Shifts in the gut microbiota of sea urchin Diadema antillarum associated with the 2022 disease outbreak.

Introduction: In recent decades, Caribbean coral reefs have lost many vital marine species due to diseases. The well-documented mass mortality event of the long-spined black sea urchin Diadema antillarum in the early 1980s stands out among these collapses. This die-off killed over 90% of D. antillarum changing the reefscape from coral to algal-dominated. Nearly 40 years later, D. antillarum populations have yet to recover. In early 2022, a new mortality event of D. antillarum was reported along the Caribbean, including Puerto Rico. Methods: This study identifies the gut microbiota changes associated with the D. antillarum during this mortality event. It contrasts them with the bacterial composition of gut samples from healthy individuals collected in 2019 by using 16S rRNA sequencing analyses. Results: Notably, the die-off group's core microbiome resembled bacteria commonly found in the human skin and gut, suggesting potential anthropogenic contamination and wastewater pollution as contributing factors to the 2022 dysbiosis. The animals collected in 2022, especially those with signs of disease, lacked keystone taxa normally found in Diadema including Photobacterium and Propionigenium. Discussion: The association between human microbes and disease stages in the long-spined urchin D. antillarum, especially in relation to anthropogenic contamination, highlights a complex interplay between environmental stressors and marine health. While these microbes might not be the direct cause of death in this species of sea urchins, their presence and proliferation can indicate underlying issues, such as immune depletion due to pollution, habitat destruction, or climate change, that ultimately compromise the health of these marine organisms.

A successful method to restore seagrass habitats in coastal areas affected by consecutive natural events.

Background: Seagrass meadows, known for providing essential ecosystem services like supporting fishing, coastline protection from erosion, and acting as carbon sinks to mitigate climate change effects, are facing severe degradation. The current deteriorating state can be attributed to the combination of anthropogenic activities, biological factors (i.e., invasive species), and natural forces (i.e., hurricanes). Indeed, the global seagrass cover is diminishing at an alarming mean rate of 7% annually, jeopardizing the health of these vital ecosystems. However, in the Island Municipality of Culebra, Puerto Rico, losses are occurring at a faster pace. For instance, hurricanes have caused over 10% of cover seagrass losses, and the natural recovery of seagrasses across Culebra's coast has been slow due to the low growth rates of native seagrasses (Thalassia testudinum and Syringodium filiforme) and the invasion of the invasive species Halophila stipulacea. Restoration programs are, thus, necessary to revitalize the native seagrass communities and associated fauna while limiting the spread of the invasive species. Methods: Here, we present the results of a seagrass meadow restoration project carried out in Punta Melones (PTM), Culebra, Puerto Rico, in response to the impact of Hurricanes Irma and María during 2017. The restoration technique used was planting propagation units (PUs), each with an area of 900 cm2 of native seagrasses Thalassia testudinum and Syringodium filiforme, planted at a depth between 3.5 and 4.5 m. A total of 688 PUs were planted between August 2021 and August 2023, and a sub-sample of 88 PUs was monitored between August 2021 and April 2023. Results: PUs showed over 95% of the seagrass survived, with Hurricane Fiona causing most of the mortalities potentially due to PUs burial by sediment movement and uplifting by wave energy. The surface area of the planting units increased by approximately 200% (i.e., 2,459 cm2), while seagrass shoot density increased by 168% (i.e., 126 shoots by PU). Additionally, flowering and fruiting were observed in multiple planting units, indicating 1) that the action taken did not adversely affect the PUs units and 2) that the project was successful in revitalizing seagrass populations. The seagrass restoration project achieved remarkable success, primarily attributed to the substantial volume of each PUs. Likely this high volume played a crucial role in facilitating the connection among roots, shoots, and microfauna while providing a higher number of undamaged and active rhizome meristems and short shoots. These factors collectively contributed to the enhanced growth and survivorship of the PUs, ultimately leading to the favorable outcome observed in the seagrass restoration project.

Uneven demographic consequences of the 2022 disease outbreak for the sea urchin Diadema antillarum in Puerto Rico.

