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Novelty, diversity, and genetic dark matter in enterococci of invertebrates.

Enterococci appear to have originated in the guts of early terrestrializing arthropods and invertebrates over 425 million years ago-hosts that are now highly diverse and widespread in nature today. Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined Enterococcus association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and Enterococcus-adapted genes, 40% (eight of 20) of the Enterococcus species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of E. casseliflavus and E. flavescens in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci-diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains.IMPORTANCEEnterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years-the "antibiotic era"-E. faecalis and E. faecium gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of Enterococcus biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.

Animals

Pseudoalteromonas is a novel symbiont of marine invertebrates that exhibits broad patterns of phylosymbiosis.

Despite growing insights into the composition of marine invertebrate microbiomes, our understanding of their ecological and evolutionary patterns remains poor, owing to limited sampling depth and low-resolution datasets. Previous studies have provided mixed results when evaluating patterns of phylosymbiosis between marine invertebrates and marine bacteria. Here, we investigated potential animal-microbe symbioses in Pseudoalteromonas, an overlooked bacterial genus consistently identified as a core microbiome taxon in diverse invertebrates. Using a pangenomic analysis of 236 free-living and invertebrate-associated bacterial strains (including two new nematode-associated isolates generated in this study), we confirm that Pseudoalteromonas is a novel symbiont with substantial evidence of phylosymbiosis across at least three marine invertebrate phyla (e.g., Nematoda, Mollusca, and Cnidaria). Patterns of symbiosis were consistent irrespective of geography (including in Antarctica), with FISH images from nematodes indicating that bacterial symbionts form biofilms in the mouth and esophagus. The evolutionary history of Pseudoalteromonas is marked by substantial host-switching and lifestyle transitions, and host-associated genomes suggest that these bacteria are facultative symbionts involved in nutritional mutualisms. In marine environments, we hypothesize that horizontally-acquired symbionts may have co-evolved with invertebrates, using host mucus as a physical niche and food source, while providing their animal hosts with Vitamin B, amino acids, and bioavailable carbon compounds in return.

Marine Invertebrates

Invertebrate collagens.

The collagens of all major invertebrate phyla have been studied, but characterization has been thorough in only a few classes and in no case in the detail (such as sequence analysis) known for vertebrate collagen. Biochemical data on insect collagen are particularly sparse. Invertebrate and vertebrate collagens are strikingly similar, with some notably unique features in annelids and nematodes. Present data do not support the suggestion that invertebrate collagens resemble vertebrate basement membrane collagen. In invertebrates, as in vertebrates, collagens of specific tissues show differenes that probably reflect individual tissue requirements.

Amino Acid Sequence

The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates.

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.

AMP Deaminase

[Tumor formations and cell proliferation in invertebrates].

Critical analysis of the present status of invertebrate oncology through an appreciation of data relative to the various origins of tumors : spontaneous formations, cell proliferations induced by viruses, so-called hereditary tumors, effects of injuries induced by carcinogenic agents, irradiations or hormones on the formation of neoplasms in invertebrates. Attempts are made to interpret these data as compared with those obtained in medical and veterinary oncology. The possible direction of studies on invertebrates towards comparative oncology is considered, emphasizing the great usefulness for the medical research to study the phylogenetic development of oncogenesis.

Animals

Relationships between specialized cells, capillaries and intermediary cytofibrillary elements. Xth Note. Biological evolution of the emonctory subsystem and stereotype in invertebrates.

The paper points out the importance of homeostasis as the most general system of the organism (Ist order system) and the 2nd order subsystem which composes it, discussing the relations between them and the biological evolution of the emonctory subsystem in protists and invertebrates. The emonctory structures, functions and stereotype and their component parts are studied in protists, spongia, coelenterata and coelomata: lower worms, annelids, their hyponeurian descendents (arthropods, molluses) and epineurian descedents echinoderms and protochordates (Stomochordata, Tunicata, Cephalochordata). The structure, functions and stereotypes of protonephridia and metanephridia and of substitutive (vicarious) emonctory organs (nephrocytes and peritoneal cells) are studied in coelomatic invertebrates. Their evolution appears to be directioned to localize within the limits of the emonctory subsystem. During their evolution, some nephridial functions are performed by the vicarious emonctory organs. The evolution of the subsystem in invertebrates appears to be based on stable characters and their relationships with variable features.

Animals

Gas-liquid chromatographics determination of Bayer 73 in fish, aquatic invertebrates, mud, and water.

