Natural Plasmodium infection of Anopheles benarrochi B (Diptera: Culicidae) in native communities of the Province of Condorcanqui, Amazonas-Peru

Natural Plasmodium infection of Anopheles benarrochi B (Diptera: Culicidae) in native communities of the Province of Condorcanqui, Amazonas-Peru | 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 Natural Plasmodium infection of Anopheles benarrochi B (Diptera: Culicidae) in native communities of the Province of Condorcanqui, Amazonas-Peru Marianella Villegas-Pingo, Jhon Zumaeta, Luis M. Rojas, Lizandro Gonzales, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3409121/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 Malaria is a severe health problem in the native communities of Condorcanqui in the Amazonas Department of Peru. Recently, the number of malaria cases has increased considerably following a Plasmodium falciparum outbreak in 2019. However, there is no information on the anopheline species acting as Plasmodium vectors in this area or its insecticide resistance status. This study aims to: i) to molecularly characterize the anopheline population from the district of Rio Santiago; ii) to determine their incrimination in malaria transmission; and iii) to evaluate mutations associated with resistance to pyrethroid insecticides and DDT in the mosquito population. Methods Mosquitoes were collected between March and September 2022, using Shannon traps, CDC light traps, and mouth aspirators. Only those morphologically identified as Anopheles sp. were subjected to molecular confirmation by PCR amplification and sequencing of the COX1 barcode region. Additionally, specimens that were molecularly confirmed as Anopheles were analyzed for the kdr region of the VGSC gene related to insecticide resistance. Likewise, the presence of human blood as a food source was detected using the β-globin marker, and the presence of P. falciparum and Plasmodium vivax was determined through a nested PCR. Results A total of 453 mosquitoes were captured, of which ninety-four were morphologically identified as female anophelines. Of the latter, sixty-six (~ 70%) specimens were molecularly confirmed as anophelines and were grouped into four species: An. benarrochi B, An. triannulatus, An. costai and An. nimbus . The sixty-six anophelines were analyzed for human β-globin and Plasmodium . It was found that twenty-three samples of An. benarrochi B (~ 35%) and one specimen of An. triannulatus were positive for human β-globin. Likewise, six (~ 9%) samples of An. benarrochi B were positive for Plasmodium parasites (four for P. falciparum and two for P. vivax ). It is worth noting that four specimens tested positive for Plasmodium parasites and human blood simultaneously, making this a robust outcome to incriminate An. benarrochi B as the main malaria vector. No specimens presented mutations associated with insecticide resistance in the kdr region. Conclusions An. benarrochi B is the dominant anopheline species in this study and plays an important role in malaria transmission. Further studies are needed to understand its feeding behavior and activity during dry and rainy seasons to fully incriminate it with malaria transmission and implement targeted vector control programs. Plasmodium Anopheles COX1 kdr Malaria Native communities Amazonas Peru Figures Figure 1 Figure 2 Figure 3 Figure 4 BACKGROUND In Peru, 26,652 malaria cases were reported in 2022, with ~ 84% Plasmodium vivax and ~ 15% P. falciparum cases [ 1 ], nevertheless, malaria infections remain uncertain in remotes areas because control activities have been postponed owing to COVID-19. Although Loreto is one of the most affected departments in the country (89% of malaria cases) [ 2 ], Amazonas, a northeastern department of Peru, has reported a significant increase in the number of malaria cases for the past six years, from 710 cases in 2018 to 1657 in 2022 [ 3 ]. In 2019, a 2.5-fold increase was reported due to an outbreak of autochthonous P. falciparum cases in native communities of Rio Santiago, in the Condorcanqui Province of Amazonas, this outbreak was triggered by an index case of P. falciparum imported from Loreto, which subsequently spread to other communities during that year [ 3 ]. However, there is limited information about the circulating vectors involved in malaria transmission in these communities. In the Peruvian Amazon, the species recognized as the main malaria vector is Anopheles darlingi Root [ 4 ]; this highly anthropophilic species has been reported in the departments of Madre de Dios [ 5 ], Loreto [ 6 , 7 ], Ucayali and San Martin [ 8 ]. Other potential malaria vectors, An. triannulatus (Neiva & Pinto) and An. benarrochi (Cova García & López) have been reported in eastern and in western Loreto, respectively [ 9 ]. Previous studies suggested that An. benarrochi is a species complex consisting in four distinct species ( An. benarrochi B, An. benarrochi G1, An. benarrochi G2 and An. benarrochi ) [ 10 ], from which An. benarrochi B has been identified as a malaria vector in northern Loreto and Madre de Dios [ 11 ]. Efforts to eliminate malaria transmission in native communities of Rio Santiago have focused on rapid diagnosis, case management, insecticide-treated nets (ITNs), and indoor and outdoor residual spraying programing by the MoH. However, this progress can be undermined by parasites´ resistance to antimalarial, and also by mosquitoes’ resistance to insecticides and vector´s behavior. According to the World Malaria Report 2022, a total of eighty-eight countries reported insecticide resistance in the last decade. Among these countries, twenty-nine documented resistance to four primary classes of insecticides (pyrethroids, organophosphates, carbamates, and organochlorines) at various locations within their territories [ 12 ]. One of the primary mechanisms related to pyrethroid resistance, known as knockdown resistance (kdr), involves mutations at the kdr region of the voltage-gated sodium channel gene (VGSC), which is the primary target of synthetic pyrethroids [ 13 ]. In South America, An. darlingi resistance to dichlorodiphenyltrichloroethane (DDT) and pyrethroids has been reported in western Colombia [ 14 ], while on the northwestern coast of Peru, An. albimanus , has shown cross-resistance to all classes of insecticides [ 15 ]. In this study, a molecular characterization of mosquitoes collected in native communities of Rio Santiago was evaluated in order to identify species of Anopheles mosquitoes, incriminate them as malarial vectors, and evaluate insecticide resistance by the kdr region marker. METHODS Study area and mosquito collection Approximately, 99% of malaria cases (1633 malaria cases in 2022) [ 16 ] in the Amazonas Department were reported in the Rio Santiago District (Condorcanqui Province). This is a remote impoverished area where native communities live on the banks of the river, with no electricity, drinking water, or road access, with the rivers as the primary means of transportation [ 3 ]. Mosquito collections were conducted in four native communities of Rio Santiago: Alianza Progreso (AP), Nueva Esperanza (NE), Chapiza (CH), and Caterpiza (CT) (Fig. 1 ), located within the ecosystem of the Amazonian Humid forests with temperatures that can reach 35ºC, an average annual rainfall of around 4,800 mm, and a relative humidity of above 90% [ 3 , 17 ]. Adult mosquitoes were collected for six nights during the rainy season between March and September of 2022.Mosquitoes were captured with three Shannon traps and then, with the help of mouth aspirators, the mosquitoes were transferred to cryovials. Likewise, two light traps from the Centers for Disease Control and Prevention (CDC) were used, last two traps placed ~ 10 m from the houses and ~ 10 m from the forest entrance between 18:00 and 22:00 [ 18 , 19 ]. Anopheles mosquitoes were identified in the field based on their morphological characteristics using entomological keys [ 20 ], then they were stored in 1.5ml cryovials containing 70% ethanol. Samples were classified and stored by date and location; additional information such as temperature and relative humidity was also collected. DNA isolation, amplification, and sequencing of COX1 and kdr Genomic DNA was extracted from the whole body of each specimen using a DNeasy Blood & Tissue kit (Qiagen) following the manufacturer's instructions. The 710bp barcode region of the mitochondrial Cytochrome Oxidase I gene (COX1) was amplified for all samples using the LCO1490 and HCO2198 primers [ 21 ]. The 25 µl PCR reaction included 1ul of extracted DNA, 0.5 µM of each primer, 1 unit of Platinum Taq DNA polymerase (Invitrogen), 0.2mM dNTP, 1X PCR buffer (Invitrogen), 2.5mM MgCl 2 and nuclease-free water. Thermocycling conditions were used according to the literature [ 22 ]. PCR amplification of segment 6 of domain II (IIS6) of the VGSC gene (approximately 225 bp), corresponding to the kdr region, was amplified using AAKDRF2 and AAKDRR primers [ 23 ]. The 25 µl PCR reaction included 5 µl of extracted DNA, 0.5 µM of each primer, 1 unit of Platinum Taq DNA polymerase (Invitrogen), 0.2 mM dNTP, 1X PCR buffer (Invitrogen), 2.0 mM MgCl2 and nuclease-free water. Thermocycling conditions were used according to the literature [ 24 ]. Amplicons were visualized on 2% agarose gels stained with SafeView™ Classic (Applied Biological Materials). PCR products were purified using the Exo-CIP™ kit (New England Biolabs), and sequencing was performed on a 3500 Genetic Analyzer (Applied Biosystems™). Bioinformatic analysis The sequence data was analyzed using the Geneious Prime (version 2022.2.1, Biomatters Inc, Newark) ( https:/www.geneious.com ), and Basic Local Alignment Search Tool (BLAST) searches were carried out for species identification with sequences available in the GenBank database. Species were determined based on query coverage (QC) and percentage identity (PI). All anopheline sequences were aligned using the MUSCLE tool, implemented in the MEGA software (version 7.2.6.1). In addition, 77 previously reported sequences of different anopheline species were also included in the phylogeny (Additional file 1: Table S1 ) [ 10 , 25 , 26 , 27 , 28 , 29 ]. A Maximum Likelihood (ML) tree was constructed using a Kimura 2-parameter model (K-2P) and the Bootstrap method with 1000 bootstrap replicates to examine phylogenetic relationships in MEGA X software [ 30 ]. The Aedes aegypti COX1 sequence (NC035159) was used as an outgroup to root the tree. An. benarrochi haplotypes from this study and from 59 previously reported sequences (Additional file 1: Table S2 ) were determined using DnaSP v6 [ 31 ], and the haplotype network was constructed using the Median-Joining algorithm in PoPART software [ 25 ]. For kdr analysis, An. darlingi (MN062262) and An . albitarsis (MW315118) were used as references. Human blood meal identification and Plasmodium detection Source of mosquitoes blood meal was assessed using a human β-globin protocol previously reported [ 32 ]; the thermal profile consisted of an initial denaturation step at 94°C for 7 min; followed by 35 cycles at 94°C for 1 minute for denaturation, 53°C for 1 minute for hybridization, and 72°C for 1 minute for extension; and then a final extension at 72°C for 5 minutes. The detection of P. falciparum and P. vivax infections in mosquitoes was performed using the 18S rRNA subunit nested PCR technique according to the protocol previously described [ 33 ]. RESULTS Molecular identification of mosquitoes The environmental conditions during the collection dates were very similar, with a temperature range from 25ºC to 28ºC and humidity levels between 75% and 87% (Table 1 ). A total of 453 individuals were captured using Shannon traps and then transferred into cryovials using mouth aspirators. None individuals were captured using CDC-LT traps. Ninety-four females were initially identified as Anopheles based on morphological characteristics (Table 1 ) and the remaining species were identified morphologically as Culicidae (detailed data not shown); After molecular confirmation, it was found that only sixty-six individuals belonged to the Anopheles genus (AP = 55, CT = 5, NE = 5, and CH = 1) and the remaining twenty-eight mosquitoes were molecularly confirmed as other non-anopheline Culicidae. Table 1 Mosquito specimens (n = 94) collected in Rio Santiago, Condorcanqui province, 2022. Alianza Progreso (AP = 81), Nueva Esperanza (NE = 5), Chapiza (CH = 1), and Caterpiza (CT = 7) Site Coordinates Date T° (°C) RH (%) Number of specimens Molecular Identification NE 196280.9 9581345.6 02/03/2022 27 75 5 An. benarrochi B CH 199060.7 9587806.4 02/03/2022 27 87 1 An. benarrochi B AP 195051.2 9592391.3 01/03/2022 26 75 13 An. benarrochi B 01/03/2022 26 75 2 An. triannulatus 23/05/2022 28.1 83 13 An. benarrochi B 23/05/2022 28.1 83 15 Coquillettidia venezuelensis 24/05/2022 26 86 15 An. benarrochi B 24/05/2022 26 86 5 Coquillettidia venezuelensis 25/05/2022 25.3 87 9 An. benarrochi B 25/05/2022 25.3 87 2 An. costai 25/05/2022 25.3 87 1 An. nimbus 25/05/2022 25.3 87 6 Coquillettidia venezuelensis CT 198182.6 9566709.0 20/09/2022 25.3 87 5 An. benarrochi B 20/09/2022 25.3 87 1 Sabethes sp 20/09/2022 25.3 87 1 Culex bastagarius An. benarrochi B was found to be the predominant species (include %, 51/66 specimens) with the presence of An. triannulatus (2/66), An. costai (2/66) and An. nimbus (1/66) (Additional file 1: Table S3). Among non-anopheline mosquitoes, Coquillettidia venezuelensis (26/28) , one Sabethes sp, and one Culex bastagarius specimens were also molecularly identified (Additional file 1: Table S4). According to the median-joining haplotype network based on COX1 (Fig. 2 ), there were thirty-six An. benarrochi B haplotypes, from which six were found in Amazonas. Haplotype 1 was found in Amazonas and also in Ecuador, which is expected due to proximity. Haplotypes 3, 5 and 6 were only found in Amazonas, while haplotypes 2 and 4 were found in Amazonas and Loreto Department. A ML K-2P tree (Fig. 3 ) further supported the correct identification of the specimens in this study. Additionally, the species clade assignment corresponded to the traditional subgenus classification [ 34 ]; specifically, species such as An. benarrochi B, An. benarrochi, An. rangeli, An. konderi, An. albimanu s and An. triannulatus are included in the Nyssorhynchus subgenus, while An . nimbus and An . kompi belonged to the Stethomyia subgenus group and An. costai to the Anopheles subgenus group. Blood meal source and Plasmodium identification in mosquitoes Anopheles spp. During morphological identification, it was observed that certain specimens presented an enlarged abdomen, which was attributed to their blood meal (Additional file 2: Fig. S1 ). Of the sixty-six anophelines molecularly confirmed, An. benarrochi B (twenty-three samples) and An. triannulatus (one sample) were positive to human β-globin. Furthermore, six specimens of An. benarrochi B were found to be positive for Plasmodium (four P. falciparum , and two P. vivax ) (Additional file 1: Table S3). Notably, four samples exhibited the presence of Plasmodium parasites and human blood, suggesting the incrimination of Anopheles benarrochi B in malaria transmission in the region. Identification of insecticide-resistance genotype in the kdr region Out of the total sixty-six anophelines, sixty-four successfully underwent amplification and genotyping for the kdr region (Fig. 4 , Additional file 1: Table S3). These sequences were further examined to identify mutations in codons 1010, 1013, and 1014 [ 24 ]. No missense mutations were detected in any of the sequences. All sequences of An. benarrochi B and An. triannulatus showed the GTT codon for valine at position 1010 (V1010), AAC codon for asparagine at position 1013 (N1013), and TTA codon for leucine at position 1014 (L1014), suggesting that the collected anopheline population has wild-type genotypes related to susceptibility to DDT and pyrethroid insecticides. Only the An. nimbus sequence showed a synonymous mutation at position V1010 (GTA). On the other hand, two An. benarrochi B haplotypes differed in a C/A substitution (at position 45); however, it should be noted that these mutations correspond to synonymous substitutions in codons unrelated to insecticide resistance. Similarly, the two An. triannulatus sequences differed in an A/G substitution in the intronic region. Further analysis of the intron downstream kdr revealed size variations among the species. The intron of An. nimbus (90 bp) was found to be larger than that of An. benarrochi B (72 bp) and An. triannulatus (75 bp), which also differed from the reference sequences ( An. darlingi wildtype and An. albitarsis mutated at 1014 codon). DISCUSSION This study is the first report on the molecular characterization of anopheline species circulating in native communities in Condorcanqui – Amazonas Department, Peru. The primary objective was to identify the malaria vector species in the area to maximize the impact of malaria control strategies. In the eastern region of Peru, An. darlingi has been recognized as the main malaria vector; however, it is plausible that other Anopheles species could be transmitting malaria in endemic areas where An. darlingi is absent [ 8 ]. For instance, An . triannulatus was reported as the dominant vector in eastern Loreto, while An. benarrochi was mostly found in western Loreto [ 8 , 35 ]. Two anopheline species, An . benarrochi B (56/66) and An. triannulatus (2/66), both belonging to the Nyssorhynchus subgenus, were successfully identified in this study. Furthermore, the network analysis and ML phylogenetic tree showed that An. benarrochi B sequences were strongly linked to previously reported sequences in Ecuador, Colombia [ 25 ], Peru [ 11 , 29 , 25 ] and Brazil [ 10 ]. The presence of An. benarrochi B as a malaria vector in an endemic area is usually associated with the absence of An. darlingi [ 36 ]. Our findings further support this hypothesis, since An. benarrochi B was identified as the predominant species in all four communities in all sampling periods. Likewise, it should be emphasized that An. benarrochi B has already been identified in other regions of Peru [ 25 , 11 , 35 ], and with the present study, it extends its distribution to the northwest of the country, including three new haplotypes. Malaria cases in these communities have been significantly on the rise in the last years. In 2020, the Chapiza Health Establishment reported a high Annual Parasite Index (API) of 122.8, which further increased to 224.8 by 2022. The cases included infections caused by both P. falciparum and P. vivax . Here, we determined the presence of these parasites in specimens of An. benarrochi B from the Alianza Progreso community, which belongs under the jurisdiction of the Chapiza Health Center. A similar situation was observed at the Caterpiza Health Establishment, where the API was 9.7 in 2020 (considered a moderate risk) and it spiked to 812.7 (classified as high risk) with only P. vivax cases in 2022. Interestingly, the anophelines captured in Caterpiza did not test positive for Plasmodium , but they did test positive for the human B-globin marker, confirming the anthropophilic behavior of An. benarrochi B. Moreover, a total of six out of sixty-six anophelines were found to be infected with Plasmodium , which represents a highly significant percentage compared to what was reported previously [ 11 , 35 , 37 ]. Considering that the specimen’s collection was made after the rainy season, with high mosquitoes' density and in areas with APIs of moderate to high risk, it may explain the high number of infected mosquitoes found in the study. A limitation of this study is the low number of entomological surveys in all communities (one sampling night in each one) preventing the calculation of reliable entomological indices. Nevertheless, the data provides evidence of the importance of Anopheles benarrochi B as a malaria vector in this unexplored area, although implication of other anopheline species should be investigated. Furthermore, since 2019, there has been a significant shift in the epidemiological landscape of malaria in Condorcanqui, given the emergence of P. falciparum [ 3 ]; it is suspected that alterations in vector composition, the high human biting rate and peak biting time (∼18:00–20:00) [ 35 ] could have also contributed to this event. On the other hand, the genetic diversity of An. costai and An. nimbus has received limited attention in previous studies. The lack of research on these species may be attributed to their lack of implication as significant malaria vectors. They have been predominantly observed in forest habitats and have often been misidentified as An. mediopunctatus , which has been found to carry P. vivax [ 38 ]. There is limited information regarding insecticide resistance in the most significant malaria vectors in Latin America [ 24 ] and conducting insecticide resistance studies in the Peruvian Amazon region is challenging due to limited accessibility, inadequate laboratory capacity, and a shortage of trained personnel [ 39 ]. In this study, genotypes linked to resistance to DDT and pyrethroids were not detected within the kdr region of any of the sixty-six Anopheles specimens captured in the Rio Santiago district. Further investigations involving a larger number of mosquitoes are essential to evidence the susceptibility status of main malaria vectors to develop successful vector surveillance programs in Peru. Additionally, noteworthy findings include the identification of synonymous mutations in An. benarrochi B (in the exonic region) and An. triannulatus (in the intronic region) and variations in the intronic size within the species. The analysis of intron sequences, including nucleotide positions and sizes, may support the hypothesis of interspecific differences and offers potential as a tool for taxonomic classification [ 23 , 24 ]. Regarding other Anopheline