Vect. In accordance with the aforementioned, Lizarazo et al. (2019). PLoS Neglect. This confirms that this arbovirus is not circulating in the urban cycle of the three sampled municipalities. The lines in each branch indicate the number of mutational steps between haplotypes, and the size of the circle is proportional to the number of individuals. 8, 542555. Subsequently, a step-by-step, second-level interaction model was executed, which included interactions between vector species and the collection area or municipality. aegypti Bora strain was provided by the Dr. Fabrice Chandre, the scientific supervisor of the Vectopole center (IRD, Montpelier, France). In this scenario, molecular identification may become more accurate (Chan et al., 2014). PubMed 2015;8:402. 2016;10:e0004351. 2019;104:91105. 2018;34:22745. Dis. However, in the municipality of Arauquita the species that is found in higher frequency is A. albopictus (Table 2). California Privacy Statement, (2013). b The same four MS spectra per species and developmental stages were analysed using the composite correlation index (CCI) tool. doi: 10.1371/journal.pntd.0002311, Sarfraz, M. S., Tripathi, N. K., Tipdecho, T., Thongbu, T., Kerdthong, P., and Souris, M. (2012). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2020.602190/full#supplementary-material. By using this website, you agree to our However, for the eastern region of Colombia, this information is limited. Since Endogenous Flaviviral Elements have been identified in the mosquito genome (Hou et al., 2019), the chosen primers have an analytical validation to avoid cross reactions. Individuals of the Aedes genus are found in areas with high arbovirus rates, which is why it is associated with an infection cycle in urban areas (Beaty et al., 2016). J. Med. 18, 200-204 doi: 10.1111/j.0269-283X.2004.00482.x, Guerbois, M., Fernandez-Salas, I., Azar, S. R., Danis-Lozano, R., Alpuche-Aranda, C. M., Leal, G., et al. (Bruker Daltonics) . Health 23, 13421349. doi: 10.4269/ajtmh.2011.11-0359, Lorenz, C., Ribeiro, A. F., and Chiaravalloti-Neto, F. (2019). Parasitology. Mem. doi: 10.1371/journal.pntd.0003301, Liang, L., Jia, P., Tan, X., Chen, J., and Chen, X. doi: 10.1093/jmedent/29.1.49, Vega-Ra, A., Zouache, K., Girod, R., Failloux, A. 33, 437442. Article For technical reasons, only the RNA of the insect could be obtained. Dis. Diarra AZ, Almeras L, Laroche M, Berenger JM, Kone AK, Bocoum Z, et al. Symp. Emerg Microbes Infect. 87, 902-909. doi: 10.4269/ajtmh.2012.12-0244, Maia, L. M. S., Bezerra, M. C. F., Costa, M. C. S., Souza, E. M., Oliveira, M. E. B., Ribeiro, A. L. M., et al. Finally, for MAYV, the only reported value is 1.75 (Diallo et al., 2014; Guerbois et al., 2016; Huerta et al., 2017; Cevallos et al., 2018; Eiras et al., 2018; Garcia-Rejon et al., 2018). (Bruker Daltonics) for data processing (smoothing, baseline subtraction, peak picking). PubMed In accordance with the above, there is a coexistence of A. aegypti and A. albopictus in urban areas of the Saravena and Arauquita municipalities (Figure 4A). 2. 2014;113:23758. Sequence cleaning and alignment was performed in Seqman software (Lasergene) and the nucleotide sequence was compared to the Nucleotide Blast database1; the species were assigned considering the percentage of identity, the coverage value and e-value. Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali. In A. aegypti, DENV presents a higher range of MIR compared to other viruses and ranges from 0.2 to 26 infected individuals per 1,000 individuals. Process. Considering the groups formed by species in the haplotype network (Figure 3A) and the clustering by species in the phylogenetic tree (Figure 3C), it is possible to determine the precision of molecular tests to identify A. aegypti and A. albopictus. Epidemiological trends of dengue disease in Colombia (2000-2011): a systematic review. From the alignments, a network of haplotypes was generated in PorpArt v1.7 software using the TCS algorithm (Clement et al., 2002), and haplotype diversity (h) was calculated in DNAsp v6 (Librado and Rozas, 2009). Salud. J. Epidemiol. Cluster analyses (MSP dendrogram) were performed based on a comparison of the MSP given by MALDI-Biotyper v3.0. 