A gas-liquid chromatographic (GLC) method is described for determining residues of Bayer 73 (2-aminoethanol salt of 2',5-dichloro-4'-nitrosalicylanilide) in fish muscle, aquatic invertebrates, mud, and water by analyzing for 2-chloro-4-nitroaniline (CNA), a hydrolysis product of Bayer 73. Bayer 73 residues are extracted from fish muscle tissue, invertebrates, and mud with acetone-formic acid (98+2), and partitioned from water samples with chloroform. After sample cleanup by solvent and acid-base partitioning, the concentrated extract is hydrolyzed with 2N NaOH and H2O2 for 10 min at 95 degrees C. The CNA is then partitioned into hexane-ethyl ether (7+3) and determined by electron capture GLC. Average recoveries were 88% for fish, 82% for invertebrates, 82% for mud, and 98% for water at 3 or more fortification levels.

Animals

DNA methylation-based ageing in a deuterostome invertebrate: an epigenetic clock for the crown-of-thorns seastar (Acanthaster cf. solaris).

Accurate and reliable ageing tools are essential for wildlife conservation and management. While DNA methylation has emerged as a promising tool for age estimation in vertebrates, its application to invertebrates remains contested and has been limited to arthropods. Here, we develop an epigenetic clock for the Pacific crown-of-thorns seastar (CoTS; Acanthaster cf. solaris), a destructive coral predator contributing to habitat degradation across Indo-Pacific reefs. Using Oxford Nanopore Technologies, we generated whole-genome DNA methylation profiles across five age groups and identified 1910 CpG sites with methylation patterns significantly associated with age. We then fitted age prediction models using elastic net regression and evaluated predictive performance with leave-one-out cross-validation (LOOCV), achieving a mean absolute error of 0.31 ± 0.22 years, corresponding to 4-6% of the CoTS lifespan (5-8 years). This accuracy suggests the potential to differentiate annual cohorts, supporting future management-relevant inference. To facilitate practical implementation, we constructed an optimized epigenetic clock from 14 CpG sites consistently selected across LOOCV iterations. Our results demonstrate that DNA methylation-based age estimation is feasible in a deuterostome invertebrate, extending epigenetic ageing approaches beyond arthropods and establishing their potential to advance age determination and management in invertebrates that lack reliable ageing methods.

Animals

Solute compatibility with enzyme function and structure: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates.

The major nitrogenous osmolytes present in the cells of marine invertebrates, notably the free amino acids glycine, alanine and proline, and trimethylamine oxide and betaine, are highly compatible with proper enzyme function and structure. These nitrogenous osmolytes display either non-perturbing or, in some cases, favorable effects on enzyme-substrate and enzyme-cofactor complex formation, catalytic velocity and protein structural stability. In contrast, inorganic salts (KCl and NaCl) and certain of the free amino acids which play only a minor osmotic role, e.g., arginine and lysine, have strongly perturbing effects on one or more of these enzymic parameters. The compatible nitrogenous solutes therefore are suitable for use at high (several tenths molar) concentrations and at widely varying concentrations in osmo-conforming species. Certain nitrogenous solutes, especially trimethylamine oxide, betaine and glutamate, offset some of the perturbing effects of inorganic ions on enzyme function. The selective accumulation of osmolytes thus involves not only the concentration of non-perturbing solutes, but also a balanced accumulation of solutes with opposing effects on enzymes. The selection of end-products of anaerobic metabolism also appears to be based, in part, on considerations of solute compatibility with enzyme function. Octopine is a non-perturbing solute, whereas arginine, which is condensed with pyruvate to form octopine, is very strongly perturbing. Succinate has marked stabilizing effects on protein structure. We conclude that the composition of the intracellular fluids of marine invertebrates reflects selection for osmolytes and end-products whose net effects create a cellular microenvironment which is conducive to optimal enzyme function and structure. The accumulation of compatible solutes may preclude the necessity for widespread changes in protein structure in adapting to concentrated or highly variable osmotic environments.

Amino Acids

Evolutionary change in invertebrate cytochrome C.

Recently published amino acid sequences are compared to those of other cytochromes c. Molecular phylogenies constructed by using an ancestral sequence method are compared to the classical biological view of invertebrate evolution. Problems associated with the analysis of sequences of different chain lengths and of high variability are discussed, and the logistics of increasing the representation of key invertebrate phyla is assessed.

Amino Acid Sequence

Ubiquinone analyses in fish tissues and in some marine invertebrates.

1. Ubiquinone contents were determined in species of marine invertebrates, and in heart, red and white muscle and liver of three species of fish. 2. Three different methods of determination were compared, based on spectrophotometry, reduction and a reaction with the dimethoxy groups of ubiquinone. 3. Using ubiquinone homologues 6-10 prepared from beef heart and commercially available microorganisms (SCP) as standards, ubiquinone 10 was found in all samples. In addition were found minor amounts of Q-9 in samples of saithe heart and red muscle. 4. Less than 10 mg/kg wet wt of ubiquinone was found in the samples of marine invertebrates and in white muscle and liver of the fish samples, with one exception: 40 mg/kg in a sample of mackerel liver. 5. Higher contents of ubiquinone were found in fish heart and red muscle tissues, ranging from 24 to 116 mg/kg wet wt. The ubiquinone contents were comparable in the two tissues. 6. A test on cellular fragments of red muscle tissue of saithe showed that the ubiquinone was concentrated in the mitochondria fraction.

