Anopheles species complexes from Guyana and Venezuela malaria endemic areas using COI sequences

Anopheles species complexes from Guyana and Venezuela malaria endemic areas using COI sequences | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anopheles species complexes from Guyana and Venezuela malaria endemic areas using COI sequences Yasmin Rubio-Palis, Claudia Corredor-Medina, Audrey E Lenhart, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7584315/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND Effective interventions for controlling Anopheles malaria vectors depend on accurately identifying the species. Venezuela has the highest malaria prevalence in the region of the Americas; however, there is very limited knowledge about the vectors, especially regarding species complexes and their distribution. OBJECTIVES To conduct molecular taxonomy and phylogenetic analysis of the Albitarsis, Nuneztovari, and Oswaldoi complexes from malaria-endemic regions of Guyana and Venezuela using mtCOI sequences. METHODS Anopheles were collected in Guyana (Potaro Region) and Venezuela (five municipalities). Species identification was carried out by analyzing mitochondrial cytochrome C oxidase ( mtCOI ) gene data using MrBayes, TCS, Posterior Probabilities of Correct Identification (P ID), Rosenberg’s P (AB) values, and P (Randomly Distinct) as species delimitation approaches. FINDINGS Four distinct taxa were identified: An. albitarsis F, An. goeldii , An. oswaldoi A and An. oswaldoi B. Anopheles oswaldoi A and An. oswaldoi B occurred sympatrically in Boca de Nichare, Bolívar State, Venezuela. CONCLUSION Anopheles goeldii is reported for the first time from Guyana and Venezuela, and discuss its role as vector of malaria parasites in both countries and its relative recent colonization. Anopheles oswaldoi A is reported for the first time from Venezuela. Anopheles albitarsis F, an important malaria vector in Colombia and Venezuela, is confirmed as the only species of the Albitarsis complex widely distributed in Venezuela. Our results will be valuable for future research aimed at clarifying the status of these significant species complexes in the region. Entomology Anopheles albitarsis F Anopheles goeldii Anopheles oswaldoi complex mtCOI Figures Figure 1 Figure 2 Figure 3 Background The genus Anopheles Meigen, 1818 comprises approximately 470 species [ 1 ], of which 43 species have been reported in Venezuela [ 2 ]. Out of these 43 species, 12 have been incriminated in the transmission of malaria parasites [ 2 – 10 ]. According to the World Health Organization (WHO) [ 11 ], Venezuela reported 135,000 malaria cases in 2023, the second highest in the Americas, accounting for 24% of the region's total of 548,000 cases, following Brazil. Nevertheless, Venezuela has the highest rate of cases in the region at 477 per 100,000, while Brazil's rate stands at 75.3 per 100,000. More than 70% of cases in Venezuela are reported from Bolívar State. The confirmed vector species in Bolívar State include An. darlingi Root 1926 [ 5 , 7 , 9 , 10 ], An. albitarsis Lynch-Arribálzaga 1878 sensu lato, An. nuneztovari Gabaldon 1940 s.l. [ 5 , 7 , 10 ], and An. (Anopheles) neomaculipalpus Curry 1931 [ 5 , 7 ]. In addition, the potential vectors, An. albitarsis F and An. oswaldoi B have been found in the malaria foci in southern Venezuela [ 12 , 13 ]. Anopheles albitarsis F [ 14 ] is a molecularly delimited species, part of the An. albitarsis complex, which currently includes five formally described species: An. albitarsis , An. marajoara Galvão and Damasceno 1942, An. deaneorum Rosa-Freitas 1989, An. oryzalimnetes Wilkerson and Motoki 2009 and An. janconnae Wilkerson and Sallum 2009. Based on molecular markers, there are an additional 5 proposed species in the complex: An. albitarsis F [ 14 ], An. albitarsis G [ 15 , 16 ], An. albitarsis I [ 15 , 17 ], An. albitarsis H [ 15 ] y An. albitarsis J [ 18 ]. Anopheles albitarsis F has been reported from western, central, and southern Venezuela [ 12 , 13 , 15 ], east of the Eastern Andean Cordillera of Colombia, and on the island of Trinidad [ 13 , 15 ]. Zúñiga et al [ 13 ] suggested that previous reports on the distribution, bionomics, and vector incrimination of An. albitarsis s.l. from northern South America corresponded to An. albitarsis F. This species is found sympatric with the mitochondrial lineage An. albitarsis I in Zulia State, Venezuela and the neighbouring Department of Norte de Santander in Colombia [ 15 ]. So far, this putative species has not been found positive for malaria parasites [ 13 ]. The species of the Anopheles nuneztovari complex have a wide distribution, encompassing eastern Panamá, Colombia, Venezuela, Guyana, Brazil, Ecuador, Perú and Bolivia [ 19 ]. Based on morphological, behavioral, cytogenetics and molecular markers ( White and CAD genes , ITS2-rDNA and DNA mtCOI ) [ 20 – 27 ], it has been suggested that the An. nuneztovari complex includes four formally described species: An. nuneztovari, An. goeldii Rozeboom and Gabaldon 1941, An. dunhami Causey 1945 and An. jamariensis Sant’Ana and Sallum 2022. Anopheles nuneztovari is distributed from western Venezuela, Colombia and eastern Panamá [ 4 , 28 , 29 ]. Anopheles goeldii has been confirmed from the States of Amazonas, Amapá and Pará in northern Brazil [ 25 , 26 ]. Anopheles dunhami is found in Colombia [ 23 ] and the Brazilian Amazon region [ 25 , 30 ]. Anopheles jamariensis was recently described from the Jamari river, municipality of Monte Negro, Rondônia State, western Amazonian region of Brazil [ 27 ]. Since these four species can be easily misidentified, it is necessary to understand their actual geographic distribution and role as vectors of malaria parasites, particularly in areas of sympatry in the Amazon basin. The Anopheles oswaldoi complex has a wide distribution extending from Costa Rica to the northern provinces of Argentina, east of the Andes, and the island of Trinidad [ 19 , 31 ]. Based on molecular markers, Marrelli et al. [ 32 ] suggested that An. oswaldoi was a complex of at least four species: An. oswaldoi (Peryassú, 1922) s.s ., An. konderi Galvão and Damasceno 1942 and two other species yet to be described. Nevertheless, other studies confirmed the genetic variability among members of the complex and taxonomic misidentification of the mosquitoes included in that study [ 33 – 35 ]. Building on genetic analyses, Ruíz-López et al. [ 36 ] utilized ITS2-rDNA and mtCOI molecular markers to study populations across Brazil, Colombia, Perú, and Trinidad and Tobago. By comparing these results with sequences archived in GenBank, their research clarified the composition of the Oswaldoi Complex, revealing three distinct species: An. oswaldoi s.s ., An . oswaldoi A, and An . oswaldoi B. Their findings indicate that An . oswaldoi s.s. and An . oswaldoi A are present in both Brazil and Colombia, whereas An . oswaldoi B is distributed in Colombia, Ecuador, and Trinidad and Tobago. Subsequently, Rubio-Palis et al. [ 12 ] confirmed the presence of An. oswaldoi B in Sucre municipality, Bolívar State, a malaria endemic area in southern Venezuela. However, the Oswaldoi Complex could be part of a larger complex, the Oswaldoi-Konderi Complex, including An. konderi s.l. [ 36 , 37 ]. To further elucidate the geographic ranges of these complexes, the present study analyzed the molecular taxonomy and phylogenetic inference for members of the Albitarsis, Nuneztovari, and Oswaldoi Complexes from malaria-endemic areas of Guyana and Venezuela using mtCOI sequences. METHODS Mosquito collections – Table 1 shows the geographical locations, dates, and methods of mosquito collection in Guyana and Venezuela. Mosquitoes were identified using the keys by Rubio-Palis [ 4 ] and González and Carrejo [ 38 ]. The collected mosquitoes were either kept dry over silica gel or pinned; only specimens collected in 1998 in Guárico were preserved in isopropanol (Table 1 ). Mosquitoes were transported to the CDC, Entomology Branch, Atlanta, USA, for molecular analysis where 35 specimens were sequenced. Table 1 Geographical locations, dates, methods of mosquito collections, and the number (n) sequenced. Country Region – State/Municipality Coordinates Date collected Collection Method N Guyana Potaro-Siparuni 05°04'16"N, 59°12'48"W Dec. 2016 Human landing 7 Apure / Múñoz 07°26'08"N, 69°19'10"W Dec. 2010 Canopy trap 4 Guárico / Francisco de Miranda 08°58'N, 67°25'W Jan. 1998 Human landing 2 Venezuela Bolívar / Sucre 07°03'43"N, 64°58'37"W Nov. 2014 UV light trap 4 Bolívar / Sucre 06°28'N, 64°45'W Aug. 2015 Mosq Magnet trap 16 Bolívar / Sifontes 07°14'50"N, 61°20'54"W Apr. 1999 Human landing 1 Bolívar / Gran Sabana 04°35'45"N, 61°06'40"W Nov. 2011 Human landing 1 TOTAL 35 Sequence generation - DNA was extracted using the commercially available DNeasy Blood & Tissue Kit (QIAgen®, Maryland, USA). Amplification of mitochondrial COI gene ( mtCOI ) was achieved using the primers UEA3 and UEA10, as described in Lunt et al. [ 39 ] Sequencing reactions were carried out in both directions using the BigDye Terminator Kit® (PE Applied BioSystems, Warrington, England) on an ABI 3730 automated sequencer (PE Applied BioSystems). Sequences were edited using Sequencher® v.5.4.6 (Genes Codes Corporation, Ann Arbor, MI) and automatically aligned in MUSCLE (Geneious 10.0.9). Sequence similarities were compared with those available in GenBank using Basic Local Alignment Search Tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and BoldSystems ( https://www.boldsystems.org/ ). Data analysis - Phylogenetic analysis was conducted on 30 unique haplotypes (n = 35) on 860 bp of the mtCOI . A Bayesian phylogenetic analysis was performed with MrBayes [ 40 ] using the Kimura 2-Parameter (K2P) distance model [ 41 ] in Geneious 10.0.9. The analysis ran for 3 million generations, with subsampling every 1000 generations and a burn-in of 300,000 generations. To assess putative species based on the posterior probabilities from the Bayesian analysis tree, species delimitation tools were applied [ 42 ]. To assess population-level genealogies, mtCOI sequences were analyzed using TCS v. 1.21 [ 43 ]. A connection limit of 95% was adopted to investigate whether An. albitarsis , An. nuneztovari , and the An. oswaldoi species complex formed a single “meta-population” indicated by a single network, or consisted of separate species, indicated by the formation of two or more independent networks. Ethical considerations Mosquito collections were conducted by the correspondence author and field technicians of the vectors control programs from Venezuela and Guyana. Results Initial morphological identifications showed the presence of four species: An . albitarsis s.l. , An . nuneztovari s.l ., An . nr . nuneztovari ( An. nunez -like), and An . oswaldoi . Upon comparing the mtCOI sequences with the GenBank and BoldSystems databases, we found similarity between 99 to 100% with An. albitarsis F (12 haplotypes), An . aff . nuneztovari A (4 haplotypes), An. goeldii (7 haplotypes), An . oswaldoi B (9 haplotypes), and An. oswaldoi A (= An. aff. oswaldoi A) (3 haplotypes) (Table 2 ). Table 2 Comparison of morphological identifications with mtCOI sequences matches from GenBank and BoldSystems. Species ID Morphology Species ID mtCOI sequences GenBank / BoldSystems access number % ID An. albitarsis complex (n = 12) An. albitarsis F JQ615028 100 An. nuneztovari s.l. (n = 4)* An. aff. nuneztovari A (n = 4) MF381680 99 An. nunez-like (n = 7) An. goeldii (n = 7) NC_037810 99 An. oswaldoi complex (n = 12) An. oswaldoi B (n = 9) KF809115 99 An. aff. oswaldoi A (n = 3) MZ014236 100 *These specimens also showed 98.7% similarity with An. goeldii (MF381656) Bayesian analysis revealed four clades (monophyletic groups), named based on morphological and mtCOI sequence matches (GenBank and BoldSystems), as follows: An . albitarsis F, An . oswaldoi B, An. oswaldoi A and An . goeldii (Fig. 1 ). Support values below 0.8 are not shown. Outgroup: Anopheles braziliensis (Chagas, 1970) (GenBank PX101434). GenBank accession numbers: An . albitarsis F (PX101437-42, PX101452-56), An . oswaldoi B (PX101460-68), An . oswaldoi A (PX101457-59) and An . goeldii (PX101435-36, PX10144351). The species delimitation analysis strongly supports the distinction of at least four taxa. Genetic distances between species and their closest relatives confirm these findings. The intra-species distance was between 0.8% ( An. oswaldoi A) to 1.4% ( An. goeldii ), and inter-species was between 7.9% to 5.4% (Table 3 ). Table 3 Results of species delimitation analysis using Geneious (Geneious 10.0.9) for species boundaries. Species Intra Distance Inter Dist. Closet Intra/Inter P ID (Strict) P ID (Liberal) Av (MRCA-tips) (1) An. albitarsis F 0.011 0.068 ( 2 ) 0.16 0.89 (0.80, 0.98) 0.96 (0.91, 1.0) 1.2E-6 (2) An. oswaldoi B 0.010 0.054 ( 3 ) 0.19 0.88 (0.79, 0.96) 0.96 (0.91, 1.0) 1.2E-6 (3) An. oswaldoi A 0.008 0.054 ( 2 ) 0.15 0.69 (0.52, 0.87) 0.92 (0.78, 1.0) 3.3E-8 (4) An. goeldii 0.014 0.079 ( 3 ) 0.18 0.86 (0.76, 0.97) 0.95 (0.89, 1.0) 2.3E-03 The closest inter-species distances show An. albitarsis F is nearest to An. oswaldoi B at 6.8%, while An. goeldii is most distant from An. aff. oswaldoi A at 7.9%. Posterior Probabilities of Correct Identification (P ID) under strict and liberal criteria were consistently high for most species, with An. albitarsis F having a strict P ID of 0.89 (liberal: 0.96) and An. oswaldoi B showing similar values (strict: 0.88, liberal: 0.96). Anopheles oswaldoi A had a lower strict P ID of 0.69 but reached 0.92 under the liberal threshold, while An. goeldii maintained strong support with strict and liberal P IDs of 0.86 and 0.95, respectively. The analysis also highlighted strong clade support for all species, with Rosenberg's P(AB) values indicating significant differentiation; An. albitarsis F = 1.2E-6, An. goeldii = 3.3E-8, An . oswaldoi B = 1.2E-6, and An. oswaldoi A = 6.1E-5. These results, coupled with P (Randomly Distinct) values and the intra/inter species distance ratios, further validate the delineation of these species. Population analysis of the mtCOI data set using TCS (43) clearly divided the data set into four independent networks (Fig. 2 ): An. albitarsis F, An. goeldii, An. oswaldoi B, and An. oswaldoi A. This analysis is consistent with the Bayesian analysis and aligns with species delimitation boundaries as shown in Fig. 1 , where clade colours represent the selected species. Discussion The subgenus Nyssorhynchus holds significant importance in public health across the Americas due to the presence of primary and secondary malaria vectors within this group [ 19 , 31 , 44 ]. With the advent of molecular analysis and the emergence of new studies in diverse geographic regions, a considerable number of species belonging to the Nyssorhynchus subgenus have been formally described or resurrected from synonymy [ 27 , 45 – 49 ]. Regardless of these efforts, some species are still provisionally designated with temporary names, such as Species A, B, I, F, G, H, and J, among others [ 14 , 15 , 16 , 18 , 36 , 50 ]. According to the Mosquito Taxonomic Inventory [ 51 ], the Nyssorhynchus subgenus comprises 45 formally described species and 14 provisionally designated extant species. In Venezuela, 13 species of the subgenus Nyssorhynchus have been reported and 3 extant species [ 2 ]. In contrast, no recent reports are available from Guyana; data from Laubach et al. [ 52 ] included five species of the subgenus Nyssorhynchus : An. aquasalis Curry, 1932, An . braziliensis , An . darlingi , An. oswaldoi and An . triannulatus collected in Mahdia, Potaro-Siparuni Region. It is worth noting that this region is a gold mining area corresponding to the inland-forest malaria ecoregion [ 53 ], similar to the Sifontes municipality in Venezuela. The report of An. aquasalis , a coastal mosquito [ 19 , 53 ], suggests that the species was misidentified. Furthermore, during our collections in Guyana (December 