Analysis of trapped mosquito excreta as a noninvasive method to reveal biodiversity and arbovirus circulation

Emerging and endemic mosquito‐borne viruses can be difficult to detect and monitor because they often cause asymptomatic infections in human or vertebrate animals or cause nonspecific febrile illness with a short recovery waiting period. Some of these pathogens circulate into complex cryptic cycles involving several animal species as reservoir or amplifying hosts. Detection of cases in vertebrate hosts can be complemented by entomological surveillance, but this method is not adapted to low infection rates in mosquito populations that typically occur in low or nonendemic areas. We identified West Nile virus circulation in Camargue, a wetland area in South of France, using a cost‐effective xenomonitoring method based on the molecular detection of virus in excreta from trapped mosquitoes. We also succeeded at identifying the mosquito species community on several sampling sites, together with the vertebrate hosts on which they fed prior to being captured using amplicon‐based metabarcoding on mosquito excreta without processing any mosquitoes. Mosquito excreta‐based virus surveillance can complement standard surveillance methods because it is cost‐effective and does not require personnel with a strong background in entomology. This strategy can also be used to noninvasively explore the ecological network underlying arbovirus circulation.

[1]  M. Jiménez-Clavero,et al.  Circulation of zoonotic flaviviruses in wild passerine birds in Western Spain. , 2022, Veterinary microbiology.

[2]  D. Roiz,et al.  A field test of the dilution effect hypothesis in four avian multi-host pathogens , 2021, PLoS pathogens.

[3]  D. Baird,et al.  Mosquito Identification From Bulk Samples Using DNA Metabarcoding: a Protocol to Support Mosquito-Borne Disease Surveillance in Canada , 2021, Journal of Medical Entomology.

[4]  David P. Parsons,et al.  Seaview Version 5: A Multiplatform Software for Multiple Sequence Alignment, Molecular Phylogenetic Analyses, and Tree Reconciliation. , 2021, Methods in molecular biology.

[5]  T. Bakonyi,et al.  West Nile virus keeps on moving up in Europe , 2020, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[6]  G. Grard,et al.  Contrasted Epidemiological Patterns of West Nile Virus Lineages 1 and 2 Infections in France from 2015 to 2019 , 2020, Pathogens.

[7]  Nicholas A. Bokulich,et al.  A total crapshoot? Evaluating bioinformatic decisions in animal diet metabarcoding analyses , 2020, Ecology and evolution.

[8]  D. Cereda,et al.  Enhanced West Nile Virus Circulation in the Emilia-Romagna and Lombardy Regions (Northern Italy) in 2018 Detected by Entomological Surveillance , 2020, Frontiers in Veterinary Science.

[9]  Steven A. Williams,et al.  Field evaluation of DNA detection of human filarial and malaria parasites using mosquito excreta/feces , 2020, PLoS neglected tropical diseases.

[10]  Guangchuang Yu,et al.  Using ggtree to Visualize Data on Tree‐Like Structures , 2020, Current protocols in bioinformatics.

[11]  M. Sharakhova,et al.  Genomic differentiation and intercontinental population structure of mosquito vectors Culex pipiens pipiens and Culex pipiens molestus , 2019, Scientific Reports.

[12]  S. Ritchie,et al.  Malaria surveillance from both ends: concurrent detection of Plasmodium falciparum in saliva and excreta harvested from Anopheles mosquitoes , 2019, Parasites & Vectors.

[13]  Z. Molnár,et al.  Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018 , 2019, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[14]  S. Ritchie,et al.  Development and Field Evaluation of a System to Collect Mosquito Excreta for the Detection of Arboviruses , 2019, Journal of Medical Entomology.

[15]  S. Ritchie,et al.  Stability of West Nile Virus (Flaviviridae: Flavivirus) RNA in Mosquito Excreta , 2019, Journal of Medical Entomology.

[16]  T. Porter,et al.  COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems , 2019, bioRxiv.

[17]  Karthik Gangavarapu,et al.  An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar , 2018, Genome Biology.

[18]  S. Ritchie,et al.  Mosquito excreta: A sample type with many potential applications for the investigation of Ross River virus and West Nile virus ecology , 2018, PLoS neglected tropical diseases.

[19]  A. Kawahara,et al.  Barcoding blood meals: New vertebrate-specific primer sets for assigning taxonomic identities to host DNA from mosquito blood meals , 2018, PLoS neglected tropical diseases.

[20]  S. Ritchie,et al.  Modifying the Biogents Sentinel Trap to Increase the Longevity of Captured Aedes aegypti , 2018, Journal of Medical Entomology.

[21]  F. Lista,et al.  Prevalence of Usutu and West Nile virus antibodies in human sera, Modena, Italy, 2012 , 2018, Journal of medical virology.

[22]  Z. Hubálek,et al.  West Nile virus in overwintering mosquitoes, central Europe , 2017, Parasites & Vectors.

[23]  O. Gascuel,et al.  SMS: Smart Model Selection in PhyML , 2017, Molecular biology and evolution.

[24]  Trevor Bedford,et al.  Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples , 2017, Nature Protocols.

[25]  B. Durand,et al.  West Nile virus epizootics in the Camargue (France) in 2015 and reinforcement of surveillance and control networks. , 2016, Revue scientifique et technique.

[26]  H. M. Tahir,et al.  The sequence divergence in cytochrome C oxidase I gene of Culex quinquefasciatus mosquito and its comparison with four other Culex species , 2016, Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis.

[27]  Paul J. McMurdie,et al.  DADA2: High resolution sample inference from Illumina amplicon data , 2016, Nature Methods.

