Larval Microbiomes Research Articles

Micromanagement: conditions influencing antipathogen function of the skin microbiome in spotted salamanders, Ambystoma maculatum

The emerging fungal pathogen Batrachochytrium salamandrivorans (Bsal) threatens the diversity of amphibians, particularly in North America where it is projected to invade. Amphibian skin defenses include a mucosal layer containing microorganisms that can potentially modulate host response to pathogens such as Bsal. In this study, we focused on the composition of the skin microbiome across life stages of spotted salamanders (Ambystoma maculatum). We also evaluated the stress hormone corticosterone and skin microbiome response to inoculations with Bsal and probiotics at both the larval and juvenile developmental stages, and the response to different environmental conditions. Results indicated that both bacterial and fungal communities found on the skin significantly differed in structure and diversity between life stages of A. maculatum. Exposure to three different probiotics (Bacillus thuringiensis, Chryseobacterium rhizoplanae, and Penicillium sp.) and Bsal evoked shifts in the microbiome of larvae and juveniles, and the metabolite profile of the larval mucosal layer of A. maculatum. Despite changes in the microbiome, all tested probiotics and Bsal were unable to persist on the skin. Larval bacterial microbiomes shifted in response to Bsal and B. thuringiensis with no significant impacts on antifungal function or bacteria richness, however fungi strongly responded to Bsal and B. thuringiensis application. This indicates that developmental shifts in the microbiome can be initiated by microbial applications such as B. thuringiensis, a widely used mosquito larvicide. Overall, experimental results indicate that life stage, growth and development, and environmental conditions appeared to be the main factors driving changes in the amphibian skin microbiome and potential anti-Batrachochytrium function.

Understanding the Ecological Robustness and Adaptability of the Gut Microbiome in Plastic-Degrading Superworms (Zophobas atratus) in Response to Microplastics and Antibiotics.

Recent discoveries indicate that several insect larvae are capable of ingesting and biodegrading plastics rapidly and symbiotically, but the ecological adaptability of the larval gut microbiome to microplastics (MPs) remains unclear. Here, we described the gut microbiome assemblage and MP biodegradation of superworms (Zophobas atratus larvae) fed MPs of five major petroleum-based polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride, and polyethylene terephthalate) and antibiotics. The shift of molecular weight distribution, characteristic peaks of C═O, and metabolic intermediates of residual polymers in egested frass proved depolymerization and biodegradation of all MPs tested in the larval intestines, even under antibiotic suppression. Superworms showed a wide adaptation to the digestion of the five polymer MPs. Antibiotic suppression negatively influenced the survival rate and plastic depolymerization patterns. The larval gut microbiomes differed from those fed MPs and antibiotics, indicating that antibiotic supplementation substantially shaped the gut microbiome composition. The larval gut microbiomes fed MPs had higher network complexity and stability than those fed MPs and antibiotics, suggesting that the ecological robustness of the gut microbiomes ensured the functional adaptability of larvae to different MPs. In addition, Mantel's test indicated that the gut microbiome assemblage was obviously related to the polymer type, the plastic degradability, antibiotic stress, and larval survival rate. This finding provided novel insights into the self-adaptation of the gut microbiome of superworms in response to different MPs.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype-dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can have carry-over effects on adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we showed that the bacterial community structure differs between the two genotypes of Ae. aegypti when given identical microbiomes, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific larval microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti. We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Partner fidelity and environmental filtering preserve stage‐specific turtle ant gut symbioses for over 40 million years