2016) in the same area, we identified An. darlingi, An. nuneztovari s.l. (= An. goeldii ), An. triannulatus s.l . and An. rangeli . Potentially, the An. aquasalis specimens reported positive for P. vivax by Laubach et al. [ 52 ] corresponded to An. goeldii . The present report updates the lists of species to include An . goeldii (Guyana and Venezuela) and An. oswaldoi A (Venezuela). Both species are sympatric with An. oswaldoi B in the municipality of Bolivar, Sucre, Venezuela (Fig. 3 ). The species identified in this research belong to three species complexes: the Albitarsis Complex ( An. albitarsis F), the Nuneztovari Complex ( An. goeldii ), and the Oswaldoi Complex ( An. oswaldoi B and An . oswaldoi A). Anopheles albitarsis F The present study contributes to broadening the distribution of An. albitarsis F in Venezuela by adding 12 new sequences and confirming the previous hypothesis by Zúñiga et al. [ 13 ] that An. albitarsis F was widely distributed in Venezuela as it had been found in Zulia, Cojedes, Portuguesa [ 15 ], Guárico [ 13 ], and Bolívar [ 12 , 13 ] States. The present study found it in Apure State, bordering Colombia, and added four more records for Bolívar State (Fig. 3 ). It is of particular interest that An. albitarsis F was confirmed from Santa Elena de Uairén, Gran Sabana municipality, Bolívar State, at an altitude of 950 m above sea level, and separated 18 km from the town of Pacaraima, State of Roraima, Brazil. It is important to mention that Ruíz-López et al. [ 15 ] reported that the only species of the complex present in Roraima was An. janconnae . We suggest that there might be an area between Santa Elena de Uairén, Venezuela, and Boa Vista, Brazil (the closest locality to Venezuela where An. janconnae was reported), where both species are sympatric. Wilkerson et al. [ 54 ] indicated that An. janconnae was present in Venezuela, although no supporting data was provided. Anopheles albitarsis F has not been formally described. Still, recent morphometric analysis of adult females from Venezuela and Brazil has shown that the ratio of the length of the prehumeral dark spot divided by the length of the humeral pale spot (PHD/HP) on the wing Costal vein is a valid diagnostic character to separate this species from the described species of the complex: An. albitarsis , An. janconnae , An. marajoara , An. deaneorum and An. oryzalimnetes [ 55 ]. Also, characters on the hind legs such as the length of dark spot on hind tarsomere 2 divided by the length of hind tarsomere 2 (DS-III 2 /Ta-III 2 ) can be used to separate these species. A complete review on the bionomics, ecology, and vector incrimination of An. albitarsis F in Venezuela was published by Zúñiga et al. [ 13 ]. Anopheles goeldii Anopheles goeldii was described by Rozeboom and Gabaldon [ 56 ] based on morphological characters in adult male genitalia, females, larvae, pupae and eggs from specimens collected near the Tapajós river, Pará State, Brazil. Later, Floch and Abonnenc [ 57 ] followed by Lane [ 58 ] synonymized An. goeldii with An. nuneztovari. Due to this controversy, Gabaldon [ 45 ] revised the morphological characters of fourth instar larvae and male genitalia that clearly could separate both species, and hence resurrecting An. goeldii from synonymy with An. nuneztovari . Calado et al. [ 46 ] confirmed An. goeldii as a valid species based on DNA sequences and morphological analyses. Subsequently, Sant’Ana et al. [ 26 ] redescribed the species based on eggs, fourth-instar larvae, pupae, male and female morphological characters, and DNA sequences from the mtCOI genome. This species distribution was restricted to northern Brazil and Suriname [ 26 , 46 , 59 ]. Eleven Anopheles specimens from our collections were identified as An. nunez -like (Table II) based on the diagnostic characters of the ratio of the length of humeral pale spot divided by the length of prehumeral dark spot (HP/PHD) (0.7–4.5) and the ratio of the length of the basal dark spot of the hind tarsomere 2 (Ta-III 2 ) divided by the length of the hind tarsomere II (0.24–0.35) which can easily separate An. nuneztovari from other species of the Oswaldoi Subgroup [ 4 , 51 ]; although we did not measure the characters, it was noticed that the range of the ratio HP/PHD was smaller, and in general, light wing scales were cream, never bright yellow as An. nuneztovari s.s. from western Venezuela. However, upon comparing these sequences with those in GenBank, we found 99% homology with An. goeldii (accession numbers: MF381655–57 by Foster et al. [ 60 ] for seven specimens from Guyana, and 89–100% homology with An. nuneztovari A (accession number: MF381680 by Foster et al.) [ 60 ] for four specimens from Bolívar State, supporting the morphological ambiguities observed when using the morphological keys for adult females of González and Carrejo [ 38 ] and Sallum et al. [ 61 ] to distinguish An. nuneztovari s.l. in Venezuela. It is important to point out that An. nuneztovari was reported from Bolívar State (Sifontes Municipality) for the first time in 2003 [ 62 ], even though regular and intensive sampling efforts occurred since 1992, suggesting a recent colonization. The species was confirmed based on key characters in adult females and male genitalia. Nevertheless, recent revision of the male genitalia showed that the characters are consistent with the description for An. goeldii , making necessary a thorough revision of the specimens preserved and new collections to include morphological and molecular analysis. Anopheles nuneztovari s.s. can be distinguished from other species in the Complex by the well-developed subapical leaflets of the aedeagus [ 26 , 63 – 65 ]. It is suggested that, so far, An. goeldii is the only species of the complex present in Bolívar State; although it is possible that other species might occur sympatrically as it has been reported in Pará State, Brazil, where An. nuneztovari s.s. , An. nuneztovari A and An. goeldii occurred sympatrically [ 65 ]. Studies on biting behavior and larval habitats of An. nuneztovari s.l . (= An. goeldii ) in Sucre municipality showed that this species has a different biting behavior to that reported for An. nuneztovari s.s . from western Venezuela [ 9 , 66 ]. Anopheles goeldii is active throughout the night with a biting peak at sunset and a second, minor peak, at sun rise (9), contrasting with the unimodal behavior for An. nuneztovari s.s . with a biting peak between 22:00 and 01:00 hrs [ 66 ]. There are no longitudinal studies data on biting behavior of An. nuneztovari s.l from Sifontes Municipality. In Sucre Municipality, An. nuneztovari s.l. (= An. goeldii ) larvae were found mainly in streams and lagoons [ 67 ] together with An. darlingi , An. triannulatus s.l ., An. oswaldoi s.l. and Chagasia bonnae (Root, 1927). In Sifontes Municipality, An. nuneztovari s.s . and/or An. goeldii were collected mainly in lagoons and rivers [ 68 ] together with An. triannulatus s.l ., An. albitarsis F, An. oswaldoi s.l ., An . darlingi and Ch. bonnae , strongly associated with rainfall (R = 0.71) and river level (R = 0.79), with an abundance peak in July-August immediately after the peak of rains [ 69 ]. To determine the actual distribution of these species in Bolívar State and characterize their bionomics, it is necessary to conduct taxonomic studies (morphological and molecular characterization), particularly in Sifontes Municipality where An. nuneztovari s.l. is a confirmed vector of malaria parasites [ 10 ]. Ruiz et al. [ 23 ] used DNA barcode sequences to propose a threshold of 2.5% genetic divergence for differentiating cryptic species within the Nuneztovari Complex ( An. nuneztovari vs. An. dunhami ). Later, McKeon et al. [ 70 ] reduced the threshold to 2% for the Albitarsis Complex, with Ruiz et al. [ 15 , 36 ] reporting similar thresholds for the Albitarsis and Oswaldoi Complexes, respectively. Our results showed an intraspecific genetic divergence of 1.4% for An. goeldii (Table III), confirming that, although the analyzed sequences exhibit moderate variation, they fall below the threshold commonly used for species delimitation within species complexes of the Nyssorhynchus subgenus. Anopheles oswaldoi complex The two formally described species belonging to Anopheles oswaldoi complex, An . oswaldoi s.s . and An . konderi s.s. , cannot be distinguished by the morphology of larvae or adult females, as all morphological characteristics are shared between them [ 71 ]. Anopheles konderi s.s . and An. oswaldoi s.s. can only be differentiated based on the morphology of male genitalia [ 71 ]. DNA sequencing is the only reliable tool available for distinguishing the five members of Oswaldoi Complex ( An . oswaldoi s.s., An . oswaldoi A, An . oswaldoi B, An . konderi s.s. and An . sp. nr. konderi ) [ 37 ]. The genetic variation we found among members of this group ranges from an intraspecific divergence of 1.0% ( An. oswaldoi B) to 0.8% ( An. oswaldoi A) and interspecific divergence of 5.4% for both species (Table III). These results are similar to those found by Ruiz et al. [ 36 ], who reported an intraspecific divergence of 1.2% and an interspecific divergence of 5.2% for all members of the Oswaldoi Complex. Anopheles oswaldoi A Based on COI sequences (DNA barcoding), Ruiz et al.[(36] identified An. oswaldoi A as a species belonging to the An. oswaldoi complex from Brazil (Acre, Amazonas, Mato Grosso, Pará, and Rondônia) and Colombia (Amazonas). Subsequently, Silva-do-Nascimento et al. [ 72 ] discovered that some specimens from Roraima, Brazil, matched the An. oswaldoi A described by Ruiz et al. [ 36 ]. However, their mTPT (Multi-rate Poisson Tree Processes) analysis revealed that these specimens formed two distinct clusters, separated by a K2P genetic distance of 2.16%, suggesting the presence of a potential new species within An . oswaldoi A. These sequences were deposited in GenBank as An . aff . oswaldoi A (accession number MZ014236). However, although the sequences of this manuscript (accession number PX101457-59) matched 100% with MZ014236, we will retain the taxon as An . oswaldoi A rather than An . aff . oswaldoi A until the new hypothesis has been resolved. The specimens collected in this study are from Boca de Nichare, Bolívar State (Venezuela) (Fig. 3 ), approximately 420 km from Parafuri, Roraima State (Brazil) where Silva-do-Nascimento et al. [ 72 ] collected mosquitoes of the Oswaldoi Complex (Fig. 3 ). Anopheles oswaldoi A has been implicated in malaria transmission in Acre, Brazil [ 73 ]. However, its role in malaria transmission in other regions where the species is present remains unclear. Sequences of three specimens collected in Boca de Nichare, Bolivar State, were compatible with An. oswaldoi A. Initially, these mosquitoes were identified as An. oswaldoi s.l. due to the distinctive key character in adult females of hind tarsomere 2 (Ta-III 2 ) with a basal dark spot 0.11–0.22 length of tarsomere [ 4 ] which can easily separate An. oswaldoi from other species of the Albimanus Section present in the study area. This character is also present in An. ininii Senevet & Abonnenc, 1939, but we did not observe the white scales on the Costa vein nor examine and measure diagnostic characters in the fore tarsomeres, which can separate An. oswaldoi from An. ininii [ 31 , 71 ]. Anopheles oswaldoi A found here exhibited 98% homology with An . ininii , based on comparisons of COI sequences in the GenBank database (accession number MT757855). These sequences of An. ininii were deposited as direct submission and lacked additional details, such as information about collection location and morphological confirmation, hence they were not included in our analysis. Anopheles oswaldoi B This species was reported in Venezuela for the first time from Jabillal, Sucre Municipality [ 12 ], approximately 120 km from Boca de Nichare. So far, An. oswaldoi B and/or An. oswaldoi s.l . have not been found positive for plasmodia in Bolivar State, probably due to the lack of mosquitoes of this species tested for sporozoites and/or the small number of mosquitoes assayed [ 9 ]. Anopheles oswaldoi s.l. was found positive for P. vivax CS protein in Barinas State (western Venezuela) [ 3 ]; nevertheless, in this area the biting pattern observed in a 14-month longitudinal study [ 66 ] contrasted with that reported for the An. oswaldoi s.l . from Sucre Municipality, Bolívar State [ 74 ]. Anopheles oswaldoi s.l. from Barinas showed a biting peak outdoors at sunset and a second peak around midnight indoors [ 66 ] whereas in Bolívar State the biting activity increased steadily through the night, reaching a plateau around 02:00 hours that extended until sunrise [ 74 ]. It has been suggested that probably in Barinas there are two different sympatric species [ 66 ], and the Oswaldoi Complex includes other species in western Venezuela yet to be identified. In the present study, An. oswaldoi A and An. oswaldoi B occurred sympatrically in Boca de Nichare (Fig. 3 ), hence the biting pattern reported for An. oswaldoi s.l. might be inaccurate, including both species. Further studies in Venezuela should consider collections of immatures and rearing of individuals to obtain all stages for morphometric and molecular identification to establish species distribution, bionomics and medical importance of members of the Oswaldoi Complex. CONCLUSIONS Anopheles goeldii is reported for the first time from Guyana and Venezuela, and discuss its role as vector of malaria parasites in both countries and its relative recent colonization. Anopheles oswaldoi A is reported for the first time from Venezuela. Anopheles albitarsis F, an important malaria vector in Colombia and Venezuela, is confirmed as the only species of the Albitarsis complex widely distributed in Venezuela. The complex taxonomic situation of the subgenus Nyssorhynchus requires further research, and future studies should incorporate multidisciplinary approaches that include morphological and molecular species identification, ecology, and vector incrimination. Our results will be valuable for future research aimed at clarifying the status of these significant species complexes in the region. Declarations Acknowledgements Special thanks are due to Horace Cox, Jean Alexandre, Brian Puno, and the entomology technicians that participated in field collections in Guyana; to Jorge Moreno for valuable discussion on An. nuneztovari from Sifontes and providing specimens for this study; to Jesús Berti for providing specimens from Bolívar State. To Simón Caura, Hernán Guzmán, Yarys Estrada, Víctor Sánchez, and Jesús González, who participated in mosquito collections. To the people of Jabillal and Boca de Nichare for logistic support and friendship. We thank Ananias Escalante for helpful comments on the methodology and manuscript. Author contributions YRP and AL conceived and designed the study; YRP conducted the fieldwork; CCM and YRP carried out the lab work; FRL analyzed the data; YRP and FRL wrote the manuscript. All authors read and approved of the final manuscript. Funding YRP, CCM, AJL and FRL received no specific funding for this work. Data availability All data generated and analysed during this study are included in this published article. The datasets generated during the current study are available in the GenBank repository, under accession numbers [PX101434 –PX101468]. Competing interests The authors declare that there are no competing interests associated with the manuscript. Disclaimer The findings and views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the Centers for Disease Control and Prevention. Author details 1 Instituto de Investigaciones Biomédicas “Dr. Francisco Triana Alonso”, Universidad de Carabobo, Maracay, Venezuela. 2 US Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria, Entomology Branch, Atlanta, GA, United States of America. 3 Programa para el Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, Medellín, Colombia. References Knols BGJ. Review of Mosquitoes of the World by Richard C. Wilkerson, Yvonne-Marie Linton, and Daniel Strickman. Parasit Vectors. 