[28]  A. Fontaine,et al.  Excretion of dengue virus RNA by Aedes aegypti allows non-destructive monitoring of viral dissemination in individual mosquitoes , 2016, Scientific Reports.

[29]  Weam I Zaky,et al.  A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria , 2016, PLoS neglected tropical diseases.

[30]  B. Nosal,et al.  West Nile Virus , 2016, Methods in Molecular Biology.

[31]  David Bryant,et al.  popart: full‐feature software for haplotype network construction , 2015 .

[32]  N. Becker,et al.  Epidemic Spread of Usutu Virus in Southwest Germany in 2011 to 2013 and Monitoring of Wild Birds for Usutu and West Nile Viruses. , 2015, Vector borne and zoonotic diseases.

[33]  A. Aldemir,et al.  Barcoding Turkish Culex mosquitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new potential vectors. , 2015, Acta tropica.

[34]  W. Johnson,et al.  Xenosurveillance: A Novel Mosquito-Based Approach for Examining the Human-Pathogen Landscape , 2015, PLoS neglected tropical diseases.

[35]  A. von Haeseler,et al.  IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies , 2014, Molecular biology and evolution.

[36]  Jean M. Macklaim,et al.  Unifying the analysis of high-throughput sequencing datasets: characterizing RNA-seq, 16S rRNA gene sequencing and selective growth experiments by compositional data analysis , 2014, Microbiome.

[37]  Jiajie Zhang,et al.  A general species delimitation method with applications to phylogenetic placements , 2013, Bioinform..

[38]  Susan Holmes,et al.  phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data , 2013, PloS one.

[39]  J. Martínez‐de la Puente,et al.  Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus , 2013, Malaria Journal.

[40]  T. Andreadis The Contribution of Culex pipiens Complex Mosquitoes to Transmission and Persistence of West Nile Virus in North America , 2012, Journal of the American Mosquito Control Association.

[41]  Brian D. Carroll,et al.  Detection of persistent west nile virus RNA in experimentally and naturally infected avian hosts. , 2012, The American journal of tropical medicine and hygiene.

[42]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[43]  A. Tenório,et al.  Phylogenetic relationships of Western Mediterranean West Nile virus strains (1996-2010) using full-length genome sequences: single or multiple introductions? , 2011, The Journal of general virology.

[44]  R. Charrel,et al.  RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests , 2011, PloS one.

[45]  S. Ritchie,et al.  Exploiting mosquito sugar feeding to detect mosquito-borne pathogens , 2010, Proceedings of the National Academy of Sciences.

[46]  A. Giovannini,et al.  Epidemiology of west nile in europe and in the mediterranean basin. , 2010, The open virology journal.

[47]  Z. Hubálek,et al.  Zoonotic mosquito-borne flaviviruses: worldwide presence of agents with proven pathogenicity and potential candidates of future emerging diseases. , 2010, Veterinary microbiology.

[48]  Hadley Wickham,et al.  ggplot2 - Elegant Graphics for Data Analysis (2nd Edition) , 2017 .

[49]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[50]  P. G. Shute,et al.  The Culex pipiens Complex. , 2009 .

[51]  J. Swaddle,et al.  Increased Avian Diversity Is Associated with Lower Incidence of Human West Nile Infection: Observation of the Dilution Effect , 2008, PloS one.

[52]  H. Zeller,et al.  West Nile virus in wild resident birds, Southern France, 2004. , 2007, Vector borne and zoonotic diseases.

[53]  D. Bicout,et al.  Bird species potentially involved in introduction, amplification, and spread of West Nile virus in a Mediterranean wetland, the Camargue (Southern France). , 2007, Vector borne and zoonotic diseases.

[54]  S. Guptill,et al.  Avian diversity and West Nile virus: testing associations between biodiversity and infectious disease risk , 2006, Proceedings of the Royal Society B: Biological Sciences.

[55]  Olivier Gascuel,et al.  PHYML Online: A Web Server for Fast Maximum Likelihood-Based Phylogenetic Inference , 2018 .

[56]  A. Dobson Population Dynamics of Pathogens with Multiple Host Species , 2004, The American Naturalist.

[57]  E. Gould,et al.  Serological evidence of West Nile virus, Usutu virus and Sindbis virus infection of birds in the UK. , 2003, The Journal of general virology.

[58]  Armando Giovannini,et al.  West Nile virus Epidemic in Horses, Tuscany Region, Italy , 2002, Emerging infectious diseases.

[59]  V. Deubel,et al.  West Nile in the Mediterranean Basin: 1950‐2000 , 2001, Annals of the New York Academy of Sciences.

[60]  K. Schmit,et al.  Dead bird surveillance as an early warning system for West Nile virus. , 2001, Emerging infectious diseases.

[61]  R. Lanciotti,et al.  West Nile virus in overwintering Culex mosquitoes, New York City, 2000. , 2001, Emerging infectious diseases.

[62]  J. P. Durand,et al.  West Nile outbreak in horses in southern France, 2000: the return after 35 years. , 2001, Emerging infectious diseases.

[63]  K. Crandall,et al.  TCS: a computer program to estimate gene genealogies , 2000, Molecular ecology.

[64]  Z. Hubálek,et al.  West Nile fever--a reemerging mosquito-borne viral disease in Europe. , 1999, Emerging infectious diseases.

[65]  C Cernescu,et al.  West Nile encephalitis epidemic in southeastern Romania , 1998, The Lancet.

[66]  R. Vrijenhoek,et al.  DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. , 1994, Molecular marine biology and biotechnology.

[67]  L Joubert,et al.  [Epidemiology of the West Nile virus: study of a focus in Camargue. IV. Meningo-encephalomyelitis of the horse]. , 1970, Annales de l'Institut Pasteur.