Evolutionary history influences the microbiomes of a female symbiotic reproductive organ in cephalopods.

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 class Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were 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) and Ruegeria (Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel test r = 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.

Gene modelling and annotation for the Hawaiian bobtail squid, Euprymna scolopes.

Coleoid cephalopods possess numerous complex, species-specific morphological and behavioural adaptations, e.g., a uniquely structured nervous system that is the largest among the invertebrates. The Hawaiian bobtail squid (Euprymna scolopes) is one of the most established cephalopod species. With its recent publication of the chromosomal-scale genome assembly and regulatory genomic data, it also emerges as a key model for cephalopod gene regulation and evolution. However, the latest genome assembly has been lacking a native gene model set. Our manuscript describes the generation of new long-read transcriptomic data and, made using this combined with a plethora of publicly available transcriptomic and protein sequence data, a new reference annotation for E. scolopes.

Emergence of novel genomic regulatory regions associated with light-organ development in the bobtail squid.

Light organs (LO) with symbiotic bioluminescent bacteria are hallmarks of many bobtail squid species. These organs possess structural and functional features to modulate light, analogous to those found in coleoid eyes. Previous studies identified four transcription factors and modulators (SIX, EYA, PAX6, DAC) associated with both eyes and light organ development, suggesting co-option of a highly conserved gene regulatory network. Using available topological, open chromatin, and transcriptomic data, we explore the regulatory landscape around the four transcription factors as well as genes associated with LO and shared LO/eye expression. This analysis revealed several closely associated and putatively co-regulated genes. Comparative genomic analyses identified distinct evolutionary origins of these putative regulatory associations, with the DAC locus showing a unique topological and evolutionarily recent organization. We discuss different scenarios of modifications to genome topology and how these changes may have contributed to the evolutionary emergence of the light organ.

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

"Failure To Launch": Development of a Reproductive Organ Linked to Symbiotic Bacteria.

Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes. In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host's natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia, are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes. IMPORTANCE Microbiota have profound effects on animal and plant development. Hosts raised axenically or without symbionts often suffer negative outcomes resulting in developmental defects or reduced organ function. Using defined experimental conditions, we demonstrate that environmental bacteria are required for the formation of a female-specific symbiotic organ in the Hawaiian bobtail squid, Euprymna scolopes. Although nascent tissues from this organ that are involved with bacterial recruitment formed initially, the mature organ failed to develop and was absent or severely reduced in sexually mature animals that were not exposed to microbiota from the host's natural environment. This is the first example of complete organ development relying on exposure to symbiotic bacteria in an animal host. This study broadens the use of E. scolopes as a model organism for studying the influence of beneficial bacteria on animal development.

Emergence of novel cephalopod gene regulation and expression through large-scale genome reorganization.

Coleoid cephalopods (squid, cuttlefish, octopus) have the largest nervous system among invertebrates that together with many lineage-specific morphological traits enables complex behaviors. The genomic basis underlying these innovations remains unknown. Using comparative and functional genomics in the model squid Euprymna scolopes, we reveal the unique genomic, topological, and regulatory organization of cephalopod genomes. We show that coleoid cephalopod genomes have been extensively restructured compared to other animals, leading to the emergence of hundreds of tightly linked and evolutionary unique gene clusters (microsyntenies). Such novel microsyntenies correspond to topological compartments with a distinct regulatory structure and contribute to complex expression patterns. In particular, we identify a set of microsyntenies associated with cephalopod innovations (MACIs) broadly enriched in cephalopod nervous system expression. We posit that the emergence of MACIs was instrumental to cephalopod nervous system evolution and propose that microsyntenic profiling will be central to understanding cephalopod innovations.

Development of the Accessory Nidamental Gland and Associated Bacterial Community in the Hawaiian Bobtail Squid, Euprymna scolopes.

