The Seasonal Microbial Ecology of Plankton and Plankton-Associated Vibrio parahaemolyticus in the Northeast United States.

Microbial ecology studies have proven to be important resources for improving infectious disease response and outbreak prevention. Vibrio parahaemolyticus is an ongoing source of shellfish-borne food illness in the Northeast United States, and there is keen interest in understanding the environmental conditions that coincide with V. parahaemolyticus disease risk, in order to aid harvest management and prevent further illness. Zooplankton and chitinous phytoplankton are associated with V. parahaemolyticus dynamics elsewhere; however, this relationship is undetermined for the Great Bay estuary (GBE), an important emerging shellfish growing region in the Northeast United States. A comprehensive evaluation of the microbial ecology of V. parahaemolyticus associated with plankton was conducted in the GBE using 3 years of data regarding plankton community, nutrient concentration, water quality, and V. parahaemolyticus concentration in plankton. The concentrations of V. parahaemolyticus associated with plankton were highly seasonal, and the highest concentrations of V. parahaemolyticus cultured from zooplankton occurred approximately 1 month before the highest concentrations of V. parahaemolyticus from phytoplankton. The two V. parahaemolyticus peaks corresponded with different water quality variables and a few highly seasonal plankton taxa. Importantly, V. parahaemolyticus concentrations and plankton community dynamics were poorly associated with nutrient concentrations and chlorophyll a, commonly applied proxy variables for assessing ecological health risks and human health risks from harmful plankton and V. parahaemolyticus elsewhere. Together, these statistical associations (or lack thereof) provide valuable insights to characterize the plankton-V. parahaemolyticus dynamic and inform approaches for understanding the potential contribution of plankton to human health risks from V. parahaemolyticus for the Northeast United States. IMPORTANCE The Vibrio-plankton interaction is a focal relationship in Vibrio disease research; however, little is known about this dynamic in the Northeast United States, where V. parahaemolyticus is an established public health issue. We integrated phototactic plankton separation with seasonality analysis to determine the dynamics of the plankton community, water quality, and V. parahaemolyticus concentrations. Distinct bimodal peaks in the seasonal timing of V. parahaemolyticus abundance from phyto- versus zooplankton and differing associations with water quality variables and plankton taxa indicate that monitoring and forecasting approaches should consider the source of exposure when designing predictive methods for V. parahaemolyticus. Helicotheca tamensis has not been previously reported in the GBE. Its detection during this study provides evidence of the changes occurring in the ecology of regional estuaries and potential mechanisms for changes in V. parahaemolyticus populations. The Vibrio monitoring approaches can be translated to aid other areas facing similar public health challenges.

Parallel Evolution of Two Clades of an Atlantic-Endemic Pathogenic Lineage of Vibrio parahaemolyticus by Independent Acquisition of Related Pathogenicity Islands.

Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States. This change coincided with a bacterial population shift toward human-pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43, and ST636) to nearshore areas off the Atlantic coast of the Northeast United States. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human-pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins or contained a chromosome I-inserted island we call VPaIß that encodes a type 3 secretion system (T3SS2ß) typical of Trh hemolysin producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaIγ that encodes both tdh and trh and that was inserted in chromosome II. VPaIγ was derived from VPaIß but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaIγ-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages that collectively cause 67% of infections in the Northeast United States as of 2016.IMPORTANCE The availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast U.S. coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaIγ along the U.S. Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease.

Use of Whole-Genome Phylogeny and Comparisons for Development of a Multiplex PCR Assay To Identify Sequence Type 36 Vibrio parahaemolyticus.

Vibrio parahaemolyticus sequence type 36 (ST36) strains that are native to the Pacific Ocean have recently caused multistate outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole-genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern U.S. sources, were used to identify diagnostic loci, one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present in only one clade of closely related strains of ST36, ST59, and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two nonclade isolates and cps in four. Based on the distribution of these loci in sequenced genomes, prp identified clade strains with >99% accuracy, but the addition of one more locus increased accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus and determines the presence of both the tdh and trh hemolysin-encoding genes, which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a 4-year period in three northeastern U.S. states and 87 environmental isolates revealed that the prp and cps amplicons were detected only in clinical isolates identified as belonging to the ST36 clade and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections.

Spontaneous phenotypic suppression of GacA-defective Vibrio fischeri is achieved via mutation of csrA and ihfA.