species, several studies have reported the absence of mutations in the kdr region associated with insecticide resistance. Wild-type kdr alleles were found in An. nuneztovari s.l. An. darlingi , and An . albimanus from Valle del Cauca, Colombia [ 24 ] and in An. albimanus populations from Guatemala, Ecuador, and Colombia [ 23 ]. On the other hand, specific mutations at codons 1010, 1014 and 1014 in the kdr region have been associated with pyrethroid resistance [ 40 ]. In Peru, resistance to pyrethroids was reported in An. darlingi in Loreto [ 41 ] and a high frequency of TCG (L1014S) and TGT (L1014C) associated with resistance in An. albimanus was revealed in Tumbes through RNA-seq analysis [ 42 ]. CONCLUSION This study reveals the presence of Anopheles benarrochi B and its natural infection with Plasmodium in unexplored native communities in the Amazonas region highlighting its role as a major malaria vector in this area, although other anopheline species might be also involved. This research also evidences the wider distribution of An. benarrochi B in northern Peru. Furthermore, the fraction of mosquito population analyzed showed wild-type/susceptible kdr genotypes in native communities of the province of Condorcanqui, providing a base line for insecticide surveillance and control interventions in the area. More entomological surveys in these communities of the province of Condorcanqui are required to characterize vector bionomics and behavior across seasons to unveil the species involved in malaria transmission. This will help in the design and implementation of specific programs to combat malaria by the Condorcanqui Health Network. Abbreviations BLAST Basic Local Alignment Search Tool CDC Centers for Disease Control and Prevention COX1 Mitochondrial Cytochrome Oxidase I gene DNA Deoxyribonucleic acid ELISA Enzyme-linked immunosorbent assay. IET Instituto de Enfermedades Tropicales KDR Knockdown resistance PCR Polymerase chain reaction. PI Percent identity rRNA Ribosomal ribonucleic acid. QC Query coverage VGSC Voltage-Gated Sodium Channel gene Declarations Acknowledgements The authors express their sincere thanks to colleagues of the Institute of Tropical Diseases (IET-UNTRM) and Dirección Regional de Salud de Amazonas. Funding Proyecto METAVEC N°: 050-2021-FONDECYT Ethics declaration National Forestry and Wildlife Service. Authorization code No AUT-IFS-2021-071 and Ethics committee approval from UNTRM CIEI-N° 011. Contributions SC and RT conceived and designed the study. LG, MM and CG, advised on interventions, study communities and coordination with local and national authorities. SC, RT, LR and LG implemented the study. MVP and JZ analysed the data. MVP, JZ, SC and RT interpreted the data. MVP & JZ wrote the first draft of the manuscript. SC, MM & CG critically revised the manuscript for important content. All authors read and approved the final version of the manuscript. References MINSA. Sala de Situación de Salud Perú a la SE 52–2022. Perú Ministerio de Salud. ; 2022. https://www.dge.gob.pe/epipublic/uploads/asis-sala/asis-sala_202252_09_234037.pdf . Accessed 23 March 2023. MINSA. Sala de Situación de Salud Perú a la SE 33–2023. Perú: Ministerio de Salud. ; 2023. https://www.dge.gob.pe/portal/docs/vigilancia/sala/2023/salaSE33.pdf . Accessed 30 August 2023. Montenegro CC, Bustamante-Chauca TP, Pajuelo Reyes C, Bernal M, Gonzales L, Tapia-Limonchi R, et al. Plasmodium falciparum outbreak in native communities of Condorcanqui, Amazonas, Perú. Malar J. 2021;20(1):88. MINSA. Sala de Situación de Malaria Perú a la SE 33–2020. Perú: Ministerio de Salud. ; 2020. http://www.dge.gob.pe/portal/docs/tools/teleconferencia/2021/SE012021/03.pdf . Accessed 30 August 2022. Tineo E, Medina A, Fallaque C, Chávez L, Quispe S, Mercado M, et al. Distribución geográfica y comportamiento estacional de la picadura del Anopheles ( Nyssorhynchus ) darlingi Root, 1926 en localidades de la frontera Perú-Bolivia, Madre de Dios, Perú. Rev Peru Med Exp Salud Publica. 2003;20(2):78–83. Reinbold-Wasson DD, Sardelis MR, Jones JW, Watts DM, Fernandez R, Carbajal F, et al. Determinants of Anopheles seasonal distribution patterns across a forest to periurban gradient near Iquitos, Peru. Am J Trop Med Hyg. 2012;86(3):459–63. Moreno M, Saavedra MP, Bickersmith SA, Lainhart W, Tong C, Alava F, et al. Implications for changes in Anopheles darlingi biting behaviour in three communities in the peri-Iquitos region of Amazonian Peru. Malar J. 2015;14(1):290. Flores-Mendoza C, Fernández R, Escobedo-Vargas KS, Vela-Perez Q, Schoeler GB. Natural Plasmodium infections in Anopheles darlingi and Anopheles benarrochi (Diptera: Culicidae) from eastern Peru. J Med Entomol. 2004;41(3):489–94. Aramburú JG, Ramal CA, Witzig R. Malaria reemergence in the Peruvian Amazon region. Emerg Infect Dis. 1999;5(2):209–15. Bourke BP, Conn JE, de Oliveira TMP, Chaves LSM, Bergo ES, Laporta GZ, et al. Exploring malaria vector diversity on the Amazon Frontier. Malar J. 2018;17(1):342. Conn JE, Moreno M, Saavedra M, Bickersmith SA, Knoll E, Fernandez R, Vera H, Burrus RG, Lescano AG, Sanchez JF, Rivera E, Vinetz JM. Molecular taxonomy of Anopheles ( Nyssorhynchus ) benarrochi (Diptera: Culicidae) and malaria epidemiology in Southern Amazonian Peru. Am J Trop Med Hyg. 2013;88(2):319–24. WHO. World malaria report 2022. Geneva: World Health Organization. ; 2022. https://www.who.int/publications/i/item/9789240064898 . Accessed 21 May 2023. Zlotkin E. The insect voltage-gated sodium channel as target of insecticides. Annu Rev Entomol. 1999;44(1):429–55. Suarez MF, Quiñones ML, Palacios JD, Carrillo A. First record of DDT resistance in Anopheles darlingi. J Am Mosq Control Assoc. 1990;6(1):72–4. Vargas FV, Córdova PO, Alvarado AA. Determinación de la resistencia a insecticidas en Aedes aegypti, Anopheles albimanus y Lutzomyia peruensis procedentes del Norte Peruano. Rev Peru Med Exp Salud Publica. 2006;23(4):259–64. MINSA. Número de casos de malaria, Perú 2019–2022. 2022. Sala de Situación de Salud Perú a la SE 52–2022. Perú Ministerio de Salud; 2022. https://www.dge.gob.pe/portal/docs/vigilancia/sala/2022/SE52/malaria.pdf Accessed 19 September 2023. Gobierno Regional de Amazonas. Estudio de diagnóstico y zonificación para el tratamiento de la demarcación territorial de la provincia Condorcanqui. Amazonas: Dirección Nacional Técnica de Demarcación Territorial; 2014. Need JT, Rogers EJ, Phillips IA, Falcon R, Fernandez R, Carbajal F, Quintana J. Mosquitoes (Diptera: Culicidae) Captured in the Iquitos Area of Peru. J Med Entomol. 1993;30(3):634–8. Neves A, Urbinatti PR, Malafronte R dos, Fernandes S, da Silva Paganini A, Natal W. Malaria outside the Amazon region: Natural Plasmodium infection in anophelines collected near an indigenous village in the Vale do Rio Branco, Itanhaém, SP, Brazil. Acta Trop. 2013;125(1):102–6. Forattini OP. Entomológica Médica. Tomo 1. Brasil: Faculdade de Higiene e Saude Publica; Sao Paulo; 1962. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3(5):294–9. Linton Y-M, Pecor JE, Porter CH, Mitchell LB, Garzón-Moreno A, Foley DH, et al. Mosquitoes of eastern Amazonian Ecuador: biodiversity, bionomics and barcodes. Mem Inst Oswaldo Cruz. 2013;108(Supp 1):100–9. Lol JC, Castellanos ME, Liebman KA, Lenhart A, Pennington PM, Padilla NR. Molecular evidence for historical presence of knock-down resistance in Anopheles albimanus, a key malaria vector in Latin America. Parasit Vectors. 2013;6(1):268. Orjuela LI, Álvarez-Diaz DA, Morales JA, Grisales N, Ahumada ML, Venegas HJ, et al. Absence of knockdown mutations in pyrethroid and DDT resistant populations of the main malaria vectors in Colombia. Malar J. 2019;18(1):384. Morales Viteri D, Herrera-Varela M, Albuja M, Quiroga C, Diaz G, Del Aguila Morante C, et al. New records of Anopheles benarrochi B (Diptera: Culicidae) in malaria hotspots in the Amazon regions of Ecuador and Peru. J Med Entomol. 2021;58(3):1234–40. Foster PG, Bergo ES, Bourke BP, Oliveira TMP, Nagaki SS, Sant’Ana DC, Sallum MAM. Phylogenetic Analysis and DNA-based Species Confirmation in Anopheles ( Nyssorhynchus ). PLoS ONE. 2013;8(2):e54063. Chan-Chable RJ, Martínez-Arce A, Mis-Avila PC, Ortega-Morales AI. DNA Barcodes and Evidence of Cryptic Diversity of Anthropophagous Mosquitoes in Quintana Roo, Mexico. Ecol Evol. 2019;9(8):4692–705. McKeon SN, Moreno M, Sallum MA, Povoa MM, Conn JE. Distinct Population Structure for Co-Occurring Anopheles goeldii and Anopheles triannulatus in Amazonian Brazil. Mem Inst Oswaldo Cruz. 2013;108(5):605–15. Prussing C, Saavedra MP, Bickersmith SA, Alava F, Guzmán M, Manrique E, et al. Malaria vector species in Amazonian Peru co-occur in larval habitats but have distinct larval microbial communities. PLoS Negl Trop Dis. 2019;13(5):e0007412. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9. Gutiérrez LA, Naranjo NJ, Cienfuegos AV, Muskus CE, Luckhart S, Conn JE, et al. Population structure analyses and demographic history of the malaria vector Anopheles albimanus from the Caribbean and the Pacific regions of Colombia. Malar J. 2009;8(1):259. Virreira M, Torrico F, Truyens C, Alonso-Vega C, Solano M, Carlier Y, et al. Comparison of polymerase chain reaction methods for reliable and easy detection of congenital Trypanosoma cruzi infection. Am J Trop Med Hyg. 2003;68(5):574–82. Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg. 1999;60(4):687–92. Foster PG, de Oliveira TMP, 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(11):170758. Conn JE, Bickersmith SA, Saavedra MP, Morales JA, Alava F, Diaz Rodriguez GA, del Aguila Morante CR, Tong CG, Alvarez-Antonio C, Daza Huanahui JM, Vinetz JM, Gamboa D. Natural Infection of Nyssorhynchus darlingi and Nyssorhynchus benarrochi B with Plasmodium during the Dry Season in the Understudied Low-Transmission Setting of Datem del Marañon Province, Amazonian Peru. Am J Trop Med Hyg. 2023;109(2):288–95. Schoeler GB, Flores-Mendoza C, Fernández R, Davila JR, Zyzak M. Geographical distribution of Anopheles darlingi in the Amazon Basin region of Peru. J Am Mosq Control Assoc. 2003;19(4):286–96. Neafsey DE, Taylor AR, MacInnis BL. Advances and opportunities in malaria population genomics. Nat Rev Genet. 2021;22(8):502–17. Sallum MA, Wilkerson RC, Forattini OP. Taxonomic study of species formerly identified as Anopheles mediopunctatus and resurrection of An. costai (Diptera: Culicidae). J Med Entomol. 1999;36(3):282–300. WHO. World malaria report 2021. Geneva: World Health Organization. ; 2021. https://www.who.int/publications/i/item/9789240040496 . Accessed 31 August 2023. Zhang H, Li M, Tan R, Deng C, Huang B, Wu Z, et al. Presence of L1014F Knockdown-Resistance Mutation in Anopheles gambiae s.s. From São Tomé and Príncipe. Front Cell Infect Microbiol. 2021;11:633905. Laboratorio de Referencia Nacional de Entomología. Centro Nacional de Salud Pública, Instituto Nacional de Salud. Vigilancia de la resistencia a los insecticidas en artrópodos vectores. Bol Inst Nac Salud. 