AbstractThe Hawaiian bobtail squid, Euprymna scolopes, has a female reproductive organ called the accessory nidamental gland that contains a symbiotic bacterial consortium. These bacteria are deposited from the accessory nidamental gland into the squid's egg cases, where the consortium prevents microbial fouling. The symbiont community is environmentally transmitted and conserved across host populations, yet little is known about how the organ develops and is colonized by bacteria. In order to understand accessory nidamental gland development in E. scolopes, we characterized the gland during maturation by using histology and confocal and transmission electron microscopy. We found that an epithelial field formed first about four weeks after hatching, followed by the proliferation of numerous pores during what we hypothesize to be the initiation of bacterial recruitment (early development). Microscopy revealed that these pores were connected to ciliated invaginations that occasionally contained bacteria. During mid development, these epithelial fields expanded, and separate colonized tubules were observed below the epithelial layer that contained the pores and invaginations. During late development, the superficial epithelial fields appeared to regress as animals approached sexual maturity and were never observed in fully mature adults (about 2-3 months post-hatching), suggesting that they help facilitate bacterial colonization of the accessory nidamental gland. An analysis of 16S rRNA gene diversity in accessory nidamental glands from females of varying size showed that the bacterial community changed as the host approached sexual maturity, increasing in community evenness and shifting from a Verrucomicrobia-dominated to an Alphaproteobacteria-dominated consortium. Given the host's relationship with the well-characterized light organ symbiont Vibrio fischeri, our work suggests that the accessory nidamental gland of E. scolopes may have similar mechanisms to recruit bacteria from the environment. Understanding the developmental and colonization processes of the accessory nidamental gland will expand the use of E. scolopes as a model organism for studying bacterial consortia in marine symbioses.

A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner.

For more than 30 years, the association between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri has been studied as a model system for understanding the colonization of animal epithelia by symbiotic bacteria. The squid-vibrio light-organ system provides the exquisite resolution only possible with the study of a binary partnership. The impact of this relationship on the partners' biology has been broadly characterized, including their ecology and evolutionary biology as well as the underlying molecular mechanisms of symbiotic dynamics. Much has been learned about the factors that foster initial light-organ colonization, and more recently about the maturation and long-term maintenance of the association. This Review synthesizes the results of recent research on the light-organ association and also describes the development of new horizons for E. scolopes as a model organism that promises to inform biology and biomedicine about the basic nature of host-microorganism interactions.

Disparities in acute decompensated heart failure.

PURPOSE OF REVIEW: The aim of this review is to discuss racial and sex disparities in the management and outcomes of patients with acute decompensated heart failure (ADHF). RECENT FINDINGS: Race and sex have a significant impact on in-hospital admissions and overall outcomes in patients with decompensated heart failure and cardiogenic shock. Black patients not only have a higher incidence of heart failure than other racial groups, but also higher admissions for ADHF and worse overall survival, while women receive less interventions for cardiogenic shock complicating acute myocardial infarction. Moreover, White patients are more likely than Black patients to be cared for by a cardiologist than a noncardiologist in the ICU, which has been linked to overall improved survival. In addition, recent data outline inherent racial and sex bias in the evaluation process for advanced heart failure therapies indicating that Black race negatively impacts referral for transplant, women are judged more harshly on their appearance, and that Black women are perceived to have less social support than others. This implicit bias in the evaluation process may impact appropriate timing of referral for advanced heart failure therapies. SUMMARY: Though significant racial and sex disparities exist in the management and treatment of patients with decompensated heart failure, these disparities are minimized when therapies are properly utilized and patients are treated according to guidelines.

Prevalence of newly diagnosed sarcoidosis in patients with ventricular arrhythmias: a cardiac magnetic resonance and 18F-FDG cardiac PET study.