2010;27:4753. Yssouf A, Parola P, Lindstrom A, Lilja T, LAmbert G, Bondesson U, et al. This shows a simultaneous circulation of these two arboviruses within one specimen, which can lead to coinfection in humans, where an increase in coinfection cases would lead to rapid genetic evolution of the virus (Caron et al., 2012). Angelini P, Macini P, Finarelli AC, Pol C, Venturelli C, Bellini R, et al. The main characteristic that permits differentiation between these two species is the pattern in the scutum (mesothorax sclerite). The absence of taxonomic keys and/or the overlapping features used for the exuviae, results in failures to reach definitive species identification based on morphology . Parasitol Res. Accordingly, the high number and diversity of haplotypes present in Saravena (h = 0.806, Figure 3B) may be due to evolutionary pressures that generate genetic changes in these dipteran species such as the use of insecticides. de Parasitologa. In accordance with our results, different models (Barmak et al., 2016; Massaro et al., 2019; Zhu et al., 2019) show that human mobility is an important factor for the increase in the incidence of DENV even over short distances, i.e., between cities. J. Trop. However, mosquito larval identification is generally complex and requires entomological expertise. Pest Manag Sci. (2015). B., and Loureno-de-Oliveira, R. (2014). Biol Lett. DNA Barcoding and Forensic Entomology: A Molecular Approach for Diptera Species Identification. The phenomena of protein degradation/digestion generating unstable MS profiles, was previously reported when blood meals from engorged mosquitoes were analyzed by MALDI-TOF MS approach . Geneve: WHO. J. Bioinform. Representative MS spectra of two exuviae from Ae. Rev. Therefore, it is important that future studies implement viral enrichment techniques that allow virus detection even at low concentrations. doi: 10.7705/biomedica.v38i0.3728, PubMed Abstract | CrossRef Full Text | Google Scholar, Azevedo, R. S., Silva, E. V., Carvalho, V. L., Rodrigues, S. G., Neto, J. P. N., Monteiro, H. A., et al. The thermal profile used for all arboviruses consisted of the following: one cycle for 50C for reverse transcriptase activation for 10 min, followed by initial denaturation at 95C for 1 min, 40 cycles of 95C for 15 s, 60C for 1 min and final extension at 72C for 10 min. World Health Organization [WHO] (2020). The intensifying storm: Domestication of Aedes aegypti, urbanization of arboviruses, and emerging insecticide resistance, in Proceedings of the Global Health Im-pacts of Vector-Borne Diseases: Workshop Summary (Washington, DC: National Academies Press). Euro Surveill. However, there are no studies with methodologies that detect natural infection for arboviruses in single insects. Our findings could become valuable to improve national control programs for arbovirosis. Infect. The sequences were aligned using the MUSCLE algorithm in the MEGA X v10.1 software (Sohpal et al., 2010). Therefore, we propose to use this complementary tool mainly in three situations; (i) generate a baseline on the proportions of individuals per species in areas where this information is not available, (ii) as a quality control of morphological identification, choosing a random percentage of individuals to be confirmed by DNA barcoding and (iii) when individuals are damaged and morphological identification cannot be performed. Longitudinal monitoring of environmental factors at Culicidae larval habitats in urban areas and their association with various mosquito species using an innovative strategy. Colomb. Baldacchino F, Caputo B, Chandre F, Drago A, Della TA, Montarsi F, et al. Detection of invasive mosquito vectors using environmental DNA (eDNA) from water samples. MS spectra profiles were firstly controlled visually with flex Analysis v3.3 software (Bruker Daltonics). This strategy will overcome the limitations of morphological and molecular identification and will avoid the use of the entire body of the specimen, as it was done previously . Nebbak A, Willcox AC, Koumare S, Berenger JM, Raoult D, Parola P, et al. For an efficient vector control at immature stages, accurate identification of mosquito species at the larval stage is compulsory. Genetic variability of Aedes aegypti in the department of sucre, colombia, by analysis of the nucleotide sequence of the mitochondrial ND4 gene. (2019). 