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ABSTRACT Functional studies of host-microbe interactions benefit from natural model systems that enable the exploration of molecular mechanisms at the host-microbe interface. BioluminescentVibrio fischericolonize the light organ of the Hawaiian bobtail squid,Euprymna scolopes, and this binary model has enabled advances in understanding host-microbe communication, colonization specificity,in vivobiofilms, intraspecific competition, and quorum sensing. The hummingbird bobtail squid,Euprymna berryi,can be generationally bred and maintained in lab settings and has had multiple genes deleted by CRISPR approaches. The prospect of expanding the utility of the light organ model system by producing multigenerational host lines led us to determine the extent to which theE. berryilight organ symbiosis parallels known processes inE. scolopes. However, the nature of theE. berryilight organ, including its microbial constituency and specificity for microbial partners, has not been examined. In this report, we isolated bacteria fromE. berryianimals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes inE. berryiandE. scolopeshatchlings. This study revealsE. berryito be a valuable comparative model to complement studies inE. scolopes.IMPORTANCEMicrobiome studies have been substantially advanced by model systems that enable functional interrogation of the roles of the partners and the molecular communication between those partners. TheEuprymna scolopes-Vibrio fischerisystem has contributed foundational knowledge, revealing key roles for bacterial quorum sensing broadly and in animal hosts, for bacteria in stimulating animal development, for bacterial motility in accessing host sites, and forin vivobiofilm formation in development and specificity of an animal’s microbiome.Euprymna berryiis a second bobtail squid host, and one that has recently been shown to be robust to laboratory husbandry and amenable to gene knockout. This study identifiesE. berryias a strong symbiosis model host due to features that are conserved with those ofE. scolopes, which will enable the extension of functional studies in bobtail squid symbioses. 

ABSTRACT Many plants and animals house symbiotic microorganisms in specialized tissues or organs. Here, we used multidimensionalin situimaging techniques to illuminate how host organ structure and bacterial microbiogeography contribute to the symbiotic function of an organ in the Hawaiian bobtail squid,Euprymna scolopes. Along with the well-studied light organ, femaleE. scolopesharbor a community of bacteria in the accessory nidamental gland (ANG). The ANG is a dense network of epithelium-lined tubules, some of which are dominated by a single bacterial taxon. These bacteria are deposited into squid eggs, where they defend the developing embryos from harmful biofouling. This study used a combination of imaging techniques to visualize different dimensions of the ANG and its bacterial communities. Imaging entire organs with light sheet microscopy revealed that the ANG is a composite tissue of individual, non-intersecting tubules that each harbor their own bacterial population. The organ is bisected, with tubules converging toward two points at the posterior end. At these points, tubules empty into a space where bacteria can mix with squid jelly to be deposited onto eggs. Observations of the symbiotic community correlated bacterial taxa with cell morphology and revealed that tubule populations varied: some tubules contained populations of mixed taxa, whereas others contained only one bacterial genus. Together, these data shed light on how bacterial populations interact within the ANG and how the host uses physical structure to maintain and employ a symbiotic bacterial population in a defensive context.IMPORTANCESequence-based microbiome studies have revealed much about how hosts interact with communities of symbiotic microbiota but often lack a spatial understanding of how microbes relate to each other and the host in which they reside. This study uses a combination of microscopy techniques to reveal how the structure of a symbiotic organ in the female bobtail squid,Euprymna scolopes, houses diverse, beneficial bacterial populations and deploys them for egg defense. These findings suggest that spatial partitioning may be key to harboring a diverse population of antimicrobial-producing bacteria and establishing a foundation for further understanding how host structures mediate symbiotic interactions. 

