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A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae).

ABSTRACT: DNA metabarcoding offers new perspectives, especially with regard to the high-throughput identification and diagnostics of pathogens. Microsporidia are an example of widely distributed, opportunistic and pathogenic microorganisms in which molecular identification is important for both environmental research and clinical diagnostics. We have developed a method for parallel detection of both microsporidian infection and the host species. We designed new primer sets: one specific for the classical Microsporidia (targeting the hypervariable V5 region of small subunit [ssu] rDNA), and a second one targeting a shortened fragment of the COI gene (standard metazoan DNA-barcode); both markers are well suited for next generation sequencing. Analysis of the ssu rDNA data set representing 607 microsporidian species (120 genera) indicated that the V5 region enables identification of >98% species in the data set (596/607). To test the method, we used microsporidians that infect mosquitoes in natural populations. Using mini-COI data, all field-collected mosquitoes were unambiguously assigned to seven species; among them almost 60% of specimens were positive for at least 11 different microsporidian species, including a new microsporidian ssu rDNA sequence (Microsporidium sp. PL01). Phylogenetic analysis showed that this species belongs to one of the two main clades in the Terresporidia. We found a high rate of microsporidian co-infections (9.4%). The numbers of sequence reads for the operational taxonomic units suggest that the occurrence of Nosema spp. in co-infections could benefit them; however, this observation should be retested using a more intensive host sampling. Our results show that DNA barcoding is a rapid and cost-effective method for deciphering sample diversity in greater resolution, including the hidden biodiversity that may be overlooked using classical methodology.

SUBMITTER: Trzebny A

PROVIDER: S-EPMC7818484 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae).

Trzebny Artur A Slodkowicz-Kowalska Anna A Becnel James J JJ Sanscrainte Neil N Dabert Miroslawa M

Molecular ecology resources 20200701 6

DNA metabarcoding offers new perspectives, especially with regard to the high-throughput identification and diagnostics of pathogens. Microsporidia are an example of widely distributed, opportunistic and pathogenic microorganisms in which molecular identification is important for both environmental research and clinical diagnostics. We have developed a method for parallel detection of both microsporidian infection and the host species. We designed new primer sets: one specific for the classical ...[more]

PMID: 32516485

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Project description:BackgroundMicrosporidia is a large group of eukaryotic obligate intracellular spore-forming parasites, of which 17 species can cause microsporidiosis in humans. Most human-infecting microsporidians belong to the genera Enterocytozoon and Encephalitozoon. To date, only five microsporidian species, including Encephalitozoon-like, have been found in hard ticks (Ixodidae) using microscopic methods, but no sequence data are available for them. Furthermore, no widespread screening for microsporidian-infected ticks based on DNA analysis has been carried out to date. Thus, in this study, we applied a recently developed DNA metabarcoding method for efficient microsporidian DNA identification to assess the role of ticks as potential vectors of microsporidian species causing diseases in humans.MethodsIn total, 1070 (493 juvenile and 577 adult) unfed host-seeking Ixodes ricinus ticks collected at urban parks in the city of Poznan, Poland, and 94 engorged tick females fed on dogs and cats were screened for microsporidian DNA. Microsporidians were detected by PCR amplification and sequencing of the hypervariable V5 region of 18S rRNA gene (18S profiling) using the microsporidian-specific primer set. Tick species were identified morphologically and confirmed by amplification and sequencing of the shortened fragment of cytochrome c oxidase subunit I gene (mini-COI).ResultsAll collected ticks were unambiguously assigned to I. ricinus. Potentially zoonotic Encephalitozoon intestinalis was identified in three fed ticks (3.2%) collected from three different dogs. In eight unfed host-seeking ticks (0.8%), including three males (1.1%), two females (0.7%) and three nymphs (0.7%), the new microsporidian sequence representing a species belonging to the genus Endoreticulatus was identified.ConclusionsThe lack of zoonotic microsporidians in host-seeking ticks suggests that I. ricinus is not involved in transmission of human-infecting microsporidians. Moreover, a very low occurrence of the other microsporidian species in both fed and host-seeking ticks implies that mechanisms exist to defend ticks against infection with these parasites.