AbstractSustaining beneficial gut symbioses presents a major challenge for animals, including holometabolous insects. Social insects may meet such challenges through partner fidelity, aided by behavioral symbiont transfer and transgenerational inheritance through colony founders. We address such potential through colony‐wide explorations across 13 eusocial, holometabolous insect species in the ant genus Cephalotes. Through amplicon sequencing, we show that previously characterized worker microbiomes are conserved in soldier castes, that adult microbiomes exhibit trends of phylosymbiosis, and that Cephalotes cospeciate with their most abundant adult‐enriched symbiont. We find, also, that winged queens harbor worker‐like microbiomes prior to colony founding, suggesting vertical inheritance as a means of partner fidelity. Whereas some adult‐abundant symbionts colonize larvae, larval gut microbiomes are uniquely characterized by environmental bacteria from the Enterobacteriales, Lactobacillales, and Actinobacteria. Distributions across Cephalotes larvae suggest more than 40 million years of conserved environmental filtering and, thus, a second sustaining mechanism behind an ancient, developmentally partitioned symbiosis.

Different Gut Microbiomes of Developmental Stages of Field-Collected Native and Invasive Western Bean Cutworm, Striacosta albicosta, in Western Nebraska.

While insects harbor gut microbial associates that perform various functions for the host, lepidopterans have not been considered as prime examples of having such relationships. The western bean cutworm, Striacosta albicosta (Lepidoptera: Noctuidae), is native to North America and has historically been a significant corn pest in its western distribution. It is currently expanding eastwards and is invasive in these new regions. Using 16S rRNA gene sequencing data, this study focused on characterizing the microbiota associated with field-collected eggs, larvae, adults, and host plant materials of S. albicosta in its native range. The diversity of microbiomes varied significantly among S. albicosta eggs, larvae, adults, and the host plant materials. Microbial diversity was highest in adult stages relative to other insect stages. Furthermore, S. albicosta eggs, larvae, and adults harbored very distinct microbial communities, indicative of stage-specific microbiomes possibly performing different functions. Bacterial taxa underscoring these differences in composition identified four phyla and thirty families across samples. Members of the Firmicutes (Unassigned Lactobacillales), Proteobacteria (Pseudomonadaceae and Moraxellaceae), Bacteroidota (Weeksellaceae), and Chloroflexi dominated across all developmental stages. In addition, cellulose-degrading Lactobacillales (phylum: Firmicutes) dominated larval microbiomes, indicative of larval plant diet. This taxon was comparatively negligible in eggs and adults. Members of Proteobacteria dominated egg and host leaf microbiomes, while members of Bacteroidota dominated nectar-feeding adult gut microbiomes. Our results suggest a possible diet-dependent stage-specific microbiome composition and the potential for using stage-specific microbes as potential biological control tools against this important pest moving forward.

Spruce budworm bacterial communities vary among sites and host tree species in a boreal landscape

AbstractAimMicrobial communities often vary spatially in how they assemble and knowledge is lacking about which factors determine the biogeography of host‐associated microbiomes. Our aim is to assess the relative importance of spatial, environmental and host‐associated factors on microbial community composition of an important defoliating insect.LocationBoreal forests in eastern Canada (Quebec, Newfoundland and Labrador).TaxonThe eastern spruce budworm (Choristoneura fumiferana) and its associated bacterial communities.MethodsWe characterized bacterial communities associated with spruce budworm larvae using bacterial 16S rRNA gene amplicon sequencing. We quantified how much of the variation in these bacterial communities could be explained by (1) environmental conditions, (2) the microbiome of foliage the larvae were eating, (3) host tree species and (4) spatial structure as quantified using Moran's Eigenvector Maps (MEMs).ResultsBudworm larval microbiomes varied significantly among sites and between host tree species. Larvae bacterial community structure was strongly correlated with the structure of bacterial communities taken from paired foliage samples. Spatial structure, foliage bacterial communities and host tree species collectively explained almost one‐sixth of the variation in budworm bacterial communities while environmental conditions did not explain variation on their own.Main conclusionsLepidopteran microbiomes primarily originate from the foliage diet. However, subtle differences in microbial communities between larvae and foliage suggest that some bacteria establish and grow in the budworm microbiome, and that dispersal of bacteria from sources other than foliage as well as differences in environmental filtering between larval bodies and foliage play a role in the assembly of the budworm microbiome. While spatial location and spatial structure were also important drivers of spruce budworm bacterial community composition, none of the environmental variables we measured could explain the variation among sites, and identifying the drivers of this spatial variation remains an open question that will need to be addressed by future studies.