Cardiac sarcoidosis (CS) is known to be associated with ventricular tachycardia (VT); however, most investigations to date have focused on patients with known extra-cardiac sarcoidosis. The presence of CS is typically evaluated using 18F-fluorodeoxyglucose (18F-FDG) uptake on cardiac positron emission tomography (PET) or late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR). In this study, we sought to determine the prevalence of primary CS and the relationship between myocardial 18F-FDG uptake and LGE in patients with VT without known sarcoidosis. We retrospectively identified 67 patients without known sarcoidosis or active ischemic heart disease (i.e. significant ischemic disease that had not been previously revascularized) referred for both CMR and PET for evaluation of VT. Standard cine- and LGE- CMR and cardiac PET protocols were used. Myocardial LGE was defined as signal intensity > 5 SDs above the mean signal intensity of normal myocardium. Cardiac PET images were considered positive if there was focal myocardial 18F-FDG uptake having greater activity than the left ventricular blood pool. 45 patients (67%) had LGE, while only 4 (6%) had myocardial FDG uptake. Nine percent of patients with LGE had FDG-uptake while none without LGE did, and 10% of the cohort had indeterminate FDG uptake presumably from poor dietary preparation. Of those with both FDG uptake and LGE, 3/4 ultimately received a clinical diagnosis of CS. 4.5% of patients without previously known sarcoidosis or active ischemic heart disease presenting with VT have newly diagnosed CS. Detection of CS can be increased using a CMR first approach followed by cardiac PET for patients with non-ischemic LGE.

In the beginning: egg-microbe interactions and consequences for animal hosts.

Microorganisms are associated with the eggs of many animals. For some hosts, the egg serves as the ideal environment for the vertical transmission of beneficial symbionts between generations, while some bacteria use the egg to parasitize their hosts. In a number of animal groups, egg microbiomes often perform other essential functions. The eggs of aquatic and some terrestrial animals are especially susceptible to fouling and disease since they are exposed to high densities of microorganisms. To overcome this challenge, some hosts form beneficial associations with microorganisms, directly incorporating microbes and/or microbial products on or in their eggs to inhibit pathogens and biofouling. Other functional roles for egg-associated microbiomes are hypothesized to involve oxygen and nutrient acquisition. Although some egg-associated microbiomes are correlated with increased host fitness and are essential for successful development, the mechanisms that lead to such outcomes are often not well understood. This review article will discuss different functions of egg microbiomes and how these associations have influenced the biology and evolution of animal hosts. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D'.

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A' (compounds 1a/1b), as well as two new derivatives, BAC-D/D' (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey's method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host's environment.IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.

Equiprobable discrete models of site-specific substitution rates underestimate the extent of rate variability.

It is standard practice to model site-to-site variability of substitution rates by discretizing a continuous distribution into a small number, K, of equiprobable rate categories. We demonstrate that the variance of this discretized distribution has an upper bound determined solely by the choice of K and the mean of the distribution. This bound can introduce biases into statistical inference, especially when estimating parameters governing site-to-site variability of substitution rates. Applications to two large collections of sequence alignments demonstrate that this upper bound is often reached in analyses of real data. When parameter estimation is of primary interest, additional rate categories or more flexible modeling methods should be considered.

Synonymous Site-to-Site Substitution Rate Variation Dramatically Inflates False Positive Rates of Selection Analyses: Ignore at Your Own Peril.

Most molecular evolutionary studies of natural selection maintain the decades-old assumption that synonymous substitution rate variation (SRV) across sites within genes occurs at levels that are either nonexistent or negligible. However, numerous studies challenge this assumption from a biological perspective and show that SRV is comparable in magnitude to that of nonsynonymous substitution rate variation. We evaluated the impact of this assumption on methods for inferring selection at the molecular level by incorporating SRV into an existing method (BUSTED) for detecting signatures of episodic diversifying selection in genes. Using simulated data we found that failing to account for even moderate levels of SRV in selection testing is likely to produce intolerably high false positive rates. To evaluate the effect of the SRV assumption on actual inferences we compared results of tests with and without the assumption in an empirical analysis of over 13,000 Euteleostomi (bony vertebrate) gene alignments from the Selectome database. This exercise reveals that close to 50% of positive results (i.e., evidence for selection) in empirical analyses disappear when SRV is modeled as part of the statistical analysis and are thus candidates for being false positives. The results from this work add to a growing literature establishing that tests of selection are much more sensitive to certain model assumptions than previously believed.

HyPhy 2.5-A Customizable Platform for Evolutionary Hypothesis Testing Using Phylogenies.