2018;24(3–4):31–3. Mackenzie-Impoinvil L, Weedall GD, Lol JC, Pinto J, Vizcaino L, Dzuris N, et al. Contrasting patterns of gene expression indicate differing pyrethroid resistance mechanisms across the range of the New World malaria vector Anopheles albimanus. PLoS ONE. 2019;14(1):e0210586. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1.TablesS1S2S3S4.xlsx Additional File 1. Table S1: Sequences available in GenBank used to construct the Maximum Likelihood (ML) phylogenetic tree. Table S2: Genbank data of Anopheles benarrochi B used for the 469-bp COI median-joining haplotype network. Table S3: Molecular identification of anophelines collected in the four native communities of Condorcanqui, Amazonas. Table S4: Molecular identification of non-anophelines collected in the study area AdditionalFile2.FigureS1.png Additional File 2. Figure S1: A) An. benarrochi B with a reduced abdomen before a blood meal. B) An. benarrochi B with an enlarged abdomen after a blood meal. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3409121","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237731314,"identity":"b6becdaf-8a89-453d-b457-89c4f60ea451","order_by":0,"name":"Marianella Villegas-Pingo","email":"","orcid":"","institution":"Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marianella","middleName":"","lastName":"Villegas-Pingo","suffix":""},{"id":237731315,"identity":"c7975ac3-b661-4663-8acb-a348d8ff5557","order_by":1,"name":"Jhon Zumaeta","email":"","orcid":"","institution":"Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jhon","middleName":"","lastName":"Zumaeta","suffix":""},{"id":237731316,"identity":"d3209a51-8876-4b18-8c2d-e1ac7b352b95","order_by":2,"name":"Luis M. Rojas","email":"","orcid":"","institution":"Dirección Regional de Salud","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luis","middleName":"M.","lastName":"Rojas","suffix":""},{"id":237731317,"identity":"ef50a52b-a773-4335-b44f-4c7e10c84a9a","order_by":3,"name":"Lizandro Gonzales","email":"","orcid":"","institution":"Dirección Regional de Salud","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lizandro","middleName":"","lastName":"Gonzales","suffix":""},{"id":237731318,"identity":"6794e2ae-fcea-49ba-b3a2-3ac7f84804d7","order_by":4,"name":"Rafael Tapia-Limochi","email":"","orcid":"","institution":"Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Tapia-Limochi","suffix":""},{"id":237731319,"identity":"953f8401-3d8e-4ee2-818a-a513dc644e6e","order_by":5,"name":"Marta Moreno","email":"","orcid":"","institution":"London School of Hygiene \u0026 Tropical Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Moreno","suffix":""},{"id":237731320,"identity":"e5d815a9-ee7e-49c0-bd7b-aa38946c9610","order_by":6,"name":"Christian R. González","email":"","orcid":"","institution":"Universidad Metropolitana de Ciencias de la Educación","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christian","middleName":"R.","lastName":"González","suffix":""},{"id":237731321,"identity":"cd390a56-d572-4fa0-bc0b-f400e65cee78","order_by":7,"name":"Stella M. Chenet","email":"data:image/png;base64,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","orcid":"","institution":"Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Stella","middleName":"M.","lastName":"Chenet","suffix":""}],"badges":[],"createdAt":"2023-10-04 02:59:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3409121/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3409121/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44321239,"identity":"ca2c7290-a6a6-40ed-9340-0637c673fdf6","added_by":"auto","created_at":"2023-10-09 21:43:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":869070,"visible":true,"origin":"","legend":"\u003cp\u003eA. Map of Peru and the geographical location of Amazonas. B. Map of Amazonas indicating the province of Condorcanqui and the district of Rio Santiago. C. Mosquito collection sites in the native communities of Rio Santiago. D. Landscape of Alianza Progreso, 2022.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/bad2edf9398ea8046153eef8.png"},{"id":44322281,"identity":"9a5939a6-548f-4db5-aa34-03805d61980e","added_by":"auto","created_at":"2023-10-09 21:51:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":335039,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAnopheles benarrochi\u003c/em\u003e B MJ network. Circles represent unique haplotypes; the size of the circle is proportional to the number of individuals sharing the haplotype. Black nodes indicate theoretical missing haplotypes, and hash marks represent mutation steps between haplotypes.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/6b1542094cc67008e55a6d72.png"},{"id":44321241,"identity":"39d75134-2aa8-4164-8c85-07913010fd11","added_by":"auto","created_at":"2023-10-09 21:43:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":363283,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood (ML) phylogenetic tree using the COX1 gene (467 bp) with a total of 144 sequences.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/34d94804efb661467d6abc75.png"},{"id":44321243,"identity":"d1423027-4a51-4a94-a82a-d2ac616cd0b0","added_by":"auto","created_at":"2023-10-09 21:43:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":364128,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of the kdr sequences, including the \u003cem\u003eAn. albitarsis \u003c/em\u003emutant L1014F (MW315118) and the \u003cem\u003eAn. darlingi \u003c/em\u003ewild type (MN062262). Mutation sites reported are enclosed in a yellow box. The blue line below indicates an intron. Primers AAKDRF2 and AAKDRR used to amplify the segment are indicated by red arrows. SNPs detected in the intron for \u003cem\u003eAn. triannulatus\u003c/em\u003e are indicated with an orange arrow, SNPs detected in the exon are indicated with a green arrow.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/ee2e2fe2506cad63240451d9.png"},{"id":61027511,"identity":"72278110-5f64-4aba-912c-cc787f390c38","added_by":"auto","created_at":"2024-07-24 18:11:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2337620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/2fc0408a-a2ca-45f6-887a-5d762c14797e.pdf"},{"id":44321240,"identity":"ecf96dae-1f3b-423b-9d5e-7d01fbcdafae","added_by":"auto","created_at":"2023-10-09 21:43:52","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":44113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1: Sequences available in GenBank used to construct the Maximum Likelihood (ML) phylogenetic tree.\u003c/p\u003e\n\u003cp\u003eTable S2: Genbank data of Anopheles benarrochi B used for the 469-bp COI median-joining haplotype network.\u003c/p\u003e\n\u003cp\u003eTable S3: Molecular identification of anophelines collected in the four native communities of Condorcanqui, Amazonas.\u003c/p\u003e\n\u003cp\u003eTable S4: Molecular identification of non-anophelines collected in the study area\u003c/p\u003e","description":"","filename":"AdditionalFile1.TablesS1S2S3S4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/fd923cb7b1db0c68b5ac4007.xlsx"},{"id":44321244,"identity":"170bb468-db50-48cc-b6fa-90960b67eef8","added_by":"auto","created_at":"2023-10-09 21:43:52","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":375162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure S1: A) \u003cem\u003eAn. benarrochi \u003c/em\u003eB with a reduced abdomen before a blood meal. B) \u003cem\u003eAn. benarrochi\u003c/em\u003eB with an enlarged abdomen after a blood meal.\u003c/p\u003e","description":"","filename":"AdditionalFile2.FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-3409121/v1/d3d0d10cdaa70fc3676ea9ca.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Natural Plasmodium infection of Anopheles benarrochi B (Diptera: Culicidae) in native communities of the Province of Condorcanqui, Amazonas-Peru","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eIn Peru, 26,652 malaria cases were reported in 2022, with ~\u0026thinsp;84% \u003cem\u003ePlasmodium vivax\u003c/em\u003e and ~\u0026thinsp;15% \u003cem\u003eP. falciparum\u003c/em\u003e cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], nevertheless, malaria infections remain uncertain in remotes areas because control activities have been postponed owing to COVID-19. Although Loreto is one of the most affected departments in the country (89% of malaria cases) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], Amazonas, a northeastern department of Peru, has reported a significant increase in the number of malaria cases for the past six years, from 710 cases in 2018 to 1657 in 2022 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In 2019, a 2.5-fold increase was reported due to an outbreak of autochthonous \u003cem\u003eP. falciparum\u003c/em\u003e cases in native communities of Rio Santiago, in the Condorcanqui Province of Amazonas, this outbreak was triggered by an index case of \u003cem\u003eP. falciparum\u003c/em\u003e imported from Loreto, which subsequently spread to other communities during that year [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, there is limited information about the circulating vectors involved in malaria transmission in these communities.\u003c/p\u003e \u003cp\u003eIn the Peruvian Amazon, the species recognized as the main malaria vector is \u003cem\u003eAnopheles darlingi\u003c/em\u003e Root [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; this highly anthropophilic species has been reported in the departments of Madre de Dios [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], Loreto [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Ucayali and San Martin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Other potential malaria vectors, \u003cem\u003eAn. triannulatus\u003c/em\u003e (Neiva \u0026amp; Pinto) and \u003cem\u003eAn. benarrochi\u003c/em\u003e (Cova Garc\u0026iacute;a \u0026amp; L\u0026oacute;pez) have been reported in eastern and in western Loreto, respectively [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Previous studies suggested that \u003cem\u003eAn. benarrochi\u003c/em\u003e is a species complex consisting in four distinct species (\u003cem\u003eAn. benarrochi\u003c/em\u003e B, \u003cem\u003eAn. benarrochi\u003c/em\u003e G1, \u003cem\u003eAn. benarrochi\u003c/em\u003e G2 and \u003cem\u003eAn. benarrochi\u003c/em\u003e) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], from which \u003cem\u003eAn. benarrochi\u003c/em\u003e B has been identified as a malaria vector in northern Loreto and Madre de Dios [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEfforts to eliminate malaria transmission in native communities of Rio Santiago have focused on rapid diagnosis, case management, insecticide-treated nets (ITNs), and indoor and outdoor residual spraying programing by the MoH. However, this progress can be undermined by parasites\u0026acute; resistance to antimalarial, and also by mosquitoes\u0026rsquo; resistance to insecticides and vector\u0026acute;s behavior. According to the World Malaria Report 2022, a total of eighty-eight countries reported insecticide resistance in the last decade. Among these countries, twenty-nine documented resistance to four primary classes of insecticides (pyrethroids, organophosphates, carbamates, and organochlorines) at various locations within their territories [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. One of the primary mechanisms related to pyrethroid resistance, known as knockdown resistance (kdr), involves mutations at the kdr region of the voltage-gated sodium channel gene (VGSC), which is the primary target of synthetic pyrethroids [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In South America, \u003cem\u003eAn. darlingi\u003c/em\u003e resistance to dichlorodiphenyltrichloroethane (DDT) and pyrethroids has been reported in western Colombia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], while on the northwestern coast of Peru, \u003cem\u003eAn. albimanus\u003c/em\u003e, has shown cross-resistance to all classes of insecticides [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, a molecular characterization of mosquitoes collected in native communities of Rio Santiago was evaluated in order to identify species of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes, incriminate them as malarial vectors, and evaluate insecticide resistance by the kdr region marker.