Molecular and morphometric study of Brazilian populations of Psychodopygus davisi.

In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low interspecific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our dataset. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Midwest. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of candidate species, which need to be validated in future studies using a more comprehensive approach with both genomic data and morphological characters.

New sand fly (Diptera, Psychodidae) records and COI DNA barcodes in the state of Maranhão, Eastern Amazon, Brazil.

The sand fly fauna and the usefulness of the DNA barcoding fragment of the cytochrome c oxidase subunit I (COI) gene were accessed in a forest fragment in the municipality of Governador Newton Bello, state of Maranhão, Brazil. We performed entomological collections in three independent campaigns in May and October 2021, and January 2023. Sand flies were morphologically-identified and then DNA barcoded. Sequences were deposited and analyzed in the BOLD System Database, and various species delimitation algorithms, to assess whether DNA sequences merge into taxonomic units in accordance with nominal species. In total, 1,524 sand flies were collected, comprising 32 nominal species. Nyssomyia antunesi was the most abundant species (31.5 %), followed by Psychodopygus davisi (27 %). We reported for the first time in the state of Maranhão, the presence of Lutzomyia evangelistai, Lutzomyia sherlocki, Pressatia equatorialis, and Psathyromyia barrettoi. We amplified and analyzed 67 COI barcodes of 23 species, which were merged with conspecific sequences extracted from GenBank. The maximum intraspecific p distances ranged from 0.0 % to 14.74 %, while the distances to the nearest neighbor varied from 1.67 % to 13.64 %. The phylogenetic gene tree and species delimitation tools clustered sequences into well-supported clades/clusters for each nominal species, except for Pressatia choti/Pr. equatorialis, which have the lowest interspecific genetic distance (1.67 %). We sequenced for the first time COI barcodes of Brumptomyia brumpti, Evandromyia monstruosa, Micropygomyia rorotaensis, Micropygomyia pilosa, Pintomyia christenseni, Pintomyia pacae, Pr. equatorialis, Pa. barrettoi, and Psathyromyia hermanlenti, which will be useful for further molecular identification and classification proposals of Neotropical species. This study updated the current list of the sand fly fauna for the state of Maranhão to 97, and demonstrated that COI barcodes are useful for specific identification.

Trichophoromyia auraensis: evidence for cryptic species and first record in the state of Maranhão, Brazil.

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogenetic gene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers.

Morphological and DNA-based description of Trichophoromyia peixotoi n. sp. (Diptera: Psychodidae), a new sand fly species from the Brazilian Amazon.

BACKGROUND: Phlebotomine sand flies of the genus Trichophoromyia Barretto, 1962 are of great relevance to public health as vectors of Leishmania protozoans. A new phlebotomine species named Trichophoromyia peixotoi n. sp. is here described based on both male morphology and COI DNA barcodes. METHODS: The sand fly specimens were collected in the Parque Nacional da Amazônia (PNA), situated in the municipality of Itaituba, state of Pará, Brazil. Morphological description was done based on 10 male specimens. Five specimens were DNA barcoded for the COI gene. RESULTS: The morphological and molecular analyses allowed the delimitation of this new species from others of Trichophoromyia. Trichophoromyia peixotoi n. sp. is closely related to other species with aedeagal ducts > 4 times the length of the sperm pump, from which it may be distinguished by the gonocoxite bristles and paramere shape. CONCLUSIONS: The description of T. peixotoi n. sp. brings the number of species of Trichophoromyia to 45, including 24 for Brazil. The integrative taxonomy effort through the analysis of COI barcodes proved to be effective in the species delimitation of some Trichophoromyia spp.

Improving the COI DNA barcoding library for Neotropical phlebotomine sand flies (Diptera: Psychodidae).

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.

DNA Barcoding Culicoides Biting Midges (Diptera: Ceratopogonidae) in Northeast Brazil.

