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.

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.

Biodiversity data supports research on human infectious diseases: Global trends, challenges, and opportunities.

The unprecedented generation of large volumes of biodiversity data is consistently contributing to a wide range of disciplines, including disease ecology. Emerging infectious diseases are usually zoonoses caused by multi-host pathogens. Therefore, their understanding may require the access to biodiversity data related to the ecology and the occurrence of the species involved. Nevertheless, despite several data-mobilization initiatives, the usage of biodiversity data for research into disease dynamics has not yet been fully leveraged. To explore current contribution, trends, and to identify limitations, we characterized biodiversity data usage in scientific publications related to human health, contrasting patterns of studies citing the Global Biodiversity Information Facility (GBIF) with those obtaining data from other sources. We found that the studies mainly obtained data from scientific literature and other not aggregated or standardized sources. Most of the studies explored pathogen species and, particularly those with GBIF-mediated data, tended to explore and reuse data of multiple species (>2). Data sources varied according to the taxa and epidemiological roles of the species involved. Biodiversity data repositories were mainly used for species related to hosts, reservoirs, and vectors, and barely used as a source of pathogens data, which was usually obtained from human and animal-health related institutions. While both GBIF- and not GBIF-mediated data studies explored similar diseases and topics, they presented discipline biases and different analytical approaches. Research on emerging infectious diseases may require the access to geographical and ecological data of multiple species. The One Health challenge requires interdisciplinary collaboration and data sharing, which is facilitated by aggregated repositories and platforms. The contribution of biodiversity data to understand infectious disease dynamics should be acknowledged, strengthened, and promoted.

Phlebotomine Fauna (Diptera: Psychodidae) and Infection by Leishmania spp. in Forest Fragments of a University Campus, Western Amazon.

Forest fragments consist of important ecosystems for the maintenance of sand fly populations and Leishmania hosts. This study sought to identify the phlebotomine fauna and its infection by Leishmania spp. in forest fragments on the campus of the Federal University of Acre (UFAC), Western Amazon. Monthly collections with CDC traps were carried out from March 2020 to June 2021, in four forest fragments of UFAC. Male and female insects were processed and identified at species level. A sample of females was subjected to polymerase chain reaction (PCR) analysis to verify the presence Leishmania DNA. In total, 465 specimens were collected, of which 238 were males and 227 were females. The most frequent species were Nyssomyia antunesi (Coutinho, 1939) (47.3%), Trichophoromyia sp. (Mangabeira, 1942) (18.70%), and Ny. whitmani (Antunes & Coutinho, 1939) (8.81%). Molecular analysis detected the presence of Leishmania (Ross, 1903) DNA in a specimen of Ny. antunesi, and another one of Evandromyia walker (Newstead, 1914). The forest fragments of the university campus harbor a diverse sand fly fauna with the presence of Leishmania DNA in these insects, in addition to the presence of other species considered incriminated vectors of Leishmania parasites.

Sciopemyia sordellii in the Neotropical region: distribution, biology, and ecology.

The species Sciopemyia sordellii (Diptera, Phlebotominae) is widely distributed throughout the American continent. This raises doubts about their identity as a single biological entity and points to the occurrence of species complex. Here, we review the geographic distribution of S. sordellii in the Neotropical region, as well as data on its biology and ecology. Records were obtained from literature and from specimens deposited in biological collections. In total, 149 records were analysed, from 12 countries in America. However, after taxonomic revision, some specimens were wrongly identified as S. sordellii, especially those from Peru, putting in doubt the occurrence of the species in that country. In Brazil, it has been recorded in all five geographic regions and collected in different biomes, besides to other ecotypes such as caves. Furthermore, its presence in an anthropized environment has also been reported. Some studies have also diagnosed Leishmania DNA in S. sordellii, indicating that parasite circulation is being maintained between the reservoir hosts. The data evaluated in our study allowed us to provide updated information on the distribution of S. sordellii in America, which may be useful in the taxonomy of this species.

