Diversity and seasonality of horse flies (Diptera: Tabanidae) in Amazon Forest fragments of Monte Negro, Rondônia, Western Amazon.

Tabanidae are considered a nuisance to humans, wild animals, and livestock due to their painful, annoying, and insistent biting. Tabanids transmit some pathogens and parasites biologically and mechanically. In humans, there are relatively few pathogens transmitted regularly. Still, tabanids serve as vectors of a number of disease agents of animals, including viruses, bacteria, protozoans, and nematodes. They are more abundant in tropical and humid regions, and their seasonal patterns are affected by habitat changes such as deforestation and fragmentation. Here, we analyze the tabanid fauna in Monte Negro, a central municipality of Rondônia, Brazil, comparing abundance, richness, and diversity in forest and pasture habitats. Traps were set for 5 days a month for 12 consecutive months. We also examined how abiotic factors (humidity, temperature, and rainfall) affected the abundance, diversity, and richness and the effectiveness of Malaise and Nzi traps as sample methods. The influence of climatic variables on the richness and abundance of the species was tested using generalized linear models, and we used non-parametric dimensional scaling (nMDS) for analysis of species composition and diversity in different traps and environments. We collect 1032 specimens of 25 species. The most abundant species were Tabanus antarcticus, Dichelacera tetradelta, Tabanus mucronatus, and Leucotabanus albovarius. Forest habitats had the highest number of tabanids, followed by pasture and the anthropized area, and there was no significant difference regarding the effectiveness of the Malaise and Nzi traps. The study provides new information on the distribution and ecology of tabanids in Brazil.

Ticks on reptiles and amphibians in Central Amazonia, with notes on rickettsial infections.

Reptiles and amphibians are exceptional hosts for different ectoparasites, including mites and ticks. In this study, we investigated tick infestations on reptiles and amphibians trapped in Central Amazonia, and also assessed the presence of rickettsial infections in the collected ticks. From September 2016 to September 2019, 385 reptiles (350 lizards, 20 snakes, 12 tortoises, and three caimans) and 120 amphibians (119 anurans and one caecilian) were captured and examined for ectoparasites. Overall, 35 (10%) lizards, three (25%) tortoises and one (0.8%) toad were parasitized by ticks (124 larvae, 32 nymphs, and 22 adults). In lizards, tick infestation varied significantly according to landscape category and age group. Based on combined morphological and molecular analyses, these ticks were identified as Amblyomma humerale (14 larvae, 12 nymphs, 19 males, and one female), Amblyomma nodosum (three larvae, one nymph, and one female), and Amblyomma rotundatum (four larvae, three nymphs, and one female), and Amblyomma spp. (103 larvae and 16 nymphs). Our study presents the first records of A. nodosum in the Amazonas state and suggests that teiid lizards are important hosts for larvae and nymphs of A. humerale in Central Amazonia. Moreover, a nymph of A. humerale collected from a common tegu (Tupinambis teguixin) was found positive for Rickettsia amblyommatis, which agrees with previous reports, suggesting that the A. humerale-R. amblyommatis relationship may be more common than currently recognized.

Presence of trypanosomatids, with emphasis on Leishmania, in Rodentia and Didelphimorphia mammals of a rural settlement in the central Amazon region.

BACKGROUND: Trypanosomatids are widespread and cause diseases - such as trypanosomiasis, sleeping sickness, Chagas disease, and cutaneous and visceral leishmaniasis - in animals and humans. These diseases occur in both rural and urban regions due to unplanned growth and deforestation. Thus, wild and synanthropic reservoir hosts living in residential areas are risk factors. OBJECTIVE: We aimed to evaluate the diversity of small mammals (rodents and marsupials), and the occurrence of trypanosomatids, especially Leishmania, in the rural settlement of Presidente Figueiredo, Amazonas. METHODS: Animals were collected using Sherman, Tomahawk, and Pitfall traps along 16 trails in four landscapes: continuous forest, forest with planting, planting, and peridomiciliar. Leishmania sp. was detected in liver samples by polymerase chain reaction targeting kDNA. FINDINGS: Diversity was higher in forests with planting and lower around residences. In total, 135 mammals (81 rodents and 54 marsupials covering 14 genera) were captured. Rodents presented infection rates (IR) of 74% and marsupials of 48%. Rodents in domicile landscapes presented a higher IR (92.9%), while marsupials showed a higher IR in forests (53.3%). MAIN CONCLUSIONS: The results suggest high prevalence of trypanosomatids across 12 mammalian genera possibly involved as reservoir hosts in the enzootic transmission of leishmaniasis in the Amazon's rural, peridomiciliar landscape.

