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.

Keywords: dissection; epidemiology; microbiome; mosquito; pooling; preservation; vector.

Figure 1

Box-plots showing the alpha-diversity of Aedes vexans and Culex pipiens microbiomes according to the number of mosquitoes pooled per sample: (A) Chao1 index and (B) Shannon index for Aedes vexans; (C) Chao1 index, and (D) Shannon index for Culex pipiens.

Figure 2

Box-plots showing the alpha-diversity of Aedes vexans microbiomes with the different processing treatments of this study: Chao1 index according to (A) preservation method and (B) dissection; Shannon index according to (C) preservation method and (D) dissection.

Figure 3

NMDS showing the beta-diversity of (A) Aedes vexans and (B) Culex pipiens microbiomes according to the number of mosquitoes pooled per sample. Confidence ellipses are shown for each group.

Figure 4

NMDS showing the beta-diversity of Aedes vexans and Culex pipiens microbiomes both from individuals and pools. Confidence ellipses are shown for each group.

Figure 5

Venn diagrams comparing the number of OTUs present in individual and pooled samples, both in total and in the core microbiome of (A) Aedes vexans and (D) Culex pipiens full data sets. Proportional representations (“Euler grids”) of the same information are given for the full data set (B,E) and the average values of the randomly sub-sampled data sets (C,F) of each species.