Screening of natural Wolbachia infection in mosquitoes (Diptera: Culicidae) from the Cape Verde islands

Funding Information: We are grateful to the National Institute of Public Health for the laboratory support in Cape Verde, and to technicians from the Ministry of Health for their assistance in field work. We would like to thank Prof. Paulo Almeida for providing DNA controls of Cx. pipiens and Cx. quinquefasciatus used in the species identification PCR assay. Funding Information: This work was funded by national funds through FCT—Fundação para a Ciência e Tecnologia, I.P., within the framework of the project ARBOMONITOR (PTDC/BIA-OUT/29477/2017. Aires da Moura was funded by the Ph.D. fellowship program of Camões I.P. Publisher Copyright: © 2023, The Author(s).Background: Wolbachia pipientis is an endosymbiont bacterium that induces cytoplasmic incompatibility and inhibits arboviral replication in mosquitoes. This study aimed to assess Wolbachia prevalence and genetic diversity in different mosquito species from Cape Verde. Methods: Mosquitoes were collected on six islands of Cape Verde and identified to species using morphological keys and PCR-based assays. Wolbachia was detected by amplifying a fragment of the surface protein gene (wsp). Multilocus sequence typing (MLST) was performed with five housekeeping genes (coxA, gatB, ftsZ, hcpA, and fbpA) and the wsp hypervariable region (HVR) for strain identification. Identification of wPip groups (wPip-I to wPip-V) was performed using PCR–restriction fragment length polymorphism (RFLP) assay on the ankyrin domain gene pk1. Results: Nine mosquito species were collected, including the major vectors Aedes aegypti, Anopheles arabiensis, Culex pipiens sensu stricto, and Culex quinquefasciatus. Wolbachia was only detected in Cx. pipiens s.s. (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens/quinquefasciatus hybrids (100%), and Culex tigripes (100%). Based on the results of MLST and wsp hypervariable region typing, Wolbachia from the Cx. pipiens complex was assigned to sequence type 9, wPip clade, and supergroup B. PCR/RFLP analysis revealed three wPip groups in Cape Verde, namely wPip-II, wPip-III, and wPip-IV. wPip-IV was the most prevalent, while wPip-II and wPip-III were found only on Maio and Fogo islands. Wolbachia detected in Cx. tigripes belongs to supergroup B, with no attributed MLST profile, indicating a new strain of Wolbachia in this mosquito species. Conclusions: A high prevalence and diversity of Wolbachia was found in species from the Cx. pipiens complex. This diversity may be related to the mosquito's colonization history on the Cape Verde islands. To the best of our knowledge, this is the first study to detect Wolbachia in Cx. tigripes, which may provide an additional opportunity for biocontrol initiatives. publishersversionpublishe

Morphological and Molecular Characterization Using Genitalia and CoxI Barcode Sequence Analysis of Afrotropical Mosquitoes with Arbovirus Vector Potential