Redescription of Hepatozoon ameivae (Carini and Rudolph, 1912) from the lizard Ameiva ameiva (Linnaeus, 1758).

Blood samples from 72 Ameiva ameiva lizards from Central Amazonian upland forests were collected, and thin smears of 40 (55.5%) animals were positive for gamonts of Hepatozoon with a mean level of intensity of infection of 14 parasites/2000 blood erythrocytes (0.73%). The gametocytes were found attached with host cells' nuclei, and their dimensions were 14.28 ± 1.05 µm in length and 4.50 ± 0.80 µm in width. Phylogenetic analyses of the 18S rRNA gene showed that the new sequences obtained from A. ameiva constitute a monophyletic sister clade to the Hepatozoon spp. from Brazilian snakes. Based on morphological features and new molecular data, we redescribe this hemogregarine as Hepatozoon ameivae. This study also provides the first molecular characterization of a Hepatozoon species from a Brazilian lizard.

Immune response in Mansonella ozzardi infection modulated by IL-6/IL-10 axis in Amazon region of Brazil.

Mansonellosis is an endemic disease in the South and Central America. In Brazil, one of the etiological agents is Mansonella ozzardi. This filarial infection is yet poorly understood, with a controversial morbity, presenting since a oligosymptoms, malaria-like signs or without complaint in humans. The knowledge of the human immune response to microfilariae infection is limited mainly by different evolutionary cycles of the parasite in the host. In addition, the prevalence of this filarial parasite infection is high in several regions of Amazonas State. A cross-sectional study was conducted in an endemic area for microfilariae of M. ozzardi (MF) infection in the Amazonas State, Brazil. Proinflammatory and regulatory cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-gamma, and IL-17A) were measured in cryopreserved serum using the Cytometric Bead Array techniques (CBA) in 54 patients diagnosed with M. ozzardi infection and 55 individuals without the infection were included in the study (Controls). The IL-4, IL-6 and IL-10 level increased in infected patients with MF infection, while IL-17A increased in control only. When we compared controls to patients with high or low parasite load, the increased level of IL-6 and IL-10 were maintained. IL-6 contributes to the proinflammatory activity and IL-10 modulates Th1, Th2 and Th17 immune response. Furthermore, IL-4 was detected as a marker in the MF infection and MF patients with low parasite load, indicating the action of the Th2 cell response. The complex network of cytokines acting during M. ozzardi infection depends on a fine balance to determine a host protective effect or filarial persistence. Therefore, these results suggest that the immune response in MF infection is modulated by IL-6/IL-10 axis.

Lutzomyia umbratilis from an area south of the Negro River is refractory to in vitro interaction with Leishmania guyanensis.

BACKGROUND: Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available. OBJECTIVES: This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River. METHODS: Procyclic and metacyclic attachment was quantified using an in vitro system. FINDINGS: Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03). MAIN CONCLUSIONS: The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon.

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes.

BACKGROUND: Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur. OBJECTIVES: In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions. METHODS: Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR. FINDINGS: The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes. MAIN CONCLUSION: Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

Sensitivity of diagnostic methods for Mansonella ozzardi microfilariae detection in the Brazilian Amazon Region.