BACKGROUND: Symbiosis defective GacA-mutant derivatives of Vibrio fischeri are growth impaired thereby creating a selective advantage for growth-enhanced spontaneous suppressors. Suppressors were isolated and characterized for effects of the mutations on gacA-mutant defects of growth, siderophore activity and luminescence. The mutations were identified by targeted and whole genome sequencing. RESULTS: Most mutations that restored multiple phenotypes were non-null mutations that mapped to conserved domains in or altered expression of CsrA, a post-transcriptional regulator that mediates GacA effects in a number of bacterial species. These represent an array of unique mutations compared to those that have been described previously. Different substitutions at the same amino acid residue were identified allowing comparisons of effects such as at the R6 residue, which conferred relative differences in luminescence and siderophore levels. The screen revealed residues not previously identified as critical for function including a single native alanine. Most csrA mutations enhanced luminescence more than siderophore activity, which was especially evident for mutations predicted to reduce the amount of CsrA. Although CsrA mutations compensate for many known GacA mutant defects, not all CsrA suppressors restore symbiotic colonization. Phenotypes of a suppressor allele of ihfA that encodes one subunit of the integration host factor (IHF) heteroduplex indicated the protein represses siderophore and activates luminescence in a GacA-independent manner. CONCLUSIONS: In addition to its established role in regulation of central metabolism, the CsrA regulator represses luminescence and siderophore as an intermediate of the GacA regulatory hierachy. Siderophore regulation was less sensitive to stoichiometry of CsrA consistent with higher affinity for the targets of this trait. The lack of CsrA null-mutant recovery implied these mutations do not enhance fitness of gacA mutants and alluded to this gene being conditionally essential. This study also suggests a role for IHF in the GacA-CsrB-CsrA regulatory cascade by potentially assisting with the binding of repressors of siderohphore and activators of luminescence. As many phosphorelay proteins reduce fitness when mutated, the documented instability used in this screen also highlights a potentially universal and underappreciated problem that, if not identified and strategically avoided, could introduce confounding variability during experimental study of these regulatory pathways.

Complete genome sequence of Vibrio fischeri strain H905, a planktonic isolate among squid symbiotic congeners.

Here we describe the genome sequence of Vibrio (Aliivibrio) fischeri H905, a non-symbiotic isolate from Kaneohe Bay, Hawaii. Despite its close phylogenetic relationship to squid symbiont strains, H905 is not adept at colonization. Its genome serves as a valuable comparator, illustrating the complex evolutionary dynamics within V. fischeri clades.

Deletion of luxI increases luminescence of Vibrio fischeri.

Bioluminescence in Vibrio fischeri is regulated by a quorum-dependent signaling system composed of LuxI and LuxR. LuxI generates N-3-oxohexanoyl homoserine lactone (3OC6-HSL), which triggers LuxR to activate transcription of the luxICDABEG operon responsible for bioluminescence. Surprisingly, a ∆luxI mutant produced more bioluminescence than the wild type in culture. In contrast, a 4 bp duplication within luxI, resulting in a frameshift mutation and null allele, decreased luminescence tenfold. A second signaling system encoded by ainSR affects bioluminescence by increasing levels of LuxR, via the transcriptional activator LitR, and the N-octanoyl homoserine lactone (C8-HSL) signal produced by AinS is considered only a weak activator of LuxR. However, ainS is required for the bright phenotype of the ∆luxI mutant in culture. When 3OC6-HSL was provided either in the medium or by expression of luxI in trans, all cultures were brighter, but the ∆luxI mutant remained significantly brighter than the luxI frameshift mutant. Taken together, these data suggest that the enhanced bioluminescence due to the LuxI product 3OC6-HSL counteracts a negative cis-acting regulatory element within the luxI gene and that when luxI is absent the C8-HSL signal is sufficient to induce luminescence. IMPORTANCE: The regulation of bioluminescence by Vibrio fischeri is a textbook example of bacterial quorum-dependent pheromone signaling. The canonical regulatory model is that an autoinducer pheromone produced by LuxI accumulates as cells achieve a high density, and this LuxI-generated signal stimulates LuxR to activate transcription of the lux operon that underlies bioluminescence. The surprising observation that LuxI is dispensable for inducing bioluminescence forces a re-evaluation of the role of luxI. More broadly, the results underscore the potential deceptiveness of complex regulatory circuits, particularly those in which bacteria produce multiple related signaling molecules.

Complete genome sequence of Vibrio parahaemolyticus ST36 strain MAVP-26, a clinical isolate from an oyster-borne human gastric infection.

A Pacific native lineage of Vibrio parahaemolyticus ST36 serotype O4:K12 was introduced into the Atlantic, which increased local source illnesses. To identify genetic determinants of virulence and ecological resiliency and track their transfer into endemic populations, we constructed a complete genome of a 2013 Atlantic-traced clinical isolate by hybrid assembly.

Gut dysbiosis was inevitable, but tolerance was not: temporal responses of the murine microbiota that maintain its capacity for butyrate production correlate with sustained antinociception to chronic voluntary morphine.