ABSTRACT Many animals contain a species-rich and diverse gut microbiota that likely contributes to several host-supportive services that include diet processing and nutrient provisioning. Loss of microbiome taxa and their associated metabolic functions as result of perturbations may result in loss of microbiome-level services and reduction of metabolic capacity. If metabolic functions are shared by multiple taxa (i.e., functional redundancy), including deeply divergent lineages, then the impact of taxon/function losses may be dampened. We examined to what degree alterations in phylotype diversity impact microbiome-level metabolic capacity. Feeding two nutritionally imbalanced diets to omnivorousPeriplaneta americanaover 8 weeks reduced the diversity of their phylotype-rich gut microbiomes by ~25% based on 16S rRNA gene amplicon sequencing, yet PICRUSt2-inferred metabolic pathway richness was largely unaffected due to their being polyphyletic. We concluded that the nonlinearity between taxon and metabolic functional losses is due to microbiome members sharing many well-characterized metabolic functions, with lineages remaining after perturbation potentially being capable of preventing microbiome “service outages” due to functional redundancy. IMPORTANCEDiet can affect gut microbiome taxonomic composition and diversity, but its impacts on community-level functional capabilities are less clear. Host health and fitness are increasingly being linked to microbiome composition and further modeling of the relationship between microbiome taxonomic and metabolic functional capability is needed to inform these linkages. Invertebrate animal models like the omnivorous American cockroach are ideal for this inquiry because they are amenable to various diets and provide high replicates per treatment at low costs and thus enabling rigorous statistical analyses and hypothesis testing. Microbiome taxonomic composition is diet-labile and diversity was reduced after feeding on unbalanced diets (i.e., post-treatment), but the predicted functional capacities of the post-treatment microbiomes were less affected likely due to the resilience of several abundant taxa surviving the perturbation as well as many metabolic functions being shared by several taxa. These results suggest that both taxonomic and functional profiles should be considered when attempting to infer how perturbations are altering gut microbiome services and possible host outcomes. 

ABSTRACT Creating and maintaining an appropriate chemical environment is essential for biomineralization, the process by which organisms precipitate minerals to form their shells or skeletons, yet the mechanisms involved in maintaining calcifying fluid chemistry are not fully defined. In particular, the role of microorganisms in facilitating or hindering animal biomineralization is poorly understood. Here, we investigated the taxonomic diversity and functional potential of microbial communities inhabiting oyster calcifying fluid. We used shotgun metagenomics to survey calcifying fluid microbial communities from three different oyster harvesting sites. There was a striking consistency in taxonomic composition across the three collection sites. We also observed archaea and viruses that had not been previously identified in oyster calcifying fluid. Furthermore, we identified microbial energy-conserving metabolisms that could influence the host’s calcification, including genes involved in sulfate reduction and denitrification that are thought to play pivotal roles in inorganic carbon chemistry and calcification in microbial biofilms. These findings provide new insights into the taxonomy and functional capacity of oyster calcifying fluid microbiomes, highlighting their potential contributions to shell biomineralization, and contribute to a deeper understanding of the interplay between microbial ecology and biogeochemistry that could potentially bolster oyster calcification. IMPORTANCEPrevious research has underscored the influence of microbial metabolisms in carbonate deposition throughout the geological record. Despite the ecological importance of microbes to animals and inorganic carbon transformations, there have been limited studies characterizing the potential role of microbiomes in calcification by animals such as bivalves. Here, we use metagenomics to investigate the taxonomic diversity and functional potential of microbial communities in calcifying fluids from oysters collected at three different locations. We show a diverse microbial community that includes bacteria, archaea, and viruses, and we discuss their functional potential to influence calcifying fluid chemistry via reactions like sulfate reduction and denitrification. We also report the presence of carbonic anhydrase and urease, both of which are critical in microbial biofilm calcification. Our findings have broader implications in understanding what regulates calcifying fluid chemistry and consequentially the resilience of calcifying organisms to 21st century acidifying oceans. 

ABSTRACT Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater, due to optical inaccessibility. Biosensors that could record transient exposure to analytes within a large time window for later retrieval represent a promising approach to solve the accessibility problem. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12-L-homoserine lactone) over 8 days (~70 generations) following analyte exposure. These biosensors sense the analyte and trigger the expression of a recombinase enzyme which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using quantitative PCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterizedPseudomonasisolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration andin situinvestigation of microbial signaling, cross-feeding, community shifts, and gene transfer beyond the reach of traditional environmental biosensors.IMPORTANCEMicrobes mediate ecological processes over timescales that can far exceed the half-lives of transient metabolites and signals that drive their collective behaviors. We investigated strategies for engineering microbes to stably record their transient exposure to a chemical over many generations through DNA rearrangements. We identify genetic architectures that improve memory biosensor performance and characterize these in wastewater isolates. Memory biosensors are expected to be useful for monitoring cell-cell signals in biofilms, detecting transient exposure to chemical pollutants, and observing microbial cross-feeding through short-lived metabolites within cryptic methane, nitrogen, and sulfur cycling processes. They will also enablein situstudies of microbial responses to ephemeral environmental changes, or other ecological processes that are currently challenging to monitor non-destructively using real-time biosensors and analytical instruments. 