Microbiomes of willow-galling sawflies: effects of host plant, gall type, and phylogeny on community structure and function.

While free-living herbivorous insects are thought to harbor microbial communities composed of transient bacteria derived from their diet, recent studies indicate that insects that induce galls on plants may be involved in more intimate host-microbe relationships. We used 16S rDNA metabarcoding to survey larval microbiomes of 20 nematine sawfly species that induce bud or leaf galls on 13 Salix species. The 391 amplicon sequence variants (ASVs) detected represented 69 bacterial genera in six phyla. Multi-variate statistical analyses showed that the structure of larval microbiomes is influenced by willow host species as well as by gall type. Nevertheless, a "core" microbiome composed of 58 ASVs is shared widely across the focal galler species. Within the core community, the presence of many abundant, related ASVs representing multiple distantly related bacterial taxa is reflected as a statistically significant effect of bacterial phylogeny on galler-microbe associations. Members of the core community have a variety of inferred functions, including degradation of phenolic compounds, nutrient supplementation, and production of plant hormones. Hence, our results support suggestions of intimate and diverse interactions between galling insects and microbes and add to a growing body of evidence that microbes may play a role in the induction of insect galls on plants.

Comparing larval microbiomes of the eastern oyster (Crassostrea virginica) raised in different hatcheries

An ongoing goal of the aquaculture industry is the improvement of oyster larval growth and production. One component of oyster hatcheries that may contribute to the health of oyster larvae is the early establishment and subsequent development of the oyster larval microbiome. The main objectives of this study were to investigate the effects of environmental conditions (hatchery types and spawning seasons) and oyster phenotypes (larval developmental stages) on the composition and diversity of the larval microbiome. In addition, the members of core larval microbiomes were identified and quantified. Microbiome composition and diversity of larval and corresponding hatchery water samples were examined using high throughput sequencing of 16S rRNA gene amplicons and represented three development stages from two different spawning events at four different hatcheries located in Chesapeake Bay tributaries. Larval and water samples from each spawning cohort were taken at 24-hours ‘D’ shape (D-stage), 1-week veliger (V-stage), and 2-weeks pediveliger (PV-stage). Larval microbiomes were significantly different from water microbiomes and were significantly influenced by hatchery and spawning, with hatchery having the greatest effect on microbiome composition. While development stage did not show a significant effect on the larval microbiomes, there was a decrease in species richness from the initial D-stage larvae through the final PV-stage and nMDS clustering patterns showed some separation between early (D- and V-) and late stages (PV-) of development, suggesting a shift towards a more selective microbiome as the larvae developed. A total of 25 members (OTU level) were identified as the core larval microbiome (core OTUs), comprising approximately one quarter of the total relative abundance of the larval microbiome. Core OTUs belonging to genera Alteronomonas and Roseobacter have been shown to offer bivalve larvae some protection against pathogens, while those belonging to family Cryomorphaceae are commonly isolated from microalgal species and most likely indicate an association with oyster larval feeding. These findings underscore the importance of environmental conditions on microbiome development of oyster larvae associated with hatchery success of larval cultivation. Further studies are needed to determine the contribution of the core larval microbiome to oyster health and disease resistance.

