Effects of Erythromycin on Nereis succinea and the Intestinal Microbiome across Different Salinity Levels.

The exposure of aquatic organisms to pollutants often occurs concomitantly with salinity fluctuations. Here, we reported the effects of erythromycin (0.250, 7.21, and 1030 µg/L) on marine invertebrate N. succinea and its intestinal microbiome under varying salinity levels (5‰, 15‰, and 30‰). The salinity elicited significant effects on the growth and intestinal microbiome of N. succinea. The susceptibility of the intestinal microbiome to erythromycin increased by 8.7- and 6.2-fold at salinities of 15‰ and 30‰, respectively, compared with that at 5‰ salinity. Erythromycin caused oxidative stress and histological changes in N. succinea intestines, and inhibited N. succinea growth in a concentration-dependent manner under 30‰ salinity with a maximum inhibition of 25%. At the intestinal microbial level, erythromycin enhanced the total cell counts at 5‰ salinity but reduced them at 15‰ salinity. Under all tested salinities, erythromycin diminished the antibiotic susceptibility of the intestinal microbiome. Two-way ANOVA revealed significant interactive effects (p < 0.05) between salinity and erythromycin on various parameters, including antibiotic susceptibility and intestinal microbial diversity. The present findings demonstrated the significant role of salinity in modulating the impacts of erythromycin, emphasizing the necessity to incorporate salinity fluctuations into environmental risk assessments.

Phytoremediation of crude oil-contaminated sediment using Suaeda heteroptera enhanced by Nereis succinea and oil-degrading bacteria.

A 150-day experiment was performed to investigate the stimulatory effect of a promising phytoremediation strategy consisting of Suaeda heteroptera (S. heteroptera), Nereis succinea (N. succinea), and oil-degrading bacteria for cleaning up total petroleum hydrocarbons (TPHs) in spiked sediment. Inoculation with oil-degrading bacteria and/or N. succinea increased plant yield and TPH accumulation in S. heteroptera plants. The highest TPH dissipation (40.5%) was obtained in the combination treatment, i.e., S. heteroptera + oil-degrading bacteria + N. succinea, in which the sediment TPH concentration decreased from an initial value of 3955 to 2355 mg/kg in 150 days. BAF, BCF, and TF confirmed the role of N. succinea and oil-degrading bacteria in the amelioration and translocation of TPHs. In addition, TPH toxicity of S. heteroptera was alleviated by N. succinea and oil-degrading bacteria addition through the reduction of oxidative stress. Therefore, S. heteroptera could be used for cleaning up oil-contaminated sediment, particularly in the presence of oil-degrading bacteria + N. succinea. Field studies on oil-degrading bacteria + N. succinea may provide new insights on the rehabilitation and restoration of sediments contaminated by TPHs.

Our study attempted to investigate the stimulatory effect of a promising phytoremediation strategy consisting of Suaeda heteroptera (S. heteroptera), Nereis succinea (N. succinea), and oil-degrading bacteria for cleaning up TPH in spiked sediment. Planting S. heteroptera can greatly increase sediment TPH removal, and its removal was enhanced greater after inoculation with oil-degrading bacteria and/or N. succinea. Moreover, the promising phytoremediation strategy developed in the current work can serve as an efficient, novel approach to removal TPH in sediment/soil. In our opinions, these findings provide insights into the assessment of their ecological risks in the environments that are of interest to broad readership of International Journal of Phytoremediation.

Nanos Is Expressed in Somatic and Germline Tissue during Larval and Post-Larval Development of the Annelid Alitta virens.

Nanos is a translational regulator that is involved in germline development in a number of diverse animals and is also involved in somatic patterning in several model organisms, including insects. Neither germline development nor somatic stem cell lines/undifferentiated multipotent cells have been characterized in the development of the annelid Alitta virens, nor is the mechanism of germ/stem-line specification generally well-understood in annelids. Here, I have cloned an Avi-nanos ortholog from A. virens and determined the spatial and temporal expression of Nanos. The results revealed that transcripts of nanos are expressed during differentiation of multiple tissues, including those that are derived from the 2d and 4d cells. In late embryonic stages and during larval development, these transcripts are expressed in the presumptive brain, ventral nerve cord, mesodermal bands, putative primordial germ cells (PGCs), and developing foregut and hindgut. During metamorphosis of the nectochaete larva into a juvenile worm, a posterior growth zone consisting of nanos-positive cells is established, and the PGCs begin to migrate. Later, the PGCs stop migrating and form a cluster of four nanos-expressing cells located immediately behind the jaws (segments 4-5). During posterior regeneration following caudal amputation, a robust Avi-nanos expression appears de novo at the site of injury and further accompanies all steps of regeneration. The obtained data suggest that blastemal cells are mostly derived from cells of the segment adjacent to the amputation site; this is consistent with the idea that the cluster of PGCs do not participate in regeneration.