2021; 14 : 341. https://doi.org/10.1186/s13071-021-04848-6. Rubio-Palis Y. Mosquitos Anophelinae de Venezuela: complejos de especies e implicaciones epidemiológicas. Salus 2022; 26(1):25-32. http://servicio.bc.uc.edu.ve/fcs/vol26n1/art05.pdf. Rubio-Palis Y, Wirtz RA, Curtis CF. Malaria entomological inoculation rates in western Venezuela. Acta Trop.1992; 52(2–3): 167–74. https://doi.org/10.1016/0001-706x(92)90033-t. Rubio-Palis Y. Anopheles ( Nyssorhynchus ) de Venezuela: taxonomía, bionomía, ecología e importancia médica . Maracay, Venezuela: Escuela de Malariología y Saneamiento Ambiental “Dr. Arnoldo Gabaldon”; 2000. 120 pp. Available from: www.iaes.edu.ve/index.php/centro-de-descargas/viewcategory/3-libros-publicaciones. Moreno JE, Rubio-Palis Y, Páez E, Pérez E, Sánchez V, Vaccari E. Anopheles ( Anopheles ) neomaculipalpus : a new malaria vector in the Amazon basin? Med Vet Entomol. 2005; 19(3): 329–32. https://doi.org/10.1111/j.13652915.2005.00572.x. Magris M, Rubio-Palis Y, Menare C, Villegas L. Vector bionomics and malaria transmission in the Upper Orinoco River, southern Venezuela. Mem Inst Oswaldo Cruz. 2007; 102: 303-311. Moreno JE, Rubio-Palis Y, Páez E, Pérez E, Sánchez V, Vaccari E. Malaria entomological inoculation rates in gold mining areas of Southern Venezuela. Mem Inst Oswaldo Cruz. 2009; 104(5): 764–8. https://doi.org/10.1590/s0074-02762009000500017. Rubio-Palis Y. Prevalencia de Plasmodium spp. en anofelinos de Venezuela. Talleres. 2009; 12: 79-83. Rubio-Palis Y, Bevilacqua M, Medina DA, Moreno JE, Cárdenas L, Sánchez V, et al. Malaria entomological risk factors in relation to land cover in the Lower Caura River Basin, Venezuela. Mem Inst Oswaldo Cruz. 2013; 108: 220-228. Abou Orm S, Moreno JE, Carrozza M, Acevedo P, Herrera F. Plasmodium spp. infection rates for some Anopheles spp. from Sifontes Municipality, Bolívar State, Venezuela. Bol Mal Sal Amb. 2017; 57(1): 17-25. World Malaria Report 2024. World malaria report 2024: addressing inequity in the global malaria response. Geneva: World Health Organization; 2024. Available from: https://www.who.int/publications/i/item/9789240104440. Rubio-Palis Y, Ruíz-López F, Guzmán H, Sánchez V, Moreno JE, Estrada Y, et al. Primer registro de Anopheles ( Nyssorhynchus ) oswaldoi B y Anopheles ( Nys .) albitarsis F en la cuenca del río Caura, Estado Bolívar, Venezuela. Bol Mal Sal Amb. 2013; 53: 68-72. Zúñiga MA, Rubio-Palis Y, Brochero H. Updating the bionomy and geographical distribution of Anopheles ( Nyssorhynchus ) albitarsis F: A vector of malaria parasites in northern South America. PLoS ONE. 2021; 16(6): e0253230. https://doi.org/10.1371/journal.pone.0253230. Brochero HL, Li C, Wilkerson RC. A newly recognized species in the Anopheles ( Nyssorhynchus ) albitarsis complex (Diptera: Culicidae) from Puerto Carreño, Colombia. Am J Trop Med Hyg. 2007; 76(6): 1113–1117. Ruíz-López F, Wilkerson RC, Conn JE, McKeon SN, Levin DM, Quiñones ML, et al. DNA barcoding reveals both known and novel taxa in the Albitarsis Group ( Anopheles : Nyssorhynchus ) of Neotropical malaria vectors. Parasit Vectors. 2012; 5: 44. https://doi.org/10.1186/1756-3305-5-44. Krzywinsky J, Li C, Morris M, Conn JE, Lima JB, Póvoa MM, et al. Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex. Mol Phylogenet Evol. 2011; 58(3): 469–77. https://doi.org/10.1016/j.ympev.2011.01.003. Gutiérrez LA, Orrego LM, Gómez GF, López A, Luckhart S, Conn JE, et al. A new mtDNA COI gene lineage closely related to Anopheles janconnae of the Albitarsis Complex in the Caribbean region of Colombia. Mem Inst Oswaldo Cruz. 2010; 105(8): 1019–25. https://doi.org/10.1590/s0074. Motoki MT, Linton YM, Conn JE, Ruíz-López F, Wilkerson RC. Phylogenetic Network of Mitochondrial COI Gene Sequences Distinguishes 10 Taxa Within the Neotropical Albitarsis Group (Diptera: Culicidae), Confirming the Separate Species Status of Anopheles albitarsis H (Diptera: Culicidae) and Revealing a Novel Lineage, Anopheles albitarsis J. J Med Entomol. 2020. https://doi.org/10.1093/jme/tjaa211. Sinka ME, Rubio-Palis Y, Manguin S, Patil AP, Temperley WH, Gething PW, et al. The dominant Anopheles vectors of human malaria in the Americas: occurrence data, distribution maps and bionomic précis. Parasit Vectors. 2010; 3: 72. https://doi.org/10.1186/1756-3305-3-72. Kitzmiller J, Kreutzer R, Tallaferro E. Chromosomal differences in populations of Anopheles nuneztovari . Bull World Health Org. 1973; 48: 435-442. Conn JE, Mitchell SE, Cockburn AF. Mitochondrial DNA analysis of the neotropical malaria vector Anopheles nuneztovari . Genome. 1998; 41:313–327. Trindade DB, Scarpassa VM. Genetic differentiation and diagnostic loci among Anopheles ( Nyssorhynchus ) rangeli , An . ( Nys. ) nuneztovari and An. ( Nys .) dunhami (Diptera: Culicidae) from the Brazilian Amazon. J Med Entomol. 2002; 39: 613–20. https://doi.org/10.1603/0022-2585-39.4.613. Ruíz F, Linton Y-M, Ponsonby DJ, Conn JE, Herrera M, Quiñones ML, et al. Molecular comparison of topotypic specimens confirms Anopheles ( Nyssorhynchus ) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon. Mem Inst Oswaldo Cruz 2010; 105: 899–903. https://doi.org/10.1590/s0074-02762010000700010. Scarpassa VM, Geurgas S, Azeredo-Espin AML, Tadei WP. Genetic divergence in mitochondrial DNA of Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia. Genet Mol Biol. 2000; 23:71–78. Scarpassa VM, Cunha-Machado AS, Saraiva JF. Evidence of new species for malaria vector Anopheles nuneztovari sensu lato in the Brazilian Amazon region. Malar J. 2016; 15: 205. https://doi.org/10.1186/s12936-016-1217-6. Sant’Ana DC, Bergo ES, Sallum MAM. Anopheles goeldii Rozeboom and Gabaldon (Diptera: Culicidae): a species of the Nuneztovari Complex of Anopheles Meigen. Rev Brasil Entomol. 2015; 59: 68–76. Sant’Ana DC, Sallum MAM. A new species of the Nuneztovari Complex of Nyssorhynchus (Diptera: Culicidae) from western Brazilian Amazon. Zootaxa. 2022; 5134(2): 275-285. https://doi.org/10.11646/zootaxa.5134.2.6. Sierra DM, Vélez IV, Linton YM. Malaria vector Anopheles ( Nyssorhynchus ) nuneztovari comprises one genetic species in Colombia based on homogeneity of nuclear ITS2 rDNA. J Med Entomol. 2004; 41(3): 302-307. Jaramillo LM, Gutiérrez LA, Luckhart S, Conn JE, Correa M. Molecular evidence for a single taxon, Anopheles nuneztovari s.l . from two endemic malaria regions in Colombia. Mem Inst Oswaldo Cruz. 2011; 106(8): 1017-1023. Peyton EL. Anopheles ( Nyssorhynchus ) dunhami , resurrected from synonymy with Anopheles nuneztovari and validated as a senior synonym of Anopheles trinkae (Diptera: Culicidae). Mosq Syst. 1993; 25(3): 151-156. Faran ME. Mosquito studies (Diptera: Culicidae). XXXIV. A revision of the Albimanus section of the subgenus Nyssorhynchus of Anopheles . Contrib Amer Entomol Inst. 1980; 15(7): 1–215. Marrelli MT, Malafronte RS, Flores-Mendoza C, Lourenco-de-Oliveira R, Kloetzel JK, Marinotti O. Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae). J Med Entomol . 1999; 36: 679-684. Ruíz F, Quiñones ML, Erazo HF, Calle DA, Alzate JF, Linton Y-M. Molecular differentiation of Anopheles ( Nyssorhynchus ) benarrochi and An. ( N .) oswaldoi from Southern Colombia. Mem Inst Oswaldo Cruz. 2005; 100(2): 155-160. Motoki MT, Linton Y-M, Ruiz F, Flores-Mendoza C, Sallum MAM. Redescription of Anopheles oswaldoi (Peryassú, 1922) (Diptera: Culicidae), with formal lectotype designation. Zootaxa. 2007; 1588: 31–51. Sallum MAM, Marrelli MT, Nagaki SS, Laporta GZ, Dos Santos CLS. Insight into Anopheles ( Nyssorhynchus ) (Diptera: Culicidae) species from Brazil. J Med Entomol. 2008; 45: 970–81. https://doi.org/10.1603/0022-2585. Ruíz-López F, Wilkerson RC, Ponsonby DJ, Herrera M, Sallum MAM, Vélez ID, et al. Systematics of the Oswaldoi Complex ( Anopheles , Nyssorhynchus ) in South America. Parasit Vectors. 2013; 6: 324. http://doi.org/10.1186/1756-3305-6-324. Saraiva JF, Souto RNP, Scarpassa VM. Molecular taxonomy and evolutionary relationships in the Oswaldoi-Konderi Complex (Anophelinae: Anopheles : Nyssorhynchus ) from the Brazilian Amazon region. PLoS ONE. 2018; 13(3): e0193591. https://doi.org/10.1371/journal.pone.0193591. González Obando R, Carrejo Gironza NS. Introducción al estudio taxonómico de Anopheles de Colombia: Claves y notas de distribución. 2nd Edit. Cali, Colombia: Universidad del Valle; 2009. 260p. https://doi.org/10.25100/peu.309. Lunt DH, Zhang DX, Szymura JM, Hewitt GM. The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol Biol . 1996; 5:153–165. Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001; 17(8): 754-755. https://doi.org/10/1093/bioinformatics/17.8.754. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111–120. Masters B C, Fan V, Ross H A. Species Delimitation - a Geneious plugin for the exploration of species boundaries. Mol Ecol Resour. 2011; 11: 154-157. Clement M, Posada D, Crandall KA. TCS: a computer program to estimate gene genealogies. Mol Ecol. 2000; 9(10): 1657–1659. Montoya-Lerma J, Solarte YA, Giraldo-Calderón GI, Quiñones ML, Ruiz-López F, Wilkerson RC, et al. Malaria vector species in Colombia: a review. Mem Inst Oswaldo Cruz. 2011; 106 (Suppl 1): 223-238. https://doi.org/10.1590/s0074-02762011000900028. Gabaldon, A. Anopheles nuneztovari : importante vector y agente de malaria refractaria en Venezuela. Bol Div Malariol San Amb. 1981; 31: 28-38. Calado DC, Foster PG, Bergo ES, Santos CL, Galardo AK, Sallum MA. Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon. Mem Inst Oswaldo Cruz. 2008; 103(8):791-799. https://doi.org/10.1590/s0074-02762008000800009. Motoki MT, Wilkerson RC, Sallum MAM. The Anopheles albitarsis complex with the recognition of Anopheles oryzalimnetes Wilkerson and Motoki, n. sp. and Anopheles janconnae Wilkerson and Sallum, n. sp. (Diptera: Culicidae). Mem Inst Oswaldo Cruz. 2009; 104(6): 823-850. Sant'Ana DC, Sallum MAM. Revision of the Strodei Subgroup of Nyssorhynchus (Diptera: Culicidae), with descriptions of 2 new species. J Med Entomol. 2024; 61(1):87-109. https://doi.org/10.1093/jme/tjad149. Sallum MA, Foster PG, Dos Santos CL, Flores DC, Motoki MT, Bergo ES. Resurrection of two species from synonymy of Anopheles ( Nyssorhynchus ) strodei Root, and characterization of a distinct morphological form from the Strodei Complex (Diptera: Culicidae). J Med Entomol. 2010; 47(4):504-526. https://doi.org/10.1603/me09229. Moreno M, Bickersmith S, Harlow W, Hildebrandt J, McKeon SN, Silva-do-Nascimento TF, et al. Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns. Parasit Vectors. 2013; 6: 47. https://doi.org/10.1186/1756-3305-6-47. Harbach RE. Mosquito Taxonomic Inventory. [ Nyssorhynchus ]. 2013. [Updated October 2024; cited 2024 Dec 3]. Available from: https://mosquito-taxonomic-inventory.myspecies.info/. Laubach HE, Validum L, Bonilla JA, Agar A, Cummings R, Mitchell C, et al. Identification of Anopheles aquasalis as a possible vector of malaria in Guyana, South America. West Indian Med J. 2001; 50(4): 319-21. Rubio-Palis Y, Zimmerman R H. Ecoregional classification of malaria vectors in the Neotropics. J Med Entomol. 1997; 34: 499-510. Wilkerson RC, Linton Y-M, Strickman D. Mosquitoes of the World. Vol. 2. Baltimore: Johns Hopkins University Press; 2021. 1308 pp. Rubio-Palis Y, Moreno JE, Moreno A, Silva J, Pérez B, Guzmán H. Análisis morfométrico soporta la presencia de Anopheles (Nyssorynchus) albitarsis F (Diptera: Culicidae) en Venezuela, importante vector de los parásitos maláricos. Entomotropica. 2022; 37: 1-17. Available from: https://sventomologia.org/wpcontent/uploads/2022/07/2022_37_1-17.pdf. Rozeboom LE, Gabaldon A. A summary of the “ tarsimaculatus ” complex of Anopheles (Diptera: Culicidae). Amer J Hyg. 1941; 33(C), 88–100. https://doi.org/10.1093/oxfordjournals.aje.a118713. Floch H, Abonnenc E. Sur A. nuñez-tovari et A. pessoai en Guyane Française. Table d’identification des Nyssorhynchus guyanais . Inst Pasteur Guyane Territ Inini Publ. 1946; 126:1-5. Lane J. Neotropical Culicidae. Vol. 1. São Paulo: Editora da Universidade de São Paulo; 1953. 548 pp. Fritz GN, Conn J, Cockburn AF, Seawright JA. Sequence analysis of the ribosomal DNA internal transcribed spacer 2 from populations of Anopheles nuneztovari (Diptera: Culicidae). Mol Biol Evol. 1994; 11: 406-416. Foster PG, Porangaba de Oliveira TM, Bergo ES, Conn JE, Sant’Ana DC, Nagaki SS, et al. Phylogeny of Anophelinae using mitochondrial protein coding genes. R Soc Open Sci. 2017; 4: 170758. http://dx.doi.org/10.1098/rsos.170758. Sallum MAM, González Obando R, Carrejo N, Wilkerson RC. Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). IV. Adult females. Parasit Vectors. 2020; 13: 584. https://doi.org/10.1186/s13071-020-04301-0. Moreno J, Rubio-Palis Y, Sánchez V, Mariany D. Primer registro de Anopheles (Nyssorhynchus) nuneztovari Gabaldon 1940 (Diptera: Culicidae) en el estado Bolívar, Venezuela y sus implicaciones eco-epidemiológicas. Entomotropica. 2004; 19(1): 55-58. Gabaldon A. Estudios sobre anofelinos. Serie I. 1. Descripción de Anopheles ( Nyssorhynchus ) núñez-tovari n.sp y consideraciones sobre una sub-división del grupo Nyssorhynchus (Diptera, Culicidae). Publ Div Malariol. 1940; 5: 3-7. Savage HM. Identification and location of the holotype and paratypes of Anopheles ( Nyssorhynchus ) nuneztovari Gabaldon (Diptera: Culicidae). Mosq Syst. 1986; 18 : 279-283. Dos Santos MMM, Sucupira IMC, Dos Santos TV, Dos Santos ACE, Lacerda RNDL, Póvoa MM, et al. Morphological identification of species of the Nuneztovari Complex of Anopheles (Diptera: Culicidae) from an area affected by a Brazilian hydroelectric plant. Zootaxa. 2019; 4565: 235-244. Rubio-Palis Y, Curtis CF. Biting and resting behavior of anophelines in western Venezuela and implications for control of malaria transmission. Med Vet Entomol. 1992; 6: 325-334. Moreno JE, Rubio-Palis Y, Bevilacqua M, Sánchez V, Guzmán H. Caracterización de hábitats larvales de anofelinos en la cuenca baja del Río Caura, estado Bolívar, Venezuela. Bol Malariol Sal Amb. 2018; 58: 32-45. Available from: http://iaes.edu.ve/iaespro/ojs/index.php/bmsa/article/view/57/29. Moreno JE, Rubio-Palis Y, Sánchez V, Martínez A. Caracterización de hábitats larvales de anofelinos en el municipio Sifontes del estado Bolívar, Venezuela. Bol Malariol Sal Amb. 2015; 55(2): 117-131. Available from: http://iaes.edu.ve/iaespro/ojs/index.php/bmsa/article/view/119/101. Moreno JE,Rubio-Palis Y , Sánchez V, Martínez A. Fluctuación poblacional y hábitat larval de anofelinos en el municipio Sifontes, estado Bolívar, Venezuela. Bol Malariol Sal Amb. 2015; 55(1): 52-68. Available from: http://iaes.edu.ve/iaespro/ojs/index.php/bmsa/article/view/107/54. McKeon SN, Lehr MA, Wilkerson RC, Ruíz JF, Sallum MA, Lima JB, et al. Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil. Malar J. 2010; 9: 271. https://doi.org/10.1186/1475-2875-9-271. Sallum MAM, González Obando R, Carrejo N, Wilkerson RC. Identification key to the Anopheles mosquitoes of South America (Diptera: Culicidae). III. Male genitalia. Parasit Vectors. 2020; 13: 542. https://doi.org/10.1186/s13071-020-04300-1. Silva-do-Nascimento TF, Sánchez-Ribas J, Oliveira TMP, Bourke BP, Oliveira-Ferreira J, Rosa-Freitas MG, et al. Molecular analysis reveals a high diversity of Anopheline mosquitoes in Yanomami lands and the Pantanal region of Brazil. Genes. 2021; 12: 1995. https://doi.org/10.3390/genes12121995. Branquinho MS, Araujo D, Natal MT, Marrelli RM, Rocha FAL, Taveira JK, et al. Anopheles oswaldoi a potential malaria vector in Acre, Brazil. Trans R Soc Trop Med Hyg. 1996; 90: 233. Rubio-Palis Y, Bravo L, Guzmán H, Caura S, Song C, Wang S, et al. Response to chemical attractants and host seeking behavior of Anopheles Meigen spp. (Diptera: Culicidae) in a malaria endemic area of Bolívar State, Venezuela. Bol Malariol Sal Amb. 2021; 61(2): 267-274. https://doi.org/10.52808/bmsa.7e5.612.016. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7584315","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513198380,"identity":"e96ffd12-cc12-4f45-ac0f-701688d1115c","order_by":0,"name":"Yasmin Rubio-Palis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBAC+xn5hz98/CORIN9/+OBnHoO6BAb2w8ck8GkxuJGTcHBmm00Cm0RasvScCokEBp60dAsCWgwO87alAbXwmDH/OQPUIqFj/oOQLUAth0FavjHntoG08JjdIOCXA4d5/wC18J/dRpwWOwmQLQ1ALQy526yJ0mIsAfJLw3+glpxn0rz/IFrwhpjhDLAWkC05bNK8YFt08GsxOHMGGMggLRJpxmAtbDxpafi1HO8HRmXD4XpgVD78zNtWV89PKCoxARtpykfBKBgFo2AUYAMADg5VV/LF3o4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0056-2540","institution":"Instituto de Investigaciones Biomedicas \"Dr Francisco Triana Alonso\", Universidad de Carabobo","correspondingAuthor":true,"prefix":"","firstName":"Yasmin","middleName":"","lastName":"Rubio-Palis","suffix":""},{"id":513198381,"identity":"0354cffe-06e0-4219-9acb-8a21a9d74161","order_by":1,"name":"Claudia Corredor-Medina","email":"","orcid":"https://orcid.org/0000-0001-8619-1618","institution":"US Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Corredor-Medina","suffix":""},{"id":513198382,"identity":"655f5d06-d551-4d73-bd2b-8c3113df5c7e","order_by":2,"name":"Audrey E Lenhart","email":"","orcid":"https://orcid.org/0000-0002-9156-4228","institution":"US Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Audrey","middleName":"E","lastName":"Lenhart","suffix":""},{"id":513198383,"identity":"55940908-c40c-4bc7-a143-c0ec347c28af","order_by":3,"name":"Freddy Ruíz-López","email":"","orcid":"https://orcid.org/0000-0001-8563-8799","institution":"Universidad de Antioquia","correspondingAuthor":false,"prefix":"","firstName":"Freddy","middleName":"","lastName":"Ruíz-López","suffix":""}],"badges":[],"createdAt":"2025-09-10 15:20:06","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7584315/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7584315/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91495791,"identity":"1a410936-2f28-4fc3-b5a4-aec487c097da","added_by":"auto","created_at":"2025-09-17 06:21:06","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117710,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian phylogenetic analysis based on 30 unique haplotypes. Support values below 0.8 are not shown. Outgroup: Anopheles braziliensis (Chagas, 1970) (GenBank PX101434). GenBank accession numbers: An. albitarsis F (PX101437-42, PX101452-56), An. oswaldoi B (PX101460-68), An. oswaldoi A (PX101457-59) and An. goeldii (PX101435-36, PX10144351).\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584315/v1/223f9d54e6535efa46d53c1a.jpeg"},{"id":91495792,"identity":"c005480b-06a8-42b7-98ef-1801f60a7331","added_by":"auto","created_at":"2025-09-17 06:21:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111629,"visible":true,"origin":"","legend":"\u003cp\u003eTCS analysis of Anopheles spp. mtCOI sequences from Guyana and Venezuela. Four networks were generated separately: An. albitarsis F, An. oswaldoi B, An. oswaldoi A and An. goeldii.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584315/v1/7e4993d799833f4b5860712e.jpeg"},{"id":91496610,"identity":"5854c27d-b89f-4545-a236-9578e0b3d0ea","added_by":"auto","created_at":"2025-09-17 06:29:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":901977,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Guyana, Venezuela and Roraima State, Brazil showing the species complex distribution: Anopheles albitarsis F (purple), An. albitarsis I (red), An. goeldii (blue), An. janconnae (brown), An. oswaldoi A (green) and An. oswaldoi B (orange).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584315/v1/98f1d3699d823a1fca6bd61f.jpeg"},{"id":91497639,"identity":"52a206bf-662e-4bfb-9eaf-aeb38d11e2d6","added_by":"auto","created_at":"2025-09-17 06:45:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2081358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7584315/v1/5fb6a7f8-a56e-4d10-a5e7-468ea0f31f5d.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAnopheles \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies complexes from Guyana and Venezuela malaria endemic areas using \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCOI \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esequences\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eThe genus \u003cem\u003eAnopheles\u003c/em\u003e Meigen, 1818 comprises approximately 470 species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], of which 43 species have been reported in Venezuela [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Out of these 43 species, 12 have been incriminated in the transmission of malaria parasites [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccording to the World Health Organization (WHO) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Venezuela reported 135,000 malaria cases in 2023, the second highest in the Americas, accounting for 24% of the region's total of 548,000 cases, following Brazil. Nevertheless, Venezuela has the highest rate of cases in the region at 477 per 100,000, while Brazil's rate stands at 75.3 per 100,000. More than 70% of cases in Venezuela are reported from Bol\u0026iacute;var State. The confirmed vector species in Bol\u0026iacute;var State include \u003cem\u003eAn. darlingi\u003c/em\u003e Root 1926 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], \u003cem\u003eAn. albitarsis\u003c/em\u003e Lynch-Arrib\u0026aacute;lzaga 1878 \u003cem\u003esensu lato, An. nuneztovari\u003c/em\u003e Gabaldon 1940 \u003cem\u003es.l.\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and \u003cem\u003eAn. (Anopheles) neomaculipalpus\u003c/em\u003e Curry 1931 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, the potential vectors, \u003cem\u003eAn. albitarsis\u003c/em\u003e F and \u003cem\u003eAn. oswaldoi\u003c/em\u003e B have been found in the malaria foci in southern Venezuela [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles albitarsis\u003c/em\u003e F [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] is a molecularly delimited species, part of the \u003cem\u003eAn. albitarsis\u003c/em\u003e complex, which currently includes five formally described species: \u003cem\u003eAn. albitarsis\u003c/em\u003e, \u003cem\u003eAn. marajoara\u003c/em\u003e Galv\u0026atilde;o and Damasceno 1942, \u003cem\u003eAn. deaneorum\u003c/em\u003e Rosa-Freitas 1989, \u003cem\u003eAn. oryzalimnetes\u003c/em\u003e Wilkerson and Motoki 2009 and \u003cem\u003eAn. janconnae\u003c/em\u003e Wilkerson and Sallum 2009. Based on molecular markers, there are an additional 5 proposed species in the complex: \u003cem\u003eAn. albitarsis\u003c/em\u003e F [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003eAn. albitarsis\u003c/em\u003e G [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eAn. albitarsis\u003c/em\u003e I [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eAn. albitarsis\u003c/em\u003e H [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] y \u003cem\u003eAn. albitarsis\u003c/em\u003e J [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles albitarsis\u003c/em\u003e F has been reported from western, central, and southern Venezuela [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], east of the Eastern Andean Cordillera of Colombia, and on the island of Trinidad [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Z\u0026uacute;\u0026ntilde;iga et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] suggested that previous reports on the distribution, bionomics, and vector incrimination of \u003cem\u003eAn. albitarsis s.l.\u003c/em\u003e from northern South America corresponded to \u003cem\u003eAn. albitarsis\u003c/em\u003e F. This species is found sympatric with the mitochondrial lineage \u003cem\u003eAn. albitarsis\u003c/em\u003e I in Zulia State, Venezuela and the neighbouring Department of Norte de Santander in Colombia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. So far, this putative species has not been found positive for malaria parasites [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe species of the \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e complex have a wide distribution, encompassing eastern Panam\u0026aacute;, Colombia, Venezuela, Guyana, Brazil, Ecuador, Per\u0026uacute; and Bolivia [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Based on morphological, behavioral, cytogenetics and molecular markers (\u003cem\u003eWhite\u003c/em\u003e and \u003cem\u003eCAD genes\u003c/em\u003e, ITS2-rDNA and DNA \u003cem\u003emtCOI\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], it has been suggested that the \u003cem\u003eAn. nuneztovari\u003c/em\u003e complex includes four formally described species: \u003cem\u003eAn. nuneztovari, An. goeldii\u003c/em\u003e Rozeboom and Gabaldon 1941, \u003cem\u003eAn. dunhami\u003c/em\u003e Causey 1945 and \u003cem\u003eAn. jamariensis\u003c/em\u003e Sant\u0026rsquo;Ana and Sallum 2022. \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e is distributed from western Venezuela, Colombia and eastern Panam\u0026aacute; [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. \u003cem\u003eAnopheles goeldii\u003c/em\u003e has been confirmed from the States of Amazonas, Amap\u0026aacute; and Par\u0026aacute; in northern Brazil [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eAnopheles dunhami\u003c/em\u003e is found in Colombia [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and the Brazilian Amazon region [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cem\u003eAnopheles jamariensis\u003c/em\u003e was recently described from the Jamari river, municipality of Monte Negro, Rond\u0026ocirc;nia State, western Amazonian region of Brazil [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Since these four species can be easily misidentified, it is necessary to understand their actual geographic distribution and role as vectors of malaria parasites, particularly in areas of sympatry in the Amazon basin.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e complex has a wide distribution extending from Costa Rica to the northern provinces of Argentina, east of the Andes, and the island of Trinidad [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Based on molecular markers, Marrelli et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] suggested that \u003cem\u003eAn. oswaldoi\u003c/em\u003e was a complex of at least four species: \u003cem\u003eAn. oswaldoi\u003c/em\u003e (Peryass\u0026uacute;, 1922) \u003cem\u003es.s\u003c/em\u003e., \u003cem\u003eAn. konderi\u003c/em\u003e Galv\u0026atilde;o and Damasceno 1942 and two other species yet to be described. Nevertheless, other studies confirmed the genetic variability among members of the complex and taxonomic misidentification of the mosquitoes included in that study [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Building on genetic analyses, Ru\u0026iacute;z-L\u0026oacute;pez et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] utilized ITS2-rDNA and \u003cem\u003emtCOI\u003c/em\u003e molecular markers to study populations across Brazil, Colombia, Per\u0026uacute;, and Trinidad and Tobago. By comparing these results with sequences archived in GenBank, their research clarified the composition of the Oswaldoi Complex, revealing three distinct species: \u003cem\u003eAn. oswaldoi s.s\u003c/em\u003e., \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A, and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B. Their findings indicate that \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi s.s.\u003c/em\u003e and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A are present in both Brazil and Colombia, whereas \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B is distributed in Colombia, Ecuador, and Trinidad and Tobago.\u003c/p\u003e\u003cp\u003eSubsequently, Rubio-Palis et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] confirmed the presence of \u003cem\u003eAn. oswaldoi\u003c/em\u003e B in Sucre municipality, Bol\u0026iacute;var State, a malaria endemic area in southern Venezuela. However, the Oswaldoi Complex could be part of a larger complex, the Oswaldoi-Konderi Complex, including \u003cem\u003eAn. konderi s.l.\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo further elucidate the geographic ranges of these complexes, the present study analyzed the molecular taxonomy and phylogenetic inference for members of the Albitarsis, Nuneztovari, and Oswaldoi Complexes from malaria-endemic areas of Guyana and Venezuela using \u003cem\u003emtCOI\u003c/em\u003e sequences.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cem\u003eMosquito collections\u003c/em\u003e \u0026ndash; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the geographical locations, dates, and methods of mosquito collection in Guyana and Venezuela. Mosquitoes were identified using the keys by Rubio-Palis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Gonz\u0026aacute;lez and Carrejo [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The collected mosquitoes were either kept dry over silica gel or pinned; only specimens collected in 1998 in Gu\u0026aacute;rico were preserved in isopropanol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mosquitoes were transported to the CDC, Entomology Branch, Atlanta, USA, for molecular analysis where 35 specimens were sequenced.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGeographical locations, dates, methods of mosquito collections, and the number (n) sequenced.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCountry\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRegion \u0026ndash; State/Municipality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoordinates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDate collected\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCollection Method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGuyana\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePotaro-Siparuni\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e05\u0026deg;04'16\"N, 59\u0026deg;12'48\"W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDec. 2016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHuman landing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eApure / M\u0026uacute;\u0026ntilde;oz\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e07\u0026deg;26'08\"N, 69\u0026deg;19'10\"W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDec. 2010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCanopy trap\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGu\u0026aacute;rico / Francisco de Miranda\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e08\u0026deg;58'N, 67\u0026deg;25'W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eJan. 1998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHuman landing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVenezuela\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBol\u0026iacute;var / Sucre\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e07\u0026deg;03'43\"N, 64\u0026deg;58'37\"W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNov. 2014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUV light trap\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBol\u0026iacute;var / Sucre\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e06\u0026deg;28'N, 64\u0026deg;45'W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAug. 2015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMosq Magnet trap\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBol\u0026iacute;var / Sifontes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e07\u0026deg;14'50\"N, 61\u0026deg;20'54\"W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApr. 1999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHuman landing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBol\u0026iacute;var / Gran Sabana\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e04\u0026deg;35'45\"N, 61\u0026deg;06'40\"W\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNov. 2011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHuman landing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTOTAL\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eSequence generation\u003c/em\u003e - DNA was extracted using the commercially available DNeasy Blood \u0026amp; Tissue Kit (QIAgen\u0026reg;, Maryland, USA). Amplification of mitochondrial \u003cem\u003eCOI\u003c/em\u003e gene (\u003cem\u003emtCOI\u003c/em\u003e) was achieved using the primers UEA3 and UEA10, as described in Lunt et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Sequencing reactions were carried out in both directions using the BigDye Terminator Kit\u0026reg; (PE Applied BioSystems, Warrington, England) on an ABI 3730 automated sequencer (PE Applied BioSystems). Sequences were edited using Sequencher\u0026reg; v.5.4.6 (Genes Codes Corporation, Ann Arbor, MI) and automatically aligned in MUSCLE (Geneious 10.0.9). Sequence similarities were compared with those available in GenBank using Basic Local Alignment Search Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and BoldSystems (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.boldsystems.org/\u003c/span\u003e\u003cspan address=\"https://www.boldsystems.