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and mosquito collection\u003c/h2\u003e \u003cp\u003eApproximately, 99% of malaria cases (1633 malaria cases in 2022) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] in the Amazonas Department were reported in the Rio Santiago District (Condorcanqui Province). This is a remote impoverished area where native communities live on the banks of the river, with no electricity, drinking water, or road access, with the rivers as the primary means of transportation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMosquito collections were conducted in four native communities of Rio Santiago: Alianza Progreso (AP), Nueva Esperanza (NE), Chapiza (CH), and Caterpiza (CT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), located within the ecosystem of the Amazonian Humid forests with temperatures that can reach 35\u0026ordm;C, an average annual rainfall of around 4,800 mm, and a relative humidity of above 90% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdult mosquitoes were collected for six nights during the rainy season between March and September of 2022.Mosquitoes were captured with three Shannon traps and then, with the help of mouth aspirators, the mosquitoes were transferred to cryovials. Likewise, two light traps from the Centers for Disease Control and Prevention (CDC) were used, last two traps placed\u0026thinsp;~\u0026thinsp;10 m from the houses and ~\u0026thinsp;10 m from the forest entrance between 18:00 and 22:00 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were identified in the field based on their morphological characteristics using entomological keys [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], then they were stored in 1.5ml cryovials containing 70% ethanol. Samples were classified and stored by date and location; additional information such as temperature and relative humidity was also collected.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA isolation, amplification, and sequencing of\u003c/b\u003e \u003cb\u003eCOX1\u003c/b\u003e \u003cb\u003eand kdr\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from the whole body of each specimen using a DNeasy Blood \u0026amp; Tissue kit (Qiagen) following the manufacturer's instructions. The 710bp barcode region of the mitochondrial Cytochrome Oxidase I gene (COX1) was amplified for all samples using the LCO1490 and HCO2198 primers [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The 25 \u0026micro;l PCR reaction included 1ul of extracted DNA, 0.5 \u0026micro;M of each primer, 1 unit of Platinum Taq DNA polymerase (Invitrogen), 0.2mM dNTP, 1X PCR buffer (Invitrogen), 2.5mM MgCl\u003csub\u003e2\u003c/sub\u003e and nuclease-free water. Thermocycling conditions were used according to the literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePCR amplification of segment 6 of domain II (IIS6) of the VGSC gene (approximately 225 bp), corresponding to the kdr region, was amplified using AAKDRF2 and AAKDRR primers [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The 25 \u0026micro;l PCR reaction included 5 \u0026micro;l of extracted DNA, 0.5 \u0026micro;M of each primer, 1 unit of Platinum Taq DNA polymerase (Invitrogen), 0.2 mM dNTP, 1X PCR buffer (Invitrogen), 2.0 mM MgCl2 and nuclease-free water. Thermocycling conditions were used according to the literature [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmplicons were visualized on 2% agarose gels stained with SafeView\u0026trade; Classic (Applied Biological Materials). PCR products were purified using the Exo-CIP\u0026trade; kit (New England Biolabs), and sequencing was performed on a 3500 Genetic Analyzer (Applied Biosystems\u0026trade;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eThe sequence data was analyzed using the Geneious Prime (version 2022.2.1, Biomatters Inc, Newark) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps:/www.geneious.com\u003c/span\u003e\u003cspan address=\"https://www.geneious.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Basic Local Alignment Search Tool (BLAST) searches were carried out for species identification with sequences available in the GenBank database. Species were determined based on query coverage (QC) and percentage identity (PI). All anopheline sequences were aligned using the MUSCLE tool, implemented in the MEGA software (version 7.2.6.1). In addition, 77 previously reported sequences of different anopheline species were also included in the phylogeny (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A Maximum Likelihood (ML) tree was constructed using a Kimura 2-parameter model (K-2P) and the Bootstrap method with 1000 bootstrap replicates to examine phylogenetic relationships in MEGA X software [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The \u003cem\u003eAedes aegypti\u003c/em\u003e COX1 sequence (NC035159) was used as an outgroup to root the tree. \u003cem\u003eAn. benarrochi\u003c/em\u003e haplotypes from this study and from 59 previously reported sequences (Additional file 1: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were determined using DnaSP v6 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and the haplotype network was constructed using the Median-Joining algorithm in PoPART software [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For kdr analysis, \u003cem\u003eAn. darlingi\u003c/em\u003e (MN062262) and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ealbitarsis\u003c/em\u003e (MW315118) were used as references.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHuman blood meal identification and\u003c/b\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003edetection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSource of mosquitoes blood meal was assessed using a human β-globin protocol previously reported [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; the thermal profile consisted of an initial denaturation step at 94\u0026deg;C for 7 min; followed by 35 cycles at 94\u0026deg;C for 1 minute for denaturation, 53\u0026deg;C for 1 minute for hybridization, and 72\u0026deg;C for 1 minute for extension; and then a final extension at 72\u0026deg;C for 5 minutes.\u003c/p\u003e \u003cp\u003eThe detection of \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e infections in mosquitoes was performed using the 18S rRNA subunit nested PCR technique according to the protocol previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular identification of mosquitoes\u003c/h2\u003e \u003cp\u003eThe environmental conditions during the collection dates were very similar, with a temperature range from 25\u0026ordm;C to 28\u0026ordm;C and humidity levels between 75% and 87% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 453 individuals were captured using Shannon traps and then transferred into cryovials using mouth aspirators. None individuals were captured using CDC-LT traps. Ninety-four females were initially identified as \u003cem\u003eAnopheles\u003c/em\u003e based on morphological characteristics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the remaining species were identified morphologically as Culicidae (detailed data not shown); After molecular confirmation, it was found that only sixty-six individuals belonged to the \u003cem\u003eAnopheles\u003c/em\u003e genus (AP\u0026thinsp;=\u0026thinsp;55, CT\u0026thinsp;=\u0026thinsp;5, NE\u0026thinsp;=\u0026thinsp;5, and CH\u0026thinsp;=\u0026thinsp;1) and the remaining twenty-eight mosquitoes were molecularly confirmed as other non-anopheline Culicidae.\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\u003eMosquito specimens (n\u0026thinsp;=\u0026thinsp;94) collected in Rio Santiago, Condorcanqui province, 2022. Alianza Progreso (AP\u0026thinsp;=\u0026thinsp;81), Nueva Esperanza (NE\u0026thinsp;=\u0026thinsp;5), Chapiza (CH\u0026thinsp;=\u0026thinsp;1), and Caterpiza (CT\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCoordinates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u0026deg; (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRH (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber of specimens\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMolecular Identification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e196280.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9581345.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e02/03/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e199060.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9587806.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e02/03/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003eAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003e195051.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003e9592391.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e01/03/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e01/03/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. triannulatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCoquillettidia venezuelensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCoquillettidia venezuelensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. costai\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. nimbus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25/05/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCoquillettidia venezuelensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e198182.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e9566709.