INVESTIGATING ASSOCIATIONS AMONG RELATEDNESS, GENETIC DIVERSITY, AND CAUSES OF MORTALITY IN SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS).

Southern sea otter (Enhydra lutris nereis) population recovery is influenced by a variety of factors, including predation, biotoxin exposure, infectious disease, oil spills, habitat degradation, and resource limitation. This population has also experienced a significant genetic bottleneck, resulting in low genetic diversity. We investigated how two metrics, familial relatedness and genetic diversity, are correlated with common causes of mortality in southern sea otters, including cardiomyopathy, acanthocephalan (Profilicollis spp.) peritonitis, systemic protozoal infection (Toxoplasma gondii and Sarcocystis neurona), domoic acid intoxication, end-lactation syndrome, and shark bite. Microsatellite genetic markers were used to examine this association in 356 southern sea otters necropsied from 1998 to 2012. Significant associations with genetic diversity or familial relatedness (P<0.05) were observed for cardiomyopathy, acanthocephalan peritonitis, and sarcocystosis, and these associations varied by sex. Adult male cardiomyopathy cases (n=86) were more related than the null expectation (P<0.049). Conversely, female acanthocephalan peritonitis controls (n=110) were more related than the null expectation (P<0.004). Including genetic diversity as a predictor for fatal acanthocephalan peritonitis in the multivariate logistic model significantly improved model fit; lower genetic diversity was associated with reduced odds of sea otter death due to acanthocephalan peritonitis. Finally, male sarcocystosis controls (n=158) were more related than the null expectation (P<0.011). Including genetic diversity in the multivariate logistic model for fatal S. neurona infection improved model fit; lower genetic diversity was associated with increased odds of sea otter death due to S. neurona. Our study suggests that genetic diversity and familial relatedness, in conjunction with other factors such as age and sex, may influence outcome (survival or death) in relation to several common southern sea otter diseases. Our findings can inform policy for conservation management, such as potential reintroduction efforts, as part of species recovery.

Structural and Functional Characterization of the FGF Signaling Pathway in Regeneration of the Polychaete Worm Alitta virens (Annelida, Errantia).

Epimorphic regeneration of lost body segments is a widespread phenomenon across annelids. However, the molecular inducers of the cell sources for this reparative morphogenesis have not been identified. In this study, we focused on the role of fibroblast growth factor (FGF) signaling in the posterior regeneration of Alitta virens. For the first time, we showed an early activation of FGF ligands and receptor expression in an annelid regenerating after amputation. The expression patterns indicate that the entire regenerative bud is competent to FGFs, whose activity precedes the initiation of cell proliferation. The critical requirement of FGF signaling, especially at early stages, is also supported by inhibitor treatments followed by proliferation assay, demonstrating that induction of blastemal cells depends on FGFs. Our results show that FGF signaling pathway is a key player in regenerative response, while the FGF-positive wound epithelium, ventral nerve cord and some mesodermal cells around the gut could be the inducing tissues. This mechanism resembles reparative regeneration of vertebrate appendages suggesting such a response to the injury may be ancestral for all bilaterians.

Complete mitochondrial genome of the marine polychaete, Nereis zonata (Phyllodocida, Nereididae) isolated from the Beaufort Sea.

Here, we present the first whole mitogenome sequence of the marine polychaete, Nereis zonata, isolated from the Beaufort Sea. The mitochondrial genome of N. zonata is 15,757 bp in length and consists of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding region that is typical of polychaetes. GC content of the N. zonata mitogenome is 37.2%. A maximum-likelihood gene tree based on the N. zonata mitogenome combined with previously published annelid mitogenome data revealed that N. zonata is clustered with Cheilonereis cyclurus, which form a sister group to Nereis sp.

Primary intracranial fibrosarcoma in a southern sea otter Enhydra lutris nereis.

