Mosquitoes reared in distinct insectaries within an institution in close spatial proximity possess significantly divergent microbiomes

Abstract

The microbiome affects important aspects of mosquito biology and differences in microbial composition can affect the outcomes of laboratory studies. To determine how the biotic and abiotic conditions in an insectary affect the composition of the bacterial microbiome of mosquitoes we reared mosquitoes from a single cohort of eggs from one genetically homogeneous inbred Aedes aegypti colony, which were split into three batches, and transferred to each of three different insectaries located within the Liverpool School of Tropical Medicine. Using three replicate trays per insectary, we assessed and compared the bacterial microbiome composition as mosquitoes developed from these eggs. We also characterised the microbiome of the mosquitoes' food sources, measured environmental conditions over time in each climate-controlled insectary, and recorded development and survival of mosquitoes. While mosquito development was overall similar between all three insectaries, we saw differences in microbiome composition between mosquitoes from each insectary. Furthermore, bacterial input via food sources, potentially followed by selective pressure of temperature stability and range, did affect the microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; and the insectary with less stable and cooler conditions resulted in slower pupation rate and higher diversity of the larval microbiome. Tray and cage effects were also seen in all insectaries, with different bacterial taxa implicated between insectaries. These results highlight the necessity of considering the variability and effects of different microbiome composition even in experiments carried out in a laboratory environment starting with eggs from one batch; and highlights the impact of even minor inconsistencies in rearing conditions due to variation of temperature and humidity.

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 were 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 dashed line box), the following samples were collected: one fish food sample, one tap water sample, three larval water samples and ten L3/L4 larvae samples collected at the same time, two sugar solution samples and ten 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 (%RH) were recorded every 15 minutes using TinyTag data loggers in insectaries A, B and C. Weekends were days five/six and 12/13 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, 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 post-hoc pairwise Dunn tests (p value ≤ alpha/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 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 ample 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 (eg ‘Asaia - 1’). Each column corresponds to a single sample, faceted by sample type. Upper colour blocks on the x axis denote 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 shade. Zero values are indicated in white.