The therapeutic benefits of opioids are compromised by the development of analgesic tolerance, which necessitates higher dosing for pain management thereby increasing the liability for dependence and addiction. Rodent models indicate opposing roles of the gut microbiota in tolerance: morphine-induced gut dysbiosis exacerbates tolerance, whereas probiotics ameliorate tolerance. Not all individuals develop tolerance which could be influenced by differences in microbiota, and yet no study has capitalized upon this natural variation to identify specific features linked to tolerance. We leveraged this natural variation in a murine model of voluntary oral morphine self-administration to elucidate the mechanisms by which microbiota influences tolerance. Although all mice shared similar and predictive morphine-driven microbiota changes that largely masked informative associations with variability in tolerance, our high-resolution temporal analyses revealed a divergence in the progression of dysbiosis that best explained differences in the development in tolerance. Mice that did not develop tolerance also maintained a higher abundance of taxa capable of producing the short-chain fatty acid (SCFA) butyrate, known to bolster intestinal barriers, suppress inflammation, and promote neuronal homeostasis. Furthermore, dietary butyrate supplementation significantly reduced the development of tolerance. These findings could inform immediate therapies to extend the analgesic efficacy of opioids.

Inoviridae prophage and bacterial host dynamics during diversification, succession, and Atlantic invasion of Pacific-native Vibrio parahaemolyticus.

IMPORTANCE: An understanding of the processes that contribute to the emergence of pathogens from environmental reservoirs is critical as changing climate precipitates pathogen evolution and population expansion. Phylogeographic analysis of Vibrio parahaemolyticus hosts combined with the analysis of their Inoviridae phage resolved ambiguities of diversification dynamics which preceded successful Atlantic invasion by the epidemiologically predominant ST36 lineage. It has been established experimentally that filamentous phage can limit host recombination, but here, we show that phage loss is linked to rapid bacterial host diversification during epidemic spread in natural ecosystems alluding to a potential role for ubiquitous inoviruses in the adaptability of pathogens. This work paves the way for functional analyses to define the contribution of inoviruses in the evolutionary dynamics of environmentally transmitted pathogens.

Deletion of arrestin-3 does not improve compulsive drug-seeking behavior in a longitudinal paradigm of oral morphine self-administration.

Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the µ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the µ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the µ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. To examine this question, we utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the µ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the µ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We created a quantitative method to define compulsivity in drug-seeking and found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Our data suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability.

Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization.

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.

Influence of seasonality on the genetic diversity of Vibrio parahaemolyticus in New Hampshire shellfish waters as determined by multilocus sequence analysis.

Risk of gastric infection with Vibrio parahaemolyticus increases with favorable environmental conditions and population shifts that increase prevalence of infective strains. Genetic analysis of New Hampshire strains revealed a unique population with some isolates similar to outbreak-causing strains and high-level diversity that increased as waters warmed.

Coastal water bacteriophages infect various sets of Vibrio parahaemolyticus sequence types.

Introduction: Gastrointestinal illnesses associated with the consumption of shellfish contaminated with Vibrio parahaemolyticus have a negative impact on the shellfish industry due to recalls and loss of consumer confidence in products. This bacterial pathogen is very diverse and specific sequence types (STs), ST631 and ST36, have emerged as prevalent causes of Vibrio foodborne disease outbreaks in the US, though other STs have been implicated in sporadic cases. We investigated whether bacteriophages could be used as a proxy to monitor for the presence of distinct V. parahaemolyticus STs in coastal waters. Methods: For this purpose, bacteriophages infecting V. parahaemolyticus were isolated from water samples collected on the Northeast Atlantic coast. The isolated phages were tested against a collection of 29 V. parahaemolyticus isolates representing 18 STs, including six clonal complexes (CC). Four distinct phages were identified based on their ability to infect different sets of V. parahaemolyticus isolates. Results and Discussion: Overall, the 29 bacterial isolates segregated into one of eight patterns of susceptibility, ranging from resistance to all four phages to susceptibility to any number of phages. STs represented by more than one bacterial isolate segregated within the same pattern of susceptibility except for one V. parahaemolyticus ST. Other patterns of susceptibility included exclusively clinical isolates represented by distinct STs. Overall, this study suggests that phages populating coastal waters could be exploited to monitor for the presence of V. parahaemolyticus STs known to cause foodborne outbreaks.

Ecology and genetic structure of a northern temperate Vibrio cholerae population related to toxigenic isolates.