Influences of claywater and greenwater on the skin microbiome of cultured larval sablefish (Anoplopoma fimbria)

BackgroundThe skin microbiome of marine fish is thought to come from bacteria in the surrounding water during the larval stages, although it is not clear how different water conditions affect the microbial communities in the water and, in turn, the composition and development of the larval skin microbiome. In aquaculture, water conditions are especially important; claywater and greenwater are often used in larval rearing tanks to increase water turbidity. Here, we explored the effects of these water additives on microbial communities in rearing water and on the skin of first-feeding sablefish larvae using 16S rRNA gene sequencing. We evaluated three treatments: greenwater, claywater, and greenwater with a switch to claywater after 1 week.ResultsWe observed additive-specific effects on rearing water microbial communities that coincided with the addition of larvae and rotifer feed to the tanks, such as an increase in Vibrionaceae in greenwater tanks. Additionally, microbial communities from experimental tank water, especially those in claywater, began to resemble larval skin microbiomes by the end of the experiment. The differential effects of the additives on larval sablefish skin microbiomes were largest during the first week, post-first feed. Bacteria associated with greenwater, including Vibrionaceae and Pseudoalteromonas spp., were found on larval skin a week after the switch to claywater. In addition to additive-specific effects, larval skin microbiomes also retained bacterial families likely acquired from their hatchery silos.ConclusionsOur results suggest that larval sablefish skin microbiomes are most sensitive to the surrounding seawater up to 1 week following the yolk-sac stage and that claywater substituted for greenwater after 1 week post-first feed does not significantly impact skin-associated microbial communities. However, the larval skin microbiome changes over time under all experimental conditions. Furthermore, our findings suggest a potential two-way interaction between microbial communities on the host and the surrounding environment. To our knowledge, this is one of the few studies to suggest that fish might influence the microbial community of the seawater.

Susceptibility of Red Mason Bee Larvae to Bacterial Threats Due to Microbiome Exchange with Imported Pollen Provisions.

Solitary bees are subject to a variety of pressures that cause severe population declines. Currently, habitat loss, temperature shifts, agrochemical exposure, and new parasites are identified as major threats. However, knowledge about detrimental bacteria is scarce, although they may disturb natural microbiomes, disturb nest environments, or harm the larvae directly. To address this gap, we investigated 12 Osmia bicornis nests with deceased larvae and 31 nests with healthy larvae from the same localities in a 16S ribosomal RNA (rRNA) gene metabarcoding study. We sampled larvae, pollen provisions, and nest material and then contrasted bacterial community composition and diversity in healthy and deceased nests. Microbiomes of pollen provisions and larvae showed similarities for healthy larvae, whilst this was not the case for deceased individuals. We identified three bacterial taxa assigned to Paenibacillus sp. (closely related to P. pabuli/amylolyticus/xylanexedens), Sporosarcina sp., and Bacillus sp. as indicative for bacterial communities of deceased larvae, as well as Lactobacillus for corresponding pollen provisions. Furthermore, we performed a provisioning experiment, where we fed larvae with untreated and sterilized pollens, as well as sterilized pollens inoculated with a Bacillus sp. isolate from a deceased larva. Untreated larval microbiomes were consistent with that of the pollen provided. Sterilized pollen alone did not lead to acute mortality, while no microbiome was recoverable from the larvae. In the inoculation treatment, we observed that larval microbiomes were dominated by the seeded bacterium, which resulted in enhanced mortality. These results support that larval microbiomes are strongly determined by the pollen provisions. Further, they underline the need for further investigation of the impact of detrimental bacterial acquired via pollens and potential buffering by a diverse pollen provision microbiome in solitary bees.

Next-Generation Sequencing reveals relationship between the larval microbiome and food substrate in the polyphagous Queensland fruit fly

Insects typically host substantial microbial communities (the ‘microbiome’) that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni (‘Queensland fruit fly’) that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect’s microbiome and that of the surrounding host tissues.

Characterization of the microbiome and bioluminescent symbionts across life stages of Ceratioid Anglerfishes of the Gulf of Mexico.