Funding Information: Eddyson Montalvo-Sabino was recipient of a grant from “Programa Nacional de Becas y Crédito Educativo” (PRONABEC), 2019—Beca Generacion del Bicentenario, from the “Ministerio de Educación” of Peru. A.P. Abilio was a recipient of a grant from Wellcome Trust (Grant WT087546MA) through SACIDS RVF and NPHI-Phase-II from the National Institute for Health of Mozambique through a cooperative agreement number [5NU14GH001237-03-00]. Marietjie Venter was a recipient of a sub-award from the Global Disease Detection Program, US-CDC award 5U19GH000571-02 with the NICD and University of Pretoria that funded vector surveillance in South Africa (2012–2015) and by the Cooperative Agreement Number (5 NU2GGH001874-02-00) with the University of Pretoria (2014–2017). Milehna M. Guarido received a studentship through this grant. A.P.G. Almeida has been a recipient of the Visiting Professor Programme by the University of Pretoria for the work in South Africa. This work received financial support from the Global Health and Tropical Medicine Center (GHTM|IHMT|NOVA), which is funded through FCT contract UID/Multi/04413/2013, Portugal. The findings and conclusions expressed in this manuscript are those of the author(s) and do not necessarily represent the official position of the funding agencies. Publisher Copyright: © 2022 by the authors.Potential arboviral Afrotropical mosquito vectors are underrepresented in public databases of CoxI barcode sequences. Furthermore, available CoxI sequences for many species are often not associated with voucher specimens to match the corresponding fine morphological characterization of specimens. Hence, this study focused on the characterization of Culicine mosquitoes from South Africa, Mozambique, and Angola and their classification using a complementary approach including a morphological analysis of specimens’ genitalia and phylogenetic study based on the analysis of CoxI barcode sequences using maximum likelihood and Bayesian phylogenetic inference methods, alongside Median-Joining Network and PCOORD analyses. Overall, 800 mosquitoes (652 males and 148 females) from 67 species, were analyzed. Genitalia from 663 specimens allowed the identification of 55 species of 10 genera. A total of 247 CoxI partial gene sequences corresponding to 65 species were obtained, 11 of which (Aedes capensis, Ae. mucidus, Culex andersoni, Cx. telesilla, Cx. inconspicuosus, Eretmapodites subsimplicipes, Er. quinquevittatus, Ficalbia uniformis, Mimomyia hispida, Uranotaenia alboabdominalis, and Ur. mashonaensis) are, to the best of our knowledge, provided here for the first time. The presence of Cx. pipiens ecotypes molestus and pipiens and their hybrids, as well as Cx. infula, is newly reported in the Afrotropical region. The rates of correct sequence identification using BOLD and BLASTn (≥95% identity) were 64% and 53%, respectively. Phylogenetic analysis revealed that, except for subgenus Eumelanomyia of Culex, there was support for tribes Aedini, Culicini, Ficalbiini, and Mansoniini. A divergence >2% was observed in conspecific sequences, e.g., Aedeomyia africana, Ae. cumminsii, Ae. unilineatus, Ae. metallicus, Ae. furcifer, Ae. caballus, and Mansonia uniformis. Conversely, sequences from groups and species complexes, namely, Ae. simpsoni, Ae. mcintoshi, Cx. bitaeniorhynchus, Cx. simpsoni, and Cx. pipiens were insufficiently separated. A contribution has been made to the barcode library of Afrotropical mosquitoes with associated genitalia morphological identifications.publishersversionpublishe

FACTORES ASOCIADOS A LA CONTAMINACIÓN DE PARQUES PÚBLICOS (HUÁNUCO, PERÚ) CON HUEVOS DE TOXOCARA CANIS Y OTROS ENDOPARASITOS DE IMPORTANCIA ZOONOTICA

The objective of this work was to evaluate the contamination of public parks in the district of Huánuco, Perú with eggs of Toxocara canis. 32 soil samples were taken in each of 11 parks for a total of 352 samples in May and June of 2014. Parks were selected at random. In each park, 2-3 kg soil was collected according to the double “W” method. Flotation with a saturated sugar solution was used. Samples were considered positive when at least one egg of T. canis was found. Of the 11 parks sampled, T. canis eggs were found in 90.9%. Seven parasites were found along with T. canis in the soil. The sequence of parasite prevalence was in the following descending order: T. canis> Strongylus type eggs of Ancylostoma caninum > A. caninum larvae = Blastocystis hominis > Trichuris vulpis= Entamoeba sp. > Capillaria sp. = Balantidium coli. Parasite richness was 2.45 ± 1.21 (1-4) parasites per public park. Asignificant positive correlation was observed between the area of public parks and parasite richness per public park. No association was found between the condition of the public park and it being positive for T. canis. A dendrogram of similarity of association of parasites present in the 11 public parks showed a greater association between the Strongylus egg type and larvae of A. caninum. Also, a greater similarity existed between T. vulpis, B. coli, Entamoeba sp. and Capillaria sp. T. canis had the lowest similarity with the other parasitic forms. An increase in the prevalence of T. canis was observed in comparison to the previous decade as well as to the presence of other infective parasite stages of public health importance. People and especially children could become contaminated while in public parks and sanitary measures should be taken to control this zoonosis.El objetivo de este trabajo fue evaluar la contaminación de parques públicos del distrito de Huánuco, Perú por huevos de Toxocara canis. En cada uno de los 11 parques fueron tomadas 32 muestras por lo que se evaluaron un total de 352 muestras de tierra en mayo y junio del 2014. La selección de los parques fue al azar. De cada parque se recolectó 2 a 3 kg de tierra, según el método de la doble “W”. Se usó el método de flotación con solución saturada de azúcar. Se consideró positivo cuando se encontró al menos un huevo de T. canis por parque evaluado. De los 11 parques muestreados se encontró huevos de T. canis en el 90,9%. Siete fueron las formas parasitarias acompañantes a T. canis en el suelo. La secuencia de prevalencia parasitaria presentó el siguiente orden descendente: T. canis > huevos tipo Strongylus de Ancylostoma caninum > larvas de A. caninum = Blastocystis hominis > Trichuris vulpis = Entamoeba sp. > Capillaria sp. = Balantidium coli. La riqueza parasitaria fue de 2,45±1,21 (1 a 4) parásitos·parque público-1. Se observó una correlación positiva significativa entre el área del parque público y la riqueza de parásitos·parque público-1. No se encontró asociación entre el estado de conservación del parque público y la positividad con T. canis. Un dendrograma de similitud de asociación de los parásitos presentes en los 11 parques públicos mostró una mayor asociación entre huevos tipo Strongylus y larvas de A. caninum. También una mayor similitud entre T. vulpis, B. coli, Entamoeba sp. y Capillaria sp. T. canis presentó la menor similitud con el resto de formas parasitarias. Se observó un aumento en la prevalencia de T. canis en comparación a la década anterior, así como la presencia de otros estadios infectivos de parásitos de importancia en salud pública. Las personas y especialmente los niños pudieran contaminarse durante su estadía en los parques públicos y deben tomarse medidas sanitarias para el control de esta zoonosis