Although Vibrio cholerae is an important human pathogen, little is known about its populations in regions where the organism is endemic but where cholera disease is rare. A total of 31 independent isolates confirmed as V. cholerae were collected from water, sediment, and oysters in 2008 and 2009 from the Great Bay Estuary (GBE) in New Hampshire, a location where the organism has never been detected. Environmental analyses suggested that abundance correlates most strongly with rainfall events, as determined from data averaged over several days prior to collection. Phenotyping, genotyping, and multilocus sequence analysis (MLSA) revealed a highly diverse endemic population, with clones recurring in both years. Certain isolates were closely related to toxigenic O1 strains, yet no virulence genes were detected. Multiple statistical tests revealed evidence of recombination among strains that contributed to allelic diversity equally as mutation. This relatively isolated population discovered on the northern limit of detection for V. cholerae can serve as a model of natural population dynamics that augments predictive models for disease emergence.

Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1.

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

Comparison of the pathogenic potentials of environmental and clinical vibrio parahaemolyticus strains indicates a role for temperature regulation in virulence.

Although the presence of pathogenic Vibrio spp. in estuarine environments of northern New England has been known for some time (C. H. Bartley and L. W. Slanetz, Appl. Microbiol. 21: 965-966, 1971, and K. R. O'Neil, S. H. Jones, and D. J. Grimes, FEMS Microbiol. Lett. 60:163-167, 1990), their virulence and the relative threat they may pose to human health has yet to be evaluated. In this study, the virulence potential of 33 Vibrio parahaemolyticus isolates collected from the Great Bay Estuary of New Hampshire was assessed in comparison to that of clinical strains. The environmental isolates lack thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by tdh and trh, respectively. Though not hemolytic, they do possess putative virulence factors, such type III secretion system 1, and are highly cytotoxic to human gastrointestinal cells. The expression of known and putative virulence-associated traits, including hemolysin, protease, motility, biofilm formation, and cytotoxicity, by clinical reference isolates correlated with increased temperature from 28°C to 37°C. In contrast, the environmental isolates did not induce their putative virulence-associated traits in response to a temperature of 37°C. We further identified a significant correlation between hemolytic activity and growth phase among clinical strains, whereby hemolysin production decreases with increasing cell density. The introduction of a tdh::gfp promoter fusion into the environmental strains revealed that they regulate this virulence-associated gene appropriately in response to temperature, indicating that their existing regulatory mechanisms are primed to manage newly acquired virulence genes.

Stress tolerance and environmental fitness of Pseudomonas fluorescens A506, which has a mutation in RpoS.

Establishment of suppressive populations of bacterial biological control agents on aerial plant surfaces is a critical phase in biologically based management of floral diseases. Periodically, biocontrol agents encounter inhospitable conditions for growth on plants; consequently, tolerance of environmental stresses may contribute to their fitness. In many gram-negative bacteria, including strains of Pseudomonas spp., the capacity to survive environmental stresses is influenced by the stationary phase sigma factor RpoS. This study focused on the role of RpoS in stress response and epiphytic fitness of Pseudomonas fluorescens A506, a well-studied bacterial biological control agent. We detected a frameshift mutation in the rpoS of A506 and demonstrated that the mutation resulted in a truncated, nonfunctional RpoS. Using site-directed mutagenesis, we deleted a nucleotide from rpoS, which then encoded a full-length, functional RpoS. We compared the stress response and epiphytic fitness of A506 with derivative strains having the functional full-length RpoS or a disrupted, nonfunctional RpoS. RpoS had little effect on stress response of A506 and no consistent influence on epiphytic population size of A506 on pear or apple leaves or flowers. Although the capacity of strain A506 to withstand exposure to environmental stresses was similar to that of other fluorescent pseudomonads, this capacity was largely independent of rpoS.

Forecasting Seasonal Vibrio parahaemolyticus Concentrations in New England Shellfish.

Seafood-borne Vibrio parahaemolyticus illness is a global public health issue facing resource managers and the seafood industry. The recent increase in shellfish-borne illnesses in the Northeast United States has resulted in the application of intensive management practices based on a limited understanding of when and where risks are present. We aim to determine the contribution of factors that affect V. parahaemolyticus concentrations in oysters (Crassostrea virginica) using ten years of surveillance data for environmental and climate conditions in the Great Bay Estuary of New Hampshire from 2007 to 2016. A time series analysis was applied to analyze V. parahaemolyticus concentrations and local environmental predictors and develop predictive models. Whereas many environmental variables correlated with V. parahaemolyticus concentrations, only a few retained significance in capturing trends, seasonality and data variability. The optimal predictive model contained water temperature and pH, photoperiod, and the calendar day of study. The model enabled relatively accurate seasonality-based prediction of V. parahaemolyticus concentrations for 2014-2016 based on the 2007-2013 dataset and captured the increasing trend in extreme values of V. parahaemolyticus concentrations. The developed method enables the informative tracking of V. parahaemolyticus concentrations in coastal ecosystems and presents a useful platform for developing area-specific risk forecasting models.