Pervasive epizootic events have had a significant impact on marine invertebrates throughout the Caribbean, leading to severe population declines and consequential ecological implications. One such event was the regional collapse of herbivory, partly caused by the Diadema antillarum mortality event in 1983-84, resulting in a trophic cascade and altering the structure of reef communities. Consequently, there was a notable decrease in coral recruitment and an increase in the coverage of macroalgae. Nearly four decades later, in early 2022, the Caribbean basin experienced another widespread mass mortality event, further reducing the populations of D. antillarum. To assess the effects of this recent mortality event on the current demographics of D. antillarum, we surveyed eight populations along the eastern, northeastern, northern, and northwestern coast of Puerto Rico from May to July 2022, estimating their population density, size distribution, and disease prevalence. Additionally, the study compared these population parameters with data from four sites previously surveyed in 2012 and 2017 to understand the impact of the recent mortality event. The survey conducted in 2022 showed varying population densities at the surveyed reefs. Some populations exhibited mean densities of nearly one individual per square meter, while others had extremely low or no living individuals per square meter. The four populations with the highest density showed no evidence of disease, whereas the four populations with the lowest D. antillarum densities exhibited moderate to high disease prevalence. However, when considering all sites, the estimated disease prevalence remained below 5%. Nevertheless, the comparison with data from 2012 and 2017 indicated that the recent mortality event had a negative impact on D. antillarum demographics at multiple sites, as the densities in 2022 were reduced by 60.19% compared to those from the previous years. However, it is still too early to determine the severity of this new mortality event compared to the 1983-84 mortality event. Therefore, it is imperative to continue monitoring these populations.

CREARE: A Course-Based Undergraduate Research Experience To Study the Responses of the Endangered Coral Acropora cervicornis to a Changing Environment.

There is mounting evidence to support that students who participate in scientific research experiences are more likely to continue on to advanced degrees and careers in science, technology, engineering, and mathematics (STEM). To introduce more students to the benefits of research, we have drawn on an ongoing project aimed at understanding how the Caribbean staghorn coral Acropora cervicornis responds to environmental fluctuations to develop a semester-long course-based undergraduate research experience (CURE), entitled CREARE (Coral Response to Environment Authentic Research Experience). The main mode of instruction in CREARE is through topic modules, and course evaluation is achieved through writing assignments. Students in CREARE perform experiments in the laboratory to measure the abundance of photo-protective proteins in coral tissue from samples collected at different depths and at different times of the year and analyze environmental data using the R programming language. CREARE participants have contributed to the progress of the research project by generating novel data and making improvements to experimental protocols. Furthermore, pre- and post-course assessment of content knowledge revealed that students perform significantly better on a written exam after participating in CREARE, while also displaying appreciable shifts in attitudes towards science in student perception surveys. In addition, through qualitative analysis of focus group interviews, we gathered evidence to suggest that mediating variables that predict students' persistence in science are bolstered through our application of the CURE modality. Overall, CREARE can serve as a model for developing more research-based courses that successfully engage students in scientific research.

Reef-Building Corals as a Tool for Climate Change Research in the Genomics Era.

Coral reef ecosystems are among the most biodiverse habitats in the marine realm. They not only contribute with a plethora of ecosystem services, but they also are beneficial to humankind via nurturing marine fisheries and sustaining recreational activities. We will discuss the biology of coral reefs and their ecophysiology including the complex bacterial microbiota associated with them.

The microbial biosphere of the coral Acropora cervicornis in Northeastern Puerto Rico.

BACKGROUND: Coral reefs are the most biodiverse ecosystems in the marine realm, and they not only contribute a plethora of ecosystem services to other marine organisms, but they also are beneficial to humankind via, for instance, their role as nurseries for commercially important fish species. Corals are considered holobionts (host + symbionts) since they are composed not only of coral polyps, but also algae, other microbial eukaryotes and prokaryotes. In recent years, Caribbean reef corals, including the once-common scleractinian coral Acropora cervicornis, have suffered unprecedented mortality due to climate change-related stressors. Unfortunately, our basic knowledge of the molecular ecophysiology of reef corals, particularly with respect to their complex bacterial microbiota, is currently too poor to project how climate change will affect this species. For instance, we do not know how light influences microbial communities of A. cervicornis, arguably the most endangered of all Caribbean coral species. To this end, we characterized the microbiota of A. cervicornis inhabiting water depths with different light regimes. METHODS: Six A. cervicornis fragments from different individuals were collected at two different depths (three at 1.5 m and three at 11 m) from a reef 3.2 km off the northeastern coast of Puerto Rico. We characterized the microbial communities by sequencing the 16S rRNA gene region V4 with the Illumina platform. RESULTS: A total of 173,137 good-quality sequences were binned into 803 OTUs with a 97% similarity. We uncovered eight bacterial phyla at both depths with a dominance of 725 Rickettsiales OTUs (Proteobacteria). A fewer number (38) of low dominance OTUs varied by depth and taxa enriched in shallow water corals included Proteobacteria (e.g. Rhodobacteraceae and Serratia) and Firmicutes (Streptococcus). Those enriched in deeper water corals featured different Proteobacterial taxa (Campylobacterales and Bradyrhizobium) and Firmicutes (Lactobacillus). DISCUSSION: Our results confirm that the microbiota of A. cervicornis inhabiting the northeastern region of Puerto Rico is dominated by a Rickettsiales-like bacterium and that there are significant changes in less dominant taxa at different water depths. These changes in less dominant taxa may potentially impact the coral's physiology, particularly with respect to its ability to respond to future increases in temperature and CO2.