Biting midges of the genus Culicoides are small insects associated with the transmission of several pathogens, which requires the correct identification of the species, for implementation of effective strategies against these insects. However, many species are difficult to identify only by morphological characters. Therefore, the use of molecular methods can help in the taxonomy and systematics of this group. Here, the DNA barcode approach was evaluated for nine species of Culicoides from the State of Maranhão, Brazil. We generated 39 sequences from a 476 bp (base pairs) fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene. To assess the usefulness of COI barcodes for the identification of these species, paired genetic distances from intra and interspecific comparisons and phylogenetic trees were generated in MEGA and RAxML/BEAST softwares, respectively. In addition, species delimitation was performed using the PTP, GMYC, and ABGD algorithms. The intra and interspecific genetic distances showed a clear distinction between them, demonstrating that, for the taxa studied, there can hardly be ambiguous identifications with barcodes. In the same sense, the phylogenetic reconstruction resulted in well-supported clades for all morphospecies analyzed.

A Review of Historical Phlebotominae Taxonomy (Diptera: Psychodidae).

Psychodidae is a group constituted by ca. 3400 species distributed in six existing and one extinct subfamily. Among them, Phlebotominae is of relevant medical and veterinary interest as vectors of pathogens, such as viruses, bacteria, and trypanosomatides, to vertebrates. The taxonomy of Phlebotominae, begun in 1786, received a great boost at the beginning of the twentieth century, when some of its species began to be implicated as vectors of leishmaniasis agents. Currently, the group consists of 1060 species/subspecies described for both hemispheres. Its taxonomy and systematics have been largely based on the morphological characters of adults in view of the limited number of species known in their immature forms, as well as by molecular approaches. This review focuses on the evolution of the phlebotomine systematics, with information regarding when the descriptions of the sand fly species/subspecies occurred, where their type localities are, the number of authors involved in their descriptions, and the researchers who have most contributed to the description of these taxa and their respective institutions. The morphological characters of adult forms used in the taxonomy of groups, in an evolutionary approach, and the current state of knowledge based on immature forms is also presented.

DNA barcoding of sand flies (Diptera, Psychodidae, Phlebotominae) from the western Brazilian Amazon.

The subfamily Phlebotominae comprises important insects for public health. The use of complementary tools such as molecular taxonomy is necessary for interspecific delimitation and/or discovery of cryptic species. Here, we evaluated the DNA barcoding tool to identify different species in the southwestern Brazilian Amazon. For this, we collected sand flies in forest fragments along the highway BR-317, in the municipality of Brasiléia, state of Acre, Brazil. The specimens were DNA-barcoded using a fragment of the cytochrome c oxidase subunit I (COI) gene. The sequences were analyzed to generate K2P pairwise genetic distances and a Neighbour-joining tree. The sand fly barcodes were also clustered into Molecular Operation Taxonomic Units (MOTU) using Automatic Barcode Gap Discovery (ABGD) approach. A total of 59 COI sequences comprising 22 nominal species and ten genera were generated. Of these, 11 species had not been sequenced before, thus being new COI sequences to science. Intraspecific genetic distances ranged between 0 and 4.9%, with Pintomyia serrana presenting the highest values of genetic distance, in addition to having been partitioned into three MOTUs. Regarding the distances to the nearest neighbour, all species present higher values in relation to the maximum intraspecific distance, in addition to forming well supported clusters in the neighbour-joining analysis. The DNA barcoding approach is useful for the molecular identification of sand flies from Brasiléia, state of Acre, and was efficient in detecting cryptic diversity of five species which can be confirmed in future studies using an integrative approach. We also generated new COI barcodes for Trichophoromyia auraensis, Nyssomyia shawi, and Psychodopygus paraensis, which may play a role in the transmission of Leishmania spp. in the Brazilian Amazon.

Spatiotemporal Distribution of Leishmaniasis in an Endemic Area of Northeast Brazil: Implications for Intervention Actions.

