Three species of axenic mosquito larvae recruit a shared core of bacteria in a common garden experiment.

In this study, we describe the generation of two new species of axenic mosquito, Aedes albopictus and Aedes triseriatus. Along with Aedes aegypti, axenic larvae of these three species were exposed to an environmental water source to document the assembly of the microbiome in a common garden experiment. Additionally, the larvae were reared either individually or combinatorially with the other species to characterize the effects of co-rearing on the composition of the microbiome. We found that the microbiome of the larvae was composed of a relatively low-diversity collection of bacteria from the colonizing water. The abundance of bacteria in the water was a poor predictor of their abundance in the larvae, suggesting the larval microbiome is made up of a subset of relatively rare aquatic bacteria. We found 11 bacterial 16S rRNA gene amplicon sequence variants (ASVs) that were conserved among ≥90% of the mosquitoes sampled, including 2 found in 100% of the larvae, pointing to a conserved core of bacteria capable of colonizing all three species of mosquito. Yet, the abundance of these ASVs varied widely between larvae, suggesting individuals harbored largely unique microbiome structures, even if they overlapped in membership. Finally, larvae reared in a tripartite mix of the host-species consistently showed a convergence in the structure of their microbiome, indicating that multi-species interactions between hosts potentially lead to shifts in the composition of their respective microbiomes. IMPORTANCE This study is the first report of the axenic (free of external microbes) rearing of two species of mosquito, Aedes albopictus and Aedes triseriatus. Our previous report of axenic Aedes aegypti brings the number of axenic species to three. We designed a method to perform a common garden experiment to characterize the bacteria the three species of axenic larvae assemble from their surroundings. Furthermore, species could be reared in isolation or in multi-species combinations to assess how host-species interactions influence the composition of the microbiome. We found all three species recruited a common core of bacteria from their rearing water, with a large contingent of rare and sporadically detected bacteria. Finally, we also show that co-rearing of mosquito larvae leads to a coalescence in the composition of their microbiome, indicating that host-species interactions potentially influence the composition of the microbiome.

Introducing an environmental microbiome to axenic Aedes aegypti mosquitoes documents bacterial responses to a blood meal.

IMPORTANCE: The blood meal of the female mosquito serves as a nutrition source to support egg development, so is an important aspect of its biology. Yet, the roles the microbiome may play in blood digestion are poorly characterized. We employed axenic mosquitoes to investigate how the microbiome differs between mosquitoes reared in the insectary versus mosquitoes that acquire their microbiome from the environment. Environmental microbiomes were more diverse and showed larger temporal shifts over the course of blood digestion. Importantly, only bacteria from the environmental microbiome performed hemolysis in culture, pointing to functional differences between bacterial populations. These data highlight that taxonomic differences between the microbiomes of insectary-reared and wild mosquitoes are potentially also related to their functional ecology. Thus, axenic mosquitoes colonized with environmental bacteria offer a way to investigate the role of bacteria from the wild in mosquito processes such as blood digestion, under controlled laboratory conditions.

Parallel decay of vision genes in subterranean water beetles.

In the framework of neutral theory of molecular evolution, genes specific to the development and function of eyes in subterranean animals living in permanent darkness are expected to evolve by relaxed selection, ultimately becoming pseudogenes. However, definitive empirical evidence for the role of neutral processes in the loss of vision over evolutionary time remains controversial. In previous studies, we characterized an assemblage of independently-evolved water beetle (Dytiscidae) species from a subterranean archipelago in Western Australia, where parallel vision and eye loss have occurred. Using a combination of transcriptomics and exon capture, we present evidence of parallel coding sequence decay, resulting from the accumulation of frameshift mutations and premature stop codons, in eight phototransduction genes (arrestins, opsins, ninaC and transient receptor potential channel genes) in 32 subterranean species in contrast to surface species, where these genes have open reading frames. Our results provide strong evidence to support neutral evolutionary processes as a major contributing factor to the loss of phototransduction genes in subterranean animals, with the ultimate fate being the irreversible loss of a light detection system.

The Axenic and Gnotobiotic Mosquito: Emerging Models for Microbiome Host Interactions.

The increasing availability of modern research tools has enabled a revolution in studies of non-model organisms. Yet, one aspect that remains difficult or impossible to control in many model and most non-model organisms is the presence and composition of the host-associated microbiota or the microbiome. In this review, we explore the development of axenic (microbe-free) mosquito models and what these systems reveal about the role of the microbiome in mosquito biology. Additionally, the axenic host is a blank template on which a microbiome of known composition can be introduced, also known as a gnotobiotic organism. Finally, we identify a "most wanted" list of common mosquito microbiome members that show the greatest potential to influence host phenotypes. We propose that these are high-value targets to be employed in future gnotobiotic studies. The use of axenic and gnotobiotic organisms will transition the microbiome into another experimental variable that can be manipulated and controlled. Through these efforts, the mosquito will be a true model for examining host microbiome interactions.

Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago.

Most subterranean animals are assumed to have evolved from surface ancestors following colonization of a cave system; however, very few studies have raised the possibility of "subterranean speciation" in underground habitats (i.e., obligate cave-dwelling organisms [troglobionts] descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raise the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister-species triplet, arr1 shared by a sympatric sister-species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function-altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenization provides a novel way of testing modes of speciation and the history of diversification in blind cave animals.

Limited influence of the microbiome on the transcriptional profile of female Aedes aegypti mosquitoes.