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20/09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. benarrochi\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20/09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eSabethes sp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20/09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCulex bastagarius\u003c/em\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\u003eAn. benarrochi\u003c/em\u003e B was found to be the predominant species (include %, 51/66 specimens) with the presence of \u003cem\u003eAn. triannulatus\u003c/em\u003e (2/66), \u003cem\u003eAn. costai\u003c/em\u003e (2/66) and \u003cem\u003eAn. nimbus\u003c/em\u003e (1/66) (Additional file 1: Table S3). Among non-anopheline mosquitoes, \u003cem\u003eCoquillettidia venezuelensis (26/28)\u003c/em\u003e, one \u003cem\u003eSabethes\u003c/em\u003e sp, and one \u003cem\u003eCulex bastagarius specimens\u003c/em\u003e were also molecularly identified (Additional file 1: Table S4). According to the median-joining haplotype network based on COX1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), there were thirty-six \u003cem\u003eAn. benarrochi\u003c/em\u003e B haplotypes, from which six were found in Amazonas. Haplotype 1 was found in Amazonas and also in Ecuador, which is expected due to proximity. Haplotypes 3, 5 and 6 were only found in Amazonas, while haplotypes 2 and 4 were found in Amazonas and Loreto Department. A ML K-2P tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) further supported the correct identification of the specimens in this study. Additionally, the species clade assignment corresponded to the traditional subgenus classification [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]; specifically, species such as \u003cem\u003eAn. benarrochi B, An. benarrochi, An. rangeli, An. konderi, An. albimanu\u003c/em\u003es and \u003cem\u003eAn. triannulatus\u003c/em\u003e are included in the \u003cem\u003eNyssorhynchus\u003c/em\u003e subgenus, while \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003enimbus\u003c/em\u003e and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ekompi\u003c/em\u003e belonged to the \u003cem\u003eStethomyia\u003c/em\u003e subgenus group and \u003cem\u003eAn. costai\u003c/em\u003e to the \u003cem\u003eAnopheles\u003c/em\u003e subgenus group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBlood meal source and\u003c/b\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003eidentification in mosquitoes\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003espp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDuring morphological identification, it was observed that certain specimens presented an enlarged abdomen, which was attributed to their blood meal (Additional file 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Of the sixty-six anophelines molecularly confirmed, \u003cem\u003eAn. benarrochi\u003c/em\u003e B (twenty-three samples) and \u003cem\u003eAn. triannulatus\u003c/em\u003e (one sample) were positive to human β-globin. Furthermore, six specimens of \u003cem\u003eAn. benarrochi\u003c/em\u003e B were found to be positive for \u003cem\u003ePlasmodium\u003c/em\u003e (four \u003cem\u003eP. falciparum\u003c/em\u003e, and two \u003cem\u003eP. vivax\u003c/em\u003e) (Additional file 1: Table S3). Notably, four samples exhibited the presence of \u003cem\u003ePlasmodium\u003c/em\u003e parasites and human blood, suggesting the incrimination of \u003cem\u003eAnopheles benarrochi\u003c/em\u003e B in malaria transmission in the region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of insecticide-resistance genotype in the kdr region\u003c/h2\u003e \u003cp\u003eOut of the total sixty-six anophelines, sixty-four successfully underwent amplification and genotyping for the kdr region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Additional file 1: Table S3). These sequences were further examined to identify mutations in codons 1010, 1013, and 1014 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. No missense mutations were detected in any of the sequences. All sequences of \u003cem\u003eAn. benarrochi\u003c/em\u003e B and \u003cem\u003eAn. triannulatus\u003c/em\u003e showed the GTT codon for valine at position 1010 (V1010), AAC codon for asparagine at position 1013 (N1013), and TTA codon for leucine at position 1014 (L1014), suggesting that the collected anopheline population has wild-type genotypes related to susceptibility to DDT and pyrethroid insecticides. Only the \u003cem\u003eAn. nimbus\u003c/em\u003e sequence showed a synonymous mutation at position V1010 (GTA). On the other hand, two \u003cem\u003eAn. benarrochi\u003c/em\u003e B haplotypes differed in a C/A substitution (at position 45); however, it should be noted that these mutations correspond to synonymous substitutions in codons unrelated to insecticide resistance. Similarly, the two \u003cem\u003eAn. triannulatus\u003c/em\u003e sequences differed in an A/G substitution in the intronic region. Further analysis of the intron downstream kdr revealed size variations among the species. The intron of \u003cem\u003eAn. nimbus\u003c/em\u003e (90 bp) was found to be larger than that of \u003cem\u003eAn. benarrochi\u003c/em\u003e B (72 bp) and \u003cem\u003eAn. triannulatus\u003c/em\u003e (75 bp), which also differed from the reference sequences (\u003cem\u003eAn. darlingi\u003c/em\u003e wildtype and \u003cem\u003eAn. albitarsis\u003c/em\u003e mutated at 1014 codon).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study is the first report on the molecular characterization of anopheline species circulating in native communities in Condorcanqui \u0026ndash; Amazonas Department, Peru. The primary objective was to identify the malaria vector species in the area to maximize the impact of malaria control strategies. In the eastern region of Peru, \u003cem\u003eAn. darlingi\u003c/em\u003e has been recognized as the main malaria vector; however, it is plausible that other \u003cem\u003eAnopheles\u003c/em\u003e species could be transmitting malaria in endemic areas where \u003cem\u003eAn. darlingi\u003c/em\u003e is absent [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For instance, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003etriannulatus\u003c/em\u003e was reported as the dominant vector in eastern Loreto, while \u003cem\u003eAn. benarrochi\u003c/em\u003e was mostly found in western Loreto [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo anopheline species, \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ebenarrochi\u003c/em\u003e B (56/66) and \u003cem\u003eAn. triannulatus\u003c/em\u003e (2/66), both belonging to the \u003cem\u003eNyssorhynchus\u003c/em\u003e subgenus, were successfully identified in this study. Furthermore, the network analysis and ML phylogenetic tree showed that \u003cem\u003eAn. benarrochi\u003c/em\u003e B sequences were strongly linked to previously reported sequences in Ecuador, Colombia [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], Peru [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Brazil [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of \u003cem\u003eAn. benarrochi\u003c/em\u003e B as a malaria vector in an endemic area is usually associated with the absence of \u003cem\u003eAn. darlingi\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our findings further support this hypothesis, since \u003cem\u003eAn. benarrochi\u003c/em\u003e B was identified as the predominant species in all four communities in all sampling periods. Likewise, it should be emphasized that \u003cem\u003eAn. benarrochi\u003c/em\u003e B has already been identified in other regions of Peru [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and with the present study, it extends its distribution to the northwest of the country, including three new haplotypes.\u003c/p\u003e \u003cp\u003eMalaria cases in these communities have been significantly on the rise in the last years. In 2020, the Chapiza Health Establishment reported a high Annual Parasite Index (API) of 122.8, which further increased to 224.8 by 2022. The cases included infections caused by both \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e. Here, we determined the presence of these parasites in specimens of \u003cem\u003eAn. benarrochi\u003c/em\u003e B from the Alianza Progreso community, which belongs under the jurisdiction of the Chapiza Health Center. A similar situation was observed at the Caterpiza Health Establishment, where the API was 9.7 in 2020 (considered a moderate risk) and it spiked to 812.7 (classified as high risk) with only \u003cem\u003eP. vivax\u003c/em\u003e cases in 2022. Interestingly, the anophelines captured in Caterpiza did not test positive for \u003cem\u003ePlasmodium\u003c/em\u003e, but they did test positive for the human B-globin marker, confirming the anthropophilic behavior of \u003cem\u003eAn. benarrochi\u003c/em\u003e B. Moreover, a total of six out of sixty-six anophelines were found to be infected with \u003cem\u003ePlasmodium\u003c/em\u003e, which represents a highly significant percentage compared to what was reported previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Considering that the specimen\u0026rsquo;s collection was made after the rainy season, with high mosquitoes' density and in areas with APIs of moderate to high risk, it may explain the high number of infected mosquitoes found in the study. A limitation of this study is the low number of entomological surveys in all communities (one sampling night in each one) preventing the calculation of reliable entomological indices. Nevertheless, the data provides evidence of the importance of \u003cem\u003eAnopheles benarrochi\u003c/em\u003e B as a malaria vector in this unexplored area, although implication of other anopheline species should be investigated. Furthermore, since 2019, there has been a significant shift in the epidemiological landscape of malaria in Condorcanqui, given the emergence of \u003cem\u003eP. falciparum\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; it is suspected that alterations in vector composition, the high human biting rate and peak biting time (\u0026sim;18:00\u0026ndash;20:00) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] could have also contributed to this event.