BACKGROUND: The human filarial worm Mansonella ozzardi is highly endemic in the large tributaries of the Amazon River. This infection is still highly neglected and can be falsely negative when microfilariae levels are low. OBJECTIVES: This study investigated the frequency of individuals with M. ozzardi in riverine communities in Coari municipality, Brazilian Amazon. METHODS: Different diagnostic methods including polymerase chain reaction (PCR), blood polycarbonate membrane filtration (PCMF), Knott's method (Knott), digital thick blood smears (DTBS) and venous thick blood smears (VTBS) were used to compare sensitivity and specificity among the methods. Data were analysed using PCMF and Bayesian latent class models (BLCM) as the gold standard. We used BLCM to calculate the prevalence of mansonelliasis based on the results of five diagnostic methods. FINDINGS: The prevalence of mansonelliasis was 35.4% by PCMF and 30.1% by BLCM. PCR and Knott methods both possessed high sensitivity. Sensitivity relative to PCMF was 98.5% [95% confidence interval (CI): 92.0 - 99.7] for PCR and 83.5% (95% CI: 72.9 - 90.5) for Knott. Sensitivity derived by BLCM was 100% (95% CI 93.7 - 100) for PCMF, 100% (95% CI: 93.7 - 100) for PCR and 98.3% (95% CI: 90.6 - 99.9) for Knott. The odds ratio of being diagnosed as microfilaremic increased with age but did not differ between genders. Microfilariae loads were higher in subjects aged 30 - 45 and 45 - 60 years. MAIN CONCLUSIONS: PCMF and PCR were the best methods to assess the prevalence of mansonelliasis in our samples. As such, using these methods could lead to higher prevalence of mansonelliasis in this region than the most commonly used method (i.e., thick blood smears).

Molecular Verification of New World Mansonella perstans Parasitemias.

We obtained ribosomal and mitochondrial DNA sequences from residents of Amazonas state, Brazil, with Mansonella parasitemias. Phylogenetic analysis of these sequences confirm that M. ozzardi and M. perstans parasites occur in sympatry and reveal the close relationship between M. perstans in Africa and Brazil, providing insights into the parasite's New World origins.

Trichophoromyia auraensis is a putative vector

The sandfly Trichophoromyia auraensis has recently evolved as a proven vector of Leishmania (Viannia) endemic to state of Acre in the north of Brazil. This note is intended to propose a correction in the report of the first occurrence of natural infection of Leishmania (Viannia) in this species. We and the other scientific groups reinforced that Tr. auraensis is a possible vector involved in the transmission of American cutaneous leishmaniasis in Acre, Brazil.

Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American Cutaneous Leishmaniasis human cases along the Brazilian border with Peru and Bolivia.

In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality.

Mansonella ozzardi in the municipality of Tefé, Amazonas, Brazil, 60 years after the first report: an epidemiologic study.

The prevalence of mansonelliasis was studied in the municipality of Tefé, state of Amazonas, Brazil. The prevalence (thick blood smear method) was 13.6% (147/1,078), higher in the Solimões River region (16.3%) than in the Tefé River region (6.3%). In the sampled communities in the Solimões River region, a higher density of cases was observed, as indicated by a kernel analysis (odds ratio 0.34; 95% confidence interval: 0.20-0.57). Males had a higher prevalence (χ2 = 31.292, p < 0.001) than women. Mansonella ozzardi prevalence was higher in retirees and farmers (28.9% and 27%, respectively). Prevalence also significantly increased with age (χ2 = -128.17, p < 0.001), with the highest numbers occurring in persons older than 67 years.

Low coverage of species constrains the use of DNA barcoding to assess mosquito biodiversity.

