Emerging infectious and chronic non-infectious diseases in wildlife are increasing in frequency and severity, largely due to human impacts on natural environments. Human activities can compound the effects of disease by altering the biotic and abiotic environment, disseminating pathogens to new localities and populations, increasing domestic host densities, and introducing chemical pollutants that cumulatively compromise immune function in wild species.

Systematic reviews of the scientific literature show increases in marine disease outbreaks in several taxa since the 1970’s including sea turtles, mammals, corals, echinoderms, and molluscs (Ward and Lafferty 2004; Simeone et al 2015) and several groups of aquatic plants (Egan et al. 2014). Major diseases in wildlife can have profound ecological consequences when foundational species (e.g. sea stars) are affected, and in some cases, can even drive extinction. Most documented marine disease outbreaks are associated with compounding factors including climate change, pollutants (nutrients, toxics and noise), aquaculture, invasive species, and habitat loss. Diseases in marine ecosystems pose a particular challenge due to several factors: lack of adequate monitoring for early detection and management, a gap in baseline data for current biodiversity and microbial assemblages in healthy states, the extent of exotic species introductions, and the sheer vastness of the ocean, which makes it particularly subject to both localized and global threats.

Aquatic Plants and Macroalgae

Disease in aquatic plants and algae are of great concern given the habitat-forming role of such organisms in marine ecosystems. Changing ocean conditions due to climate appears to be a major driver of marine disease, as changing climatic conditions can induce higher physiological stress, thereby increasing host susceptibility or altering pathogen dynamics such as growth rates and virulence. Identifying pathogens and linking pathogen presence and growth to environmental variables should be a priority in research. eDNA-based monitoring and surveillance should be established for a variety of marine aquatic plants and macroalgae.

Kelps

Macroalgae are the primary habitat-forming plants along temperate coastlines and play critical roles in food webs. Kelp forests are disappearing from major sections of temperate coastline around the world, and climate induced disease is increasingly recognized as one potential driver of population declines (Egan et al., 2013). Bleaching is a common phenotype of potentially diseased states that leads to reduced fitness. A single-strand DNA virus has been identified as a potential causative pathogen for bleaching in Ecklonia radiata along the Australian coast (Beattie et al., 2018), although in several instances, disease appears to be the outcome of not a single pathogen but of distinct combinations of microbial organisms (Egan et al., 2013; Wahl et al., 2015; Kumar et al., 2016). In general, however, disease conditions in kelp are associated with climatic variables (Campbell et al., 2011), suggesting that climate change will continue to exacerbate disease issues in kelp.

Seagrasses and Eelgrasses

Similar to kelp forests, seagrasses have important roles in coastal ecosystems that are increasingly compromised by anthropogenic threats (Orth et al., 2006). Wasting disease, caused by a slime mold in the genus Labyrinthula, is an increasing threat to seagrass species worldwide that may operate in tandem with other major threats to drive species extinction (Orth et al., 2006). For example, Labyrinthula zosterae has been shown to be more highly virulent at warmer temperatures (Eisenlord et al., 2017).

Marine Invertebrates

Disease is an increasing threat to corals, molluscs, sponges, and echinoderms. Many affected species play critical roles in ecosystem function which can have ecologic and economic consequences following disease outbreaks. Climate change is again likely causing increased disease occurrence in marine invertebrates, suggesting that disease impacts will likely continue to increase without intervention. Genomic research should aim to uncover mechanisms of high thermal and low pH tolerance, with the potential for gene editing or pharmaceutical approaches to conserve vulnerable populations.

Corals

As discussed at length earlier in this chapter, coral disease is a pervasive conservation issue, with several studies supporting the hypothesis that coral disease outbreaks are increasing in frequency and prevalence (Sutherland et al., 2004; Ward and Lafferty 2004; Willis et al., 2004). A large body of research is dedicated to characterizing microbial communities and pathogens associated with diseased states using molecular techniques (Littman et al., 2011; Hadaidi et al., 2018). Coral bleaching is associated with a variety of anthropogenic stressors including thermal stress due to climate change (Maynard et al., 2015), sedimentation plumes caused by dredging (Pollock et al., 2014), nutrient enrichment (Vega et al., 2014), and pollution (Lamb et al., 2018). Coral colonies exposed to temperature stress showed variable transcriptomic responses in a subset of genes associated with resistance to bleaching, which largely operate within the unfolded protein response (Traylor-Knowles et al 2017).