HYpothesis testing using PHYlogenies (HyPhy) is a scriptable, open-source package for fitting a broad range of evolutionary models to multiple sequence alignments, and for conducting subsequent parameter estimation and hypothesis testing, primarily in the maximum likelihood statistical framework. It has become a popular choice for characterizing various aspects of the evolutionary process: natural selection, evolutionary rates, recombination, and coevolution. The 2.5 release (available from www.hyphy.org) includes a completely re-engineered computational core and analysis library that introduces new classes of evolutionary models and statistical tests, delivers substantial performance and stability enhancements, improves usability, streamlines end-to-end analysis workflows, makes it easier to develop custom analyses, and is mostly backward compatible with previous HyPhy releases.

Shielding the Next Generation: Symbiotic Bacteria from a Reproductive Organ Protect Bobtail Squid Eggs from Fungal Fouling.

The importance of defensive symbioses, whereby microbes protect hosts through the production of specific compounds, is becoming increasingly evident. Although defining the partners in these associations has become easier, assigning function to these relationships often presents a significant challenge. Here, we describe a functional role for a bacterial consortium in a female reproductive organ in the Hawaiian bobtail squid, Euprymna scolopes Bacteria from the accessory nidamental gland (ANG) are deposited into the egg jelly coat (JC), where they are hypothesized to play a defensive role during embryogenesis. Eggs treated with an antibiotic cocktail developed a microbial biomass primarily composed of the pathogenic fungus Fusarium keratoplasticum that infiltrated the JC, resulting in severely reduced hatch rates. Experimental manipulation of the eggs demonstrated that the JC was protective against this fungal fouling. A large proportion of the bacterial strains isolated from the ANG or JC inhibited F. keratoplasticum in culture (87.5%), while a similar proportion of extracts from these strains also exhibited antifungal activity against F. keratoplasticum and/or the human-pathogenic yeast Candida albicans (72.7%). Mass spectral network analyses of active extracts from bacterial isolates and egg clutches revealed compounds that may be involved in preventing microbial overgrowth. Several secondary metabolites were identified from ANG/JC bacteria and egg clutches, including the known antimicrobial lincomycin as well as a suite of glycerophosphocholines and mycinamicin-like compounds. These results shed light on a widely distributed but poorly understood symbiosis in cephalopods and offer a new source for exploring bacterial secondary metabolites with antimicrobial activity.IMPORTANCE Organisms must have strategies to ensure successful reproduction. Some animals that deposit eggs protect their embryos from fouling/disease with the help of microorganisms. Although beneficial bacteria are hypothesized to contribute to egg defense in some organisms, the mechanisms of this protection remain largely unknown, with the exception of a few recently described systems. Using both experimental and analytical approaches, we demonstrate that symbiotic bacteria associated with a cephalopod reproductive gland and eggs inhibit fungi. Chemical analyses suggest that these bacteria produce antimicrobial compounds that may prevent overgrowth from fungi and other microorganisms. Given the distribution of these symbiotic glands among many cephalopods, similar defensive relationships may be more common in aquatic environments than previously realized. Such defensive symbioses may also be a rich source for the discovery of new antimicrobial compounds.

Diverse deep-sea anglerfishes share a genetically reduced luminous symbiont that is acquired from the environment.

Deep-sea anglerfishes are relatively abundant and diverse, but their luminescent bacterial symbionts remain enigmatic. The genomes of two symbiont species have qualities common to vertically transmitted, host-dependent bacteria. However, a number of traits suggest that these symbionts may be environmentally acquired. To determine how anglerfish symbionts are transmitted, we analyzed bacteria-host codivergence across six diverse anglerfish genera. Most of the anglerfish species surveyed shared a common species of symbiont. Only one other symbiont species was found, which had a specific relationship with one anglerfish species, Cryptopsaras couesii. Host and symbiont phylogenies lacked congruence, and there was no statistical support for codivergence broadly. We also recovered symbiont-specific gene sequences from water collected near hosts, suggesting environmental persistence of symbionts. Based on these results we conclude that diverse anglerfishes share symbionts that are acquired from the environment, and that these bacteria have undergone extreme genome reduction although they are not vertically transmitted.