Southern sea otters Enhydra lutris nereis, a threatened marine mammal species, face numerous environmental and infectious disease challenges in their native habitat of coastal California, USA. However, there are few published cases describing neoplasia in sea otters despite their relatively long life span when cared for in aquarium settings. An 18 yr old neutered male southern sea otter, born and raised in human care, presented with an acute onset of seizures and dull mentation. Magnetic resonance imaging of the head revealed a large, central brain lesion. After no improvement with treatment, euthanasia was elected due to a poor prognosis. Grossly, a poorly demarcated, granular, tan mass expanded the cranial meninges in the longitudinal fissure at the level of the cruciate sulcus and extended into the underlying gray matter and superficial white matter. Histologically, the mass was composed of spindle cells, forming haphazardly arranged interlacing bundles and herringbone patterns, with a high mitotic count, moderate cellular pleomorphism, and prominent vascularization. Neoplastic cells demonstrated positive immunoreactivity for vimentin and negative immunoreactivity for smooth muscle actin, factor VIII-related antigen, S100, melan-A, E-cadherin, desmin, glial fibrillary acidic protein, and cytokeratin AE1/AE3. Based on gross, histologic, and immunohistochemical findings, the mass was most consistent with a primary intracranial fibrosarcoma (PIF). PIFs are a rare neoplasm in both humans and other animals with few reports in the veterinary literature. This is the first recorded case of a PIF in a sea otter.

Type X strains of Toxoplasma gondii are virulent for southern sea otters (Enhydra lutris nereis) and present in felids from nearby watersheds.

Why some Toxoplasma gondii-infected southern sea otters (Enhydra lutris nereis) develop fatal toxoplasmosis while others have incidental or mild chronic infections has long puzzled the scientific community. We assessed robust datasets on T. gondii molecular characterization in relation to detailed necropsy and histopathology results to evaluate whether parasite genotype influences pathological outcomes in sea otters that stranded along the central California coast. Genotypes isolated from sea otters were also compared with T. gondii strains circulating in felids from nearby coastal regions to assess land-to-sea parasite transmission. The predominant T. gondii genotypes isolated from 135 necropsied sea otters were atypical Type X and Type X variants (79%), with the remainder (21%) belonging to Type II or Type II/X recombinants. All sea otters that died due to T. gondii as a primary cause of death were infected with Type X or X-variant T. gondii strains. The same atypical T. gondii strains were detected in sea otters with fatal toxoplasmosis and terrestrial felids from watersheds bordering the sea otter range. Our results confirm a land-sea connection for virulent T. gondii genotypes and highlight how faecal contamination can deliver lethal pathogens to coastal waters, leading to detrimental impacts on marine wildlife.

Cat Gets Its Tern: A Case Study of Predation on a Threatened Coastal Seabird.

Domestic cats have a cosmopolitan distribution, commonly residing in urban, suburban and peri-urban environments that are also critical for biodiversity conservation. This study describes the impact of a desexed, free-roaming cat on the behavior of a threatened coastal seabird, the Australian Fairy Tern, Sternula nereis nereis, in Mandurah, south-western Australia. Wildlife cameras and direct observations of cat incursions into the tern colony at night, decapitated carcasses of adult terns, dead, injured or missing tern chicks, and cat tracks and scats around the colony provided strong evidence of cat predation, which led to an initial change in nesting behavior and, ultimately, colony abandonment and the reproductive failure of 111 nests. The death of six breeding terns from the population was a considerable loss for this threatened species and had the potential to limit population growth. This study highlights the significant negative impacts of free-roaming cats on wildlife and the need for monitoring and controlling cats at sites managed for species conservation. It also provides strong evidence against the practice of trap-neuter-release programs and demonstrates that desexed cats can continue to negatively impact wildlife post-release directly through predation, but also indirectly through fundamental changes in prey behavior and a reduction in parental care.

Serine Protease from Nereis virens Inhibits H1299 Lung Cancer Cell Proliferation via the PI3K/AKT/mTOR Pathway.

This study explores the in vitro anti-proliferative mechanism between Nereis Active Protease (NAP) and human lung cancer H1299 cells. Colony formation and migration of cells were significantly lowered, following NAP treatment. Flow cytometry results suggested that NAP-induced growth inhibition of H1299 cells is linked to apoptosis, and that NAP can arrest the cells at the G0/G1 phase. The ERK/MAPK and PI3K/AKT/mTOR pathways were selected for their RNA transcripts, and their roles in the anti-proliferative mechanism of NAP were studied using Western blots. Our results suggested that NAP led to the downregulation of p-ERK (Thr 202/Tyr 204), p-AKT (Ser 473), p-PI3K (p85), and p-mTOR (Ser 2448), suggesting that NAP-induced H1299 cell apoptosis occurs via the PI3K/AKT/mTOR pathway. Furthermore, specific inhibitors LY294002 and PD98059 were used to inhibit these two pathways. The effect of NAP on the downregulation of p-ERK and p-AKT was enhanced by the LY294002 (a PI3K inhibitor), while the inhibitor PD98059 had no obvious effect. Overall, the results suggested that NAP exhibits antiproliferative activity by inducing apoptosis, through the inhibition of the PI3K/AKT/mTOR pathway.