Sea Stars

Sea star wasting disease has caused mass mortality events in a top predator of intertidal environments across the Pacific Northwest and California coast. These top predators play a critical role in intertidal ecosystems by controlling foraging pressure on kelp (Schielbelhut et al., 2018). Higher temperature has been demonstrated in laboratory settings to increase susceptibility to this disease (Bates et al., 2009; Kohl et al., 2016). While the disease has been shown to be infectious, a causative pathogen has yet to be identified, although a densovirus is a strong candidate (Hewson et al., 2014). Genomic approaches targeting transcriptome profiling of diseased and resistant individuals are underway (Dawson Lab, UC Merced-personal communication). Further work is needed to identify the causative pathogen(s) and environmental causes before intervention can be considered.

Molluscs

Molluscs provide critical ecosystem services through filter feeding, essentially clearing the water of biotic and abiotic contaminants. As such, they can accumulate very high pathogen and toxin loads and are susceptible to a wide range of diseases. Several such diseases, including Dermo, MSX, Marteliliosis, and OsHV-1 herpes virus, are associated with outbreaks with warmer temperature (Guo and Ford, 2016), suggesting that thermal stress due to climate change may increase mollusc disease occurrence in the future.

Sponges

Disease in sponges are being increasingly recognized and are geographically widespread, with high rates of mortality affecting both ecological and economic value of sponges (Luter and Webster 2017). Specific pathogens vary widely but disease is most frequently associated with bacterial agents, fungal species and general shifts in the cyanobacterial communities (Luter and Webster 2017). Elevated ocean temperatures may play an important role in disease outbreaks in sponges (Cerrano et al., 2000; Luter and Webster, 2017) but research is limited.

Marine Vertebrates

Sea turtles and sea mammals are highly vulnerable to demographic impacts of disease, as they are generally very long lived and have slow reproductive rates. Conserved evolution of vertebrate immune systems makes marine vertebrates an ideal system to develop precision medicine techniques for wildlife, as immune proteins/pathways are well characterized for human and mice, and bioinformatics can be used to identify potential antigen and host immune cell receptor compatibility.

Fishes

Although reports of disease occurrence in fishes have not increased in the literature (Ward and Lafferty, 2014), this is possibly due to poor monitoring and insufficient baseline data.  Aquaculture potentially intensifies disease occurrence and transmission in wild populations, as well as introduces novel pathogens into wild environments. In a list of 67 marine diseases that have direct economic impact, 49 percent were diseases of fishes, including viral (22 percent), bacterial (45 percent), protist (9 percent), and metazoan (24 percent) pathogens and parasites (Lafferty et al., 2015).

Sea Turtles

Of the seven species of sea turtle, five are currently listed on the US Endangered Species Act and the remaining two are known to be declining in numbers across at least some part of their range. In addition, five of these species are known to be affected by fibropapillomatosis, a tumor-forming disease potentially associated with a herpes virus chHV5 (Lawrance et al., 2018). Transcriptome analysis showed shared genomic drivers with certain human cancers, which has led to advances in treatments and novel therapies following surgery on turtles afflicted by orbital tumors (Duffy et al., 2018).

Mammals

A systematic review of disease-related publications in marine mammals (Simeone et al. 2015) showed 65 percent of published cases from 1972-2012 occurred in California, with the peak number of cases occurring between 1998-2004. While this may reflect a significant reporting bias due to the presence of two major Marine Mammal rescue centers in the state that serve as major sources of disease data, such patterns may also reflect heavy coastal burden due to high human population densities across the California coast. The majority of pathogens were bacterial or caused by biotoxicosis associated with algal blooms. Fungal, viral, and protozoal disease made up a smaller proportion of reported cases, but all are increasing in frequency. Interestingly, there are broad scale geographic patterns in marine mammal disease cases. For example, protozoal diseases appear to be increasing, particularly in otter populations in California and the Pacific Northwest; while viral infections appear to be increasingly more common on the Atlantic coast (e.g. morbillivirus in bottlenose dolphins). Read More

