Mosquitoes Reared in Nearby Insectaries at the Same Institution Have Significantly Divergent Microbiomes

Abstract

The microbiome influences critical aspects of mosquito biology and variations in microbial composition can impact the outcomes of laboratory studies. To investigate how biotic and abiotic conditions in an insectary affect the composition of the mosquito microbiome, a single cohort of Aedes aegypti eggs was divided into three batches and transferred to three different climate-controlled insectaries within the Liverpool School of Tropical Medicine. The bacterial microbiome composition was compared as mosquitoes developed, the microbiome of the mosquitoes' food sources was characterised, environmental conditions over time in each insectary were measured, and mosquito development and survival were recorded. While developmental success was similar across all three insectaries, differences in microbiome composition were observed between mosquitoes from each insectary. Environmental conditions and bacterial input via food sources varied between insectaries, potentially contributing to the observed differences in microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; the insectary with less stable and cooler conditions resulted in a slower pupation rate and higher diversity of the larval microbiome. These findings underscore that even minor inconsistencies in rearing conditions can affect the composition of the mosquito microbiome, which may influence experimental outcomes.

Keywords: Aedes; development; diversity; environment; humidity; microbiome; temperature.

FIGURE 1

Layout of the insectaries used in this experiment and experimental setup. (a) Schematic showing the layouts of each individual insectary used in this experiment, with (i) placement locations of mosquito trays and cages and (ii) map showing locations of the three buildings where insectaries are located. (b) Experimental setup. (i) Conventionally reared Ae. aegytpi (Liverpool line) that had been continually reared in ‘Insectary A’ at the Liverpool School of Tropical Medicine (LSTM) were allowed to lay eggs under standard conditions. (ii) One cohort of eggs was vacuum‐hatched in the laboratory. (iii) The resulting L1 larvae were divided into nine trays of 150 larvae. (iv) Three replicate trays were transferred into each of three insectaries at LSTM: The original insectary ‘Insectary A’, and two further insectaries ‘Insectary B’ and ‘Insectary C’. Here, the cohorts were reared to adulthood according to standard conditions, recording the number of individuals that successfully developed to pupal and adult life stages. Recordings were always made between 09:00 and 12:00. TinyTag data loggers were used to measure the temperature and humidity throughout the experiment. (v) For each of the three replicates in each of the three insectaries (shown in the dashed line box), the following samples were collected: 1 fish food sample, 1 tap water sample, 3 larval water samples and 10 L3/L4 larvae samples collected at the same time, 2 sugar solution samples and 10 adult females. One additional tap water sample was also collected from each insectary. Samples were then stored at −80°C, before (vi) DNA extraction along with an additional extraction blank per batch and 16S rRNA sequencing. Panel (a ii) was created with QGIS version: Version 3.28, https://www.gqis.org/ Basemap: Positron, Map tiles by CartoDB, under CC BY 3.0. Data by OpenStreetMap, under ODbL. Panel (b) was created with Biorender.com.

FIGURE 2

Environmental conditions and mosquito development in each insectary over the course of the experiment. (a) Temperature (°C) and humidity (% relative humidity RH) were recorded every 15 min using TinyTag data loggers in Insectaries A, B and C. Days 5/6 and 12/13 represent weekends, and there were no public holidays during this time. (b) Average and spread of recorded temperature (i) and humidity (iii) in each insectary. (c) Time taken for individuals to develop to the pupal stage in each insectary. (d) Mosquito development in each replicate tray, faceted by insectary, showing numbers of individuals successfully developed to the pupal and adult stages from an initial 150 larvae/tray.

FIGURE 3

Microbial diversity amongst sample types from different insectaries. (a) Alpha diversity calculated as Shannon's index for each sample type, grouped by insectary (A, B and C). Statistically significant pairwise differences between samples from the three different insectaries, within sample types, are denoted by asterisks and are calculated using Kruskal Wallace tests with posthoc pairwise Dunn tests (p ≤ α/2). (b) PCoA plots showing beta diversity calculated as (i, ii) Bray–Curtis and (iii, iv) unweighted Unifrac dissimilarity metrics. Diversity was calculated using all samples passing quality thresholds, and coloured according to sample type (i, iii) Diversity metrics were then recalculated on the data subset by sample type and coloured to visualise the distribution of samples originating from each of the three insectaries (ii, iv). p values show results of PERMANOVA analyses to determine differences between sample types (i, iii) insectary within each sample type (ii, iv).

FIGURE 4

Taxonomic composition of the microbiome across sample types and insectaries. (a) Relative abundance of the top 20 most abundant genera in the data set averaged according to whether they were from Insectary A, B or C, for each sample type (tap water, fish food, larval water, larvae, sugar and adult females). All other genera were grouped together as ‘Other’. Detailed per‐sample composition is shown in Figure S2. (b) Heat map showing the relative abundance of ASVs in each sample, including all ASVs present at ≥ 5% relative abundance in at least one sample. Each row corresponds to a single ASV and is labelled on the y‐axis according to genus if known or, if unknown, the lowest taxonomic ranking known. Where there are taxonomic groups containing more than one ASV present at ≥ 5% relative abundance in at least one sample, the labels are suffixed with a number (e.g., ‘Asaia − 1’). Each column corresponds to a single sample, faceted by sample type. Upper colour blocks on the x‐axis denote the insectary of origin. Lower colour blocks denote tray/cage number within each insectary for larval water, larvae and adult female samples. Tap water, fish food and sugar samples were collected before being provided to trays/cages. Relative abundance is indicated by the blue gradient, with more highly abundant ASVs in darker shades. Zero values are indicated in white. Relative abundance values in the legend are shown as proportion data, where a relative abundance of 1 would equal 100%.