The SISBIOTA-Diptera Brazilian Network: A long-term survey of Diptera from unexplored Brazilian Western Arc of Amazon, Cerrado, and Pantanal

ABSTRACT The SISBIOTA-BRASIL was a three-year multimillion-dollar research program of the Brazilian government to document plants and animals in endangered/understudied areas and biomes in Brazil. Distributional patterns and the historical events that generated them are extensively unknown regarding Brazilian fauna and flora. This deficiency hinders the development of conservation policies and the understanding of evolutionary processes. Conservation decisions depend on precise knowledge of the taxonomy and geographic distribution of species. Given such a premise, we proposed to research the diversity of Diptera of the Brazilian western arc of Amazon, Cerrado, and Pantanal in the states of Mato Grosso, Mato Grosso do Sul, and Rondônia. Three important biomes of the South American continent characterize these Brazilian states: Amazon forest, Cerrado (Brazilian Savannah), and Pantanal. Besides their ecological relevance, these biomes historically lack intensive entomological surveys. Therefore, they are much underrepresented in the Brazilian natural history collections and in the scientific literature, which is further aggravated by the fact that these areas are being exponentially and rapidly converted to commercial lands. Our project involved over 90 collaborators from 24 different Brazilian institutions and one from Colombia among researchers, postdocs, graduate and undergraduate students, and technicians. We processed and analyzed nearly 300,000 specimens from ~60 families of Diptera collected with a large variety of methods in the sampled areas. Here, we provide a detailed overview of the genera and species diversity of 41 families treated. Our results point to a total of 2,130 species and 514 genera compiled and identified for the three states altogether, with an increase of 41% and 29% in the numbers of species and genera known for the three states combined, respectively. Overall, the 10 most species-rich families were Tachinidae, Cecidomyiidae, Tabanidae, Psychodidae, Sarcophagidae, Stratiomyidae, Bombyliidae, Syrphidae, Tephritidae, and Asilidae. The 10 most diverse in the number of genera were Tachinidae, Stratiomyidae, Asilidae, Mycetophilidae, Syrphidae, Tabanidae, Muscidae, Dolichopodidae, Sarcophagidae, and Chloropidae. So far, 111 scientific papers were published regarding taxonomic, phylogenetic, and biogeographical aspects of the studied families, with the description of 101 new species and three new genera. We expect that additional publications will result from this investigation because several specimens are now curated and being researched by specialists.

Publishing data to support the fight against human vector-borne diseases.

Vector-borne diseases are responsible for more than 17% of human cases of infectious diseases. In most situations, effective control of debilitating and deadly vector-bone diseases (VBDs), such as malaria, dengue, chikungunya, yellow fever, Zika and Chagas requires up-to-date, robust and comprehensive information on the presence, diversity, ecology, bionomics and geographic spread of the organisms that carry and transmit the infectious agents. Huge gaps exist in the information related to these vectors, creating an essential need for campaigns to mobilise and share data. The publication of data papers is an effective tool for overcoming this challenge. These peer-reviewed articles provide scholarly credit for researchers whose vital work of assembling and publishing well-described, properly-formatted datasets often fails to receive appropriate recognition. To address this, GigaScience's sister journal GigaByte partnered with the Global Biodiversity Information Facility (GBIF) to publish a series of data papers, with support from the Special Programme for Research and Training in Tropical Diseases (TDR), hosted by the World Health Organisation (WHO). Here we outline the initial results of this targeted approach to sharing data and describe its importance for controlling VBDs and improving public health.