The technology era has introduced a wealth of tools to enable monitoring of wildlife through citizen science efforts (i.e. iNaturalist; CALeDNA) and centralized reporting. Some examples exist of citizen-based monitoring in oceans. For example, Ocean Sanctuaries allows non-researchers to upload photos to document wild species, enhancing baseline measures to monitor changing ocean conditions. Similarly, the NOAA sponsored Marine Debris Monitoring and Assessment Project is a citizen science initiative that engages partner organizations and volunteers nationwide in completing shoreline marine debris surveys. Cumulative effects and impacts to marine ecosystems, fish and wildlife are hard to track and monitor but these new efforts are a good step in attempting to track marine disease.

Centralized databases have been established in a variety of countries such as Canada and Australia to monitor and report disease incidence in wildlife. In the United States, the USGS has collated data for all wildlife health incidence reporting and established a framework to report and query wildlife health incidences. However, these are focused predominantly on terrestrial and freshwater ecosystems.  Additionally, a major fallback of government sponsored projects is that they are subject to political events such as government shutdowns and administration changes, which can impair their efficacy and long-term viability. In marine environments, efforts have been established to establish a centralized database to monitor marine mammal health, but the project lacks sufficient support and is not updated regularly. Further, disease is a widespread conservation challenge, and given the complexity of ecosystem interactions, a more powerful strategy would be to monitor and report disease across taxa in order to track changes with respect to environmental variables over time.

The use of eDNA has proven successful in identifying pathogen presence in freshwater systems, aiding in the management of chytridiomycosis in amphibians, one of the most devastating wildlife diseases known (Schmidt et al., 2013), as well as parasite presence in amphibians (Huver et al., 2015). However, no such efforts specific to disease have been reported in marine environments, although eDNA is a demonstrated tool for biodiversity monitoring in oceans (Stat et al., 2017).

Genomics has revolutionized medicine in humans. Gene expression and genome wide association studies have provided suites of genes that are useful in the early detection, prognosis, and therapy design for cancers (Kuderer et al., 2011) while several drug therapies have been developed to treat autoimmune disease based on genomic insights (Leiding et al., 2018). To date, genomics has aided in disease ecology primarily through a focus on the pathogen, resulting in the identification of putative virulence factors, geographic origin, and transmission dynamics (Ren et al., 2003; Picardeau et al., 2008; den Baaker et al., 2011; Rosenblum et al., 2012; Valdazo-González et al., 2012; Kamath et al., 2016). Increasingly, however, precision medicine is being explored as a viable option to manage emerging infectious disease in wildlife and to conserve species (Whilde et al., 2017; Duffy et al., 2018).  For example, transcriptome sequencing of fibropapilloma tumors in sea turtles revealed similarities with human cancers and therefore informed a viable treatment option to prevent tumor regrowth following surgery (Duffy et al., 2018). Additionally, comparative genomics of cetacean morbillivirus (CeMV) strains have yielded insights into transmission and host-variability (Rima et al. 2005), and several candidate genes underlying resistance and susceptibility have been identified in bottlenose dolphins (Batley et al., 2018).

Vaccines in marine wildlife are relatively underexplored. Preliminary trials for CeMV targeting the fusion (F) and hemagglutinin (H) genes have been conducted in US Naval trained dolphins with some success (Vaughan et al., 2007).  However, these vaccines are DNA vaccines, which carry several risks such as inducing antibody production against DNA or affecting genes controlling cell growth. These risks are minimal compared to using live attenuated viruses (where the virus has been mutated to inactivate virulence), indicating the potential of a mutation in the vaccine virus to reinstate virulence and cause an inadvertent disease crisis (Duignan et al., 2014). Such an outcome could be catastrophic in a wild species, particularly in an ocean ecosystem where transmission could occur rapidly over broad geographic scales and across species. Trial vaccinations against morbillivirus in Hawaiian monk seals have been implemented in recent years using a ferret recombinant vaccine (Robinson et al., 2017). However, manufacturer availability is a challenge, and the efficacy of the vaccine in this species remains uncertain.