\u003c/p\u003e \u003cp\u003eOn the other hand, the genetic diversity of \u003cem\u003eAn. costai\u003c/em\u003e and \u003cem\u003eAn. nimbus\u003c/em\u003e has received limited attention in previous studies. The lack of research on these species may be attributed to their lack of implication as significant malaria vectors. They have been predominantly observed in forest habitats and have often been misidentified as \u003cem\u003eAn. mediopunctatus\u003c/em\u003e, which has been found to carry \u003cem\u003eP. vivax\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is limited information regarding insecticide resistance in the most significant malaria vectors in Latin America [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and conducting insecticide resistance studies in the Peruvian Amazon region is challenging due to limited accessibility, inadequate laboratory capacity, and a shortage of trained personnel [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, genotypes linked to resistance to DDT and pyrethroids were not detected within the kdr region of any of the sixty-six \u003cem\u003eAnopheles\u003c/em\u003e specimens captured in the Rio Santiago district. Further investigations involving a larger number of mosquitoes are essential to evidence the susceptibility status of main malaria vectors to develop successful vector surveillance programs in Peru. Additionally, noteworthy findings include the identification of synonymous mutations in \u003cem\u003eAn. benarrochi\u003c/em\u003e B (in the exonic region) and \u003cem\u003eAn. triannulatus\u003c/em\u003e (in the intronic region) and variations in the intronic size within the species. The analysis of intron sequences, including nucleotide positions and sizes, may support the hypothesis of interspecific differences and offers potential as a tool for taxonomic classification [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding other \u003cem\u003eAnopheline\u003c/em\u003e species, several studies have reported the absence of mutations in the kdr region associated with insecticide resistance. Wild-type kdr alleles were found in \u003cem\u003eAn. nuneztovari s.l. An. darlingi\u003c/em\u003e, and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003ealbimanus\u003c/em\u003e from Valle del Cauca, Colombia [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and in \u003cem\u003eAn. albimanus\u003c/em\u003e populations from Guatemala, Ecuador, and Colombia [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. On the other hand, specific mutations at codons 1010, 1014 and 1014 in the kdr region have been associated with pyrethroid resistance [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In Peru, resistance to pyrethroids was reported in \u003cem\u003eAn. darlingi\u003c/em\u003e in Loreto [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and a high frequency of TCG (L1014S) and TGT (L1014C) associated with resistance in \u003cem\u003eAn. albimanus\u003c/em\u003e was revealed in Tumbes through RNA-seq analysis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study reveals the presence of \u003cem\u003eAnopheles benarrochi\u003c/em\u003e B and its natural infection with \u003cem\u003ePlasmodium\u003c/em\u003e in unexplored native communities in the Amazonas region highlighting its role as a major malaria vector in this area, although other anopheline species might be also involved. This research also evidences the wider distribution of \u003cem\u003eAn. benarrochi\u003c/em\u003e B in northern Peru. Furthermore, the fraction of mosquito population analyzed showed wild-type/susceptible kdr genotypes in native communities of the province of Condorcanqui, providing a base line for insecticide surveillance and control interventions in the area. More entomological surveys in these communities of the province of Condorcanqui are required to characterize vector bionomics and behavior across seasons to unveil the species involved in malaria transmission. This will help in the design and implementation of specific programs to combat malaria by the Condorcanqui Health Network.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBLAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasic Local Alignment Search Tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCenters for Disease Control and Prevention\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOX1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial Cytochrome Oxidase I gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent assay.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstituto de Enfermedades Tropicales\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKnockdown resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePercent identity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibosomal ribonucleic acid.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuery coverage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVGSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVoltage-Gated Sodium Channel gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere thanks to colleagues of the Institute of Tropical Diseases (IET-UNTRM) and\u0026nbsp;Direcci\u0026oacute;n Regional de Salud\u0026nbsp;de Amazonas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProyecto\u0026nbsp;METAVEC N\u0026deg;: 050-2021-FONDECYT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Forestry and Wildlife Service.\u0026nbsp;Authorization code No AUT-IFS-2021-071 and Ethics committee approval from UNTRM \u0026nbsp;CIEI-N\u0026deg; 011.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSC and RT conceived and designed the study. LG, MM and CG, advised on interventions, study communities and coordination with local and national authorities. SC, RT, LR and LG implemented the study. MVP and JZ analysed the data. MVP, JZ, SC and RT interpreted the data. MVP \u0026amp; JZ wrote the first draft of the manuscript. SC, MM \u0026amp; CG critically revised the manuscript for important content. All authors read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMINSA. Sala de Situaci\u0026oacute;n de Salud Per\u0026uacute; a la SE 52\u0026ndash;2022. Per\u0026uacute; Ministerio de Salud. ; 2022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dge.gob.pe/epipublic/uploads/asis-sala/asis-sala_202252_09_234037.pdf\u003c/span\u003e\u003cspan address=\"https://www.dge.gob.pe/epipublic/uploads/asis-sala/asis-sala_202252_09_234037.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 23 March 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMINSA. Sala de Situaci\u0026oacute;n de Salud Per\u0026uacute; a la SE 33\u0026ndash;2023. Per\u0026uacute;: Ministerio de Salud. ; 2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dge.gob.pe/portal/docs/vigilancia/sala/2023/salaSE33.pdf\u003c/span\u003e\u003cspan address=\"https://www.dge.gob.pe/portal/docs/vigilancia/sala/2023/salaSE33.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 30 August 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontenegro CC, Bustamante-Chauca TP, Pajuelo Reyes C, Bernal M, Gonzales L, Tapia-Limonchi R, et al. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e outbreak in native communities of Condorcanqui, Amazonas, Per\u0026uacute;. Malar J. 2021;20(1):88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMINSA. Sala de Situaci\u0026oacute;n de Malaria Per\u0026uacute; a la SE 33\u0026ndash;2020. Per\u0026uacute;: Ministerio de Salud. ; 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.dge.gob.pe/portal/docs/tools/teleconferencia/2021/SE012021/03.pdf\u003c/span\u003e\u003cspan address=\"http://www.dge.gob.pe/portal/docs/tools/teleconferencia/2021/SE012021/03.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 30 August 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTineo E, Medina A, Fallaque C, Ch\u0026aacute;vez L, Quispe S, Mercado M, et al. Distribuci\u0026oacute;n geogr\u0026aacute;fica y comportamiento estacional de la picadura del \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003edarlingi\u003c/em\u003e Root, 1926 en localidades de la frontera Per\u0026uacute;-Bolivia, Madre de Dios, Per\u0026uacute;. Rev Peru Med Exp Salud Publica. 2003;20(2):78\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReinbold-Wasson DD, Sardelis MR, Jones JW, Watts DM, Fernandez R, Carbajal F, et al. Determinants of Anopheles seasonal distribution patterns across a forest to periurban gradient near Iquitos, Peru. Am J Trop Med Hyg. 2012;86(3):459\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreno M, Saavedra MP, Bickersmith SA, Lainhart W, Tong C, Alava F, et al. Implications for changes in Anopheles darlingi biting behaviour in three communities in the peri-Iquitos region of Amazonian Peru. Malar J. 2015;14(1):290.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlores-Mendoza C, Fern\u0026aacute;ndez R, Escobedo-Vargas KS, Vela-Perez Q, Schoeler GB. Natural \u003cem\u003ePlasmodium\u003c/em\u003e infections in \u003cem\u003eAnopheles darlingi\u003c/em\u003e and \u003cem\u003eAnopheles benarrochi\u003c/em\u003e (Diptera: Culicidae) from eastern Peru. J Med Entomol. 2004;41(3):489\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArambur\u0026uacute; JG, Ramal CA, Witzig R. Malaria reemergence in the Peruvian Amazon region. Emerg Infect Dis. 1999;5(2):209\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourke BP, Conn JE, de Oliveira TMP, Chaves LSM, Bergo ES, Laporta GZ, et al. Exploring malaria vector diversity on the Amazon Frontier. Malar J. 2018;17(1):342.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConn JE, Moreno M, Saavedra M, Bickersmith SA, Knoll E, Fernandez R, Vera H, Burrus RG, Lescano AG, Sanchez JF, Rivera E, Vinetz JM. Molecular taxonomy of \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e) \u003cem\u003ebenarrochi\u003c/em\u003e (Diptera: Culicidae) and malaria epidemiology in Southern Amazonian Peru. Am J Trop Med Hyg. 2013;88(2):319\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. World malaria report 2022. Geneva: World Health Organization. ; 2022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/publications/i/item/9789240064898\u003c/span\u003e\u003cspan address=\"https://www.who.int/publications/i/item/9789240064898\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 21 May 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZlotkin E. The insect voltage-gated sodium channel as target of insecticides. Annu Rev Entomol. 1999;44(1):429\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuarez MF, Qui\u0026ntilde;ones ML, Palacios JD, Carrillo A. First record of DDT resistance in Anopheles darlingi. J Am Mosq Control Assoc. 1990;6(1):72\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVargas FV, C\u0026oacute;rdova PO, Alvarado AA. Determinaci\u0026oacute;n de la resistencia a insecticidas en \u003cem\u003eAedes aegypti, Anopheles albimanus\u003c/em\u003e y \u003cem\u003eLutzomyia peruensis\u003c/em\u003e procedentes del Norte Peruano. Rev Peru Med Exp Salud Publica. 2006;23(4):259\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMINSA. N\u0026uacute;mero de casos de malaria, Per\u0026uacute; 2019\u0026ndash;2022. 2022. Sala de Situaci\u0026oacute;n de Salud Per\u0026uacute; a la SE 52\u0026ndash;2022. Per\u0026uacute; Ministerio de Salud; 2022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dge.gob.pe/portal/docs/vigilancia/sala/2022/SE52/malaria.pdf\u003c/span\u003e\u003cspan address=\"https://www.dge.gob.pe/portal/docs/vigilancia/sala/2022/SE52/malaria.