Microbiota identified from preserved Anopheles

Abstract

Background Mosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution. Methods Microbial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks. Results Elizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices. Conclusions This study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

Microbiota Variation Across Life Stages of European Field-Caught Anopheles atroparvus and During Laboratory Colonization: New Insights for Malaria Research

“…atroparvus , the vast majority from the phylum Proteobacteria . Most of the bacterial taxa herein reported have been described as part of the microbiota in culicid mosquitoes ( Dada et al, 2014 ; Muturi et al, 2016 ; Rocha-David et al, 2016 ; Hegde et al, 2018 ; Kang et al, 2020 ) including Anopheles from different geographic regions ( Rani et al, 2009 ; Djadid et al, 2011 ; Wang et al, 2011 ; Boissiere et al, 2012 ; Gimonneau et al, 2014 ; Ngo et al, 2015 ; Bogale et al, 2020 ; Galeano-Castañeda et al, 2020 ; Zoure et al, 2020 ; Feng et al, 2021 ; Silva et al, 2021 ).…”

“…In adult mosquitoes, diverse dietary regimes (e.g., plant sap and nectar or blood) may alter the composition of the microbiota and incorporate diversity into the microbial communities ( Rani et al, 2009 ). In fact, descriptive studies that have characterized the microbiota of several field populations and/or laboratory colonies of culicid mosquitoes have suggested geographical, species, sex, and even individual variation ( Yadav et al, 2015 ; Akorli et al, 2016 ; Muturi et al, 2016 ; Bascuñan et al, 2018 ; Rodriguez-Ruano et al, 2020 ; Saab et al, 2020 ; Tainchum et al, 2020 ; Silva et al, 2021 ).…”

Malaria-Transmitting Vectors Microbiota: Overview and Interactions With Anopheles Mosquito Biology

“…funestus and An. arabiensis , the presence of a number of bacteria even weeks after mosquitoes’ preservation on silica or in RNAlater ® solution ( E Silva et al, 2021 ). The authors found that the midgut of An.…”

“…arabiensis was mainly colonized by Bacteroidetes and Proteobacteria , with the latter being the predominant one colonizing An. funestus midgut ( E Silva et al, 2021 ). Elizabethkingia and Serratia were found in both An.…”

Introduction to Molecular Diagnostics of Insects

“…Альтернативой криоконсервации для особо ценных образцов могут служить различные коммерческие реагенты, например, Allprotect Tissue Reagent (Qiagen) или RNAlater (Ambion), позволяющие без использования жидкого азота и сухого льда фиксировать ДНК образцов до 1 суток при 37 °C, до 7 суток при комнатной температуре, до 4 недель при 2-8 °C или неограниченно при -20 или -80 °C (Gorokhova, 2005;Nagy, 2010;Peña-Llopis, Brugarolas, 2013;Silva et al, 2021). Важно, что повторная разморозка/ заморозка образца не нарушает сохранность ДНК, однако морфологические структуры могут пострадать.…”