177, 7480. Six traps were installed in the municipality of Saravena, 5 in Tame and 4 in Arauquita. The LSVs ranged between 1.652.72 for fourth-instar exuviae and 1.232.70 for pupal exuviae (Fig. Virol. Genetic variability of the Aedes aegypti (Diptera: Culicidae) mosquito in El Salvador, vector of dengue, yellow fever, chikungunya and Zika. Spatial dynamics of Chikungunya virus, Venezuela, 2014. Table 2 shows the arbovirus infection rates in each species, and there was a higher infection rate observed in individuals of the species A. albopictus. This interaction is shown in the models that show an association between land use and the presence of A. aegypti and A. albopictus and the prevalence of arboviruses. Clin. Parasit Vectors. Development of guidelines for the surveillance of invasive mosquitoes in Europe. Int. MALDI-TOF MS biotyping emerged recently as a relevant alternative solution, to dispense with morphological and molecular biology identification tools . Furthermore, the global diversity of haplotypes for all individuals captured in Arauca was h = 0.639. The next step will be the validation of this innovative strategy on exuviae from aedine larvae collected in the field and laboratory bred until adulthood. Encyclop. Unfortunately, for species identification by MS, the euthanasia of the mosquito specimen is required. Dis. We observed two groups that corresponded to the species A. aegypti (Orange) and A. albopictus (Purple) and are differentiated by 36 bp (Figure 3A). Approximately 300 structural cuticular proteins from Anopheles gambiae were recently found . Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Muller P, et al. BMC Publ. As two MS peaks from exuviae of Aedes species were shared, a total of 18 MS peaks were selected corresponding to the top five mass peak lists per sample group (Fig. IV., Bennett, K. E., Gorrochtegui-Escalante, N., Barillas-Mury, C. V., Fernndez-Salas, I., de Lourdes Muoz, M., et al. In this study was described the precision of taxonomic keys and DNA barcoding to classify individuals of the genus Aedes. *Correspondence: Juan David Ramrez, firstname.lastname@example.org, Biology, Systematics, Taxonomy, and Evolution of Insect Vectors, View all The parameters of Main effects model and Second level interaction model. Am J Trop Med Hyg. To control matrix quality (i.e. This corroborates the influence that the movement of people between these two countries has on the prevalence of infection in insects. albopictus MRS strain originated from a field collection of larvae of Marseille city (south of France) . A. Entomol. 2018;12:e0006845. (2018). In addition, the infection by DENV-1, CHIKV, ZIKV, and MAYV was attempted. CAS Unreliable species identification may contribute to unsuitable vector control efforts which could conduct to VBDs outbreaks. LA conceived and designed the experiments, performed the experiments and analyzed the data. 651, 969978. In this way, exuviae from the fourth-instar and pupal stages of Aedes albopictus and Aedes aegypti were submitted to MALDI-TOF MS analysis. Molecular identification of mosquito vectors using genomic DNA isolated from eggshells, larval and pupal exuvium. These sites were chosen due to the high incidence of arboviral cases. The analysis of exuviae MS spectra revealed that protein profiles are singular between these two aedine species but also between exuviae of the fourth-instar and pupal stages for each species. However, to produce a correct estimate, it is necessary to obtain a sampling 1,000 individuals and sizes of small pools. software was also used, to assess the spectral variations within and between sample groups, as previously described [20, 26]. Res. Total Environ. Regarding the analysis of the COI sequences, a global diversity of haplotypes value of 0.639 was evident for species A. aegypti and A. albopictus. albopictus (a, b) and Ae. Vect. Vet Sci. albopictus and Ae. aegypti (c, d) at fourth-instar. Boletn Epidemiolgico Semanal (BES) Semana Epidemiolgica 22, 24 al 30 de mayo (2020). The reproducibility of MS spectra was confirmed by CCI matrix highlighting the good correlation between exuviae of the same species and stage (meanSD: 0.800.14; Fig. 2014;7:142. Trends Parasitol. Oswaldo Cruz 115: e190437. Prevalence and distribution of dengue virus in Aedes aegypti in yogyakarta city before deployment of Wolbachia infected Aedes aegypti. 