Methodological Insight Into Mosquito Microbiome Studies

Abstract

Symbiotic bacteria affect competence for pathogen transmission in insect vectors, including mosquitoes. However, knowledge on mosquito-microbiome-pathogen interactions remains limited, largely due to methodological reasons. The current, cost-effective practice of sample pooling used in mosquito surveillance and epidemiology prevents correlation of individual traits (i.e., microbiome profile) and infection status. Moreover, many mosquito studies employ laboratory-reared colonies that do not necessarily reflect the natural microbiome composition and variation in wild populations. As a consequence, epidemiological and microbiome studies in mosquitoes are to some extent uncoupled, and the interactions among pathogens, microbiomes, and natural mosquito populations remain poorly understood. This study focuses on the effect the pooling practice poses on mosquito microbiome profiles, and tests different approaches to find an optimized low-cost methodology for extensive sampling while allowing for accurate, individual-level microbiome studies. We tested the effect of pooling by comparing wild-caught, individually processed mosquitoes with pooled samples. With individual mosquitoes, we also tested two methodological aspects that directly affect the cost and feasibility of broad-scale molecular studies: sample preservation and tissue dissection. Pooling affected both alpha-and beta-diversity measures of the microbiome, highlighting the importance of using individual samples when possible. Both RNA and DNA yields were higher when using inexpensive reagents such as NAP (nucleic acid preservation) buffer or absolute ethanol, without freezing for short-term storage. Microbiome alpha-and beta-diversity did not show overall significant differences between the tested treatments compared to the controls (freshly extracted samples or dissected guts). However, the use of standardized protocols is highly recommended to avoid methodological bias in the data.

Ontogeny, species identity, and environment dominate microbiome dynamics in wild populations of kissing bugs (Triatominae)

“…Considering the amongindividual compositional variability we observed, it is clear that pooling samples may have significantly distorted the profiles. A similar methodological artefact has been shown in mosquitoes [118]. The lack of ontogenetic differences in Waltmann et al [47] could reflect the sample source (faeces) and incompleteness of the ontogenetic spectrum (L3 to adults only) rather than a real biological pattern in natural populations.…”

Considerations for mosquito microbiome research from the Mosquito Microbiome Consortium

“…When individual mosquitoes are pooled for processing, biological replicates of pools should ideally be processed. Collecting and processing biological replicates can provide insights into variation within groups or populations, but analyzing individual samples (an adequate number to provide enough statistical power) where possible would be most ideal and informative [ 20 ]. This particular decision is of utmost relevance, as the microbiome data will have three inherent characteristics that are directly affected by the pooling scheme: dimensionality (pooling creates new groups), compositionality (revealed patterns and conclusions drawn from pools may not reflect individual composition), and zero-inflated counts (low frequency and/or undetected taxa may not be uniformly distributed across pools) [ 21 ].…”

“…This particular decision is of utmost relevance, as the microbiome data will have three inherent characteristics that are directly affected by the pooling scheme: dimensionality (pooling creates new groups), compositionality (revealed patterns and conclusions drawn from pools may not reflect individual composition), and zero-inflated counts (low frequency and/or undetected taxa may not be uniformly distributed across pools) [ 21 ]. Ultimately, and as tested by Rodríguez-Ruano et al [ 20 ], the microbial diversity indices will significantly be influenced by the decision taken at this methodological crossroad.…”

“…Thus, the preservation method used (e.g., buffer, preservative, temperature) along with storage duration, including any freeze-thaw cycles and/ or transportation chains (e.g., transportation on ice/dry ice/ambient temperature), should be recorded for each study. Study objectives and available resources would influence the choice of sample storage and handling, and previous research on how different methods affect insect microbiomes, e.g., [20,64,65], could inform these decisions. Irrespective of chosen methods, each study should ideally use a single storage method to prevent the introduction of batch effects.…”

Identification and Removal of Potential Contaminants in 16S rRNA Gene Sequence Datasets from Low Microbial Biomass Samples: An Example from the Mosquito Gut

“…Interest in standardizing the methodologies has grown for mosquito microbiota research [17,18], recognizing that mosquito tissues are low-biomass samples, expected to be prone to sequencing artifacts [17]. However, no consensus approach has been developed for the identi cation and removal of possible contaminating sequences from mosquito tissues to date.…”