This study aimed to analyze the spatiotemporal distribution of leishmaniases, and contribute to the knowledge of their epidemiological dynamics from 2007 to 2017 in the municipality of Caxias, Maranhão, Northeast Brazil. Data on American tegumentary leishmaniasis (ATL) and human visceral leishmaniasis (HVL) were obtained in the Epidemiological Surveillance Sector of Caxias, while data on canine visceral leishmaniasis (CVL) were obtained in the Zoonoses Surveillance Unit. For data analysis and spatial representation of leishmaniasis cases, the geoprocessing of the data was performed, and the geometric features of the state of Maranhão, Caxias, and the disease registration sites were obtained from the shapefile database of the Brazilian Institute of Geography and Statistics. Geostatistics was used to create maps based on the Kernel density method, starting from the points, producing a raster file for each case with several data frames, allowing the instantaneous comparison of the phenomena. During the study period, ATL, HVL, and CVL were reported in Caxias, accounting for 114,304 and 8,498 cases, respectively. The geoprocessing analysis showed that leishmaniasis is widely distributed in the urban area of Caxias. However, there are risk areas for the transmission of these diseases to humans and dogs, associated with deforestation and urban expansion, and may vary over time. Preventive measures must focus on risk areas, including conservation efforts and urban planning, in order to reduce the transmission of leishmaniases.

Molecular taxonomy of phlebotomine sand flies (Diptera, Psychodidae) with emphasis on DNA barcoding: A review.

The taxonomy and systematics of sand flies (Diptera, Psychodidae, Phlebotominae) are one of the pillars of research aimed to identifying vector populations and the agents transmitted by these insects. Traditionally, the use of morphological traits has been the main line of evidence for the definition of species, but the use of DNA sequences is useful as an integrative approach for their delimitation. Here, we discuss the current status of the molecular taxonomy of sand flies, including their most sequenced molecular markers and the main results. Only about 37% of all sand fly species have been processed for any molecular marker and are publicly available in the NCBI GenBank or BOLD Systems databases. The genera Phlebotomus, Nyssomyia, Psathyromyia and Psychodopygus are well-sampled, accounting for more than 56% of their sequenced species. However, less than 34% of the species of Sergentomyia, Lutzomyia, Trichopygomyia and Trichophoromyia have been sampled, representing a major gap in the knowledge of these groups. The most sequenced molecular markers are those within mtDNA, especially the DNA barcoding fragment of the cytochrome c oxidase subunit I (coi) gene, which has shown promising results in detecting cryptic diversity within species. Few sequences of conserved genes have been generated, which hampers higher-level phylogenetic inferences. We argue that sand fly species should be sequenced for at least the coi DNA barcoding marker, but multiple markers with different mutation rates should be assessed, whenever possible, to generate multilocus analysis.

DNA Barcoding of genus Culicoides biting midges (Diptera: Ceratopogonidae) in the Brazilian Amazon.

Culicoides biting midges are capable to transmit Oropouche virus, Bluetongue virus and Mansonella spp. This study aimed to assess the utility of DNA barcode as an alternative method in the Culicoides species identification. The study was conducted in Jamari National Forest. Biting midges were collected using HP light traps during four months, February, April, August and October 2018. Insects were morphologically identified to the species level, and rest of the body were subjected to DNA extraction and PCR targeting a fragment of the cytochrome c oxidase subunit I (COI) gene, which were analyzed and deposited in GenBank. A phylogenetic gene tree was reconstructed using RAxML software, and the sequences were assigned at Molecular Operational Taxonomic Unit (MOTU) level by species delimitation algorithms. According to morphological approach, 18 species of 2 subgenera and 7 species groups were identified. A total of 191 new COI barcodes from 18 species were generated. Of these, fifteen species have been deposited for the first time in all datasets in the world. These sequences allowed the correct identification of 188 and 187 specimens according to the BM and BCM criteria, respectively. The intraspecific genetic distances ranged from 0 to 16.5%, while the interspecific ones ranged from 2.1 to 27.1%. The nominal species Culicoides glabellus and C. tetrathyris splitted into three and two MOTUs, respectively, except for mPTP, indicating a cryptic diversity in these species. Also, sequences of C. pseudodiabolicus formed two MOTUs using all algorithms, except for PTP and ABGD, suggesting the existence of two potential species. In contrast, some barcodes of C. quasiparaensis and C. paraensis merged into a single MOTU, which can be explained by the complex characteristics of the paraensis group, since these species have similar morphological characters. Here, we provided the first COI barcodes for biting midges in Rondônia and Brazil, and demonstrates that these are sufficient to discriminate between some species.

