania infection patterns of the sand fly Psathyromyia cratifer in Southern Mexico.

BACKGROUND: Localized cutaneous leishmaniasis (LCL) is a serious public health problem in Southern Mexico. Six species of Phlebotominae (Diptera: Psychodidae) have been found to be infected with Leishmania (Leishmania) mexicana, the causative agent of LCL in the region. However, little is known about the biology and potential participation of Psathyromyia cratifer in the Leishmania transmission cycle in Mexico, and the Americas. The present study provides evidence of temporal infection caused by Leishmania in Psathyromyia cratifer as well as data on its population dynamics in a LCL endemic area during the well-known transmission cycle of Leishmania in Southern Mexico. METHODOLOGY/PRINCIPAL FINDINGS: Individual specimens of Psathyromyia cratifer were collected in four sites over the course of five months (from November 2020 through March 2021) using animal-baited, human-baited, and light traps. The temporal activity pattern (month + hour) of Psathyromyia cratifer was assessed along with its relationship with environmental variables. Moreover, Leishmania DNA and blood meals were analyzed and detected in female sand flies. This evidenced an infection rate ranging from 8% to 83%, and the record of Homo sapiens and Ototylomys phyllotis as blood hosts of this sand fly species. High abundances of these sand flies in human-baited traps were recorded which revealed the marked anthropophilic behavior of Psathyromyia cratifer. As regards the transmission dynamics of the parasite within the region, it was observed that the potential highest epidemiological risk for Leishmania transmission by Psathyromyia cratifer occurred during the months of January and March. CONCLUSION: This is the first contribution ever made to both the population dynamic and the temporal Leishmania prevalence patterns in Psathyromyia cratifer. The resulting findings suggest that this sand fly specimen is the sixth potential vector of L. (L.) mexicana in Southern Mexico. Nonetheless, various biology, behavior, and ecology strands are yet to be addressed. The latter, to determine the role it plays in the transmission dynamics of the parasite within the region, and other areas of the country.

Entomological aspects of leishmaniasis transmission in the urban area of Teófilo Otoni, a municipality endemic for the disease in the Brazilian state of Minas Gerais.

Brazil is endemic for both visceral (VL) and cutaneous (CL) clinical forms of leishmaniasis, poverty-associated diseases with worldwide distribution. Leishmania parasites are the etiological agents of leishmaniases, which are transmitted to humans through the bites of infected phlebotomine sand flies. From 2018 to 2023, 15 cases of VL and 129 cases of CL were reported in Téofilo Otoni, an important economic center in the Brazilian state of Minas Gerais. Owing to the lack of data on the entomological fauna, the present study aimed to clarify this main aspect of leishmaniasis. From May, 2021 to April, 2023, entomological captures were performed monthly in ten neighborhoods in Teófilo Otoni. The influence of bioclimatic variables on insect populations was evaluated, and natural infection by Leishmania spp. was investigated using molecular methods. A total of 306 specimens of 12 species of phlebotomine sand fly were collected. The majority (91.6%) were proven or putative vectors of leishmaniasis agents. The population of insects tended to increase during the cooler and drier months. Although Leishmania infection was not detected in any of the samples, the presence of vectors provides conditions for the maintenance and expansion of the transmission cycle of leishmaniasis in Teófilo Otoni.

[Distribution and seasonal fluctuation of visceral leishmaniasis vectors sandflies in Henan Province in 2023].

OBJECTIVE: To investigate the geographical distribution and seasonal fluctuations of visceral leishmaniasis vectors sandflies in Henan Province in 2023, so as to provide insights into the prevention and control of visceral leishmaniasis vectors. METHODS: A total of 23 counties (districts) were sampled from 18 cities of Henan Province from May to September, 2023 as sandfly surveillance sites, and sandflies were captured using human capture and light trapping methods. Following morphological identification, the changes in the sandfly density were calculated at different months and in different breeding habitats. RESULTS: A total of 406 light traps were set at sandfly surveillance sites in Henan Province from May to September, 2023, and a total of 3 137 female sandlies were captured, with an average density of 7.73 sandlies/(light·night). A total of 1 494 Phlebotomus chinensis sandflies were captured, including 1 222 female sandflies, with an average density of 3.01 sandflies/(light·night), and the highest density of P. chinensis was found in Gongyi City [17.00 sandflies/(light·night)]. A total of 5 544 sandflies were captured using the human capture method, including 230 P. chinensis, and the density of P. chinensis appeared a unimodal distribution, with a peak in early July [5.81 sandflies/(light·night)]. Among different breeding habitats, the highest P. chinensis density was detected in pigpens [4.50 sandflies/(light·night)]. CONCLUSIONS: P. chinensis was predominantly distributed in hilly areas of northern and central-western Henan Province in 2023, and the sandfly density appeared a unimodal distribution. Intensified monitoring of visceral leishmaniasis vectors is recommended.