There are three key opportunities to improve conservation efforts directed at marine disease using technology and genomic tools. These include the:

1. establishment of a centralized database for marine disease reporting across taxa;
2. establishment of 5 mobile laboratories equipped with real time sequencing technologies to rapidly characterize and monitor microbial diversity and associations with disease in kelp; and
3. development of a precision medicine model for wildlife species through comparative “omics” research to inform vaccine and therapy design.

Genomic technologies hold great potential to improve conservation measures for marine wildlife threatened by infectious disease (Whilde et al., 2017; DeCandia et al., 2018). Specifically, pathogen sequencing initiatives can vastly advance the development of vaccines or enable discovery of exploitable weaknesses using a methodology termed “reverse vaccinology” (Del Tordelo et al., 2017), while large-scale whole genome resequencing of host populations can help to identify resistant populations and genetic variation associated with resistance that can potentially be engineered to protect vulnerable populations. Understanding genetic variation linked to resistance can aid in disease modelling, particularly where such variation can be spatially mapped against probable transmission corridors. It can also accelerate the development of disease-resistant stocks through marker-assisted selection (Guo and Ford 2016), which may be critical for coral conservation in the face of coral bleaching disease, for example. Finally, whole transcriptome sequencing studies in both controlled experimental settings and in wild populations can help to understand genomic pathways involved in the host response to stress and disease (Fraser et al., 2018). In summary, we need better monitoring and genomic resources for hosts, pathogen, and the environment to identify drivers of disease outbreaks.

Centralized Reporting and Monitoring Database

What is needed is the establishment of  a centralized reporting database designed to enable observations from multiple stakeholders, researchers, and public and to link key research and sequencing data generated to understand drivers of emerging disease in marine wildlife.

In 2011, the Ecology of Marine Infectious Disease workshop identified the following priorities for data sharing:

• Improve baseline data collection by standardization of protocols and reporting methods.
• Support a coordinated expansion of existing data sources (e.g., USGS Wildlife disease information network database, wildlife disease.nbii.gov, environmental health tracking networks, coral disease registry).
• Share and archive all data from EID funded projects in appropriate data center(s) with a web portal created for purposes of disseminating information.
• Authorize resources for EID projects in collaboration with existing studies (e.g., NSF Long-Term Ecological Research (LTER) Network and the National Ecological Observatory Network (NEON)) to add pathogen and disease-relevant data.

Seemingly, none of these priorities have been implemented, and there remains no centralized repository for information and reporting of diseases in oceans. Such a database could link research efforts globally, help identify important drivers of disease based on common patterns or conditions during outbreaks, and can facilitate early action and response to a disease outbreak, all of which will greatly improve conservation outcomes. Further, NSF funding for the EMID research coordination network is set to expire in 2019, suggesting opportunity for alternate funding sources to help the program meet its original objectives.

In partnership with the EMID Research Coordination Network, a centralized reporting database specific to marine diseases should be designed and implemented. This database should be public domain and provide opportunity for scientists, citizen scientists and marine rehabilitation centers to report eDNA studies, monitoring efforts and data, disease outbreaks, and anomalous mortality and morbidity events in sea life. The database would be linked to a genomics repository, where eDNA monitoring results, pathogen sequencing and phylogeography data, and genomic variants associated with resistance will be identified and mapped.

The initial investment will be into coordinating a working group meeting to identify key reporting priorities, establish a framework for the database, and determine roles. Subsequent investment will be in purchasing domain space or cloud storage as a repository for the data and development of the database infrastructure.

Environmental DNA Mobile Monitoring Laboratories to Support Global Kelp Forest Health

Establishing an eDNA monitoring and surveillance program designed specifically for characterizing microbial communities across space and time in kelp forests.