2018;45:393407. 2019;6:40. Royal Soc. 93.8% (135/144) of exuviae LSVs reached the threshold value (LSVs>1.8) for reliable identification [24, 27], among which 100% of the samples were correctly classified at the species and stage levels. This shows the susceptibility of this species to infection by arboviruses. Nevertheless, the molecular biology approach for mosquito identification remains laborious, time-consuming and costly . doi: 10.1016/j.sste.2014.05.002. Med. Entomol. The dotted lines mark the inconsistencies where individuals were initially assigned by morphology to a different species compared to the one detected by molecular methods, and the latter is the technique by which individuals were ultimately assigned for subsequent sequence analysis. Reinert JF, Kaiser PE, Seawright JA. Acta tropica 198:105093. doi: 10.1016/j.actatropica.2019.105093, Lounibos, L. P., and Kramer, L. D. (2016). Zamora-Delgado, J., Castao, J. C., and Hoyos-Lpez, R. (2015). Marchand E, Prat C, Jeannin C, Lafont E, Bergmann T, Flusin O, et al. Pest Manag Sci. doi: 10.7554/eLife.08347, Lanciotti, R. S., Kosoy, O. L., Laven, J. J., Panella, A. J., Velez, J. O., Lambert, A. J., et al. 9: e0003499. The insects or pools were stored in Invitrogen RNAlater (Salehi and Najafi, 2014) in jars marked with the coordinates and information regarding the house and/or the collection site and transported to the microbiology laboratory of the Universidad del Rosario. 85, 750757. Google Scholar. These patterns are described as susceptible to blurring during insect storage processing (Patsoula et al., 2006). Paras. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Terms and Conditions, Study of Aedes aegypti population with emphasis on the gonotrophic cycle length and identification of arboviruses: implications for vector management in cemeteries. Vector competence of European mosquitoes for West Nile virus. CCI matrix was calculated using MALDI-Biotyper v3.0. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Inconsistencies were mainly evident in the identification of 13 individuals (7.7%), where they were initially identified through morphology as A. aegypti while the molecular marker analysis indicated that they were A. albopictus. The black dot indicates the urban area where the capture of individuals was made. 112, 124128. J Am Mosq Control Assoc. Reproducible and specific MS spectra according to aedine species and stage of exuviae were observed which were objectified by cluster analyses, composite correlation index (CCI) tool and principal components analysis (PCA). Methods 195, 194204. Loaiza JR, Almanza A, Rojas JC, Mejia L, Cervantes ND, Sanchez-Galan JE, et al. Among them, the expression of some cuticular proteins was restricted to a single metamorphic stage, which could explain the singularity of MS profiles between exuviae from the same species. Distrib. The parameter settings in ClinProTools software for spectrum preparation were as follows: a resolution of 300; a noise threshold of 2.00; a maximum peak shift of 800 ppm and a match to calibrating agent peaks of 10%. The current strategy for mosquito identification by this proteomic tool at the immature stages is done on whole specimens. The creation of reference MS spectra DB for mosquitoes covering the different developmental stages from eggs to the adult stage are essential for the wide use of this innovative proteomic tool. Dis. The PCA revealed a clear separation of the dots corresponding to MS spectra from exuviae stages of Aedes species (Fig. doi: 10.1371/journal.pntd.0003499, Walter, SD, Hildreth, SWY and Beaty, BJ. 8:e3301. Likewise, a longer sampling time that could allow us a greater capture of individuals and a more general view of the behavior of the infection of arboviruses in A. aegypti and A. albopictus. Biomdica 37, 193200. Supplementary Table 1 | Logistic regression. Environ Sci Technol. The dengue virus mosquito vector Aedes aegypti at high elevation in Mexico. Google Scholar. (2019). albopictus and Ae. Rev. (2020) shows that under laboratory conditions, A. aegypti and A. albopictus have the ability to become infected and the potential to transmit MAYV genotype D. Likewise, it is possible to infect mosquitoes with the virus from the saliva of individuals who initially tested negative. https://doi.org/10.1186/s13071-020-04029-x, DOI: https://doi.org/10.1186/s13071-020-04029-x. (2018). 