Occurrence records and metadata for sand flies (Diptera, Psychodidae, Phlebotominae) collected in the lands of indigenous people in the Brazilian Amazon

In order to contribute to knowledge of the epidemiology of American cutaneous leishmaniasis (ACL) among indigenous people living in sylvatic regions, we studied the sand fly fauna collected in areas of disease transmission in the Brazilian Amazon. Our two datasets reported here are comprised of occurrence data for sand flies from the Suruwaha Indigenous Land in the state of Amazonas collected between 2012-1013, and the Wajãpi Indigenous Land in the state of Amapá collected between 2013-2014. Sand flies were collected using unbaited CDC-like light traps at various sites within each study area and were identified to species-level by taxonomists with expertise in Amazonian fauna. A total of 4,646 records are reported: 1,428 from the Suruwaha and 3,218 from the Wajãpi. These records will contribute to a better understanding of ACL transmission dynamics, as well as the distribution of insect vectors, in these areas.

Sand fly (Diptera Psychodidae Phlebotominae) records in Acre, Brazil a Dataset

Amazonian phlebotomine fauna is considered one of the most diverse in the world. In addition, the region is the most prevalent for leishmaniasis in Brazil and South America. The state of Acre, which belongs to this region, also stands out in terms of the diversity of the sand fly fauna, as well as the occurrence of American tegumentary leishmaniasis. In this context, the present Dataset comprise a bibliographic review of sand fly species recorded in Acre State, Amazon region, Brazil. A total of 1,096 observations from Material Citation type (and two of preserved specimens) were presented using 32 variables according to Darwin Core terms. The bibliographic review was performed in PubMed, Google Scholar, SciELO, BVS (Biblioteca Virtual em Saúde) and bibliographic references cited in related scientific articles. Thus, the present study is a solid report for further studies on sand fly studies in Acre and other Amazon states.

Online catalogue of the Coleção de Flebotomíneos (FIOCRUZ/COLFLEB), a biological collection of American sand flies (Diptera: Psychodidae, Phlebotominae) held at Fiocruz Minas, Brazil

The "Coleção de Flebotomíneos" (FIOCRUZ/COLFLEB) held at Fiocruz Minas is a curated biological collection comprised of approximately 80,000 individual specimens of 370 species of sand flies (Diptera: Psychodidae, Phlebotominae) mostly from the Americas collected over the last 80 years by entomologists interested in understanding and controlling the vector-borne disease leishmaniases. Since 2010, the metadata of each of the individual biological specimens held in FIOCRUZ/COLFLEB, including the back catalogue of those deposited in previous decades, has been digitized. Here, our resulting electronic catalogue, containing records for 72,624 of the specimens, including all of the available provenance information associated with each of them, is published online through the speciesLink network <http://www.splink.org.br/search?collectioncode=FIOCRUZ11COLFLEB&group=animais&lang=pt&action=openform> and the Sistema de Informação sobre a Biodiversidade Brasileira (SiBBr) <http://ipt.fiocruz.br/ipt/resource?r=fiocruz_colfleb>.

The "Coleção de Flebotomíneos" (FIOCRUZ/COLFLEB) held at Fiocruz Minas is a curated biological collection comprised of approximately 80,000 individual specimens of 370 species of sand flies (Diptera: Psychodidae, Phlebotominae) mostly from the Americas collected over the last 80 years by entomologists interested in understanding and controlling the vector-borne disease leishmaniases. Since 2010, the metadata of each of the individual biological specimens held in FIOCRUZ/COLFLEB, including the back catalogue of those deposited in previous decades, has been digitized. Here, our resulting electronic catalogue, containing records for 72,624 of the specimens, including all of the available provenance information associated with each of them, is published online through the speciesLink network <http://www.splink.org.br/search?collectioncode=FIOCRUZ11COLFLEB&group=animais&lang=pt&action=openform> and the Sistema de Informação sobre a Biodiversidade Brasileira (SiBBr) <http://ipt.fiocruz.br/ipt/resource?r=fiocruz_colfleb>.

Online catalogue of the Coleção de Flebotomíneos (FIOCRUZ/COLFLEB), a biological collection of American sand flies (Diptera: Psychodidae, Phlebotominae) held at Fiocruz Minas, Brazil.