Comparison of chemical compounds associated with sclerites from healthy and diseased sea fan corals (Gorgonia ventalina).

BACKGROUND: The roles of gorgonian sclerites as structural components and predator deterrents have been widely studied. Yet their role as barriers against microbes has only recently been investigated, and even less is known about the diversity and roles of the chemical compounds associated with sclerites. METHODS: Here, we examine the semi-volatile organic compound fraction (SVOCs) associated with sclerites from healthy and diseased Gorgonia ventalina sea fan corals to understand their possible role as a stress response or in defense of infection. We also measured the oxidative potential of compounds from diseased and healthy G. ventalina colonies. RESULTS: The results showed that sclerites harbor a great diversity of SVOCs. Overall, 70 compounds were identified, the majority of which are novel with unknown biological roles. The majority of SVOCs identified exhibit multiple immune-related roles including antimicrobial and radical scavenging functions. The free radical activity assays further confirmed the anti-oxidative potential of some these compounds. The anti-oxidative activity was, nonetheless, similar across sclerites regardless of the health condition of the colony, although sclerites from diseased sea fans display slightly higher anti-oxidative activity than the healthy ones. DISCUSSION: Sclerites harbor great SVOCs diversity, the majority of which are novel to sea fans or any other corals. Yet the scientific literature consulted showed that the roles of compounds found in sclerites vary from antioxidant to antimicrobial compounds. However, this study fell short in determine the origin of the SVOCs identified, undermining our capacity to determine the biological roles of the SVOCs on sclerites and sea fans.

Analysis of human mitochondrial DNA sequences from fecally polluted environmental waters as a tool to study population diversity.

Mitochondrial signature sequences have frequently been used to study human population diversity around the world. Traditionally, this requires obtaining samples directly from individuals which is cumbersome, time consuming and limited to the number of individuals that participated in these types of surveys. Here, we used environmental DNA extracts to determine the presence and sequence variability of human mitochondrial sequences as a means to study the diversity of populations inhabiting in areas nearby a tropical watershed impacted with human fecal pollution. We used high-throughput sequencing (Illumina) and barcoding to obtain thousands of sequences from the mitochondrial hypervariable region 2 (HVR2) and determined the different haplotypes present in 10 different water samples. Sequence analyses indicated a total of 19 distinct variants with frequency greater than 5%. The HVR2 sequences were associated with haplogroups of West Eurasian (57.6%), Sub-Saharan African (23.9%), and American Indian (11%) ancestry. This was in relative accordance with population census data from the watershed sites. The results from this study demonstrates the potential value of mitochondrial sequence data retrieved from fecally impacted environmental waters to study the population diversity of local municipalities. This environmental DNA approach may also have other public health implications such as tracking background levels of human mitochondrial genes associated with diseases. It may be possible to expand this approach to other animal species inhabiting or using natural water systems.

The role of coral colony health state in the recovery of lesions.

Coral disease literature has focused, for the most part, on the etiology of the more than 35 coral afflictions currently described. Much less understood are the factors that underpin the capacity of corals to regenerate lesions, including the role of colony health. This lack of knowledge with respect to the factors that influence tissue regeneration significantly limits our understanding of the impact of diseases at the colony, population, and community level. In this study, we experimentally compared tissue regeneration capacity of diseased versus healthy fragments of Gorgonia ventalina colonies at 5 m and 12 m of depth. We found that the initial health state of colonies (i.e., diseased or healthy) had a significant effect on tissue regeneration (healing). All healthy fragments exhibited full recovery regardless of depth treatment, while diseased fragments did not. Our results suggest that being diseased or healthy has a significant effect on the capacity of a sea fan colony to repair tissue, but that environmental factors associated with changes in depth, such as temperature and light, do not. We conclude that disease doesn't just compromise vital functions such as growth and reproduction in corals but also compromises their capacity to regenerate tissue and heal lesions.