Mosquitoes (Culicidae) represent the main vector insects globally, and they also inhabit many of the terrestrial and aquatic habitats of the world. DNA barcoding and metabarcoding are now widely used in both research and routine practices involving mosquitoes. However, these methodologies rely on information available in databases consisting of barcode sequences representing taxonomically identified voucher specimens. In this study, we assess the availability of public data for mosquitoes in the main online databases, focusing specifically on the two most widely used DNA barcoding markers in Culicidae: COI and ITS2. In addition, we test hypotheses on possible factors affecting species coverage (i.e., the percentage of species covered in the online databases) for COI in different countries and the occurrence of the DNA barcode gap for COI. Our findings showed differences in the data publicly available in the repositories, with a taxonomic or species coverage of 28.4-30.11% for COI in BOLD + GenBank, and 12.32% for ITS2 in GenBank. Afrotropical, Australian and Oriental biogeographic regions had the lowest coverages, while Nearctic, Palearctic and Oceanian had the highest. The Neotropical region had an intermediate coverage. In general, countries with a higher diversity of mosquitoes and higher numbers of medically important species had lower coverage. Moreover, countries with a higher number of endemic species tended to have a higher coverage. Although our DNA barcode gap analyses suggested that the species boundaries need to be revised in half of the mosquito species available in the databases, additional data must be gathered to confirm these results and to allow explaining the occurrence of the DNA barcode gap. We hope this study can help guide regional species inventories of mosquitoes and the completion of a publicly available reference library of DNA barcodes for all mosquito species.

Description of a new phlebotomine species (Diptera: Psychodidae, Phlebotominae) and new records of sand flies from the State of Acre, northern Brazil.

Groundbreaking studies of phlebotomine sand fly populations in Assis Brasil, State of Acre, Brazil, resulted in the collection of 13 new records of phlebotomine sand flies and one previously undescribed species. Lutzomyia naiffi sp. nov. is described here. The new species is similar to Lutzomyia columbiana (Ristorcelli & Van Ty) in measurements and other morphological characters.

Inaccurate recording of Mansonella perstans in free-ranging primates outside its endemic area in Brazil?

In the study undertaken by Souza et al. [Primates 64(1):153-159, 2022; https://doi.org/10.1007/s10329-022-01038-5 ], published in the most recent volume of this journal, the blood samples of two Alouatta guariba clamitans (Primates, Atelidae) from two municipalities in the state of Rio Grande do Sul, southern Brazil were reported to be positive for Mansonella perstans. This is the first reported finding of M. perstans in A. guariba clamitans, as well as the first time that M. perstans has been recorded in Brazil outside the Amazon region. We would like to express our concern about this finding, specifically with respect to the geographical distribution of M. perstans in Brazil, as, up until this study, this filaria had only been found in the upper Rio Negro region in São Gabriel da Cachoeira, Amazonas, Brazil. Moreover, species identification was performed using partial sequences of three gene fragments, namely internal transcribed spacer 2, 12S, and 18S, yet neither the phylogenetic trees nor the BLAST alignments of these sequences provided supporting evidence that they belong to M. perstans.

Change in the faunal composition of mosquitoes (Diptera: Culicidae) along a heterogeneous landscape gradient in the Brazilian Amazon.

This study aimed to evaluate the influence of different anthropic landscape profiles on the diversity and distribution of mosquito species in a rural settlement of the Brazilian Amazon. Eight field collections were conducted at 18 sampling points interspersed throughout 2020-2021. Plastic containers, bamboo internodes, and tires were used as traps to capture immature mosquitoes in three distinct habitats: forest, forest edge, and peridomicile. A total of 15,547 individuals, distributed in 26 species of culicids, were collected. The most abundant species were Culex urichii (8,376 specimens), Culex (Melanoconion) (2,473 specimens), and Aedes albopictus (1,252 specimens). Forest habitat showed the highest abundance, and forest edge showed the highest species richness. Different types of environments influenced both the abundance and richness of mosquitoes. The species composition was also significantly different between the analyzed sites, mainly between forest and peridomicile environments. The change in species dominance could largely explain this change in mosquito community composition. Haemagogus janthinomys, an important sylvatic arbovirus vector, was found in peridomicile habitats and Ae. albopictus, a vector associated with human environments, was found in forest habitats, thus providing evidence of species spillover. Our results indicated that landscape changes affect mosquito communities, influencing their richness and abundance. These changes may have implications for future arboviral outbreaks in this rural settlement due to the possible establishment of sylvatic vector species in anthropic environments.