The Coleção de Flebotomíneos ("Phlebotomine Collection"; FIOCRUZ/COLFLEB) held at Fiocruz Minas is a curated biological collection comprising approximately 80 000 individual specimens of 370 species of sand flies (Diptera: Psychodidae, Phlebotominae). These were mostly collected from the Americas in the last 80 years by entomologists interested in understanding and controlling the vector-borne disease leishmaniases. Since 2010, the metadata of each biological specimen held in FIOCRUZ/COLFLEB, including the back catalogue of those deposited in previous decades, has been digitized. Here, we present the resulting electronic catalog containing records for 72,624 of the specimens, including all of the available provenance information associated with each specimen. The catalog is published online through the speciesLink network and the Sistema de Informação sobre a Biodiversidade Brasileira (SiBBr).

Sand fly (Diptera: Psychodidae: Phlebotominae) records in Acre, Brazil: a dataset.

Amazonian phlebotomine sand fly fauna is one of the most diverse in the world. The Amazon region is also the most prevalent for leishmaniasis in Brazil and South America. The state of Acre, in this region, also stands out in terms of the diversity of sand fly fauna, as well as the occurrence of American tegumentary leishmaniasis. In this context, the present dataset comprises a bibliographic review of sand fly species recorded in Acre state, Brazil. A total of 1,094 observations from material citations and two of preserved specimens are presented using 33 variables according to Darwin Core terms. The bibliographic review was performed in PubMed, Google Scholar, SciELO, Biblioteca Virtual em Saúde, and references cited in related scientific articles. Thus, this report will be valuable for further studies on sand flies in Acre and other Amazon states.

Occurrence records and metadata for sand flies (Diptera, Psychodidae, Phlebotominae) collected in the lands of indigenous people in the Brazilian Amazon.

To contribute to knowledge of the epidemiology of American cutaneous leishmaniasis (ACL) among indigenous people living in sylvatic regions, we studied the sand fly fauna collected in areas of disease transmission in the Brazilian Amazon. Here we report two datasets comprising occurrence data for sand flies from the Suruwaha Indigenous Land in the state of Amazonas collected in 2012-2013, and the Wajãpi Indigenous Land in the state of Amapá collected in 2013-2014. Sand flies were collected using unbaited CDC-like light traps at various sites within each study area and were identified to either genus or species-level by taxonomists with expertise in Amazonian fauna. A total of 4,646 records are reported: 1,428 from the Suruwaha and 3,218 from the Wajãpi. These records will contribute to a better understanding of ACL transmission dynamics, as well as the distribution of insect vectors, in these areas.

Large-scale Degradation of the Tocantins-Araguaia River Basin.

The Tocantins-Araguaia Basin is one of the largest river systems in South America, located entirely within Brazilian territory. In the last decades, capital-concentrating activities such as agribusiness, mining, and hydropower promoted extensive changes in land cover, hydrology, and environmental conditions. These changes are jeopardizing the basin's biodiversity and ecosystem services. Threats are escalating as poor environmental policies continue to be formulated, such as environmentally unsustainable hydropower plants, large-scale agriculture for commodity production, and aquaculture with non-native fish. If the current model persists, it will deepen the environmental crisis in the basin, compromising broad conservation goals and social development in the long term. Better policies will require thought and planning to minimize growing threats and ensure the basin's sustainability for future generations.

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).

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil.