Detection of Wolbachia in Mosquitoes (Diptera: Culicidae) in the State of Maranhão, Brazil.

Recently, the endobacteria Wolbachia has emerged as a biological tool for the control of arboviruses. Thus, we investigated the rate of natural infection by Wolbachia in Culicidae species from Maranhão, Brazil. For this, we amplified the Wolbachia surface protein gene (wsp) from mosquitoes collected in six localities of Maranhão, and positive samples were subjected to new analysis using group-specific primers. In total, 448 specimens comprising 6 genera and 18 species of mosquitoes were analyzed. Wolbachia DNA was PCR-detected in 7 species, three of which are new records: Aedes scapularis (Rondani, 1848), Coquillettidia juxtamansonia (Chagas, 1907) and Cq. venezuelensis (Theobald, 1912), in addition to Ae. albopictus (Skuse, 1894) and Culex quinquefasciatus Say, 1823, which are commonly described as permissive to maintain this bacterium in natural environments, and two species of the subgenera Anopheles (Nyssorhynchus) Blanchard, 1902 and Culex (Melanoconion) Theobald, 1903 which could not be identified at species level. The infection rate of all species ranged from 0 to 80%, and the average value was 16.5%. This study increases the knowledge about the prevalence of Wolbachia in the culicid fauna and may help in selecting strains for biological control purposes.

Identification of Bloodmeals from Sand Flies (Diptera: Psychodidae) Collected in the Parque Nacional do Viruá, State of Roraima, Brazil.

The transmission of pathogens that cause leishmaniases occurs by the bite of female sand flies (Diptera: Psychodidae) in their vertebrate hosts, which makes the identification of their bloodmeal sources an important step for the control and epidemiology of these diseases. In Brazil, the state of Roraima has a great diversity of sand flies, vertebrate hosts, and protozoan Leishmania, but little is known about the host blood-feeding preferences of sand flies. Thus, we evaluated the bloodmeal sources of sand flies collected from their sylvatic habitats in Parque Nacional do Viruá, Roraima. Fieldwork was carried-out between 13th and 18th August 2019 using CDC light traps. Sand flies were slide-mounted and morphologically identified using the head and last segments of the abdomen. Engorged females had their DNA extracted, followed by amplification and sequencing of the cytochrome b (cytb) molecular marker for vertebrates. Sequences were analyzed and compared with those from GenBank using the BLASTn search tool, in addition to the reconstruction of a phylogenetic tree to demonstrate the clustering pattern of these sequences. A total of 1,209 sand flies were identified, comprising 20 species, in which the most abundant were Psychodopygus ayrozai (Barretto and Coutinho) (42.10%) and Psychodopygus chagasi (Costa Lima) (26.22%). Bloodmeal source identification was successfully performed for 34 sand flies, that confirm four vertebrate species, being the most abundant the armadillo Dasypus novemcinctus Linnaeus, 1758 (Cingulata: Dasypodidae).

DNA Barcoding Reveals Hidden Diversity of Sand Flies (Diptera: Psychodidae) at Fine and Broad Spatial Scales in Brazilian Endemic Regions for Leishmaniasis.