FoxA expression pattern in two polychaete species, Alitta virens and Platynereis dumerilii: Examination of the conserved key regulator of the gut development from cleavage through larval life, postlarval growth, and regeneration.

BACKGROUND: foxA orthologs are involved in various processes from embryo patterning to regulation of metabolism. Since foxA conserved role in the development of the gut of errant annelids has never been thoroughly studied, we used a candidate gene approach to unravel the molecular profile of the alimentary canal in two closely related nereid worms with a trochophore-type lecithotrophic larva. RESULTS: The character of foxA expression in the two polychaetes was similar but not identical. The genes were successively activated first in blastoporal cells, then in the stomodeum, the midgut, and hindgut primordia, and in the cells of central and peripheral nervous system. Before the start of active feeding of nectochaetes, we observed a short phase of foxA expression in the entire digestive tract. After amputation of posterior segments, foxA expression was established de novo in the new terminal part of the intestine, and then in the developing hindgut and the anus. CONCLUSIONS: We discovered an early marker of endoderm formation previously unknown in errant annelids. Its expression dynamics provided valuable insights into the gut development. Comparative analysis of foxA activity suggests its primary role in gastrulation morphogenesis independently of its type and in midgut and foregut specification. Developmental Dynamics 248:728-743, 2019. © 2018 Wiley Periodicals, Inc.

Measures of effective population size in sea otters reveal special considerations for wide-ranging species.

Conservation genetic techniques and considerations of the evolutionary potential of a species are increasingly being applied to species conservation. For example, effective population size (N e) estimates are useful for determining the conservation status of species, yet accurate estimates of current N e remain difficult to obtain. The effective population size can contribute to setting federal delisting criteria, as was done for the southern sea otter (Enhydra lutris nereis). After being hunted to near extinction during the North Pacific fur trade, the southern sea otter has recovered over part of its former range, but remains at relatively low numbers, making it desirable to obtain accurate and consistent estimates of N e. Although theoretical papers have compared the validity of several methods, comparisons of estimators using empirical data in applied conservation settings are limited. We combined thirteen years of demographic and genetic data from 1,006 sea otters to assess multiple N e estimators, as well as temporal trends in genetic diversity and population genetic structure. Genetic diversity was low and did not increase over time. There was no evidence for distinct genetic units, but some evidence for genetic isolation by distance. In particular, estimates of N e based on demographic data were much larger than genetic estimates when computed for the entire range of the population, but were similar at smaller spatial scales. The discrepancy between estimates at large spatial scales could be driven by cryptic population structure and/or individual differences in reproductive success. We recommend the development of new delisting criteria for the southern sea otter. We advise the use of multiple estimates of N e for other wide-ranging species, species with overlapping generations, or with sex-biased dispersal, as well as the development of improved metrics of genetic assessments of populations.

Chronic exposure to copper and zinc induces DNA damage in the polychaete Alitta virens and the implications for future toxicity of coastal sites.

Copper and zinc are metals that have been traditionally thought of as past contamination legacies. However, their industrial use is still extensive and current applications (e.g. nanoparticles and antifouling paints) have become additional marine environment delivery routes. Determining a pollutant's genotoxicity is an ecotoxicological priority, but in marine benthic systems putative substances responsible for sediment genotoxicity have rarely been identified. Studies that use sediment as the delivery matrix combined with exposures over life-history relevant timescales are also missing for metals. Here we assess copper and zinc's genotoxicity by exposing the ecologically important polychaete Alitta virens to sediment spiked with environmentally relevant concentrations for 9 months. Target bioavailable sediment and subsequent porewater concentrations reflect the global contamination range for coasts, whilst tissue concentrations, although elevated, were comparable with other polychaetes. Survival generally reduced as concentrations increased, but monthly analyses show that growth was not significantly different between treatments. The differential treatment mortality may have enabled the surviving worms in the high concentration treatments to capture more food thus removing any concentration treatment effects for biomass. Using the alkaline comet assay we confirm that both metals via the sediment are genotoxic at concentrations routinely found in coastal regions and this is supported by elevated DNA damage in worms from field sites. However, combined with the growth data it also highlights the tolerance of A. virens to DNA damage. Finally, using long term (decadal) monitoring data we show stable or increasing sediment concentrations of these metals for many areas. This will potentially mean coastal sediment is a significant mutagenic hazard to the benthic community for decades to come. An urgent reappraisal of the current input sources for these 'old pollutants' is, therefore, required.