The microbiome is an assemblage of microorganisms living in association with a multicellular host. Numerous studies have identified a role for the microbiome in host physiology, development, immunity, and behaviour. The generation of axenic (germ-free) and gnotobiotic model systems has been vital to dissecting the role of the microbiome in host biology. We have previously reported the generation of axenic Aedes aegypti mosquitoes, the primary vector of several human pathogenic viruses, including dengue virus and Zika virus. In order to better understand the influence of the microbiome on mosquitoes, we examined the transcriptomes of axenic and conventionally reared Ae. aegypti before and after a blood meal. Our results suggest that the microbiome has a much lower effect on the mosquito's gene expression than previously thought with only 170 genes influenced by the axenic state, while in contrast, blood meal status influenced 809 genes. The pattern of expression influenced by the microbiome is consistent with transient changes similar to infection rather than sweeping physiological changes. While the microbiome does seem to affect some pathways such as immune function and metabolism, our data suggest the microbiome is primarily serving a nutritional role in development with only minor effects in the adult.

Refining trophic dynamics through multi-factor Bayesian mixing models: A case study of subterranean beetles.

Food web dynamics are vital in shaping the functional ecology of ecosystems. However, trophic ecology is still in its infancy in groundwater ecosystems due to the cryptic nature of these environments. To unravel trophic interactions between subterranean biota, we applied an interdisciplinary Bayesian mixing model design (multi-factor BMM) based on the integration of faunal C and N bulk tissue stable isotope data (δ13C and δ15N) with radiocarbon data (Δ14C), and prior information from metagenomic analyses. We further compared outcomes from multi-factor BMM with a conventional isotope double proxy mixing model (SIA BMM), triple proxy (δ13C, δ15N, and Δ14C, multi-proxy BMM), and double proxy combined with DNA prior information (SIA + DNA BMM) designs. Three species of subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis, and Paroster microsturtensis) and their main prey items Chiltoniidae amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis), cyclopoids and harpacticoids from a calcrete in Western Australia were targeted. Diet estimations from stable isotope only models (SIA BMM) indicated homogeneous patterns with modest preferences for amphipods as prey items. Multi-proxy BMM suggested increased-and species-specific-predatory pressures on amphipods coupled with high rates of scavenging/predation on sister species. SIA + DNA BMM showed marked preferences for amphipods AM1 and AM2, and reduced interspecific scavenging/predation on Paroster species. Multi-factorial BMM revealed the most precise estimations (lower overall SD and very marginal beetles' interspecific interactions), indicating consistent preferences for amphipods AM1 in all the beetles' diets. Incorporation of genetic priors allowed crucial refining of the feeding preferences, while integration of more expensive radiocarbon data as a third proxy (when combined with genetic data) produced more precise outcomes but close dietary reconstruction to that from SIA + DNA BMM. Further multidisciplinary modeling from other groundwater environments will help elucidate the potential behind these designs and bring light to the feeding ecology of one the most vital ecosystems worldwide.

Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome.

The emerging and increasing prevalence of bacterial antibiotic resistance is a significant public health challenge. To begin to tackle this problem, it will be critical to not only understand the origins of this resistance but also document environmental reservoirs of antibiotic resistance. In this study we investigated the possibility that both colony and field caught mosquitoes could harbor antibiotic resistant bacteria. Specifically, we characterized the antibiotic resistant bacterial populations from colony-reared Aedes aegypti larvae and adults and two field caught mosquito species Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial populations were dominated by isolates belonging to the class Gammaproteobacteria. Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and clustering into Operational Taxonomic Units (OTUs; 99% sequence identity). 27 antibiotic resistant OTUs were identified, although members of an OTU did not always share the same resistance profile. This suggests the clustering was either not sensitive enough to distinguish different bacteria taxa or different antibiotic resistant sub-populations exist within an OTU. Finally, the antibiotic selection opened up a niche to culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) were identified. Two fungal OTUs both classified to the class Microbotryomycetes were commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate that antibiotic resistant bacteria and certain fungi are common and conserved mosquito microbiome members. These observations highlight the potential of invertebrates to serve as vehicles for the spread of antibiotic resistance throughout the environment.

Opsin transcripts of predatory diving beetles: a comparison of surface and subterranean photic niches.

The regressive evolution of eyes has long intrigued biologists yet the genetic underpinnings remain opaque. A system of discrete aquifers in arid Australia provides a powerful comparative means to explore trait regression at the genomic level. Multiple surface ancestors from two tribes of diving beetles (Dytiscidae) repeatedly invaded these calcrete aquifers and convergently evolved eye-less phenotypes. We use this system to assess transcription of opsin photoreceptor genes among the transcriptomes of two surface and three subterranean dytiscid species and test whether these genes have evolved under neutral predictions. Transcripts for UV, long-wavelength and ciliary-type opsins were identified from the surface beetle transcriptomes. Two subterranean beetles showed parallel loss of all opsin transcription, as expected under 'neutral' regressive evolution. The third species Limbodessus palmulaoides retained transcription of a long-wavelength opsin (lwop) orthologue, albeit in an aphotic environment. Tests of selection on lwop indicated no significant differences between transcripts derived from surface and subterranean habitats, with strong evidence for purifying selection acting on L. palmulaoides lwop. Retention of sequence integrity and the lack of evidence for neutral evolution raise the question of whether we have identified a novel pleiotropic role for lwop, or an incipient phase of pseudogene development.