Sand fly (Diptera: Psychodidae) taxonomy is complex and time-consuming, which hampers epidemiological efforts directed toward controlling leishmaniasis in endemic regions such as northeastern Brazil. Here, we used a fragment of the mitochondrial cytochrome c oxidase I (COI) gene to identify sand fly species in Maranhão State (northeastern Brazil) and to assess cryptic diversity occurring at different spatial scales. For this, we obtained 148 COI sequences of 15 sand fly species (10 genera) from Maranhão (fine spatial scale), and joined them to COI sequences from other Brazilian localities (distant about 2,000 km from Maranhão, broad spatial scale) available in GenBank. We revealed cases of cryptic diversity in sand flies both at fine (Lutzomyia longipalpis (Lutz and Neiva) and Evandromyia termitophila (Martins, Falcão and Silva)) and broad spatial scales (Migonemyia migonei (França), Pressatia choti (Floch and Abonnenc), Psychodopygus davisi (Root), Sciopemyia sordellii (Shannon and Del Ponte), and Bichromomyia flaviscutellata (Mangabeira)). We argue that in the case of Bi. flaviscutellata, the cryptic diversity is associated with a putative new species. Cases in which DNA taxonomy was not as effective as morphological identification possibly involved recent speciation and/or introgressive hybridization, highlighting the need for integrative approaches to identify some sand fly species. Finally, we provide the first barcode sequences for four species (Brumptomyia avellari (Costa Lima), Evandromyia infraspinosa (Mangabeira), Evandromyia evandroi (Costa Lima and Antunes), and Psychodopygus complexus (Mangabeira)), which will be useful for further molecular identification of neotropical species.

Detection of Leishmania DNA and Blood Meal Identification in Sand Flies (Diptera: Psychodidae) From Lençois Maranhenses National Park Region, Brazil.

To elucidate portions of the transmission cycles of American tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) occurring in the region surrounding the Lençóis Maranhenses National Park, an important tourist center in Brazil, the present study objectives were to determine the rate of natural infection by Leishmania spp. and the blood meal in caught sand flies species in the region. Sand flies were captured over 36 mo in 62 locations of the municipality of Barreirinhas, Maranhão with notifications of disease incidence. Species identification of parasites was performed with internal transcribed spacer 1 (ITS1) PCR-RFLP using HaeIII enzyme. Blood meal identification was performed with cytochrome b (cytb) gene PCR-RFLP using HaeIII and MboI enzyme. The species Lutzomyia longipalpis (Lutz and Neiva 1912) presented a positivity rate of 3.7% for Leishmania infantum. Species not considered vectors of this parasite such as Lu. lenti (Mangabeira 1938) and Lu. whitmani (Antunes & Coutinho 1939) showed infection rates of 0.6% and 0.9%, respectively. Among the vectors of Leishmania spp. was Lu. whitmani with detection rate of 0.3% for Le. braziliensis and Lu. flaviscutellata (Mangabeira 1942) with a detection rate of 8% for Le. amazonensis. After restriction of amplification product encoding a 359bp sequence of the cytb recognized in as follows: pigs (37.9%); dogs (27.4%); chickens (20.9%); horses (9%), rodents (3.3%), and humans (1.4%). The presence of Leishmania DNA in sand flies fed with human blood and domestic animals in villages with transmission of VL and TL suggests that transmission could be occurring in the locations of the infected patients.

Detection of Leishmania amazonensis and Leishmania braziliensis in Culicoides (Diptera, Ceratopogonidae) in an endemic area of cutaneous leishmaniasis in the Brazilian Amazonia.

Biting midges in the genus Culicoides act as vectors of arboviruses throughout the world and as vectors of filariasis in Latin America, the Caribbean, and parts of Africa. Although Culicoides spp. are currently not considered to be vectors of Leishmania protozoa, the high abundance of biting midges in areas with active cutaneous leishmaniasis transmission points to the possibility of Culicoides infection by these pathogens. We used PCR to test captured Culicoides species for natural infection with Leishmania spp. We tested 450 Culicoides females, divided into 30 pools of 15 individuals each, as follows: nine pools of C. foxi (135 specimens), seven pools of C. filariferus (105), seven pools of C. insignis (105), five pools of C. ignacioi (75), and two pools of C. flavivenula (30). PCR confirmed the presence of Leishmania braziliensis DNA in C. ignacioi (0.14%), C. insignis (0.14%), and C. foxi (0.11); and Le. amazonensis DNA in C. filariferus (0.14%) and C. flavivenula (0.50%). We conclude that these Culicoides species can be naturally infected, but vector competence and transmission capability must be confirmed in future studies. Our results warrant further investigation into the role of these biting midge species in the leishmaniasis epidemiological cycle.