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eData analysis\u003c/em\u003e - Phylogenetic analysis was conducted on 30 unique haplotypes (n\u0026thinsp;=\u0026thinsp;35) on 860 bp of the \u003cem\u003emtCOI\u003c/em\u003e. A Bayesian phylogenetic analysis was performed with MrBayes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] using the Kimura 2-Parameter (K2P) distance model [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] in Geneious 10.0.9. The analysis ran for 3\u0026nbsp;million generations, with subsampling every 1000 generations and a burn-in of 300,000 generations. To assess putative species based on the posterior probabilities from the Bayesian analysis tree, species delimitation tools were applied [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo assess population-level genealogies, \u003cem\u003emtCOI\u003c/em\u003e sequences were analyzed using TCS v. 1.21 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A connection limit of 95% was adopted to investigate whether \u003cem\u003eAn. albitarsis\u003c/em\u003e, \u003cem\u003eAn. nuneztovari\u003c/em\u003e, and the \u003cem\u003eAn. oswaldoi\u003c/em\u003e species complex formed a single \u0026ldquo;meta-population\u0026rdquo; indicated by a single network, or consisted of separate species, indicated by the formation of two or more independent networks.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthical considerations\u003c/h2\u003e\u003cp\u003eMosquito collections were conducted by the correspondence author and field technicians of the vectors control programs from Venezuela and Guyana.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eInitial morphological identifications showed the presence of four species: \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ealbitarsis s.l.\u003c/em\u003e, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003enuneztovari s.l\u003c/em\u003e., \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003enr\u003c/em\u003e. \u003cem\u003enuneztovari\u003c/em\u003e (\u003cem\u003eAn. nunez\u003c/em\u003e-like), and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e. Upon comparing the \u003cem\u003emtCOI\u003c/em\u003e sequences with the GenBank and BoldSystems databases, we found similarity between 99 to 100% with \u003cem\u003eAn. albitarsis\u003c/em\u003e F (12 haplotypes), \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eaff\u003c/em\u003e. \u003cem\u003enuneztovari\u003c/em\u003e A (4 haplotypes), \u003cem\u003eAn. goeldii\u003c/em\u003e (7 haplotypes), \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B (9 haplotypes), and \u003cem\u003eAn. oswaldoi\u003c/em\u003e A (=\u0026thinsp;\u003cem\u003eAn. aff. oswaldoi\u003c/em\u003e A) (3 haplotypes) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of morphological identifications with \u003cem\u003emtCOI\u003c/em\u003e sequences matches from GenBank and BoldSystems.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies ID Morphology\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpecies ID \u003cem\u003emtCOI\u003c/em\u003e sequences\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGenBank / BoldSystems access number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e% ID\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAn. albitarsis\u003c/em\u003e complex (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAn. albitarsis\u003c/em\u003e F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJQ615028\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAn. nuneztovari s.l.\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4)*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAn. aff. nuneztovari\u003c/em\u003e A (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMF381680\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAn. nunez-like\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAn. goeldii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNC_037810\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eAn. oswaldoi\u003c/em\u003e complex (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAn. oswaldoi\u003c/em\u003e B (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKF809115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAn. aff. oswaldoi\u003c/em\u003e A (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMZ014236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e*These specimens also showed 98.7% similarity with \u003cem\u003eAn. goeldii\u003c/em\u003e (MF381656)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eBayesian analysis revealed four clades (monophyletic groups), named based on morphological and \u003cem\u003emtCOI\u003c/em\u003e sequence matches (GenBank and BoldSystems), as follows: \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ealbitarsis\u003c/em\u003e F, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B, \u003cem\u003eAn. oswaldoi\u003c/em\u003e A and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003egoeldii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSupport values below 0.8 are not shown. Outgroup: \u003cem\u003eAnopheles braziliensis\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(Chagas, 1970) (GenBank PX101434). GenBank accession numbers: \u003cem\u003eAn\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003ealbitarsis\u003c/em\u003e F (PX101437-42, PX101452-56), \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B (PX101460-68),\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A (PX101457-59) and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003egoeldii\u003c/em\u003e (PX101435-36, PX10144351).\u003c/p\u003e\u003cp\u003eThe species delimitation analysis strongly supports the distinction of at least four taxa. Genetic distances between species and their closest relatives confirm these findings. The intra-species distance was between 0.8% (\u003cem\u003eAn. oswaldoi\u003c/em\u003e A) to 1.4% (\u003cem\u003eAn. goeldii\u003c/em\u003e), and inter-species was between 7.9% to 5.4% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of species delimitation analysis using Geneious (Geneious 10.0.9) for species boundaries.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIntra Distance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInter Dist.\u003c/p\u003e\u003cp\u003eCloset\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIntra/Inter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP ID (Strict)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP ID (Liberal)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAv (MRCA-tips)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e(1)\u003c/b\u003e \u003cem\u003eAn. albitarsis\u003c/em\u003e F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.068 (\u003cb\u003e2\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.89 (0.80, 0.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.96 (0.91, 1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.2E-6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e(2)\u003c/b\u003e \u003cem\u003eAn. oswaldoi\u003c/em\u003e B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.054 (\u003cb\u003e3\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.88 (0.79, 0.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.96 (0.91, 1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.2E-6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e(3)\u003c/b\u003e \u003cem\u003eAn. oswaldoi\u003c/em\u003e A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.054 (\u003cb\u003e2\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.69 (0.52, 0.87)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.92 (0.78, 1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.3E-8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e(4)\u003c/b\u003e \u003cem\u003eAn. goeldii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.079 (\u003cb\u003e3\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.86 (0.76, 0.97)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.95 (0.89, 1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.3E-03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe closest inter-species distances show \u003cem\u003eAn. albitarsis\u003c/em\u003e F is nearest to \u003cem\u003eAn. oswaldoi\u003c/em\u003e B at 6.8%, while \u003cem\u003eAn. goeldii\u003c/em\u003e is most distant from \u003cem\u003eAn. aff. oswaldoi\u003c/em\u003e A at 7.9%. Posterior Probabilities of Correct Identification (P ID) under strict and liberal criteria were consistently high for most species, with \u003cem\u003eAn. albitarsis\u003c/em\u003e F having a strict P ID of 0.89 (liberal: 0.96) and \u003cem\u003eAn. oswaldoi\u003c/em\u003e B showing similar values (strict: 0.88, liberal: 0.96). \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e A had a lower strict P ID of 0.69 but reached 0.92 under the liberal threshold, while \u003cem\u003eAn. goeldii\u003c/em\u003e maintained strong support with strict and liberal P IDs of 0.86 and 0.95, respectively. The analysis also highlighted strong clade support for all species, with Rosenberg's P(AB) values indicating significant differentiation; \u003cem\u003eAn. albitarsis\u003c/em\u003e F\u0026thinsp;=\u0026thinsp;1.2E-6, \u003cem\u003eAn. goeldii\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.3E-8, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B\u0026thinsp;=\u0026thinsp;1.2E-6, and \u003cem\u003eAn. oswaldoi\u003c/em\u003e A\u0026thinsp;=\u0026thinsp;6.1E-5. These results, coupled with P (Randomly Distinct) values and the intra/inter species distance ratios, further validate the delineation of these species.\u003c/p\u003e\u003cp\u003ePopulation analysis of the \u003cem\u003emtCOI\u003c/em\u003e data set using TCS\u003csup\u003e(43)\u003c/sup\u003e clearly divided the data set into four independent networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e): \u003cem\u003eAn. albitarsis\u003c/em\u003e F, \u003cem\u003eAn. goeldii, An. oswaldoi\u003c/em\u003e B, and \u003cem\u003eAn. oswaldoi\u003c/em\u003e A. This analysis is consistent with the Bayesian analysis and aligns with species delimitation boundaries as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, where clade colours represent the selected species.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe subgenus \u003cem\u003eNyssorhynchus\u003c/em\u003e holds significant importance in public health across the Americas due to the presence of primary and secondary malaria vectors within this group [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. With the advent of molecular analysis and the emergence of new studies in diverse geographic regions, a considerable number of species belonging to the \u003cem\u003eNyssorhynchus\u003c/em\u003e subgenus have been formally described or resurrected from synonymy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Regardless of these efforts, some species are still provisionally designated with temporary names, such as Species A, B, I, F, G, H, and J, among others [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. According to the Mosquito Taxonomic Inventory [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], the \u003cem\u003eNyssorhynchus\u003c/em\u003e subgenus comprises 45 formally described species and 14 provisionally designated extant species.\u003c/p\u003e\u003cp\u003eIn Venezuela, 13 species of the subgenus \u003cem\u003eNyssorhynchus\u003c/em\u003e have been reported and 3 extant species [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In contrast, no recent reports are available from Guyana; data from Laubach et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] included five species of the subgenus \u003cem\u003eNyssorhynchus\u003c/em\u003e: \u003cem\u003eAn. aquasalis\u003c/em\u003e Curry, 1932, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ebraziliensis\u003c/em\u003e, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003edarlingi\u003c/em\u003e, \u003cem\u003eAn. oswaldoi\u003c/em\u003e and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003etriannulatus\u003c/em\u003e collected in Mahdia, Potaro-Siparuni Region. It is worth noting that this region is a gold mining area corresponding to the inland-forest malaria ecoregion [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], similar to the Sifontes municipality in Venezuela. The report of \u003cem\u003eAn. aquasalis\u003c/em\u003e, a coastal mosquito [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], suggests that the species was misidentified. Furthermore, during our collections in Guyana (December 2016) in the same area, we identified \u003cem\u003eAn. darlingi, An. nuneztovari s.l.\u003c/em\u003e (=\u0026thinsp;\u003cem\u003eAn. goeldii\u003c/em\u003e), \u003cem\u003eAn. triannulatus s.l\u003c/em\u003e. and \u003cem\u003eAn. rangeli\u003c/em\u003e. Potentially, the \u003cem\u003eAn. aquasalis\u003c/em\u003e specimens reported positive for \u003cem\u003eP. vivax\u003c/em\u003e by Laubach et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] corresponded to \u003cem\u003eAn. goeldii\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe present report updates the lists of species to include \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003egoeldii\u003c/em\u003e (Guyana and Venezuela) and \u003cem\u003eAn. oswaldoi\u003c/em\u003e A (Venezuela). Both species are sympatric with \u003cem\u003eAn. oswaldoi\u003c/em\u003e B in the municipality of Bolivar, Sucre, Venezuela (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe species identified in this research belong to three species complexes: the Albitarsis Complex (\u003cem\u003eAn. albitarsis\u003c/em\u003e F), the Nuneztovari Complex (\u003cem\u003eAn. goeldii\u003c/em\u003e), and the Oswaldoi Complex (\u003cem\u003eAn. oswaldoi\u003c/em\u003e B and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnopheles albitarsis\u003c/b\u003e \u003cb\u003eF\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe present study contributes to broadening the distribution of \u003cem\u003eAn. albitarsis\u003c/em\u003e F in Venezuela by adding 12 new sequences and confirming the previous hypothesis by Z\u0026uacute;\u0026ntilde;iga et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] that \u003cem\u003eAn. albitarsis\u003c/em\u003e F was widely distributed in Venezuela as it had been found in Zulia, Cojedes, Portuguesa [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], Gu\u0026aacute;rico [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and Bol\u0026iacute;var [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] States. The present study found it in Apure State, bordering Colombia, and added four more records for Bol\u0026iacute;var State (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It is of particular interest that \u003cem\u003eAn. albitarsis\u003c/em\u003e F was confirmed from Santa Elena de Uair\u0026eacute;n, Gran Sabana municipality, Bol\u0026iacute;var State, at an altitude of 950 m above sea level, and separated 18 km from the town of Pacaraima, State of Roraima, Brazil. It is important to mention that Ru\u0026iacute;z-L\u0026oacute;pez et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] reported that the only species of the complex present in Roraima was \u003cem\u003eAn. janconnae\u003c/em\u003e. We suggest that there might be an area between Santa Elena de Uair\u0026eacute;n, Venezuela, and Boa Vista, Brazil (the closest locality to Venezuela where \u003cem\u003eAn. janconnae\u003c/em\u003e was reported), where both species are sympatric. Wilkerson et al. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] indicated that \u003cem\u003eAn. janconnae\u003c/em\u003e was present in Venezuela, although no supporting data was provided.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles albitarsis\u003c/em\u003e F has not been formally described. Still, recent morphometric analysis of adult females from Venezuela and Brazil has shown that the ratio of the length of the prehumeral dark spot divided by the length of the humeral pale spot (PHD/HP) on the wing Costal vein is a valid diagnostic character to separate this species from the described species of the complex: \u003cem\u003eAn. albitarsis\u003c/em\u003e, \u003cem\u003eAn. janconnae\u003c/em\u003e, \u003cem\u003eAn. marajoara\u003c/em\u003e, \u003cem\u003eAn. deaneorum\u003c/em\u003e and \u003cem\u003eAn. oryzalimnetes\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Also, characters on the hind legs such as the length of dark spot on hind tarsomere 2 divided by the length of hind tarsomere 2 (DS-III\u003csub\u003e2\u003c/sub\u003e/Ta-III\u003csub\u003e2\u003c/sub\u003e) can be used to separate these species.\u003c/p\u003e\u003cp\u003eA complete review on the bionomics, ecology, and vector incrimination of \u003cem\u003eAn. albitarsis\u003c/em\u003e F in Venezuela was published by Z\u0026uacute;\u0026ntilde;iga et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAnopheles goeldii\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eAnopheles goeldii\u003c/em\u003e was described by Rozeboom and Gabaldon [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] based on morphological characters in adult male genitalia, females, larvae, pupae and eggs from specimens collected near the Tapaj\u0026oacute;s river, Par\u0026aacute; State, Brazil. Later, Floch and Abonnenc [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] followed by Lane [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] synonymized \u003cem\u003eAn. goeldii\u003c/em\u003e with \u003cem\u003eAn. nuneztovari.\u003c/em\u003e Due to this controversy, Gabaldon [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] revised the morphological characters of fourth instar larvae and male genitalia that clearly could separate both species, and hence resurrecting \u003cem\u003eAn. goeldii\u003c/em\u003e from synonymy with \u003cem\u003eAn. nuneztovari\u003c/em\u003e. Calado et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] confirmed \u003cem\u003eAn. goeldii\u003c/em\u003e as a valid species based on DNA sequences and morphological analyses. Subsequently, Sant\u0026rsquo;Ana et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] redescribed the species based on eggs, fourth-instar larvae, pupae, male and female morphological characters, and DNA sequences from the \u003cem\u003emtCOI\u003c/em\u003e genome. This species distribution was restricted to northern Brazil and Suriname [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEleven \u003cem\u003eAnopheles\u003c/em\u003e specimens from our collections were identified as \u003cem\u003eAn. nunez\u003c/em\u003e-like (Table II) based on the diagnostic characters of the ratio of the length of humeral pale spot divided by the length of prehumeral dark spot (HP/PHD) (0.7\u0026ndash;4.5) and the ratio of the length of the basal dark spot of the hind tarsomere 2 (Ta-III\u003csub\u003e2\u003c/sub\u003e) divided by the length of the hind tarsomere II (0.24\u0026ndash;0.35) which can easily separate \u003cem\u003eAn. nuneztovari\u003c/em\u003e from other species of the Oswaldoi Subgroup [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]; although we did not measure the characters, it was noticed that the range of the ratio HP/PHD was smaller, and in general, light wing scales were cream, never bright yellow as \u003cem\u003eAn. nuneztovari s.s.\u003c/em\u003e from western Venezuela. However, upon comparing these sequences with those in GenBank, we found 99% homology with \u003cem\u003eAn. goeldii\u003c/em\u003e (accession numbers: MF381655\u0026ndash;57 by Foster et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] for seven specimens from Guyana, and 89\u0026ndash;100% homology with \u003cem\u003eAn. nuneztovari\u003c/em\u003e A (accession number: MF381680 by Foster et al.) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] for four specimens from Bol\u0026iacute;var State, supporting the morphological ambiguities observed when using the morphological keys for adult females of Gonz\u0026aacute;lez and Carrejo [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and Sallum et al. [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] to distinguish \u003cem\u003eAn. nuneztovari s.l.\u003c/em\u003e in Venezuela. It is important to point out that \u003cem\u003eAn. nuneztovari\u003c/em\u003e was reported from Bol\u0026iacute;var State (Sifontes Municipality) for the first time in 2003 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], even though regular and intensive sampling efforts occurred since 1992, suggesting a recent colonization. The species was confirmed based on key characters in adult females and male genitalia. Nevertheless, recent revision of the male genitalia showed that the characters are consistent with the description for \u003cem\u003eAn. goeldii\u003c/em\u003e, making necessary a thorough revision of the specimens preserved and new collections to include morphological and molecular analysis. \u003cem\u003eAnopheles nuneztovari s.s.\u003c/em\u003e can be distinguished from other species in the Complex by the well-developed subapical leaflets of the aedeagus [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. It is suggested that, so far, \u003cem\u003eAn. goeldii\u003c/em\u003e is the only species of the complex present in Bol\u0026iacute;var State; although it is possible that other species might occur sympatrically as it has been reported in Par\u0026aacute; State, Brazil, where \u003cem\u003eAn. nuneztovari s.s.\u003c/em\u003e, \u003cem\u003eAn. nuneztovari\u003c/em\u003e A and \u003cem\u003eAn. goeldii\u003c/em\u003e occurred sympatrically [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStudies on biting behavior and larval habitats of \u003cem\u003eAn. nuneztovari s.l\u003c/em\u003e. (=\u0026thinsp;\u003cem\u003eAn. goeldii\u003c/em\u003e) in Sucre municipality showed that this species has a different biting behavior to that reported for \u003cem\u003eAn. nuneztovari s.s\u003c/em\u003e. from western Venezuela [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. \u003cem\u003eAnopheles goeldii\u003c/em\u003e is active throughout the night with a biting peak at sunset and a second, minor peak, at sun rise (9), contrasting with the unimodal behavior for \u003cem\u003eAn. nuneztovari s.s\u003c/em\u003e. with a biting peak between 22:00 and 01:00 hrs [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. There are no longitudinal studies data on biting behavior of \u003cem\u003eAn. nuneztovari s.l\u003c/em\u003e from Sifontes Municipality.\u003c/p\u003e\u003cp\u003eIn Sucre Municipality, \u003cem\u003eAn. nuneztovari s.l.\u003c/em\u003e (=\u0026thinsp;\u003cem\u003eAn. goeldii\u003c/em\u003e) larvae were found mainly in streams and lagoons [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] together with \u003cem\u003eAn. darlingi\u003c/em\u003e, \u003cem\u003eAn. triannulatus s.l\u003c/em\u003e., \u003cem\u003eAn. oswaldoi s.l.\u003c/em\u003e and \u003cem\u003eChagasia bonnae\u003c/em\u003e (Root, 1927). In Sifontes Municipality, \u003cem\u003eAn. nuneztovari s.s\u003c/em\u003e. and/or \u003cem\u003eAn. goeldii\u003c/em\u003e were collected mainly in lagoons and rivers [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] together with \u003cem\u003eAn. triannulatus s.l\u003c/em\u003e., \u003cem\u003eAn. albitarsis\u003c/em\u003e F, \u003cem\u003eAn. oswaldoi s.l\u003c/em\u003e., \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003edarlingi\u003c/em\u003e and \u003cem\u003eCh. bonnae\u003c/em\u003e, strongly associated with rainfall (R\u0026thinsp;=\u0026thinsp;0.71) and river level (R\u0026thinsp;=\u0026thinsp;0.79), with an abundance peak in July-August immediately after the peak of rains [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. To determine the actual distribution of these species in Bol\u0026iacute;var State and characterize their bionomics, it is necessary to conduct taxonomic studies (morphological and molecular characterization), particularly in Sifontes Municipality where \u003cem\u003eAn. nuneztovari s.l.\u003c/em\u003e is a confirmed vector of malaria parasites [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRuiz et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] used DNA barcode sequences to propose a threshold of 2.5% genetic divergence for differentiating cryptic species within the Nuneztovari Complex (\u003cem\u003eAn. nuneztovari\u003c/em\u003e vs. \u003cem\u003eAn. dunhami\u003c/em\u003e). Later, McKeon et al. [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] reduced the threshold to 2% for the Albitarsis Complex, with Ruiz et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] reporting similar thresholds for the Albitarsis and Oswaldoi Complexes, respectively. Our results showed an intraspecific genetic divergence of 1.4% for \u003cem\u003eAn. goeldii\u003c/em\u003e (Table III), confirming that, although the analyzed sequences exhibit moderate variation, they fall below the threshold commonly used for species delimitation within species complexes of the \u003cem\u003eNyssorhynchus\u003c/em\u003e subgenus.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnopheles oswaldoi\u003c/b\u003e \u003cb\u003ecomplex\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe two formally described species belonging to \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e complex, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi s.s\u003c/em\u003e. and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ekonderi s.s.\u003c/em\u003e, cannot be distinguished by the morphology of larvae or adult females, as all morphological characteristics are shared between them [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. \u003cem\u003eAnopheles konderi s.s\u003c/em\u003e. and \u003cem\u003eAn. oswaldoi s.s.\u003c/em\u003e can only be differentiated based on the morphology of male genitalia [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. DNA sequencing is the only reliable tool available for distinguishing the five members of Oswaldoi Complex (\u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e s.s., \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e B, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ekonderi s.s.\u003c/em\u003e and \u003cem\u003eAn\u003c/em\u003e. sp. nr. \u003cem\u003ekonderi\u003c/em\u003e) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The genetic variation we found among members of this group ranges from an intraspecific divergence of 1.0% (\u003cem\u003eAn. oswaldoi\u003c/em\u003e B) to 0.8% (\u003cem\u003eAn. oswaldoi\u003c/em\u003e A) and interspecific divergence of 5.4% for both species (Table III). These results are similar to those found by Ruiz et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], who reported an intraspecific divergence of 1.2% and an interspecific divergence of 5.2% for all members of the Oswaldoi Complex.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eAnopheles oswaldoi\u003c/b\u003e \u003cb\u003eA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBased on \u003cem\u003eCOI\u003c/em\u003e sequences (DNA barcoding), Ruiz et al.[(36] identified \u003cem\u003eAn. oswaldoi\u003c/em\u003e A as a species belonging to the \u003cem\u003eAn. oswaldoi\u003c/em\u003e complex from Brazil (Acre, Amazonas, Mato Grosso, Par\u0026aacute;, and Rond\u0026ocirc;nia) and Colombia (Amazonas). Subsequently, Silva-do-Nascimento et al. [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] discovered that some specimens from Roraima, Brazil, matched the \u003cem\u003eAn. oswaldoi\u003c/em\u003e A described by Ruiz et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, their mTPT (Multi-rate Poisson Tree Processes) analysis revealed that these specimens formed two distinct clusters, separated by a K2P genetic distance of 2.16%, suggesting the presence of a potential new species within \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A. These sequences were deposited in GenBank as \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eaff\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A (accession number MZ014236). However, although the sequences of this manuscript (accession number PX101457-59) matched 100% with MZ014236, we will retain the taxon as \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A rather than \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eaff\u003c/em\u003e. \u003cem\u003eoswaldoi\u003c/em\u003e A until the new hypothesis has been resolved. The specimens collected in this study are from Boca de Nichare, Bol\u0026iacute;var State (Venezuela) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), approximately 420 km from Parafuri, Roraima State (Brazil) where Silva-do-Nascimento et al. [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] collected mosquitoes of the Oswaldoi Complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e A has been implicated in malaria transmission in Acre, Brazil [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. However, its role in malaria transmission in other regions where the species is present remains unclear.\u003c/p\u003e\u003cp\u003eSequences of three specimens collected in Boca de Nichare, Bolivar State, were compatible with \u003cem\u003eAn. oswaldoi\u003c/em\u003e A. Initially, these mosquitoes were identified as \u003cem\u003eAn. oswaldoi s.l.\u003c/em\u003e due to the distinctive key character in adult females of hind tarsomere 2 (Ta-III\u003csub\u003e2\u003c/sub\u003e) with a basal dark spot 0.11\u0026ndash;0.22 length of tarsomere [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] which can easily separate \u003cem\u003eAn. oswaldoi\u003c/em\u003e from other species of the Albimanus Section present in the study area. This character is also present in \u003cem\u003eAn. ininii\u003c/em\u003e Senevet \u0026amp; Abonnenc, 1939, but we did not observe the white scales on the Costa vein nor examine and measure diagnostic characters in the fore tarsomeres, which can separate \u003cem\u003eAn. oswaldoi\u003c/em\u003e from \u003cem\u003eAn. ininii\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eAnopheles oswaldoi\u003c/em\u003e A found here exhibited 98% homology with \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eininii\u003c/em\u003e, based on comparisons of \u003cem\u003eCOI\u003c/em\u003e sequences in the GenBank database (accession number MT757855). These sequences of \u003cem\u003eAn. ininii\u003c/em\u003e were deposited as direct submission and lacked additional details, such as information about collection location and morphological confirmation, hence they were not included in our analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnopheles oswaldoi\u003c/b\u003e \u003cb\u003eB\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis species was reported in Venezuela for the first time from Jabillal, Sucre Municipality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], approximately 120 km from Boca de Nichare. So far, \u003cem\u003eAn. oswaldoi\u003c/em\u003e B and/or \u003cem\u003eAn. oswaldoi s.l\u003c/em\u003e. have not been found positive for plasmodia in Bolivar State, probably due to the lack of mosquitoes of this species tested for sporozoites and/or the small number of mosquitoes assayed [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eAnopheles oswaldoi s.l.