This study aimed to describe the sand fly fauna and detect trypanosomatids in these insects from Casa Branca, state of Minas Gerais, Brazil, an endemic area of both visceral (VL) and tegumentary leishmaniasis (TL). Sand flies were collected bimonthly from May 2013 to July 2014, using automatic light traps exposed for three consecutive nights in peridomiciliary areas of nine houses with previous reports of VL and TL. ITS1-PCR and DNA sequencing were performed for trypanosomatids identification. A total of 16,771 sand flies were collected belonging to 23 species. The most abundant species was Nyssomyia whitmani (Antunes & Coutinho, 1939) (70.9%), followed by Lutzomyia longipalpis (Lutz & Neiva, 1912) (15.2%) and Migonemyia migonei (França, 1920) (9.1%). Leishmania amazonensis DNA was detected in Ny. whitmani (four pools) and Le. braziliensis DNA was detected in Psychodopygus lloydi (one pool). In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for Leishmania DNA, the parasite species was not determined due to the low quality of the sequences. Moreover, DNA of Herpetomonas spp. was detected in Ny. whitmani (two pools) and Cortelezzii complex (one pool). DNA of Crithidia spp. was detected in Ny. whitmani and Ps. lloydi (both one pool). Our results suggest that Ny. whitmani may be involved in the transmission of Le. amazonensis in the study area. The molecular detection of Le. amazonensis suggests the presence of this species in a sylvatic cycle between vertebrate and invertebrate hosts in the region of Casa Branca. Our data also reveal the occurrence of other non-Leishmania trypanosomatids in sand flies in Casa Branca District.

New Records of Phlebotomine Sand Flies (Diptera: Psychodidae) From the Western Brazilian Amazon and the Description of the Female of Pintomyia fiocruzi.

An entomological survey was conducted in several localities of Rondônia State, and new records were obtained for seven sand fly species: Evandromyia apurinan Shimabukuro, Figueira & Silva, 2013, Evandromyia carmelinoi (Ryan, Fraiha, Lainson & Shaw, 1986), Micropygomyia echinatopharynx Andrade-Filho, Galati, Andrade & Falcão, 2004, Nyssomyia urbinattii Galati & Galvis, 2012, Pintomyia duckei Oliveira, Alencar & Freitas, 2018, Psathyromyia pradobarrientosi (Le Pont, Matias, Martinez & Dujardin, 2004), and Sciopemyia vattierae (Le Pont & Desjeux, 1992). Herein, we also describe the female of Pintomyia fiocruzi Pereira Júnior, Pessoa, Marivalva & Medeiros, 2019. The female has spermathecae with a detached apical ring similar to the spermathecae of Pintomyia serrana (Damasceno & Arouck, 1949), Pintomyia odax (Fairchild & Hertig, 1961), Pintomyia ottolinai (Ortiz & Scorza, 1963), and Pintomyia robusta (Galati, Cáceres & Le Pont, 1995). Here, we provide characters to separate Pi. fiocruzi from Pi. serrana and Pi. odax. Pintomyia ottolinai has a trans-Andean distribution and Pi. robusta has not been found in Brazil and in our studies, we associated the females found in this study with Pi. fiocruzi males found at the same collection sites. Our findings highlight the importance of entomological fauna surveys and demonstrate the need for taxonomists to study species diversity in Rondônia. This study increases the number of sand fly species recorded in Rondônia to 143.

Evidence of a sylvatic enzootic cycle of Leishmania infantum in the State of Amapá, Brazil

Abstract INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.

Evidence of a sylvatic enzootic cycle of Leishmania infantum in the State of Amapá, Brazil.

INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.

Geometric morphometry of the head in sand flies (Diptera: Psychodidae: Phlebotominae), an alternative approach to taxonomy studies.

Almost 30% of female American sand flies are morphologically indistinguishable from one another. These females belong to species-complexes or groups with closely related species, making species-level identification difficult or even impossible. This fact has implications for public health since several of these groups include species which are vectors of human pathogens. Thus, new tools are needed to minimize species-level identification problems. In this context, our research sought to use geometric morphometry in the study of the head of females of closely related species of the genus Psychodopygus, Chagasi series, which includes the following species: P. chagasi, P. complexus, P. squamiventris maripaensis, P. squamiventris squamiventris, and P. wellcomei. We have used ten landmarks distributed on the head of these species. All species were clearly distinguished using the centroid size and shape together. These results highlight the head analysis as an effective tool for future geometric morphometry studies in sand flies.