ABSTRACT Many female squids and cuttlefishes have a symbiotic reproductive organ called the accessory nidamental gland (ANG) that hosts a bacterial consortium involved with egg defense against pathogens and fouling organisms. While the ANG is found in multiple cephalopod families, little is known about the global microbial diversity of these ANG bacterial symbionts. We used 16S rRNA gene community analysis to characterize the ANG microbiome from different cephalopod species and assess the relationship between host and symbiont phylogenies. The ANG microbiome of 11 species of cephalopods from four families (superorder: Decapodiformes) that span seven geographic locations was characterized. Bacteria of classAlphaproteobacteria, Gammaproteobacteria, andFlavobacteriiawere found in all species, yet analysis of amplicon sequence variants by multiple distance metrics revealed a significant difference between ANG microbiomes of cephalopod families (weighted/unweighted UniFrac, Bray–Curtis,P= 0.001). Despite being collected from widely disparate geographic locations, members of the family Sepiolidae (bobtail squid) shared many bacterial taxa including (~50%)Opitutae(Verrucomicrobia) andRuegeria(Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel testr= 0.7). These data suggest that closely related sepiolids select for distinct symbionts from similar bacterial taxa. Overall, the ANGs of different cephalopod species harbor distinct microbiomes and thus offer a diverse symbiont community to explore antimicrobial activity and other functional roles in host fitness. IMPORTANCEMany aquatic organisms recruit microbial symbionts from the environment that provide a variety of functions, including defense from pathogens. Some female cephalopods (squids, bobtail squids, and cuttlefish) have a reproductive organ called the accessory nidamental gland (ANG) that contains a bacterial consortium that protects eggs from pathogens. Despite the wide distribution of these cephalopods, whether they share similar microbiomes is unknown. Here, we studied the microbial diversity of the ANG in 11 species of cephalopods distributed over a broad geographic range and representing 15–120 million years of host divergence. The ANG microbiomes shared some bacterial taxa, but each cephalopod species had unique symbiotic members. Additionally, analysis of host–symbiont phylogenies suggests that the evolutionary histories of the partners have been important in shaping the ANG microbiome. This study advances our knowledge of cephalopod–bacteria relationships and provides a foundation to explore defensive symbionts in other systems. 

ABSTRACT Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides. IMPORTANCE This work connects specific uncultured bacterial taxa with distinct polysaccharide digestion capabilities lacking in their marine vertebrate hosts, providing fresh insights into poorly understood processes for deconstructing complex sulfated polysaccharides and potential evolutionary mechanisms for microbial acquisition of expanded macroalgal utilization gene functions. Several thousand new marine-specific candidate enzyme sequences for polysaccharide utilization have been identified. These data provide foundational resources for future investigations into suppression of coral reef macroalgal overgrowth, fish host physiology, the use of macroalgal feedstocks in terrestrial and aquaculture animal feeds, and the bioconversion of macroalgae biomass into value-added commercial fuel and chemical products. 

ABSTRACT As rising temperatures threaten biodiversity across the globe, tropical ectotherms are thought to be particularly vulnerable due to their narrow thermal tolerance ranges. Nevertheless, physiology-based models highlighting the vulnerability of tropical organisms rarely consider the contributions of their gut microbiota, even though microbiomes influence numerous host traits, including thermal tolerance. We combined field and lab experiments to understand the response of the slender anole lizard ( Anolis apletophallus ) gut microbiome to climatic shifts of various magnitude and duration. First, to examine the effects of long-term climate warming in the wild, we transplanted lizards from the mainland Panama to a series of warmer islands in the Panama Canal and compared their gut microbiome compositions after three generations of divergence. Next, we mimicked the effects of a short-term “heat-wave” by using a greenhouse experiment and explored the link between gut microbiome composition and lizard thermal physiology. Finally, we examined variation in gut microbiomes in our mainland population in the years both before and after a naturally occurring drought. Our results suggest that slender anole microbiomes are surprisingly resilient to short-term warming. However, both the taxonomic and predicted functional compositions of the gut microbiome varied by sampling year across all sites, suggesting that the drought may have had a regional effect. We provide evidence that short-term heat waves may not substantially affect the gut microbiota, while more sustained climate anomalies may have effects at broad geographic scales. IMPORTANCE As climate change progresses, it is crucial to understand how animals will respond to shifts in their local environments. One component of this response involves changes in the microbial communities living in and on host organisms. These “microbiomes” can affect many processes that contribute to host health and survival, yet few studies have measured changes in the microbiomes of wild organisms experiencing novel climatic conditions. We examined the effects of shifting climates on the gut microbiome of the slender anole lizard ( Anolis apletophallus ) by using a combination of field and laboratory studies, including transplants to warm islands in the Panama Canal. We found that slender anole microbiomes remain stable in response to short-term warming but may be sensitive to sustained climate anomalies, such as droughts. We discuss the significance of these findings for a species that is considered highly vulnerable to climate change. 