\u003c/em\u003e was found positive for \u003cem\u003eP. vivax\u003c/em\u003e CS protein in Barinas State (western Venezuela) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; nevertheless, in this area the biting pattern observed in a 14-month longitudinal study [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] contrasted with that reported for the \u003cem\u003eAn. oswaldoi s.l\u003c/em\u003e. from Sucre Municipality, Bol\u0026iacute;var State [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. \u003cem\u003eAnopheles oswaldoi s.l.\u003c/em\u003e from Barinas showed a biting peak outdoors at sunset and a second peak around midnight indoors [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] whereas in Bol\u0026iacute;var State the biting activity increased steadily through the night, reaching a plateau around 02:00 hours that extended until sunrise [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. It has been suggested that probably in Barinas there are two different sympatric species [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], and the Oswaldoi Complex includes other species in western Venezuela yet to be identified. In the present study, \u003cem\u003eAn. oswaldoi\u003c/em\u003e A and \u003cem\u003eAn. oswaldoi\u003c/em\u003e B occurred sympatrically in Boca de Nichare (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), hence the biting pattern reported for \u003cem\u003eAn. oswaldoi s.l.\u003c/em\u003e might be inaccurate, including both species. Further studies in Venezuela should consider collections of immatures and rearing of individuals to obtain all stages for morphometric and molecular identification to establish species distribution, bionomics and medical importance of members of the Oswaldoi Complex.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003e\u003cem\u003eAnopheles goeldii\u003c/em\u003e is reported for the first time from Guyana and Venezuela, and discuss its role as vector of malaria parasites in both countries and its relative recent colonization. \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e A is reported for the first time from Venezuela. \u003cem\u003eAnopheles albitarsis\u003c/em\u003e F, an important malaria vector in Colombia and Venezuela, is confirmed as the only species of the Albitarsis complex widely distributed in Venezuela. The complex taxonomic situation of the subgenus \u003cem\u003eNyssorhynchus\u003c/em\u003e requires further research, and future studies should incorporate multidisciplinary approaches that include morphological and molecular species identification, ecology, and vector incrimination. Our results will be valuable for future research aimed at clarifying the status of these significant species complexes in the region.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecial thanks are due to Horace Cox, Jean Alexandre, Brian Puno, and the entomology technicians that participated in field collections in Guyana; to Jorge Moreno for valuable discussion on \u003cem\u003eAn. nuneztovari\u003c/em\u003e from Sifontes and providing specimens for this study; to Jes\u0026uacute;s Berti for providing specimens from Bol\u0026iacute;var State. To Sim\u0026oacute;n Caura, Hern\u0026aacute;n Guzm\u0026aacute;n, Yarys Estrada, V\u0026iacute;ctor S\u0026aacute;nchez, and Jes\u0026uacute;s Gonz\u0026aacute;lez, who participated in mosquito collections. To the people of Jabillal and Boca de Nichare for logistic support and friendship. We thank Ananias Escalante for helpful comments on the methodology and manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYRP and AL conceived and designed the study; YRP conducted the fieldwork; CCM and YRP carried out the lab work; FRL analyzed the data; YRP and FRL wrote the manuscript. All authors read and approved of the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYRP, CCM, AJL and FRL\u0026nbsp;received no specific funding for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analysed during this study are included in this published article.\u0026nbsp;The datasets generated during the current study are available in the GenBank repository, under accession numbers [PX101434 \u0026ndash;PX101468].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests associated with the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe findings and views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the Centers for Disease Control and Prevention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Instituto de Investigaciones Biom\u0026eacute;dicas \u0026ldquo;Dr. Francisco Triana Alonso\u0026rdquo;, Universidad de Carabobo, Maracay, Venezuela.\u003csup\u003e\u0026nbsp;2\u0026nbsp;\u003c/sup\u003eUS Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria, Entomology Branch, Atlanta, GA, United States of America.\u003csup\u003e\u0026nbsp;3\u003c/sup\u003ePrograma para el Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, Medellín, Colombia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKnols BGJ. Review of Mosquitoes of the World by Richard C. Wilkerson, Yvonne-Marie Linton, and Daniel Strickman. Parasit Vectors. 2021; 14\u003cstrong\u003e: \u003c/strong\u003e341. https://doi.org/10.1186/s13071-021-04848-6.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y. Mosquitos Anophelinae de Venezuela: complejos de especies e implicaciones epidemiol\u0026oacute;gicas. Salus 2022; 26(1):25-32. http://servicio.bc.uc.edu.ve/fcs/vol26n1/art05.pdf.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Wirtz RA, Curtis CF. Malaria entomological inoculation rates in western Venezuela. Acta Trop.1992; 52(2\u0026ndash;3): 167\u0026ndash;74. https://doi.org/10.1016/0001-706x(92)90033-t.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y. \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) de Venezuela: taxonom\u0026iacute;a, bionom\u0026iacute;a, ecolog\u0026iacute;a e importancia m\u0026eacute;dica\u003cem\u003e.\u003c/em\u003e Maracay, Venezuela: Escuela de Malariolog\u0026iacute;a y Saneamiento Ambiental \u0026ldquo;Dr. Arnoldo Gabaldon\u0026rdquo;; 2000. 120 pp. Available from: www.iaes.edu.ve/index.php/centro-de-descargas/viewcategory/3-libros-publicaciones.\u003c/li\u003e\n\u003cli\u003eMoreno JE, Rubio-Palis Y, P\u0026aacute;ez E, P\u0026eacute;rez E, S\u0026aacute;nchez V, Vaccari E. \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eAnopheles\u003c/em\u003e) \u003cem\u003eneomaculipalpus\u003c/em\u003e: a new malaria vector in the Amazon basin? Med Vet Entomol. 2005; 19(3): 329\u0026ndash;32. https://doi.org/10.1111/j.13652915.2005.00572.x.\u003c/li\u003e\n\u003cli\u003eMagris M, Rubio-Palis Y, Menare C, Villegas L. Vector bionomics and malaria transmission in the Upper Orinoco River, southern Venezuela. Mem Inst Oswaldo Cruz. 2007; 102: 303-311.\u003c/li\u003e\n\u003cli\u003eMoreno JE, Rubio-Palis Y, P\u0026aacute;ez E, P\u0026eacute;rez E, S\u0026aacute;nchez V, Vaccari E. Malaria entomological inoculation rates in gold mining areas of Southern Venezuela. Mem Inst Oswaldo Cruz. 2009; 104(5): 764\u0026ndash;8. https://doi.org/10.1590/s0074-02762009000500017.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y. Prevalencia de \u003cem\u003ePlasmodium\u003c/em\u003e spp. en anofelinos de Venezuela. Talleres. 2009; 12: 79-83.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Bevilacqua M, Medina DA, Moreno JE, C\u0026aacute;rdenas L, S\u0026aacute;nchez V, et al. Malaria entomological risk factors in relation to land cover in the Lower Caura River Basin, Venezuela. Mem Inst Oswaldo Cruz. 2013; 108: 220-228.\u003c/li\u003e\n\u003cli\u003eAbou Orm S, Moreno JE, Carrozza M, Acevedo P, Herrera F. \u003cem\u003ePlasmodium\u003c/em\u003e spp. infection rates for some \u003cem\u003eAnopheles\u003c/em\u003e spp. from Sifontes Municipality, Bol\u0026iacute;var State, Venezuela. Bol Mal Sal Amb. 2017; 57(1): 17-25.\u003c/li\u003e\n\u003cli\u003eWorld Malaria Report 2024. World malaria report 2024: addressing inequity in the global malaria response. Geneva: World Health Organization; 2024. Available from: https://www.who.int/publications/i/item/9789240104440.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Ru\u0026iacute;z-L\u0026oacute;pez F, Guzm\u0026aacute;n H, S\u0026aacute;nchez V, Moreno JE, Estrada Y, et al. Primer registro de \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003eoswaldoi\u003c/em\u003e B y \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eNys\u003c/em\u003e.) \u003cem\u003ealbitarsis \u003c/em\u003eF en la cuenca del r\u0026iacute;o Caura, Estado Bol\u0026iacute;var, Venezuela. Bol Mal Sal Amb. 2013; 53: 68-72. \u003c/li\u003e\n\u003cli\u003eZ\u0026uacute;\u0026ntilde;iga MA, Rubio-Palis Y, Brochero H. Updating the bionomy and geographical distribution of \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003ealbitarsis\u003c/em\u003e F: A vector of malaria parasites in northern South America. PLoS ONE. 2021; 16(6): e0253230. https://doi.org/10.1371/journal.pone.0253230.\u003c/li\u003e\n\u003cli\u003eBrochero HL, Li C, Wilkerson RC. A newly recognized species in the \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003ealbitarsis\u003c/em\u003e complex (Diptera: Culicidae) from Puerto Carre\u0026ntilde;o, Colombia. Am J Trop Med Hyg. 2007; 76(6): 1113\u0026ndash;1117.\u003c/li\u003e\n\u003cli\u003eRu\u0026iacute;z-L\u0026oacute;pez F, Wilkerson RC, Conn JE, McKeon SN, Levin DM, Qui\u0026ntilde;ones ML, et al. DNA barcoding reveals both known and novel taxa in the Albitarsis Group (\u003cem\u003eAnopheles\u003c/em\u003e: \u003cem\u003eNyssorhynchus\u003c/em\u003e) of Neotropical malaria vectors. Parasit Vectors. 2012; 5: 44. https://doi.org/10.1186/1756-3305-5-44.\u003c/li\u003e\n\u003cli\u003eKrzywinsky J, Li C, Morris M, Conn JE, Lima JB, P\u0026oacute;voa MM, et al. Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex. Mol Phylogenet Evol. 2011; 58(3): 469\u0026ndash;77. https://doi.org/10.1016/j.ympev.2011.01.003.\u003c/li\u003e\n\u003cli\u003eGuti\u0026eacute;rrez LA, Orrego LM, G\u0026oacute;mez GF, L\u0026oacute;pez A, Luckhart S, Conn JE, et al. A new mtDNA COI gene lineage closely related to \u003cem\u003eAnopheles janconnae\u003c/em\u003e of the Albitarsis Complex in the Caribbean region of Colombia. Mem Inst Oswaldo Cruz. 2010; 105(8): 1019\u0026ndash;25. https://doi.org/10.1590/s0074.\u003c/li\u003e\n\u003cli\u003eMotoki MT, Linton YM, Conn JE, Ru\u0026iacute;z-L\u0026oacute;pez F, Wilkerson RC. Phylogenetic Network of Mitochondrial COI Gene Sequences Distinguishes 10 Taxa Within the Neotropical Albitarsis Group (Diptera: Culicidae), Confirming the Separate Species Status of \u003cem\u003eAnopheles albitarsis\u003c/em\u003e H (Diptera: Culicidae) and Revealing a Novel Lineage, \u003cem\u003eAnopheles albitarsis\u003c/em\u003e J. J Med Entomol. 2020. https://doi.org/10.1093/jme/tjaa211.\u003c/li\u003e\n\u003cli\u003eSinka ME, Rubio-Palis Y, Manguin S, Patil AP, Temperley WH, Gething PW, et al. The dominant \u003cem\u003eAnopheles\u003c/em\u003e vectors of human malaria in the Americas: occurrence data, distribution maps and bionomic pr\u0026eacute;cis. Parasit Vectors. 2010; 3: 72. https://doi.org/10.1186/1756-3305-3-72.\u003c/li\u003e\n\u003cli\u003eKitzmiller J, Kreutzer R, Tallaferro E. Chromosomal differences in populations of \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e. Bull World Health Org. 1973; 48: 435-442.\u003c/li\u003e\n\u003cli\u003eConn JE, Mitchell SE, Cockburn AF. Mitochondrial DNA analysis of the neotropical malaria vector \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e. Genome. 1998; 41:313\u0026ndash;327.\u003c/li\u003e\n\u003cli\u003eTrindade DB, Scarpassa VM. Genetic differentiation and diagnostic loci among \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003erangeli\u003c/em\u003e, \u003cem\u003eAn\u003c/em\u003e. (\u003cem\u003eNys.\u003c/em\u003e) \u003cem\u003enuneztovari\u003c/em\u003e and \u003cem\u003eAn.\u003c/em\u003e (\u003cem\u003eNys\u003c/em\u003e.) \u003cem\u003edunhami\u003c/em\u003e (Diptera: Culicidae) from the Brazilian Amazon. J Med Entomol. 2002; 39: 613\u0026ndash;20. https://doi.org/10.1603/0022-2585-39.4.613.\u003c/li\u003e\n\u003cli\u003eRu\u0026iacute;z F, Linton Y-M, Ponsonby DJ, Conn JE, Herrera M, Qui\u0026ntilde;ones ML, et al. Molecular comparison of topotypic specimens confirms \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003edunhami \u003c/em\u003eCausey (Diptera: Culicidae) in the Colombian Amazon. Mem Inst Oswaldo Cruz 2010; 105: 899\u0026ndash;903. https://doi.org/10.1590/s0074-02762010000700010.\u003c/li\u003e\n\u003cli\u003eScarpassa VM, Geurgas S, Azeredo-Espin AML, Tadei WP. Genetic divergence in mitochondrial DNA of \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e (Diptera: Culicidae) from Brazil and Colombia. Genet Mol Biol. 2000; 23:71\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eScarpassa VM, Cunha-Machado AS, Saraiva JF. Evidence of new species for malaria vector \u003cem\u003eAnopheles nuneztovari sensu lato\u003c/em\u003e in the Brazilian Amazon region. Malar J. 2016; 15: 205. https://doi.org/10.1186/s12936-016-1217-6.\u003c/li\u003e\n\u003cli\u003eSant\u0026rsquo;Ana DC, Bergo ES, Sallum MAM. \u003cem\u003eAnopheles goeldii\u003c/em\u003e Rozeboom and Gabaldon (Diptera: Culicidae): a species of the Nuneztovari Complex of \u003cem\u003eAnopheles\u003c/em\u003e Meigen. Rev Brasil Entomol. 2015; 59: 68\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eSant\u0026rsquo;Ana DC, Sallum MAM. A new species of the Nuneztovari Complex of \u003cem\u003eNyssorhynchus\u003c/em\u003e (Diptera: Culicidae) from western Brazilian Amazon. Zootaxa. 2022; 5134(2): 275-285. https://doi.org/10.11646/zootaxa.5134.2.6.\u003c/li\u003e\n\u003cli\u003eSierra DM, V\u0026eacute;lez IV, Linton YM. Malaria vector \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003enuneztovari \u003c/em\u003ecomprises one genetic species in Colombia based on homogeneity of nuclear ITS2 rDNA. J Med Entomol. 2004; 41(3): 302-307.\u003c/li\u003e\n\u003cli\u003eJaramillo LM, Guti\u0026eacute;rrez LA, Luckhart S, Conn JE, Correa M. Molecular evidence for a single taxon, \u003cem\u003eAnopheles nuneztovari s.l\u003c/em\u003e. from two endemic malaria regions in Colombia. Mem Inst Oswaldo Cruz. 2011; 106(8): 1017-1023.\u003c/li\u003e\n\u003cli\u003ePeyton EL. \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003edunhami\u003c/em\u003e, resurrected from synonymy with \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e and validated as a senior synonym of \u003cem\u003eAnopheles trinkae\u003c/em\u003e (Diptera: Culicidae). Mosq Syst. 1993; 25(3): 151-156.\u003c/li\u003e\n\u003cli\u003eFaran ME. Mosquito studies (Diptera: Culicidae). XXXIV. A revision of the Albimanus section of the subgenus \u003cem\u003eNyssorhynchus\u003c/em\u003e of \u003cem\u003eAnopheles\u003c/em\u003e. Contrib Amer Entomol Inst. 1980; 15(7): 1\u0026ndash;215.\u003c/li\u003e\n\u003cli\u003eMarrelli MT, Malafronte RS, Flores-Mendoza C, Lourenco-de-Oliveira R, Kloetzel JK, Marinotti O. Sequence analysis of the second internal transcribed spacer of ribosomal DNA in \u003cem\u003eAnopheles oswaldoi \u003c/em\u003e(Diptera: Culicidae). J Med Entomol\u003cem\u003e.\u003c/em\u003e 1999; 36: 679-684.\u003c/li\u003e\n\u003cli\u003eRu\u0026iacute;z F, Qui\u0026ntilde;ones ML, Erazo HF, Calle DA, Alzate JF, Linton Y-M. Molecular differentiation of \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003ebenarrochi \u003c/em\u003eand \u003cem\u003eAn.