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Accessed 19 September 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGobierno Regional de Amazonas. Estudio de diagn\u0026oacute;stico y zonificaci\u0026oacute;n para el tratamiento de la demarcaci\u0026oacute;n territorial de la provincia Condorcanqui. Amazonas: Direcci\u0026oacute;n Nacional T\u0026eacute;cnica de Demarcaci\u0026oacute;n Territorial; 2014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeed JT, Rogers EJ, Phillips IA, Falcon R, Fernandez R, Carbajal F, Quintana J. Mosquitoes (Diptera: Culicidae) Captured in the Iquitos Area of Peru. J Med Entomol. 1993;30(3):634\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeves A, Urbinatti PR, Malafronte R dos, Fernandes S, da Silva Paganini A, Natal W. Malaria outside the Amazon region: Natural Plasmodium infection in anophelines collected near an indigenous village in the Vale do Rio Branco, Itanha\u0026eacute;m, SP, Brazil. Acta Trop. 2013;125(1):102\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForattini OP. Entomol\u0026oacute;gica M\u0026eacute;dica. Tomo 1. Brasil: Faculdade de Higiene e Saude Publica; Sao Paulo; 1962.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3(5):294\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLinton Y-M, Pecor JE, Porter CH, Mitchell LB, Garz\u0026oacute;n-Moreno A, Foley DH, et al. Mosquitoes of eastern Amazonian Ecuador: biodiversity, bionomics and barcodes. Mem Inst Oswaldo Cruz. 2013;108(Supp 1):100\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLol JC, Castellanos ME, Liebman KA, Lenhart A, Pennington PM, Padilla NR. Molecular evidence for historical presence of knock-down resistance in Anopheles albimanus, a key malaria vector in Latin America. Parasit Vectors. 2013;6(1):268.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrjuela LI, \u0026Aacute;lvarez-Diaz DA, Morales JA, Grisales N, Ahumada ML, Venegas HJ, et al. Absence of knockdown mutations in pyrethroid and DDT resistant populations of the main malaria vectors in Colombia. Malar J. 2019;18(1):384.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorales Viteri D, Herrera-Varela M, Albuja M, Quiroga C, Diaz G, Del Aguila Morante C, et al. New records of \u003cem\u003eAnopheles benarrochi\u003c/em\u003e B (Diptera: Culicidae) in malaria hotspots in the Amazon regions of Ecuador and Peru. J Med Entomol. 2021;58(3):1234\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoster PG, Bergo ES, Bourke BP, Oliveira TMP, Nagaki SS, Sant\u0026rsquo;Ana DC, Sallum MAM. Phylogenetic Analysis and DNA-based Species Confirmation in \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eNyssorhynchus\u003c/em\u003e). PLoS ONE. 2013;8(2):e54063.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan-Chable RJ, Mart\u0026iacute;nez-Arce A, Mis-Avila PC, Ortega-Morales AI. DNA Barcodes and Evidence of Cryptic Diversity of Anthropophagous Mosquitoes in Quintana Roo, Mexico. Ecol Evol. 2019;9(8):4692\u0026ndash;705.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKeon SN, Moreno M, Sallum MA, Povoa MM, Conn JE. Distinct Population Structure for Co-Occurring \u003cem\u003eAnopheles goeldii\u003c/em\u003e and \u003cem\u003eAnopheles triannulatus\u003c/em\u003e in Amazonian Brazil. Mem Inst Oswaldo Cruz. 2013;108(5):605\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrussing C, Saavedra MP, Bickersmith SA, Alava F, Guzm\u0026aacute;n M, Manrique E, et al. Malaria vector species in Amazonian Peru co-occur in larval habitats but have distinct larval microbial communities. PLoS Negl Trop Dis. 2019;13(5):e0007412.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuti\u0026eacute;rrez LA, Naranjo NJ, Cienfuegos AV, Muskus CE, Luckhart S, Conn JE, et al. Population structure analyses and demographic history of the malaria vector \u003cem\u003eAnopheles albimanus\u003c/em\u003e from the Caribbean and the Pacific regions of Colombia. Malar J. 2009;8(1):259.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVirreira M, Torrico F, Truyens C, Alonso-Vega C, Solano M, Carlier Y, et al. Comparison of polymerase chain reaction methods for reliable and easy detection of congenital \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e infection. Am J Trop Med Hyg. 2003;68(5):574\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg. 1999;60(4):687\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoster PG, de Oliveira TMP, 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(11):170758.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConn JE, Bickersmith SA, Saavedra MP, Morales JA, Alava F, Diaz Rodriguez GA, del Aguila Morante CR, Tong CG, Alvarez-Antonio C, Daza Huanahui JM, Vinetz JM, Gamboa D. Natural Infection of \u003cem\u003eNyssorhynchus darlingi\u003c/em\u003e and \u003cem\u003eNyssorhynchus benarrochi\u003c/em\u003e B with \u003cem\u003ePlasmodium\u003c/em\u003e during the Dry Season in the Understudied Low-Transmission Setting of Datem del Mara\u0026ntilde;on Province, Amazonian Peru. Am J Trop Med Hyg. 2023;109(2):288\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchoeler GB, Flores-Mendoza C, Fern\u0026aacute;ndez R, Davila JR, Zyzak M. Geographical distribution of Anopheles darlingi in the Amazon Basin region of Peru. J Am Mosq Control Assoc. 2003;19(4):286\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeafsey DE, Taylor AR, MacInnis BL. Advances and opportunities in malaria population genomics. Nat Rev Genet. 2021;22(8):502\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSallum MA, Wilkerson RC, Forattini OP. Taxonomic study of species formerly identified as Anopheles mediopunctatus and resurrection of An. costai (Diptera: Culicidae). J Med Entomol. 1999;36(3):282\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. World malaria report 2021. Geneva: World Health Organization. ; 2021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/publications/i/item/9789240040496\u003c/span\u003e\u003cspan address=\"https://www.who.int/publications/i/item/9789240040496\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 31 August 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Li M, Tan R, Deng C, Huang B, Wu Z, et al. Presence of L1014F Knockdown-Resistance Mutation in Anopheles gambiae s.s. From S\u0026atilde;o Tom\u0026eacute; and Pr\u0026iacute;ncipe. Front Cell Infect Microbiol. 2021;11:633905.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaboratorio de Referencia Nacional de Entomolog\u0026iacute;a. Centro Nacional de Salud P\u0026uacute;blica, Instituto Nacional de Salud. Vigilancia de la resistencia a los insecticidas en artr\u0026oacute;podos vectores. Bol Inst Nac Salud. 2018;24(3\u0026ndash;4):31\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMackenzie-Impoinvil L, Weedall GD, Lol JC, Pinto J, Vizcaino L, Dzuris N, et al. Contrasting patterns of gene expression indicate differing pyrethroid resistance mechanisms across the range of the New World malaria vector Anopheles albimanus. PLoS ONE. 2019;14(1):e0210586.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Plasmodium Anopheles, COX1, kdr, Malaria, Native communities, Amazonas, Peru","lastPublishedDoi":"10.21203/rs.3.rs-3409121/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3409121/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMalaria is a severe health problem in the native communities of Condorcanqui in the Amazonas Department of Peru. Recently, the number of malaria cases has increased considerably following a \u003cem\u003ePlasmodium falciparum\u003c/em\u003e outbreak in 2019. However, there is no information on the \u003cem\u003eanopheline\u003c/em\u003e species acting as \u003cem\u003ePlasmodium\u003c/em\u003e vectors in this area or its insecticide resistance status. This study aims to: i) to molecularly characterize the anopheline population from the district of Rio Santiago; ii) to determine their incrimination in malaria transmission; and iii) to evaluate mutations associated with resistance to pyrethroid insecticides and DDT in the mosquito population.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMosquitoes were collected between March and September 2022, using Shannon traps, CDC light traps, and mouth aspirators. Only those morphologically identified as \u003cem\u003eAnopheles\u003c/em\u003e sp. were subjected to molecular confirmation by PCR amplification and sequencing of the COX1 barcode region. Additionally, specimens that were molecularly confirmed as \u003cem\u003eAnopheles\u003c/em\u003e were analyzed for the kdr region of the VGSC gene related to insecticide resistance. Likewise, the presence of human blood as a food source was detected using the β-globin marker, and the presence of \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e was determined through a nested PCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 453 mosquitoes were captured, of which ninety-four were morphologically identified as female anophelines. Of the latter, sixty-six (~\u0026thinsp;70%) specimens were molecularly confirmed as anophelines and were grouped into four species: \u003cem\u003eAn. benarrochi\u003c/em\u003e B, \u003cem\u003eAn. triannulatus, An. costai\u003c/em\u003e and \u003cem\u003eAn. nimbus\u003c/em\u003e. The sixty-six anophelines were analyzed for human β-globin and \u003cem\u003ePlasmodium\u003c/em\u003e. It was found that twenty-three samples of \u003cem\u003eAn. benarrochi\u003c/em\u003e B (~\u0026thinsp;35%) and one specimen of \u003cem\u003eAn. triannulatus\u003c/em\u003e were positive for human β-globin. Likewise, six (~\u0026thinsp;9%) samples of \u003cem\u003eAn. benarrochi\u003c/em\u003e B were positive for \u003cem\u003ePlasmodium\u003c/em\u003e parasites (four for \u003cem\u003eP. falciparum\u003c/em\u003e and two for \u003cem\u003eP. vivax\u003c/em\u003e). It is worth noting that four specimens tested positive for \u003cem\u003ePlasmodium\u003c/em\u003e parasites and human blood simultaneously, making this a robust outcome to incriminate \u003cem\u003eAn. benarrochi\u003c/em\u003e B as the main malaria vector. No specimens presented mutations associated with insecticide resistance in the kdr region.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAn. benarrochi\u003c/em\u003e B is the dominant anopheline species in this study and plays an important role in malaria transmission. Further studies are needed to understand its feeding behavior and activity during dry and rainy seasons to fully incriminate it with malaria transmission and implement targeted vector control programs.\u003c/p\u003e","manuscriptTitle":"Natural Plasmodium infection of Anopheles benarrochi B (Diptera: Culicidae) in native communities of the Province of Condorcanqui, Amazonas-Peru","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-09 21:43:47","doi":"10.21203/rs.3.rs-3409121/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":"f57c9e99-9e56-415c-be54-d75cefe8fc33","owner":[],"postedDate":"October 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-24T18:03:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-09 21:43:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3409121","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3409121","identity":"rs-3409121","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}