2008;78:4928. Mech. 2016;85:87. aList of MS peaks used to distinct exuviae stages from two Aedes species based on the genetic algorithm model analysis of ClinProTools. doi: 10.1016/j.actatropica.2016.03.010, Turell, M. J., Beaman, J. R., and Tammariello, R. F. (1992). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Trop. Parasit Vectors. 4). 10:311. doi: 10.1186/1743-422x-10-311. 2016;50:185967. Copyright 2020 Martnez, Hernndez, Muoz, Armesto, Cuervo and Ramrez. aegypti (g, h) at the pupal stage. a Four exuviae per species (i.e. However, the absence of individuals infected by MAYV may be because the insects processed in this study are part of the urban cycle and no studies demonstrate the efficiency of viral transmission in this cycle. Field application of MALDI-TOF MS on mosquito larvae identification. Sousa CA, Clairouin M, Seixas G, Viveiros B, Novo MT, Silva AC, et al. Nebbak A, Koumare S, Willcox AC, Berenger JM, Raoult D, Almeras L, et al. Parasit Vectors. (2014). albopictus and Ae. Top five mass peak list per Aedes species using exuviae from immature stages as biological material. For the collection of exuviae, mosquito larvae at third-instar were transferred individually to a small jar and reared until the larvae molted into fourth-instar, pupae and adults. The invasive mosquito Aedes japonicus japonicus is spreading in northeastern Italy. 2c), comforting the specificity of MS profiles between these sample groups. The sturdiness of the cuticle for to homogenization and the quick degradation of the exuviae are factors which could decrease the abundance of proteins and peptides for resulting MS spectra. 2022 BioMed Central Ltd unless otherwise stated. The raw data of exuviae MS spectra from Ae. Proteomics. Asymptomatic humans transmit dengue virus to mosquitoes. The top five mass peaks per sample group are indicated in bold. The lower intensity (<2000 a.u.) The topology of the phylogenetic tree obtained from the COI sequence alignment revealed two well-supported clusters (bootstrap 90) that correspond to A. aegypti and A. albopictus (Figure 3C) with a genetic distance of 0.107 between the species. Lastly, we demonstrated the coexistence of A. aegypti and A. albopictus in urban areas of Saravena and Arauquita. 8:602190. doi: 10.3389/fevo.2020.602190. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes. Google Scholar. Lionel Almeras. However, it was experimentally shown that A. albopictus is a vector more susceptible to infection than A. aegypti because the arbovirus dissemination rates were higher in nearly all strains of A. albopictus tested (Turell et al., 1992). Endogenous non-retroviral elements in genomes of Aedes mosquitoes and vector competence. Zhou Y, Badgett MJ, Orlando R, Willis JH. aegypti) and developmental stage (i.e. Dis. Expression of the natural infection data using infection rates to obtain a value per individual on arbovirus infection is proposed which increased the infection rate and the finding of viral co-infections. Dis. (2016). J. aegypti at the fourth-instar and pupal stage added to the MS reference database. 41, 218-227, Huerta, H., Gonzlez-Roldn, J. F., Snchez-Tejeda, G., Correa-Morales, F., Romero-Contreras, F. E., Crdenas-Flores, R., et al. (2010). Environmental conditions influence eDNA persistence in aquatic systems. 15:1830. doi: 10.3201/eid1511.090461, Barbosa, R. M. R., Melo-Santos, M. A. V. D., Silveira, Jr, J. C., Silva-Filha, M. H. N. L., Souza, W. V., Oliveira, C. M. F. D., et al. Venezuelas humanitarian crisis, resurgence of vector-borne diseases, and implications for spillover in the region. Only 5 individuals of A. aegypti were found coinfected by DENV-1 and CHIKV. 7, 112. Lee H, Halverson S, Ezinwa N. Mosquito-borne diseases. 