Immune response to a pathogen in corals.

The sea fan coral (Gorgonia ventalina), one of the most abundant gorgonians in the tropical and subtropical Atlantic waters, have suffered several diseases that have diminished its abundance throughout their range. In this study, we present a model that analyzes the capacity of G. ventalina to eradicate a micro-pathogen under three immune responses: strong, moderate, and very weak. The model assumes that: (1) polyps are the main unit of the coral; (2) the population of polyps is homogeneously distributed; and (3) the immune system is activated by a signal. When an endosymbiont exceeds a density threshold, it becomes pathogenic, increasing polyp mortality. As a consequence, the colony emits a signal to its stem cells to differentiate into phagocytic and humoral cells, both of which combat the pathogen. Given a strong immune response, the pathogen is rapidly eradicated by the immune cells, and the coral polyp population returns to an equilibrium state. With a moderate immune response, polyps and pathogen coexist, but the maximum capacity of polyp density is never reached. An immunologically compromised colony offering a weak immune response is unable to stop pathogen growth, and the colony dies. This analysis suggests an alternative explanation for the spatial and temporal variability in disease incidence and mortality, which is based on the strength of the immune system of hosts rather than the virulence of the pathogen.

Determining hot spots of fecal contamination in a tropical watershed by combining land-use information and meteorological data with source-specific assays.

The objective of this study was to combine knowledge of environmental, topographical, meteorological, and anthropologic factors in the Río Grande de Arecibo (RGA) watershed in Puerto Rico with information provided by microbial source tracking (MST) to map hot spots (i.e., likely sources) of fecal contamination. Water samples were tested for the presence of human and bovine fecal contamination in addition to fecal indicator bacteria and correlated against several land uses and the density of septic tanks, sewers, and latrines. Specifically, human sources were positively correlated with developed (r = 0.68), barren land uses (r = 0.84), density of septic tanks (r = 0.78), slope (r = 0.63), and the proximity to wastewater treatment plants (WWTPs) (r = 0.82). Agricultural land, the number of upstream National Pollution Discharge Elimination System (NPDES) facilities, and density of latrines were positively associated with the bovine marker (r = 0.71; r = 0.74; and r = 0.68, respectively). Using this information, we provided a hot spot map, which shows areas that should be closely monitored for fecal contamination in the RGA watershed. The results indicated that additional bovine assays are needed in tropical regions. We concluded that meteorological, topographical, anthropogenic, and land cover data are needed to evaluate and verify the performance of MST assays and, therefore, to identify important sources of fecal contamination in environmental waters.

Tracking the primary sources of fecal pollution in a tropical watershed in a one-year study.

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.

Development of quantitative PCR assays targeting the 16S rRNA genes of Enterococcus spp. and their application to the identification of enterococcus species in environmental samples.

The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

Microbial quality of tropical inland waters and effects of rainfall events.

Novel markers of fecal pollution in tropical waters are needed since conventional methods recommended for other geographical regions may not apply. To address this, the prevalence of thermotolerant coliforms, enterococci, coliphages, and enterophages was determined by culture methods across a watershed. Additionally, human-, chicken-, and cattle-specific PCR assays were used to identify potential fecal pollution sources in this watershed. An enterococcus quantitative PCR (qPCR) assay was tested and correlated with culture methods at three sites since water quality guidelines could incorporate this technique as a rapid detection method. Various rainfall events reported before sample collection at three sites were considered in the data analyses. Thermotolerant coliforms, enterococci, coliphages, and enterophages were detected across the watershed. Human-specific Bacteroides bacteria, unlike the cattle- and chicken-specific bacteria, were detected mostly at sites with the corresponding fecal impact. Enterococci were detected by qPCR as well, but positive correlations with the culture method were noted at two sites, suggesting that either technique could be used. However, no positive correlations were noted for an inland lake tested, suggesting that qPCR may not be suitable for all water bodies. Concentrations of thermotolerant coliforms and bacteriophages were consistently lower after rainfall events, pointing to a possible dilution effect. Rainfall positively correlated with enterococci detected by culturing and qPCR, but this was not the case for the inland lake. The toolbox of methods and correlations presented here could be potentially applied to assess the microbial quality of various water types.

Comparison of gull feces-specific assays targeting the 16S rRNA genes of Catellicoccus marimammalium and Streptococcus spp.

Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.