Caracterização morfológica e molecular de mosquitos afrotropicais, potenciais vetores de arbovírus

Muitas das espécies de mosquitos (Diptera: Culicidae) são vetores de patógenos responsáveis por doenças que constituem sérios problemas de saúde pública. Para o estudo da epidemiologia destas, e implementação de estratégias de controlo de vetores, é necessário o reconhecimento correto das espécies de mosquitos. Muitas destas, só são identificáveis com segurança, através de características da genitália dos machos; acresce que muitos espécimes coletados no campo tendem a danificar-se quer no processo de colheita ou de transporte até ao laboratório, perdendo caracteres de identificação importantes. Por outro lado, a integração dos métodos de identificação morfológica com o estudo de marcadores moleculares, será a melhor abordagem para a caracterização de espécies. O objetivo deste estudo foi caracterizar morfologicamente mosquitos Afrotropicais da subfamília Culicinae, nomeadamente através da genitália, bem como através do marcador barcode do gene mitocondrial CoxI, para uma identificação complementar e um estudo filogenético pelos métodos de máxima verosimilhança e Bayesiana. Além disso para o complexo pipiens usou-se o gene ACE2 para diferenciar Cx. pipiens e Cx. quinquefasciatus, e amplificou-se a região flanqueadora do microssatélite CQ11 de espécimes Cx. pipiens para diferenciar os ecotipos pipiens e molestus. Resultados: foram analisados 800 mosquitos, 652 (81,5 %) machos e 148 (18,5 %) fêmeas, de 67 espécies, tendo-se dissecado as genitálias de mosquitos machos, e algumas fêmeas, permitindo a correta identificação de 663 exemplares, 652 (98,3 %) machos e 11 (1,7 %) fêmeas, de 55 espécies de 9 géneros, Aedeomyia, Aedes, Coquillettidia, Culex, Eretmapodites, Ficalbia, Lutzia, Mansonia, Mimomyia e Uranotania. Obtiveram-se 247 sequências do gene CoxI de 65 espécies, das quais, as de Ae. capensis, Ae. mucidus, Cx. andersoni, Cx. telesilla, Cx. inconspicuosus, Er. subsimplicipes, Er. quinquevittatus, Fi. uniformis, Mi. hispida, Ur. alboabdominalis e Ur. mashonaensis, tanto quanto sabemos, foram obtidas pela primeira vez. A análise filogenética sugere que, Ad. africana apresenta duas populações na região Afrotropical; no género Aedes os subgéneros: Neomelaniconion, Stegomyia, Catageiomyia, Fredwardsius, Ochlerotatus e Mucidus formaram clados monofiléticos, enquanto os subgéneros Diceromyia, Aedimorphus e Albuginosus não, no entanto, os dois últimos formaram grupos informais; no género Culex, os subgéneros Eumelanomyia, Oculomyia e Culiciomyia formaram clados monofiléticos, no entanto, o subgénero Culex organizou-se em grupos informais, sem formar um clado monofilético; o género Lutzia formou um clado monofilético inserido dentro do género Culex; reportamos pela primeira vez, os ecotipos molestus e pipiens, e híbridos mol-pip na África do Sul; no género Mansonia, a população de Ma. uniformis, da região Afrotropical diverge da região Indomalaya; as espécies dos géneros: Eretmapodites, Coquillettidia, Ficalbia, Mimomyia e Uranotaenia agruparam-se de acordo com a sistemática. Pensamos ter contribuído para corrigir a falta de confirmação diagnosticante de sequências depositadas nas bases de dados, ao associar neste estudo quer a identificação morfológica, quer a identificação através da genitália, com as sequências respetivas do marcador CoxI. Contudo, este revelou-se insuficiente para separar complexos, e alguns grupos, de espécies, nomeadamente, Ae. simpsoni, Ae. mcintoshi, Cx. bitaeniorhynchus, Cx. simpsoni/sinaiticus, e Cx. pipiens, sendo necessários mais estudos com outros marcadores.Many mosquito species (Diptera: Culicidae) are vectors of pathogens responsible for diseases that constitute serious public health problems. For the study of their epidemiology, and implementation of vector control strategies, it is necessary to correctly recognize the species of