Project description:BackgroundIn the context of climate change, a growing concern is that vector-pathogen or host-parasite interactions may be correlated with climatic factors, especially increasing temperatures. In the present study, we used a mosquito-microsporidian model to determine the impact of environmental factors such as temperature, humidity, wind and rainfall on the occurrence rates of opportunistic obligate microparasites (Microsporidia) in hosts from a family that includes important disease vectors (Culicidae).MethodsIn our study, 3000 adult mosquitoes collected from the field over 3 years were analysed. Mosquitoes and microsporidia were identified using PCR and sequencing of the hypervariable V5 region of the small subunit ribosomal RNA gene and a shortened fragment of the cytochrome c oxidase subunit I gene, respectively.ResultsDNA metabarcoding was used to identify nine mosquito species, all of which were hosts of 12 microsporidian species. The prevalence of microsporidian DNA across all mosquito samples was 34.6%. Microsporidian prevalence in mosquitoes was more frequent during warm months (> 19 °C; humidity < 65%), as was the co-occurrence of two or three microsporidian species in a single host individual. During warm months, microsporidian occurrence was noted 1.6-fold more often than during the cold periods. Among the microsporidians found in the mosquitoes, five (representing the genera Enterocytospora, Vairimorpha and Microsporidium) were positively correlated with an increase in temperature, whereas one (Hazardia sp.) was significantly correlated with a decrease in temperature. Threefold more microsporidian co-occurrences were recorded in the warm months than in the cold months.ConclusionsThese results suggest that the susceptibility of mosquitoes to parasite occurrence is primarily determined by environmental conditions, such as, for example, temperatures > 19 °C and humidity not exceeding 62%. Collectively, our data provide a better understanding of the effects of the environment on microsporidian-mosquito interactions.

Project description:The animal gut microbiota consist of many different microorganisms, mainly bacteria, but archaea, fungi, protozoans, and viruses may also be present. This complex and dynamic community of microorganisms may change during parasitic infection. In the present study, we investigated the effect of the presence of microsporidians on the composition of the mosquito gut microbiota and linked some microbiome taxa and functionalities to infections caused by these parasites. We characterised bacterial communities of 188 mosquito females, of which 108 were positive for microsporidian DNA. To assess how bacterial communities change during microsporidian infection, microbiome structures were identified using 16S rRNA microbial profiling. In total, we identified 46 families and four higher taxa, of which Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae and Pseudomonadaceae were the most abundant mosquito-associated bacterial families. Our data suggest that the mosquito gut microbial composition varies among host species. In addition, we found a correlation between the microbiome composition and the presence of microsporidians. The prediction of metagenome functional content from the 16S rRNA gene sequencing suggests that microsporidian infection is characterised by some bacterial species capable of specific metabolic functions, especially the biosynthesis of ansamycins and vancomycin antibiotics and the pentose phosphate pathway. Moreover, we detected a positive correlation between the presence of microsporidian DNA and bacteria belonging to Spiroplasmataceae and Leuconostocaceae, each represented by a single species, Spiroplasma sp. PL03 and Weissella cf. viridescens, respectively. Additionally, W. cf. viridescens was observed only in microsporidian-infected mosquitoes. More extensive research, including intensive and varied host sampling, as well as determination of metabolic activities based on quantitative methods, should be carried out to confirm our results.

Project description:A transversal survey of immature mosquitoes was conducted on Mayotte Island (France) in the Comoros Archipelago, western Indian Ocean, with the aim to inventory the Culicidae and to document inter-species relationships in different habitats. In total 420 habitats were sampled for larvae and/or pupae mosquitoes, resulting in more than 6,000 specimens. Forty species belonging to 15 genera were collected, with eight taxa integrated for the first time to the Mayotte mosquito list. The most frequently recorded species were Stegomyia aegypti, St. albopicta, Anopheles gambiae and Eretmapodites subsimplicipes, the first three species being known vectors of viruses and parasites transmitted to humans. Mean species richness in habitats ranged from 1.00 to 3.29, with notable differences between habitats. For example, water-filled axils of banana leaves, tree-holes and crab-holes had low species richness, while cut bamboo, water pools, abandoned tires and marsh and swamp water had notably higher species richness. Twenty-seven mosquito species belonging to 12 genera were routinely collected (in ≥20% of at least one type of larval habitat) suggesting that multiple species play a role in the biocenosis of these aquatic habitats. Multispecies association was observed in 52% of the habitats. The co-occurrence of up to six species belonging to five genera was recorded in a single habitat. The mosquitoes of Mayotte show notable biogeographical affinities to those of Madagascar, as compared to the African continent. These two potential source areas are nearly equidistant from Mayotte, which in turn indicates biased dispersal from east to west. Our findings suggest that with relatively short-term intensive sampling in different habitats, it is possible to approach exhaustive species inventories based on collection of larvae. Mayotte, with its modest elevation range and land surface, has a notable species richness of mosquitoes with 45 well-documented species belonging to 15 genera.

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A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae).

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A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae).

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