[Distribution and seasonal fluctuation of visceral leishmaniasis vectors sandflie in Lüliang City of Shanxi Province in 2023].

OBJECTIVE: To investigate the distribution and seasonal fluctuations of visceral leishmaniasis vectors sandflies in Lüliang City, Shanxi Province, so as to provide insights into assessment of the visceral leishmaniasis transmission risk and formulation of visceral leishmaniasis control measures. METHODS: A total of 12 natural villages were sampled from Shilou County, Lishi District, Lanxian County, Linxian County and Wenshui County in Lüliang City, Shanxi Province from June to September, 2023, and sandflies were captured using light traps from 7 breeding habitats, including farmers' houses, sheep pens, cattle pens, chicken coops, pig pens, mule and horse pens, and loess-cave dwellings. Following morphological identification of the sandfly species, the distribution of sandflies and the seasonal fluctuations of the sandfly density were analyzed. In addition, the Leishmania was detected in sandflies using a real-time fluorescence quantitative PCR assay. RESULTS: A total of 2 831 sandflies were captured with 156 light traps in Lüliang City from June to September, 2023, including 2 638 female sandflies (93.18%) and 193 male sandflies (6.82%), and the average density was 16.91 sandflies/(light-night). The seasonal fluctuations of the sandfly density all appeared a unimodal distribution in all survey sites, and the sandfly density peaked in July and then declined rapidly. Among all types of breeding habitats, the greatest sandfly density was found in sheep pens [39.04 sandflies/(light-night)]. In addition, 4.08% (2/49) of the sandfly samples were tested positive for Leishmania nucleic acid as revealed by the real-time fluorescence quantitative PCR assay. CONCLUSIONS: Sandflies were widely distributed in Lüliang City, Shanxi Province in 2023, and the peak of the sandfly density was observed in July, which had a visceral leishmaniasis transmission risk. Intensified surveillance of visceral leishmaniasis and sandfly vectors is required and targeted vector control is recommended.

Climate change-induced degradation of expert range maps drawn for kissing bugs (Hemiptera: Reduviidae) and long-standing current and future sampling gaps across the Americas.

BACKGROUND: Kissing bugs are the vectors of Trypanosoma cruzi, the etiological agent of Chagas disease (CD). Despite their epidemiological relevance, kissing bug species are under sampled in terms of their diversity and it is unclear what biases exist in available kissing bug data. Under climate change, range maps for kissing bugs may become less accurate as species shift their ranges to track climatic tolerance. OBJECTIVES: Quantify inventory completeness in available kissing bug data. Assess how well range maps are at conveying information about current distributions and potential future distributions subject to shift under climate change. Intersect forecasted changes in kissing bug distributions with contemporary sampling gaps to identify regions for future sampling of the group. Identify whether a phylogenetic signal is present in expert range knowledge as more closely related species may be similarly well or lesser understood. METHODS: We used species distribution models (SDM), specifically constructed from Bayesian additive regression trees, with Bioclim variables, to forecast kissing bug distributions into 2100 and intersect these with current sampling gaps to identify priority regions for sampling. Expert range maps were assessed by the agreement between the expert map and SDM generated occurrence probability. We used classical hypothesis testing methods as well as tests of phylogenetic signal to meet our objectives. FINDINGS: Expert range maps vary in their quality of depicting current kissing bug distributions. Most expert range maps decline in their ability to convey information about kissing bug occurrence over time, especially in under sampled areas. We found limited evidence for a phylogenetic signal in expert range map performance. MAIN CONCLUSIONS: Expert range maps are not a perfect account of species distributions and may degrade in their ability to accurately convey distribution knowledge under future climates. We identify regions where future sampling of kissing bugs will be crucial for completing biodiversity inventories.

Bartonella spp. in Phlebotominae Sand Flies, Brazil.

Bartonella spp. are opportunistic, vectorborne bacteria that can cause disease in both animals and humans. We investigated the molecular occurrence of Bartonella spp. in 634 phlebotomine sand fly specimens, belonging to 44 different sand fly species, sampled during 2017-2021 in north and northeastern Brazil. We detected Bartonella sp. DNA in 8.7% (55/634) of the specimens by using a quantitative real-time PCR targeting the 16S-23S internal transcribed spacer intergenic region. Phylogenetic analysis positioned the Lutzomyia longipalpis sand fly-associated Bartonella gltA gene sequence in the same subclade as Bartonella ancashensis sequences and revealed a Bartonella sp. sequence in a Dampfomyia beltrani sand fly from Mexico. We amplified a bat-associated Bartonella nuoG sequence from a specimen of Nyssomyia antunesi sand fly. Our findings document the presence of Bartonella DNA in sand flies from Brazil, suggesting possible involvement of these insects in the epidemiologic cycle of Bartonella species.