Animals

Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.

Animals

The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...

Adenosine Triphosphate

Cell fusion induced by invertebrate virus. Brief report.

Inoculation of invertebrate cells with Chilo Iridescent Virus (CIV) leads rapidly to massive formation of syncytia. This polykaryocytosis occurs at a high multiplicity of infection and does not require viral genome expression. Thus, cell fusion appears in non permissive conditions, and CIV suspensions rendered non infections by UV irradiation still retain the ability to produce rapid and extensive cell fusion. In the same way, some polykaryocytes could be detected in vertebrate cells (CV1 cell line). However, this process is more effective and faster in invertebrate cell systems.

Aedes

Paramyosin in invertebrate muscles. I. Identification and localization.

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion, we identified paramyosin in two smooth invertebrate "catch" muscles (Mytilus anterior byssus retractor and Mercenaria opaque adductor) and five invertebrate striated muscles (Limulus telson levator, Homarus claw muscle, Balanus scutal depressor, Lethocerus air tube retractor, and Aequipecten striated adductor). We show that (a) the paramyosins in all of these muscles have the same chain weights and (b) they are immunologically similar. We stained all of these muscles with specific antibody to Limulus paramyosin using the indirect fluorescent antibody technique. Paramyosin was localized to the A bands of the glycerinated striated muscles, and diffus fluorescence was seen throughout the glycerinated fibers of the smooth catch muscles. The presence of paramyosin in Homarus claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor is shown here for the first time. Of the muscles in this study, Limulus telson levator is the only one for which the antiparamyosin staining pattern has been previously reported.

Animals

Invertebrate gap junctions.

Both glycerol and glutaraldehyde, the two most commonly used chemical aids in freeze-fractue studies, have been shown individually to affect the structure of certain membranes as observed in freeze-fracture replicas. The present investigation studied the effect of glycerol on the gap junctions found in a number of tissues from several invertebrate phyla. Glycerol was shown, in some of these tissues, to affect both the spatial arrangement of the particles within the membrane and their fracturing properties if the tissue had not been prefixed with glutaraldehyde. It is suggested that the effect of glycerol on invertebrate gap junctions may be of diagnostic use in enabling them to be separated into a number, or spectrum, of types. Previously B-type gap junctions have been reported only in Arthropoda. This study has extended knowledge of their occurrence into the phyla Coelenterata, Platyhelminthes and Annelida. The B-type gap junctions appear consistently to have a 12-nm particle separation, which is larger than the 10-nm separation found in vertebrate and molluscan A-type gap junctions.

Animals

Subcellular calcium localization and AT0-dependent Ca2+-uptake by smooth endoplasmic reticulum in an invertebrate photoreceptor cell. An ultrastrucutral, cytochemical and X-ray microanalytical study.

In Hirudo medicinalis an extensive and highly elaborate three dimensional network of smooth endoplasmic reticulum cisternae is found in very close structural relationship to the receptive (microvillar) membrane, as reported for many other invertebrates. A variant of the potassium pyroantimonate technique showed that these submicrovillar endoplasmic reticulum cisternae (SMC) and mitochondria are major intracellular calcium stores. Furthermore, using saponine-skinned photoreceptors for an in situ accumulation experiment, calcium oxalate precipitates in SMC demonstrate that this organelle is able to accumulate Ca2+ from a concentration of 2 x 10(-5) M, when ATP, Mg2+, and oxalate ions are present in the accumulation medium. This result provides direct evidence for the hypothesis that SMC may play a particularly important role in the regulation of intracellular ionized calcium in invertebrate photoreceptor cells. Morphological evidence supports this view.

Animals

Comparative studies on different small intestinal oligosaccharidase activities in some vertebrates and invertebrates like molluscs.

Oligosaccharidase activities of the small intestinal mucosal homogenates were measured in vertebrates viz fish, toad, garden-lizard (calotes), pigeon, rat and some invertebrates viz, molluscsa. Maximum activities of the enzymes Lactase, Sucrase and Maltase were found in the mammalian species rat, whereas much less activities were found in the non-mammalian vertebrates among which toad shows the highest values and garden lizard the lowest. Among the invertebrates Pila globosa shows higher values of all the enzymes than Achatina fulica. The results obtained have been discussed in the lights of phylogeny and diet habits.

Amphibians