ABSTRACTThe interdependence of diverse organisms through symbiosis reaches even the deepest parts of the oceans. As part of the DEEPEND project (deependconsortium.org) research on deep Gulf of Mexico biodiversity, we profiled the bacterial communities (‘microbiomes’) and luminous symbionts of 36 specimens of adult and larval deep-sea anglerfishes of the suborder Ceratioidei using 16S rDNA. Transmission electron microscopy was used to characterize the location of symbionts in adult light organs (esca). Whole larval microbiomes, and adult skin and gut microbiomes, were dominated by bacteria in the genera Moritella and Pseudoalteromonas. 16S rDNA sequencing results from adult fishes corroborate the previously published identity of ceratioid bioluminescent symbionts and support the findings that these symbionts do not consistently exhibit host specificity at the host family level. Bioluminescent symbiont amplicon sequence variants were absent from larval ceratioid samples, but were found at all depths in the seawater, with a highest abundance found at mesopelagic depths. As adults spend the majority of their lives in the meso- and bathypelagic zones, the trend in symbiont abundance is consistent with their life history. These findings support the hypothesis that bioluminescent symbionts are not present throughout host development, and that ceratioids acquire their bioluminescent symbionts from the environment.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P.downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P.downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Bacterial communities of oceanic sea star (Asteroidea: Echinodermata) larvae

Planktotrophic sea star larvae of several species are abundant in oligotrophic waters of the Gulf Stream, western Sargasso Sea, and Caribbean Sea. One abundant larval morphotype at the bipinnaria stage of development is unusual in its ability to constitutively produce clones and in harboring a community of auto-fluorescent bacteria. We hypothesized that the bacterial community would be distinct in these larvae compared to those that do not consistently reproduce clonally. Three sea star larval morphotypes were collected in the Gulf Stream off the coast of Florida. We used DNA-based maximum likelihood phylogenetic analyses to taxonomically classify the larvae and 16S rDNA profiling by deep sequencing to characterize the bacterial communities harbored within. The cloning bipinnaria and non-cloning brachiolaria morphotypes were determined to be a single species of Asteroidea in the family Oreasteridae. The third morphotype, a non-cloning bipinnaria, was identified as Mithrodia clavigera. With bacterial 16S rDNA profiling, we found that the two species of larvae harbor bacterial communities distinct from each other. The Oreasteridae bacterial community at both developmental stages has a photosynthetic Cyanobacteria Synechococcus sp. as the most abundant bacteria. The M. clavigera larvae host a flora consisting primarily of Gammaproteobacteria. The identification of the larval microbiomes is a step toward understanding their host–microbe interactions. Specifically, the association of photosynthetic bacteria with cloning larvae allows for future assessments of whether the bacteria supplement the nutrition of the larvae during extended periods of development and clonal reproduction in open ocean regions where phytoplankton food for the larvae may be in limited supply.

Culex quinquefasciatus larval microbiomes vary with instar and exposure to common wastewater contaminants.

Like many insects, mosquitoes, rely on endosymbionts to grow and develop. These can be acquired from the environment. We used next generation 454 pyrosequencing to discern the whole-body microbiome of the mosquito species Culex quinquefasciatus in various larval stadia and following exposure to common pharmaceutical and personal care products (PPCPs) found in wastewater. PPCP treatments included environmentally-relevant concentrations; 1) a combination of common antibiotics, 2) a combination of mammalian hormones, 3) a mixture of the antibiotic and hormone treatments plus acetaminophen and caffeine and, 4) an untreated control. Within control groups, the predominant families of bacterial symbionts change with each larval instar despite consistent diets and rearing conditions. This trend was also seen in hormone treatments but not in the antibiotic or the mixture treatments. Richness and evenness were reduced in both antibiotic and mixture treatments, suggesting that antibiotics remove certain bacteria or inhibit them from increasing to proportions seen in the control treatment. Interestingly, the mixture treatments had greater richness and evenness compared to antibiotic alone treatments, possibly due to the other contaminants facilitating growth of different bacteria. These findings illuminate the complexity of the microbiome of C. quinquefasciatus and may have implications for more effective control strategies.