\u003c/em\u003e (\u003cem\u003eN\u003c/em\u003e.) \u003cem\u003eoswaldoi\u003c/em\u003e from Southern Colombia. Mem Inst Oswaldo Cruz. 2005; 100(2): 155-160.\u003c/li\u003e\n\u003cli\u003eMotoki MT, Linton Y-M, Ruiz F, Flores-Mendoza C, Sallum MAM. Redescription of \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e (Peryass\u0026uacute;, 1922) (Diptera: Culicidae), with formal lectotype designation. Zootaxa. 2007; 1588: 31\u0026ndash;51.\u003c/li\u003e\n\u003cli\u003eSallum MAM, Marrelli MT, Nagaki SS, Laporta GZ, Dos Santos CLS. Insight into \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) (Diptera: Culicidae) species from Brazil. J Med Entomol. 2008; 45: 970\u0026ndash;81. https://doi.org/10.1603/0022-2585.\u003c/li\u003e\n\u003cli\u003eRu\u0026iacute;z-L\u0026oacute;pez F, Wilkerson RC, Ponsonby DJ, Herrera M, Sallum MAM, V\u0026eacute;lez ID, et al. Systematics of the Oswaldoi Complex (\u003cem\u003eAnopheles\u003c/em\u003e, \u003cem\u003eNyssorhynchus\u003c/em\u003e) in South America. Parasit Vectors. 2013; 6: 324. http://doi.org/10.1186/1756-3305-6-324.\u003c/li\u003e\n\u003cli\u003eSaraiva JF, Souto RNP, Scarpassa VM. Molecular taxonomy and evolutionary relationships in the Oswaldoi-Konderi Complex (Anophelinae: \u003cem\u003eAnopheles\u003c/em\u003e: \u003cem\u003eNyssorhynchus\u003c/em\u003e) from the Brazilian Amazon region. PLoS ONE. 2018; 13(3): e0193591. https://doi.org/10.1371/journal.pone.0193591.\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez Obando R, Carrejo Gironza NS. Introducci\u0026oacute;n al estudio taxon\u0026oacute;mico de \u003cem\u003eAnopheles\u003c/em\u003e de Colombia: Claves y notas de distribuci\u0026oacute;n. 2nd Edit. Cali, Colombia: Universidad del Valle; 2009. 260p. https://doi.org/10.25100/peu.309.\u003c/li\u003e\n\u003cli\u003eLunt DH, Zhang DX, Szymura JM, Hewitt GM. The insect \u003cem\u003ecytochrome oxidase I \u003c/em\u003egene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol Biol\u003cem\u003e.\u003c/em\u003e 1996; 5:153\u0026ndash;165.\u003c/li\u003e\n\u003cli\u003eHuelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001; 17(8): 754-755. https://doi.org/10/1093/bioinformatics/17.8.754.\u003c/li\u003e\n\u003cli\u003eKimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111\u0026ndash;120. \u003c/li\u003e\n\u003cli\u003eMasters B C, Fan V, Ross H A. Species Delimitation - a Geneious plugin for the exploration of species boundaries. Mol Ecol Resour. 2011; 11: 154-157.\u003c/li\u003e\n\u003cli\u003eClement M, Posada D, Crandall KA. TCS: a computer program to estimate gene genealogies. Mol Ecol. 2000; 9(10): 1657\u0026ndash;1659. \u003c/li\u003e\n\u003cli\u003eMontoya-Lerma J, Solarte YA, Giraldo-Calder\u0026oacute;n GI, Qui\u0026ntilde;ones ML, Ruiz-L\u0026oacute;pez F, Wilkerson RC, et al. Malaria vector species in Colombia: a review. Mem Inst Oswaldo Cruz. 2011; 106 (Suppl 1): 223-238. https://doi.org/10.1590/s0074-02762011000900028.\u003c/li\u003e\n\u003cli\u003eGabaldon, A. \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e: importante vector y agente de malaria refractaria en Venezuela. Bol Div Malariol San Amb. 1981; 31: 28-38.\u003c/li\u003e\n\u003cli\u003eCalado DC, Foster PG, Bergo ES, Santos CL, Galardo AK, Sallum MA. Resurrection of \u003cem\u003eAnopheles goeldii\u003c/em\u003e from synonymy with \u003cem\u003eAnopheles nuneztovari\u003c/em\u003e (Diptera, Culicidae) and a new record for \u003cem\u003eAnopheles dunhami\u003c/em\u003e in the Brazilian Amazon. Mem Inst Oswaldo Cruz. 2008; 103(8):791-799. https://doi.org/10.1590/s0074-02762008000800009.\u003c/li\u003e\n\u003cli\u003eMotoki MT, Wilkerson RC, Sallum MAM. The \u003cem\u003eAnopheles albitarsis\u003c/em\u003e complex with the recognition of \u003cem\u003eAnopheles oryzalimnetes\u003c/em\u003e Wilkerson and Motoki, n. sp. and \u003cem\u003eAnopheles janconnae\u003c/em\u003e Wilkerson and Sallum, n. sp. (Diptera: Culicidae). Mem Inst Oswaldo Cruz. 2009; 104(6): 823-850.\u003c/li\u003e\n\u003cli\u003eSant\u0026apos;Ana DC, Sallum MAM. Revision of the Strodei Subgroup of \u003cem\u003eNyssorhynchus \u003c/em\u003e(Diptera: Culicidae), with descriptions of 2 new species. J Med Entomol. 2024; 61(1):87-109. https://doi.org/10.1093/jme/tjad149. \u003c/li\u003e\n\u003cli\u003eSallum MA, Foster PG, Dos Santos CL, Flores DC, Motoki MT, Bergo ES. Resurrection of two species from synonymy of \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003estrodei\u003c/em\u003e Root, and characterization of a distinct morphological form from the Strodei Complex (Diptera: Culicidae). J Med Entomol. 2010; 47(4):504-526. https://doi.org/10.1603/me09229.\u003c/li\u003e\n\u003cli\u003eMoreno M, Bickersmith S, Harlow W, Hildebrandt J, McKeon SN, Silva-do-Nascimento TF, et al. Phylogeography of the neotropical \u003cem\u003eAnopheles triannulatus\u003c/em\u003e complex (Diptera: Culicidae) supports deep structure and complex patterns. Parasit Vectors. 2013; 6: 47. https://doi.org/10.1186/1756-3305-6-47.\u003c/li\u003e\n\u003cli\u003eHarbach RE. Mosquito Taxonomic Inventory. [\u003cem\u003eNyssorhynchus\u003c/em\u003e]. 2013. [Updated October 2024; cited 2024 Dec 3]. Available from: https://mosquito-taxonomic-inventory.myspecies.info/.\u003c/li\u003e\n\u003cli\u003eLaubach\u003csup\u003e \u003c/sup\u003eHE, Validum L, Bonilla JA, Agar A, Cummings R, Mitchell C, et al. Identification of \u003cem\u003eAnopheles aquasalis\u003c/em\u003e as a possible vector of malaria in Guyana, South America. West Indian Med J. 2001; 50(4): 319-21.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Zimmerman R H. Ecoregional classification of malaria vectors in the Neotropics. J Med Entomol.\u003cem\u003e \u003c/em\u003e1997; 34: 499-510.\u003c/li\u003e\n\u003cli\u003eWilkerson RC, Linton Y-M, Strickman D. Mosquitoes of the World. Vol. 2. Baltimore: Johns Hopkins University Press; 2021. 1308 pp.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Moreno JE, Moreno A, Silva J, P\u0026eacute;rez B, Guzm\u0026aacute;n H. An\u0026aacute;lisis morfom\u0026eacute;trico soporta la presencia de \u003cem\u003eAnopheles (Nyssorynchus) albitarsis\u003c/em\u003e F (Diptera: Culicidae) en Venezuela, importante vector de los par\u0026aacute;sitos mal\u0026aacute;ricos. Entomotropica. 2022; 37: 1-17. Available from: https://sventomologia.org/wpcontent/uploads/2022/07/2022_37_1-17.pdf.\u003c/li\u003e\n\u003cli\u003eRozeboom LE, Gabaldon A. A summary of the \u0026ldquo;\u003cem\u003etarsimaculatus\u003c/em\u003e\u0026rdquo; complex of \u003cem\u003eAnopheles \u003c/em\u003e(Diptera: Culicidae). Amer J Hyg. 1941; 33(C), 88\u0026ndash;100. https://doi.org/10.1093/oxfordjournals.aje.a118713.\u003c/li\u003e\n\u003cli\u003eFloch H, Abonnenc E. Sur \u003cem\u003eA. nu\u0026ntilde;ez-tovari \u003c/em\u003eet \u003cem\u003eA. pessoai \u003c/em\u003een Guyane Fran\u0026ccedil;aise. Table d\u0026rsquo;identification des \u003cem\u003eNyssorhynchus guyanais\u003c/em\u003e. Inst Pasteur Guyane Territ Inini Publ.\u003cem\u003e \u003c/em\u003e1946; 126:1-5.\u003c/li\u003e\n\u003cli\u003eLane J. Neotropical Culicidae. Vol. 1. S\u0026atilde;o Paulo: Editora da Universidade de S\u0026atilde;o Paulo; 1953. 548 pp.\u003c/li\u003e\n\u003cli\u003eFritz GN, Conn J, Cockburn AF, Seawright JA. Sequence analysis of the ribosomal DNA internal transcribed spacer 2 from populations of \u003cem\u003eAnopheles nuneztovari \u003c/em\u003e(Diptera: Culicidae). Mol Biol Evol. 1994; 11:\u003cem\u003e \u003c/em\u003e406-416.\u003c/li\u003e\n\u003cli\u003eFoster PG, Porangaba de Oliveira TM, Bergo ES, Conn JE, Sant\u0026rsquo;Ana DC, Nagaki SS, et al. Phylogeny of Anophelinae using mitochondrial protein coding genes. R Soc Open Sci. 2017; 4: 170758. http://dx.doi.org/10.1098/rsos.170758.\u003c/li\u003e\n\u003cli\u003eSallum MAM, Gonz\u0026aacute;lez Obando R, Carrejo N, Wilkerson RC. Identification keys to the \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes of South America (Diptera: Culicidae). IV. Adult females. Parasit Vectors. 2020; 13: 584. https://doi.org/10.1186/s13071-020-04301-0.\u003c/li\u003e\n\u003cli\u003eMoreno J, Rubio-Palis Y, S\u0026aacute;nchez V, Mariany D. Primer registro de \u003cem\u003eAnopheles\u003c/em\u003e\u003cem\u003e (Nyssorhynchus) nuneztovari \u003c/em\u003eGabaldon\u003cem\u003e \u003c/em\u003e1940 (Diptera: Culicidae) en el estado Bol\u0026iacute;var, Venezuela y sus implicaciones eco-epidemiol\u0026oacute;gicas. Entomotropica. 2004; 19(1): 55-58.\u003c/li\u003e\n\u003cli\u003eGabaldon A. Estudios sobre anofelinos. Serie I. 1. Descripci\u0026oacute;n de \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003en\u0026uacute;\u0026ntilde;ez-tovari\u003c/em\u003e n.sp y consideraciones sobre una sub-divisi\u0026oacute;n del grupo Nyssorhynchus (Diptera, Culicidae). Publ Div Malariol. 1940; 5: 3-7.\u003c/li\u003e\n\u003cli\u003eSavage HM. Identification and location of the holotype and paratypes of \u003cem\u003eAnopheles \u003c/em\u003e(\u003cem\u003eNyssorhynchus\u003c/em\u003e)\u003cem\u003e nuneztovari \u003c/em\u003eGabaldon (Diptera: Culicidae). Mosq Syst. 1986; 18\u003cem\u003e: \u003c/em\u003e279-283.\u003c/li\u003e\n\u003cli\u003eDos Santos MMM, Sucupira IMC, Dos Santos TV, Dos Santos ACE, Lacerda RNDL, P\u0026oacute;voa MM, et al. Morphological identification of species of the Nuneztovari Complex of \u003cem\u003eAnopheles\u003c/em\u003e (Diptera: Culicidae) from an area affected by a Brazilian hydroelectric plant. Zootaxa. 2019; 4565: 235-244.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Curtis CF. Biting and resting behavior of anophelines in western Venezuela and implications for control of malaria transmission. Med Vet Entomol. 1992; 6: 325-334. \u003c/li\u003e\n\u003cli\u003eMoreno\u003csup\u003e \u003c/sup\u003eJE, Rubio-Palis\u003csup\u003e \u003c/sup\u003eY, Bevilacqua\u003csup\u003e \u003c/sup\u003eM, S\u0026aacute;nchez\u003csup\u003e \u003c/sup\u003eV, Guzm\u0026aacute;n H. Caracterizaci\u0026oacute;n de h\u0026aacute;bitats larvales de anofelinos en la cuenca baja del R\u0026iacute;o Caura, estado Bol\u0026iacute;var, Venezuela. Bol Malariol Sal Amb. 2018; 58: 32-45. Available from: http://iaes.edu.ve/iaespro/ojs/index.php/bmsa/article/view/57/29.\u003c/li\u003e\n\u003cli\u003eMoreno JE, Rubio-Palis Y, S\u0026aacute;nchez V, Mart\u0026iacute;nez A. Caracterizaci\u0026oacute;n de h\u0026aacute;bitats larvales de anofelinos en el municipio Sifontes del estado Bol\u0026iacute;var, Venezuela.\u003cem\u003e \u003c/em\u003eBol Malariol Sal Amb. 2015;\u003cem\u003e \u003c/em\u003e55(2): 117-131. Available from: http://iaes.edu.ve/iaespro/ojs/index.php/bmsa/article/view/119/101.\u003c/li\u003e\n\u003cli\u003eMoreno JE,Rubio-Palis Y\u003cstrong\u003e, \u003c/strong\u003eS\u0026aacute;nchez V, Mart\u0026iacute;nez A. Fluctuaci\u0026oacute;n poblacional y h\u0026aacute;bitat larval de anofelinos en el municipio Sifontes, estado Bol\u0026iacute;var, Venezuela. Bol Malariol Sal Amb. 2015; 55(1): 52-68. Available from: http://iaes.edu.ve/iaespro/ojs/index.php/bmsa/article/view/107/54.\u003c/li\u003e\n\u003cli\u003eMcKeon SN, Lehr MA, Wilkerson RC, Ru\u0026iacute;z JF, Sallum MA, Lima JB, et al. Lineage divergence detected in the malaria vector \u003cem\u003eAnopheles marajoara\u003c/em\u003e (Diptera: Culicidae) in Amazonian Brazil. Malar J. 2010; 9: 271. https://doi.org/10.1186/1475-2875-9-271. \u003c/li\u003e\n\u003cli\u003eSallum MAM, Gonz\u0026aacute;lez Obando R, Carrejo N, Wilkerson RC. Identification key to the \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes of South America (Diptera: Culicidae). III. Male genitalia. Parasit Vectors. 2020; 13: 542. https://doi.org/10.1186/s13071-020-04300-1.\u003c/li\u003e\n\u003cli\u003eSilva-do-Nascimento TF, S\u0026aacute;nchez-Ribas J, Oliveira TMP, Bourke BP, Oliveira-Ferreira J, Rosa-Freitas MG, et al. Molecular analysis reveals a high diversity of Anopheline mosquitoes in Yanomami lands and the Pantanal region of Brazil. Genes. 2021; 12: 1995. https://doi.org/10.3390/genes12121995.\u003c/li\u003e\n\u003cli\u003eBranquinho MS, Araujo D, Natal MT, Marrelli RM, Rocha FAL, Taveira JK, et al. \u003cem\u003eAnopheles oswaldoi \u003c/em\u003ea potential malaria vector in Acre, Brazil. Trans R Soc Trop Med Hyg. 1996; 90: 233.\u003c/li\u003e\n\u003cli\u003eRubio-Palis Y, Bravo L, Guzm\u0026aacute;n H, Caura S, Song C, Wang S, et al. Response to chemical attractants and host seeking behavior of \u003cem\u003eAnopheles\u003c/em\u003e Meigen spp. (Diptera: Culicidae) in a malaria endemic area of Bol\u0026iacute;var State, Venezuela. Bol Malariol Sal Amb. 2021; 61(2): 267-274. https://doi.org/10.52808/bmsa.7e5.612.016.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"No particular funding","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anopheles albitarsis F, Anopheles goeldii, Anopheles oswaldoi complex, mtCOI","lastPublishedDoi":"10.21203/rs.3.rs-7584315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7584315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND \u003c/strong\u003eEffective interventions for controlling \u003cem\u003eAnopheles \u003c/em\u003emalaria vectors depend on accurately identifying the species. Venezuela has the highest malaria prevalence in the region of the Americas; however, there is very limited knowledge about the vectors, especially regarding species complexes and their distribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOBJECTIVES \u003c/strong\u003eTo conduct molecular taxonomy and phylogenetic analysis of the Albitarsis, Nuneztovari, and Oswaldoi complexes from malaria-endemic regions of Guyana and Venezuela using \u003cem\u003emtCOI\u003c/em\u003e sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS \u003c/strong\u003e\u003cem\u003eAnopheles\u003c/em\u003e were collected in Guyana (Potaro Region) and Venezuela (five municipalities). Species identification was carried out by analyzing mitochondrial cytochrome C oxidase (\u003cem\u003emtCOI\u003c/em\u003e) gene data using MrBayes, TCS, Posterior Probabilities of Correct Identification (P ID), Rosenberg’s P (AB) values, and P (Randomly Distinct) as species delimitation approaches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFINDINGS \u003c/strong\u003eFour distinct taxa were identified: \u003cem\u003eAn. albitarsis\u003c/em\u003e F, \u003cem\u003eAn. goeldii\u003c/em\u003e, \u003cem\u003eAn. oswaldoi\u003c/em\u003e A and \u003cem\u003eAn. oswaldoi\u003c/em\u003eB. \u003cem\u003eAnopheles oswaldoi\u003c/em\u003e A and \u003cem\u003eAn. oswaldoi\u003c/em\u003e B occurred sympatrically in Boca de Nichare, Bolívar State, Venezuela.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION \u003c/strong\u003e\u003cem\u003eAnopheles goeldii\u003c/em\u003e is reported for the first time from Guyana and Venezuela, and discuss its role as vector of malaria parasites in both countries and its relative recent colonization. \u003cem\u003eAnopheles oswaldoi \u003c/em\u003eA is reported for the first time from Venezuela.\u003cem\u003e Anopheles albitarsis\u003c/em\u003e F, an important malaria vector in Colombia and Venezuela, is confirmed as the only species of the Albitarsis complex widely distributed in Venezuela. Our results will be valuable for future research aimed at clarifying the status of these significant species complexes in the region.\u003c/p\u003e","manuscriptTitle":"Anopheles species complexes from Guyana and Venezuela malaria endemic areas using COI sequences","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 06:21:01","doi":"10.21203/rs.3.rs-7584315/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"baa7a591-822b-4f41-becd-ca95f8bc180c","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54520210,"name":"Entomology"}],"tags":[],"updatedAt":"2025-09-17T06:21:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 06:21:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7584315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7584315","identity":"rs-7584315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}