Tabanidae (Diptera) captured on horses in Amazon Forest fragments of the state of Rondônia, Brazil.

Tabanidae is one of the most diverse families of hematophagous dipterans. Tabanids, in general, are mechanical vectors of some pathogens. Given the vector importance and the lack of knowledge of the tabanid fauna in horses in Amazon Forest fragments of the state of Rondônia, this work aimed to determine the season that the different species of horse flies prefer to carryout hematophagy on horses and verify whether the horse fly community remains the same throughout the year. The sampling areas for tabanid captures were in the municipality of Monte Negro, Western Amazon, Brazil. Four new occurrences were recorded for the state of Rondônia: Stenotabanus albilinearis, Tabanus fuscofasciatus, T. macquarti and T. restrepoensis, which increases the number of species for the state to 109. The horse flies were most frequently collected on the hind leg (43.15%) and front leg (31.11%), followed by the belly (7.41%) and the ear (5.18%). In the other anatomical regions, the collection frequency was 13.15% of the remaining individuals.

Influence of meteorological and seasonal parameters on the activity of Culicoides paraensis (Diptera: Ceratopogonidae), an annoying anthropophilic biting midge and putative vector of Oropouche Virus in Rondônia, Brazilian Amazon.

Biting midges of the genus Culicoides are insects of proven medical and veterinary importance, because of their role in the transmission of viruses, bacteria, protozoa and nematodes. Culicoides paraensis has been considered the main vector of the Oropouche Virus (OROV) in the urban cycle of the disease in the neotropics. Due to the great abundance of Culicoides spp. in the State of Rondônia and its epidemiological history of OROV, we investigated the biting activity in humans, the abundance as a function of meteorological parameters and seasonality, and the detection of OROV. Entomological collections occurred in three municipalities from Brazilian State of Rondônia: Porto Velho, Ariquemes, and Ouro Preto do Oeste. GLMM's were used to determine if Culicoides spp. abundance was predicted by seasonal, diurnal, and meteorological factors. Total RNA was extracted from insects and viral RNA detection was performed using the S segment as the target region of OROV via RT-qPCR. In total, 7315 individuals were captured and identified as C. paraensis. In the dry season, 1488 individuals (24.5%) were recorded, 4591 (75.5%) in the rainy season, with peaks of biting activity between 4pm and 6pm. All variables showed a significative effect on the midge abundance. The rainy season, temperature between 30 °C and 32 °C and relative air humidity between 75% and 85% were the main predictive parameters for capturing the highest average number of insects. Our results confirm diurnal activity of C. paraensis and its greatest abundance in rainy periods. No sample was positive for the OROV, which could be explained by the virus absence in local human populations, C. paraensis as a minor vector species in the sampled localities, and probable low rate of infection of biting midges. Our findings on hourly and seasonal biting activities can provide support to intervention actions regarding vector control and surveillance of this species. This was the first study to collect and analyze biting midges in a region where human OROV cases had already been detected, but without previous information on entomovirological surveillance.

Dirofilaria immitis is endemic in rural areas of the Brazilian Amazonas state capital, Manaus.

The canine filarial parasite Dirofilaria immitis has not been reported in Brazil´s Amazonas state capital, Manaus, for over a century. Here, we report one imported and 27 autochthonous D. immitis infections from a microfilarial survey of 766 domestic dog blood samples collected between 2017 and 2021 in Manaus. An Overall prevalence estimate of 15.44% (23/149) was calculated from our two rural collection sites; a prevalence of 1.22% (4/328) was estimated at our periurban collection site, and an overall prevalence of 0.35% (1/289) was calculated from our two urban clinic collections. Our data suggest that in the urban areas of Manaus, where the parasites are very likely vectored by the same species of mosquito that historically vectored Wuchereria bancrofti (Culex quinquefasciatus), prevalence levels are very low and possibly maintained by an influx from rural areas where sylvatic reservoirs and/or more favorable vector transmission dynamics maintain high prevalences.

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.