Interplay between flow and bioturbation enhances metal efflux from low-permeability sediments.

Understanding the interplay effects between processes such as hydrodynamic forcing, sediment resuspension, and bioturbation is key to assessment of contaminated sediments. In the current study, effects of hydrodynamic forcing, sediment resuspension, and bioturbation by the marine polychaete Nereis virens were evaluated both independently and together in a six-month flume experiment. The results show that hydrodynamic forcing without resuspension or worm action slightly enhanced efflux of dissolved Cu to the water column, sediment resuspension released considerable amounts of dissolved Cu, and interactions between hydrodynamics and worm burrowing further enhanced Cu efflux. In non-bioturbated sediments, fine particles were only resuspended to the overlying water under the highest imposed shear stress, 0.58Pa. However, bioturbated sediments were resuspended under all shear stresses tested (0.11-0.58Pa), indicating that bioturbation destabilized the sediment bed. Further, increases in fluid shear following bioturbation caused rapid releases of dissolved Cu to the overlying water within a few hours. Cu efflux under fluid shears of 0.47Pa and 0.58Pa were 360× and 15× greater after the introduction of worms compared with the same flow conditions without their presence. Overall, our results indicate that the release of metals from low-permeability sediments is greatly enhanced by interactions between flow and bioturbation.

Assessing the impacts of bait collection on inter-tidal sediment and the associated macrofaunal and bird communities: The importance of appropriate spatial scales.

Bait collection is a multibillion dollar worldwide activity that is often managed ineffectively. For managers to understand the impacts on protected inter-tidal mudflats and waders at appropriate spatial scales macrofaunal surveys combined with video recordings of birds and bait collectors were undertaken at two UK sites. Dug sediment constituted approximately 8% of the surveyed area at both sites and is less muddy (lower organic content) than undug sediment. This may have significant implications for turbidity. Differences in the macrofaunal community between dug and undug areas if the same shore height is compared as well as changes in the dispersion of the community occurred at one site. Collection also induces a 'temporary loss of habitat' for some birds as bait collector numbers negatively correlate with wader and gull abundance. Bait collection changes the coherence and ecological structure of inter-tidal mudflats as well as directly affecting wading birds. However, as ß diversity increased we suggest that management at appropriate hectare/site scales could maximise biodiversity/function whilst still supporting collection.

A Purified Serine Protease from Nereis virens and Its Impaction of Apoptosis on Human Lung Cancer Cells.

Nereis active protease (NAP) is a novel fibrinolytic active serine protease from the polychaete Nereis virens. In this study, NAP was purified from Nereis virens and the effects of NAP on human lung cancer cells were investigated. Our results indicated that NAP inhibited the proliferation and induced apoptosis of H1299 cells in a time- and dose-dependent manner. The loss of mitochondrial membrane potential, the activation of Bax and cleaved-caspase 3/9, the release of cytochrome C, and the suppression of Bcl-2 and poly-ADP ribose polymerase were observed in NAP-treated H1299 cells by flow cytometry and Western blotting. Moreover, the expression levels of Bax and Bcl-2 mRNA were determined by real-time quantitative polymerase chain reaction and the Bax/Bcl-2 expression ratio was increased in the NAP-treated cell lines. The results indicated that NAP-induced apoptosis may be related to mitochondria mediated apoptosis and occurs through caspase-dependent pathways. Then, the effects of NAP on tumor growth in animal models were observed, where 5 or 10 mg/kg of NAP noticeably reduced tumor volume and weight and increased apoptosis as determined by Western blotting when compared to the negative control group. Therefore, our findings suggest that NAP could be a hopeful anticancer medicine for its propensity to inhibit growth and induce of apoptosis in human lung cancer cells.

Serologic Detection of Subtype-specific Antibodies to Influenza A Viruses in Southern Sea Otters (Enhydra lutris nereis).