mosquitoes. Many of these, are only reliably identified through features of male genitalia; in addition, many specimens collected in the field tend to be damaged either in the process of collection or transport to the laboratory, losing important identification characters. On the other hand, the integration of morphological identification methods with the study of molecular markers will be the best approach for species characterization. The aim of this study was to morphologically characterize Afrotropical mosquitoes of the Culicinae subfamily, namely through the genitalia, as well as through the barcode marker of the CoxI mitochondrial gene, for a complementary identification and a phylogenetic study by the maximum likelihood and Bayesian methods. In the pipiens complex, the ACE2 gene was used to differentiate Cx. pipiens and Cx. quinquefasciatus, and the flanquing region of the microsatellite CQ11 specimens was amplified to differentiate the pipiens and molestus ecotypes of Cx. pipiens. Results: 800 mosquitoes were analyzed, 652 (81.5%) males and 148 (18.5%) females, of 67 species, and the genitalia of male and some female mosquitoes were dissected, allowing the correct identification of 663 specimens, 648 (98.3%) males and 11 (1.7%) females, from 55 species of 9 genera, Aedeomyia, Aedes, Coquillettidia, Culex, Eretmapodites, Ficalbia, Lutzia, Mansonia, Mimomyia and Uranotania. 247 sequences of the CoxI gene from 65 species were obtained, of which those of Ae. capensis, Ae. mucidus, Cx. andersoni, Cx. telesilla, Cx. inconspicuosus, Er. subsimplicipes, Er. quinquevittatus, Fi. uniformis, Mi. hispida, Ur. alboabdominalis and Ur. mashonaensis, to the best of our knowledge, were obtained for the first time. Phylogenetic analysis suggests that Ad. africana has two populations in the Afrotropical region; in the genus Aedes the subgenera: Neomelaniconion, Stegomyia, Catageiomyia, Fredwardsius, Ochlerotatus and Mucidus formed monophyletic clades, while the subgenera Diceromyia, Aedimorphus and Albuginosus did not, however, the last two formed informal groups; in the genus Culex, the subgenus Eumelanomyia, Oculomyia and Culiciomyia formed monophyletic clades, however, the subgenus Culex organized into informal groups, failing to form a monophyletic clade; the genus Lutzia formed a monophyletic clade within the genus Culex; we report for the first time, the molestus and pipiens ecotypes, and mol-pip hybrids in South Africa; in the genus Mansonia, the population of Ma. uniformis, from the Afrotropical region diverges from the Indomalaya region; the species of the genera: Eretmapodites, Coquillettidia, Ficalbia, Mimomyia and Uranotaenia were grouped according to systematics. We believe to have contributed to improve the lack of diagnostic confirmation of sequences deposited in the databases, by associating in this study both morphological identification and confirmation through the genitalia, with the respective sequences of the CoxI marker. However, it was insufficient to separate complexes, and some groups, of species, namely, Ae. simpsoni, Ae. mcintoshi, Cx. bitaeniorhynchus, Cx. simpsoni/sinaiticus, and Cx. pipiens, therefore, further studies with other markers are needed

Additional file 2 of Screening of natural Wolbachia infection in mosquitoes (Diptera: Culicidae) from the Cape Verde islands

Additional file 2: Table S4. Mosquito species collected on each island and tested for Wolbachia using wsp

Additional file 1 of Screening of natural Wolbachia infection in mosquitoes (Diptera: Culicidae) from the Cape Verde islands

Additional file 1: Table S1. Primer sequences used for molecular identification of mosquito species collected in Cape Verde islands. Table S2. Primers used for PCR detection of Wolbachia and genotyping of wPip I–V groups by PCR-RFLP. Table S3. Primers used for Wolbachia MLST loci and wsp hypervariable region amplification and sequence analysis