14:726736 doi: 10.4257/oeco.2010.1403.08, Garcia-Rejon, J. E., Ulloa-Garcia, A., Cigarroa-Toledo, N., Pech-May, A., Machain-Williams, C., Cetina-Trejo, R. C., et al. Mayaro virus distribution in South America. PubMed Central Alternatively, ZIKV has a MIR value ranging from 3 to 17 individuals and CHIKV ranges from 2 to 3 individuals. The present tool is not dedicated to list mosquito fauna from an aquatic site by analyzing their exuviae, but rather to identify specimens at immature stages collected in the field and reared to adulthood in the laboratory. The global distribution of the arbovirus vectors Aedes aegypti and Ae. For this reason, the concept of the DNA barcoding emerges as a new tool for species discrimination. Mosquito-borne diseases: advances in modelling climate-change impacts. Arboviruses in Viral Infections of Humans. 13, 764767. This is why different studies propose viral enrichment techniques that allow the viral sequences to increase, which is corroborated by next generation sequencing (NGS) (Hall et al., 2014). Entomological surveillance of mosquito vectors becomes a necessity to deal with the rapid spread of MBDs, currently operating in different parts of the world. Rolo, E. S. A. MS spectra for raw exuviae from Ae. Mosquito exuviae were already used for morphological species identification [16, 36]. doi: 10.1016/j.jviromet.2013.08.035, Higa, Y., Yen, N. T., Kawada, H., Son, T. H., Hoa, N. T., and Takagi, M. (2010). 2016;11:e0162493. Instit. (B) The percentage of individuals infected by the municipalities of Arauquita, Saravena, and Tame. doi: 10.1093/molbev/msu300, Patsoula, E., Samanidou-Voyadjoglou, A., Spanakos, G., Kremastinou, J., Nasioulas, G., and Vakalis, N. C. (2006). Tahir D, Davoust B, Almeras L, Berenger JM, Varloud M, Parola P. Anti-feeding and insecticidal efficacy of a topical administration of dinotefuran-pyriproxyfen-permethrin spot-on (Vectra 3D) on mice against Stegomyia albopicta (= Aedes albopictus). Parasites Vectors 13, 161 (2020). Exuviae from fourth-instar are highly delicate and their collection in the field is very challenging. B., et al. However, A. albopictus, which is only present in the municipalities of Saravena and Arauquita in both urban and rural areas (Figure 4A), showed a frequency of 74.1% (43/58) in the municipality of Arauquita. Schaffner F, Kaufmann C, Pfluger V, Mathis A. koreicus (Korean mosquito) and Ae. TCS: estimating gene genealogies. So Paulo: Faculdade de Higiene e Sade Pblica, Depto. Euro Surveill. Miyagi I, Toma T, Mogi M, Yotopranoto S, Arifin Z, et al. By keeping the specimen intact, complementary analysis on the same specimen is possible, such as insecticide susceptibility or vector competence. were obtained for more than 95% (153/160) of exuviae from the two Aedes species. Google Scholar. This cross-checking of specimen identification will be particularly imperative to distinguish closely related species with different vector competence living sympatrically . 35. Nevertheless, the rates of correct identification at the pupal stages were lower than at late-instar stages using MALDI-TOF MS biotyping [22, 30]. Infestation of an endemic arbovirus area by sympatric populations of Aedes aegypti and Aedes albopictus in Brazil. Exuviae from the same species and developmental stage clustered on the same branch revealing the reproducibility and specificity of the protein profiles (Fig. 447, 129140. CAS Parasit Vectors. 2018;145:67787. This value is similar to that reported by Caldera et al. (2019). We would like to thank the Direccin de Investigacin e Innovacin from Universidad del Rosario for funding this study and providing the editing English service of ENAGO and cover the publication fees. Therefore, this study morphologically and molecularly identifies Aedes species circulating in three municipalities of the department of Arauca (Colombia) and obtained sequences that were used to assess the precision of the molecular test used to identify A. aegypti and A. albopictus. The Ae. 3). Provided by the Springer Nature SharedIt content-sharing initiative. Therefore, the importance of conducting surveillance of arboviruses in the individual vector in areas where there is no information on natural infection in the vectors is important. doi: 10.1371/journal.pntd.0001369. Experimental transmission of Mayaro virus by Aedes aegypti. Rev. Article Effectively, the lower protein abundance, for instance, in early larval stages  or eggs  from mosquitoes conducted to lower MS spectra, thus affecting the rate of reliable identification. Ae. Moreover, global warming facilitated the colonization of new areas by mosquitoes participating to the rapid change of the mosquito-borne diseases (MBDs) epidemiology [3, 4].