Morphological description and genetic analysis of a new black fly species (Diptera: Simuliidae) in the subgenus Asiosimulium from central Thailand.

BACKGROUND: Black flies are among the most medically and veterinary important insects, as adult females of certain species are the sole vector of Onchocerca volvulus. Here, a new black fly species belonging to the subgenus Asiosimulium Takaoka & Choochote, 2005, is described and formally named as Simulium (Asiosimulium) kittipati sp. nov. METHODS: Pupae and larvae of black flies were collected from available substrates in the stream from central Thailand. Pupae were individually separated in plastic tubes and maintained until adult flies emerged. The emerged adult flies associated with their pupal exuviae and cocoon as well as mature larvae preserved in 85% ethanol were used to describe the new species based on an integrated approach of morphological examination and molecular analysis of the COI gene. RESULTS: The new species is characterized in the female by the medium-long sensory vesicle with a medium-sized opening apically, scutum with three faint longitudinal vittae, and the ellipsoidal spermatheca; in the male by the number of upper-eye (large) facets in 20 vertical columns and 21 horizontal rows, hind basitarsus slender, nearly parallel-sided, and median sclerite much wider and upturned apically; in the pupa by the head and thoracic integument densely covered with tiny tubercles, and the pupal gill of arborescent type with 28-30 filaments; and in the larva by the postgenal cleft deep, nearly reaching the posterior margin of the hypostoma, and dark pigmented sheath of the subesophageal ganglion. The DNA barcode successfully differentiated the new species from its congeners with an interspecific genetic divergence of 1.74-18.72%, confirming the morphological identification that the species is a new member of the subgenus Asiosimulium. Phylogenetic analyses also indicated that the new species is genetically closely related to Simulium phurueaense Tangkawanit, Wongpakam & Pramual, 2018, further supporting its morphological classification. CONCLUSIONS: This is the ninth species assigned to the subgenus Asiosimulium within the genus Simulium Latreille, 1802. Taxonomic notes and identification keys are given to distinguish this new species from the eight known species members in its same subgenus. Additionally, a distribution map of all species members in this subgenus occurring in Thailand and other countries is provided.

A study on the diversity of phlebotomine sand flies (Diptera, Psychodidae) in karstic limestone areas in Vientiane Province, Laos, with a description of two new species of Sergentomyia França and & Parrot.

BACKGROUND: Southeast Asia is well known as a hotspot of biodiversity. However, very little is known about cave-dwelling hematophagous insects that are medically important. Taxonomic knowledge and ecology of phlebotomine sand flies are very poorly studied in Laos, as well as in other countries in the region. Herein, we report species diversity data and some notes on the ecology of the detected species from these karstic limestone areas of Laos. METHODS: Phlebotomine sand flies were collected using Centers for Disease Control and Prevention (CDC) light traps from limestone cave locations in three districts of Vientiane Province, Laos. Both morphological and molecular techniques were used for sand fly identification. Species diversity and abundance were analyzed according to sites, locations, collection seasons, and trapping positions. RESULTS: A total of 6564 sand flies, of which 5038 were females and 1526 were males, were morphologically identified into 20 species belonging to five genera (Chinius, Idiophlebotomus, Phlebotomus, Sergentomyia, and Grassomyia). The most abundant species were Chinius eunicegalatiae, Phlebotomus stantoni, Sergentomyia hivernus, Se. siamensis, and Idiophlebotomus longiforceps. Cytochrome b analysis results supported the morphological identification and revealed that Se. siamensis was separated from other members of the Se. barraudi group. Two new species, Se. dvoraki n. sp. and Se. marolii n. sp., were described. Sand fly density was generally high except in a cave in Vangvieng, with species richness ranging from 14 to 18 across different caves. Outside caves had higher species richness (R = 20) and diversity (H = 2.50) than cave entrances (R = 18, H = 2.41) and interiors (R = 16, H = 2.13). Seasonal variations showed high sand fly density in Feung and Hinheup during both dry and rainy seasons, while Vangvieng had a notable decrease in density during the dry season (D = 6.29). CONCLUSIONS: This study revealed that the diversity of phlebotomine sand fly fauna in Laos, particularly in karstic limestone areas, is greater than previously known. However, the taxonomic status of many species in Laos, as well as Southeast Asia, still needs more in-depth study using both morphological characters and molecular methods. Many species could be found from inside, at the entrance, and outside of caves, indicating a wide range of host-seeking behavior or possible natural breeding in the karstic cave areas.