A major priority for managing wildlife disease in oceans is early detection. Thus, there is a compelling need to develop tools that will better characterize microbial environments in stressed vs. non-stressed systems and potentially identify novel pathogens based on molecular and phylogenetic signatures of pathogenicity (Stobbe et al., 2014; Bass et al., 2015). We have discussed the power of eDNA to be used as a monitoring and surveillance tool. Establishing an eDNA program specific to assessing health in marine systems, where samples are collected longitudinally in strategic locations and in a standardized manner could vastly improve disease management efforts. We propose establishing several mobile marine laboratories equipped with on-site sequencing tools (such as the Oxford Nanopore MinION©) designated at key functional sites where disease outbreaks are likely to occur or would have the greatest impact. We propose to focus on foundational species such as kelp, as autogenic ecosystems, but increasingly recognized as major conservation priorities that would benefit from increased monitoring programs (Krumhansl et al., 2016). These laboratories will also enable direct sequencing of kelp on-site for rapid and coordinated studies to identify adaptive resistance variation. Such laboratories could also facilitate targeted PCR assessment of pathogen presence in focal species such as cetaceans, turtles, and pinnipeds sampled opportunistically or in coordinated efforts with nearby rehabilitation facilities to better characterize the ecology of other important wildlife diseases.

We propose establishing five mobile laboratories to facilitate long term eDNA-based monitoring of kelp forests in five ecoregions identified as most threatened (Krumhansl et al., 2016) in collaboration with global researchers studying kelp forest dynamics and microbial assemblages.

Precision Medicine and Reverse Vaccinology for Disease in Marine Vertebrates

For species of high conservation concern, develop a model for precision medicine in marine vertebrates informed by comparative genomic studies, pathogen sequencing, and recombinant protein technology. The selection of species and pathogens would need to be carefully considered with objective factors like the ecological role of the species and the source or cause of the pathogen. A model for precision medicine targeted at focal taxa based on the conservation genomics toolkit will greatly advance the treatment and prevention of priority wildlife disease. Two systems for evaluating this application of genomics in wildlife disease are currently ready for development.

1. California Sea Lion

   Several diseases are afflicting California sea lions.  Understanding the ecology of these diseases will benefit greatly from a better understanding of genetic processes influencing susceptibility.  For example, urogenital carcinoma in California sea lions is highly associated with homozygosity at a single microsatellite locus which maps to intron 9 of heparanase 2 gene [HPSE2] (Browning et al., 2014). Additionally, disease outcome to leptospirosis infection appears to be related to the number of MHC II DRB genes, but with surprising results (Acevedo-Whitehouse et al., 2018).

   Until recently, there was no reference genome for California sea lions. Having a good quality, high coverage genome is an important first step. However, this should be immediately followed by comparative transcriptome and whole genome resequencing of diseased and healthy individuals. Then, identification of specific sequence variants that can recognize pathogen epitopes can be used to guide vaccine development.

   California Sea Lions would make an ideal model for several reasons. First, they are not endangered, and in fact, populations appear stable or increasing, therefore providing ample opportunity for sampling and therapy testing without demographic impacts. Ultimately, treatments developed for California sea lion may be readily adapted to rarer species of concern. Second, since California sea lions are frequently treated for a variety of maladies, including disease, at marine rehabilitation centers. A coordinated sampling effort involving collection of RNA-stabilized blood samples could help evaluate transcriptional changes in response to infection.

2. Morbillivirus in Marine Mammals
   

   Several morbillivirus’ have emerged as conservation challenges in recent decades, such as phocine distemper (PDV) and cetacean morbillivirus (CeMV). These viruses are easily transmitted across different host species and thus may directly impact endangered populations. Endangered species, such as the Hawaiian monk seal, could be driven to extinction in the event of a morbillivirus outbreak. Research into recombinant protein vaccines (only proteins are used to elicit immunologic memory as opposed to live virus), guided by comparative genomics and bioinformatic analyses, could pave the way for vaccine development that is safe and effective for wild species.