There are approximately 3,000 southern sea otters (Enhydra lutris nereis) in the nearshore environment along the California coast, US, and the species is classified as Threatened under the Endangered Species Act. We tested sera from 661 necropsied southern sea otters sampled from 1997 to 2015 to determine overall exposure to influenza A viruses (IAVs) and to identify subtype-specific antibody responses. Using an enzyme-linked immunosorbent assay (ELISA), antibodies to IAV nucleoproteins were detected in 160 (24.2%) otters, with seropositive animals found in every year except 2008. When the ELISA-positive samples were tested by virus microneutralization, antibody responses were detected to avian-origin hemagglutinin subtypes H1, H3, H4, H5, H6, H7, H9, and H11. Strong antibody responses to pandemic H1N1 (pdmH1N1) were also detected, indicating that epizootic transmission of pdmH1N1 occurred among the southern sea otter population after the emergence of this human-origin virus in 2009. We conclude that southern sea otters are susceptible to infection with avian and human-origin IAV and that exposure to a wide array of subtypes likely occurs during a given otter's 10- to 15-yr life span. Important unanswered questions include what effect, if any, IAV infection has on sea otter health, and how these animals become infected in their nearshore environment.

ENDEMIC INFECTION OF STRANDED SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) WITH NOVEL PARVOVIRUS, POLYOMAVIRUS, AND ADENOVIRUS.

Over the past century, the southern sea otter (SSO; Enhydra lutris nereis) population has been slowly recovering from near extinction due to overharvest. The SSO is a threatened subspecies under federal law and a fully protected species under California law, US. Through a multiagency collaborative program, stranded animals are rehabilitated and released, while deceased animals are necropsied and tissues are cryopreserved to facilitate scientific study. Here, we processed archival tissues to enrich particle-associated viral nucleic acids, which we randomly amplified and deeply sequenced to identify viral genomes through sequence similarities. Anelloviruses and endogenous retroviral sequences made up over 50% of observed viral sequences. Polyomavirus, parvovirus, and adenovirus sequences made up most of the remaining reads. We characterized and phylogenetically analyzed the full genome of sea otter polyomavirus 1 and the complete coding sequence of sea otter parvovirus 1 and found that the closest known viruses infect primates and domestic pigs ( Sus scrofa domesticus), respectively. We tested archived tissues from 69 stranded SSO necropsied over 14 yr (2000-13) by PCR. Polyomavirus, parvovirus, and adenovirus infections were detected in 51, 61, and 29% of examined animals, respectively, with no significant increase in frequency over time, suggesting endemic infection. We found that 80% of tested SSO were infected with at least one of the three DNA viruses, whose tissue distribution we determined in 261 tissue samples. Parvovirus DNA was most frequently detected in mesenteric lymph node, polyomavirus DNA in spleen, and adenovirus DNA in multiple tissues (spleen, retropharyngeal and mesenteric lymph node, lung, and liver). This study describes the virome in tissues of a threatened species and shows that stranded SSO are frequently infected with multiple viruses, warranting future research to investigate associations between these infections and observed lesions.

Ion Effect and Metal-Coordinated Cross-Linking for Multiscale Design of Nereis Jaw Inspired Mechanomutable Materials.

The Nvjp-1 protein is a key component in the jaws of Nereis virens, a species of marine worm. It contains over 25 mol % of histidine, which is believed to play a key role in the metal-coordinated cross-linking responsible for the structural stability and exceptional mechanical performance of the worm jaw. Understanding the nanoscale mechanism behind this cross-linking and its pathway in affecting the macroscopic mechanical behavior of the material is crucial to develop bioinspired mechanomutable materials based on Nvjp-1. Here, we use a combination of multiscale modeling and experimental synthesis to understand the behavior of this heterologous-expressed protein from the nano- to the macroscale. We have built a bottom-up molecular-based model, which includes electronic-based density functional theory calculations, atomistic simulation of the nanoscale properties with replica exchange molecular dynamics, and an elastic network model for describing the macroscale behavior at different pHs. This multiscale modeling supports the experimental synthesis of a photo-cross-linked Nvjp-1 hydrogel by proving both the nanoscale mechanisms and mechanical behavior predictions. Our theoretical results agree well with the experimental observations, showing that Nvjp-1 forms a more compact structure in the presence of Zn2+ ions with a suitable pH environment, leading to the formation of more stable intramolecular metal-coordinated cross-links. These metal-coordinated cross-links induce nanoscale aggregation of Nvjp-1, which is responsible for the hydrogel contraction observed in experiments and predicted by the model.

ISOLATION AND CHARACTERIZATION OF A NOVEL MARINE BRUCELLA FROM A SOUTHERN SEA OTTER (ENHYDRA LUTRIS NEREIS), CALIFORNIA, USA.

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.