ABSTRACT Blooms of many dinoflagellates, including several harmful algal bloom (HAB) species, are seeded and revived through the germination of benthic resting cysts. Temperature is a key determinant of cysts’ germination rate, and temperature–germination rate relationships are therefore fundamental to understanding species’ germling cell production, cyst bed persistence, and resilience to climate warming. This study measured germination by cysts of the HAB dinoflagellate Alexandrium catenella using a growing degree-day ( DD ) approach that accounts for the time and intensity of warming above a critical temperature. Time courses of germination at different temperatures were fit to lognormal cumulative distribution functions for the estimation of the median days to germination. As temperature increased, germination times decreased hyperbolically. DD scaling collapsed variability in germination times between temperatures after cysts were oxygenated. A parallel experiment demonstrated stable temperature–rate relationships in cysts collected during different phases of seasonal temperature cycles in situ over three years. DD scaling of the results from prior A. catenella germination studies showed consistent differences between populations across a wide range of temperatures and suggests selective pressure for different germination rates. The DD model provides an elegant approach to quantify and compare the temperature dependency of germination among populations, between species, and in response to changing environmental conditions. IMPORTANCE Germination by benthic life history stages is the first step of bloom initiation in many, diverse phytoplankton species. This study outlines a growing degree-day ( DD ) approach for comparing the temperature dependence of germination rates measured in different populations. Germination by cysts of Alexandrium catenella , a harmful algal bloom dinoflagellate that causes paralytic shellfish poisoning, is shown to require a defined amount of warming, measured in DD after cysts are aerated. Scaling by DD , the time integral of temperature difference from a critical threshold, enabled direct comparison of rates measured at different temperatures and in different studies. 

ABSTRACT Cyanobacterial harmful algal blooms (cyanoHABs) degrade freshwater ecosystems globally. Microcystis aeruginosa often dominates cyanoHABs and produces microcystin (MC), a class of hepatotoxins that poses threats to human and animal health. Microcystin toxicity is influenced by distinct structural elements across a diversity of related molecules encoded by variant mcy operons. However, the composition and distribution of mcy operon variants in natural blooms remain poorly understood. Here, we characterized the variant composition of mcy genes in western Lake Erie Microcystis blooms from 2014 and 2018. Sampling was conducted across several spatial and temporal scales, including different bloom phases within 2014, extensive spatial coverage on the same day (2018), and frequent, autonomous sampling over a 2-week period (2018). Mapping of metagenomic and metatranscriptomic sequences to reference sequences revealed three Microcystis mcy genotypes: complete (all genes present [ mcyA–J ]), partial (truncated mcyA , complete mcyBC , and missing mcyD–J ), and absent (no mcy genes). We also detected two different variants of mcyB that may influence the production of microcystin congeners. The relative abundance of these genotypes was correlated with pH and nitrate concentrations. Metatranscriptomic analysis revealed that partial operons were, at times, the most abundant genotype and expressed in situ , suggesting the potential biosynthesis of truncated products. Quantification of genetic divergence between genotypes suggests that the observed strains are the result of preexisting heterogeneity rather than de novo mutation during the sampling period. Overall, our results show that natural Microcystis populations contain several cooccurring mcy genotypes that dynamically shift in abundance spatiotemporally via strain succession and likely influence the observed diversity of the produced congeners. IMPORTANCE Cyanobacteria are responsible for producing microcystins (MCs), a class of potent and structurally diverse toxins, in freshwater systems around the world. While microcystins have been studied for over 50 years, the diversity of their chemical forms and how this variation is encoded at the genetic level remain poorly understood, especially within natural populations of cyanobacterial harmful algal blooms (cyanoHABs). Here, we leverage community DNA and RNA sequences to track shifts in mcy genes responsible for producing microcystin, uncovering the relative abundance, expression, and variation of these genes. We studied this phenomenon in western Lake Erie, which suffers annually from cyanoHAB events, with impacts on drinking water, recreation, tourism, and commercial fishing.