   Reverse Vaccinology and Bioinformatic Therapy Design in wild species would involve 4 components:

   1. Comparative genomics and transcriptomics of host (requires sequenced and annotated reference genome). Whole genome/transcriptome sequencing susceptible vs. resistant hosts can identify key immune sequences involved in the response to infection and can direct bioinformatic and simulation modeling for antigen recognition and vaccine design.
   2. Comparative genomics of the pathogen. Pathogenic vs. non-pathogenic strains can be compared to identify virulence factors that can be targeted by drug therapy. Bioinformatic analyses of the pathogen can also be used to scan for probable antigens.
   3. Antigen epitopes (pathogen) and antigen receptors (host) can be modeled in silico to predict binding efficiency and antigenic response in hosts.
   4. Recombinant protein research to produce a viable vaccine.

    

There are few risks associated with establishing a marine disease reporting database. This would provide an invaluable resource for documenting and identifying marine disease outbreaks, thereby identifying environmental and ecological associations with disease and providing researchers and managers with predictive power and early warning signs to improve disease management outcomes. Designing and implementing such a global database will be a challenge. The initial investment will be into coordinating a working group meeting to identify key reporting priorities, establish a framework for the database, and determine roles. Subsequent investment will be in purchasing domain space or cloud storage as a repository for the data and development of the database infrastructure. Another major challenge will be coordinating and training the global research community to utilize the database.

While there may be few risks associated with establishing an eDNA program for oceans, there are several challenges. First, ocean chemistries can significantly shorten the longevity of DNA in the marine environment. Sampling would therefore need to occur with high regularity depending on the purpose of the monitoring. This creates a significant data management challenge. Second, sample storage and processing will require adequate space and technical expertise and a dedicated lab space. Third, access to on-site technology capable of collecting and generating eDNA sequence data would greatly improve eDNA monitoring. Investment into mobile laboratories incorporating portable sequencing tools such as the nanopore ion would greatly improve program outcomes. Finally, the potential benefits of such a program will not be immediate. To maximize conservation potential, the program would need to be developed with long-term goals in mind, as one of the primary objectives would be to establish baseline measures of diversity. However, with coverage across a broad geographic scope (i.e. global), patterns may emerge rapidly that will provide important conservation insights.

Developing precision medicine for wildlife poses the greatest challenges for several reasons. First, a substantial initial investment will be necessary to generate the genomic resources necessary.  While a genome can be sequenced, assembled and annotated relatively quickly and at low cost, it may take substantially more time to facilitate enough samples for gene expression studies or comparative genomic analysis. Second, insights from any comparative genomics would then need to be vetted extensively and verified in the lab using an accessible and logical model system. Third, disseminating and monitoring vaccine efficacy would be the greatest challenge, as these are wild species and typically only opportunistically available for treatment. Vaccine often require boosters for long term protective immunity to be established.  Therefore, tracking devices would be necessary at the launch of any vaccine evaluation, such that individuals can be relocated as necessary. In some instances, captive populations may be available for initial testing, however, this raises ethical challenges against experimentally infecting captive animals with a dangerous pathogen. Precision medicine has yet to be fully realized even in human disease, and the development of such a model will take time and substantial investment. Selecting the appropriate model is key, and such endeavors or only appropriate for species that are of high conservation concern.

Smithsonian Environmental Research Center, Dr. Katrina Lohan
Effects of anthropogenic activities on parasite dispersal and invasion; functional genomics of disease, parasite diversity; and relationship between biodiversity and disease.

Ecology of Marine Infectious Disease Research Coordination Network
NSF-funded science network that includes many universities and PIs to apply science to management of marine infectious disease

Marine Science Institute, University of California, Santa Barbara, Dr. Kevin Lafferty

Marine Mammal Center, Sausalito, CA
Several researchers are directly involved in research directed at disease in marine mammals, specifically leptospirosis and urogenital carcinoma in sea lions and morbillivirus in cetaceans.

Dr. Torsten Thomas (Professor, University of New South Wales) Specializes in microbial community dynamics, using high-throughput DNA sequencing and bioinformatics to make predictions about functional and ecological properties of bacterial communities

Dr. Andrew Rassweiler (Project Scientist, Marine Science Institute) Specializes in modeling kelp forest dynamics during transition states using long term ecological monitoring data.

Dr. J. A. Vásquez (Departamento de Biología Marina, Universidad Católica del Norte) Specializes in kelp monitoring and restoration in Chile.