James D. Moore

Withering Syndrome in Farmed Red Abalone Haliotis rufescens: Thermal Induction and Association with a Gastrointestinal Rickettsiales-like Prokaryote

Withering syndrome (WS) is a chronic wasting disease responsible for mass mortality in wild populations of black abalone Haliotis cracherodii. The etiology of WS is uncertain with limited evidence for the role of a gastrointestinal Rickettsiales-like prokaryote (RLP). We documented for the first time the occurrence of animals with clinical signs of WS and associated morphological changes in another haliotid species, the red abalone H. rufescens. In this study, 60 juvenile red abalone (8 cm) were randomly selected from a farmed population raised at 14°C that was known to have low-intensity RLP infections but lacked clinical signs of WS. The abalone were held in triplicate containers receiving water of approximately 14.7°C (Control, Co) or 18.5°C (elevated temperature, ET) and were fed equally for 220 d. Survival was 100% (30/30) for the Co group and 67% (20/30) for the ET group. The ET group animals had higher RLP infection intensities and showed more clinical signs (mantle retraction, lower weight gain, lower condition index) and morphological changes (digestive gland degeneration) associated with WS. In trials conducted immediately before termination of the experiment, ET group animals fed at half the rate of Co group animals. Among ET group animals, the intensity of RLP infections in the posterior portion of the esophagus was positively correlated with WS clinical signs and morphological changes, whereas no correlations were present among Co group animals. During 1997-1998 and in conjunction with elevated seawater temperatures associated with El Niño, several abalone farms in California experienced a dramatic increase in the proportion of red abalone showing signs of WS. Examination of 66 red abalone from five commercial farms revealed that animals with more advanced RLP infections had more severe WS clinical signs and associated morphological changes. Collectively, these data demonstrate that RLP infection plays a key role in the etiology of WS in red abalone and that warm water enhances the severity of the pathogenic effects of RLP infection.

OCEAN WARMING EFFECTS ON GROWTH, REPRODUCTION, AND SURVIVORSHIP OF SOUTHERN CALIFORNIA ABALONE

Traditional fisheries management in southern California has failed, in part because it is based on an assumption of an unvarying environment and is focused on size limits rather than insuring the persistence of aggregations of large fecund individuals. The combined effect of low frequency climatic variability and anthropogenic perturbations can have dramatic consequences for abalone in southern California. Abalone species are tightly linked to kelp forest ecosystems that, besides furnishing habitat, also provide the main food source for abalone. In southern California, kelp canopies are very sensitive to oceanographic climate because the kelp depend upon high nutrients in the water column. Oceanic warming, in turn, results in decreased nutrients in the surface water, and this is correlated with marked reductions in giant kelp biomass. Here we address the additive effects of ocean warming on two species of California abalone (the red abalone, Haliotis rufescens; and the green abalone, H. fulgens) by subjecting them to varied environmental conditions similar to cool, normal, and warm phases of the California current in the southern California Bight. Our experimental design simultaneously tested the synergistic effects of temperature and food quantity and quality on survivorship, growth, and reproduction. For red abalone, warm temperatures increased the onset of with- ering syndrome, a fatal abalone disease, and halted growth and reproduction. In contrast, green abalone survivorship, growth, and reproduction were relatively robust irrespective of temperature, while their growth and reproduction were most strongly influenced by food quantity. We found clear evidence suggesting that, combined with overfishing, California abalone populations are adversely affected by ecosystem responses to ocean warming: Cool- water red abalone suffer stronger consequences in warm water than do green abalone. Conservation, restoration, and recovery plans of remnant California abalone populations

Characterizing the metabolic actions of natural stresses in the California red abalone, Haliotis rufescens using 1H NMR metabolomics

Withering syndrome in California red abalone (Haliotis rufescens) is caused by the Rickettsiales-like prokaryote (WS-RLP) Candidatus Xenohaliotis californiensis. WS-RLP infection is not sufficient to cause withering syndrome, and for reasons not yet well understood additional stressors such as elevated water temperature appear to influence disease development. Using nuclear magnetic resonance (NMR) based metabolomics, we have investigated the influence of food availability, temperature, and bacterial infection, both individually and in combination, on the metabolic status of the red abalone. Food limitation caused dramatic reductions in all observed classes of foot muscle metabolites, while at the same time metabolite levels within the digestive gland were preserved or increased. We also found that food limitation in combination with elevated temperature led to greater metabolic perturbations in both tissue types than those observed under food limitation alone. WS-RLP infection and food-limitation resulted in many of the same metabolic changes within the tissues studied, although the effects of infection were less severe. We observed increased levels of homarine in the digestive gland of both food-limited and WS-RLP-infected animals, yet only observed increased homarine levels in the foot muscle of infected abalone. These results further support the recently established glucose-to-homarine ratio in foot muscle as a potential marker for differentiating WS-RLP-infected animals from those of both healthy and food limited abalone. Furthermore, we found that the NMR metabolic data correlates well with histological measurements supporting the use of the metabolomics approach for characterizing both normal and pathological events in marine species, particularly during periods of environmentally relevant stress.

Response of Red Abalone Reproduction to Warm Water, Starvation, and Disease Stressors: Implications of Ocean Warming

ABSTRACT Changes in ocean temperature can have direct and indirect effects on the population dynamics of marine invertebrates. We examined the impacts of warm water, starvation, and disease on reproduction in red abalone (Haliotis rufescens). We found that sperm production was highly sensitive to warm water and starvation, suggesting there may be a dramatic temperature threshold above which sperm production fails. Wild males from northern (72%) and southern (81%) California had sperm. In contrast, only 30% of the males exposed to warm water (18°C) for 6 mo or starvation for 13 mo had sperm, with spermatogenesis dropping dramatically from 300,000 presperm cells/mm3 (wild) to 46,000 presperm cells/mm3 (warm water) and 84,000 presperm cells/mm3 (starvation). In a longer warm-water experiment (12 mo), males had total reproductive failure in temperatures greater than 16°C, irrespective of food treatment. Egg production was less sensitive to warm water, but was impacted more by starvation, especially food quantity relative to quality. Wild females from northern (97%) and southern (100%) California had mature oocytes averaging 3 million eggs and 21 million eggs, respectively. Females exposed to 18°C water for 6 mo had diminished fecundity, averaging only 400,000 mature eggs whereas females in the starvation experiment did not produce any mature eggs. Normal sperm and egg production was found in abalone testing positive for Rickettsiales-like-prokaryote (RLP), the agent of Withering Syndrome in cool water. However, abalone with RLP also exposed to warm water developed the disease withering syndrome and did not produce any mature gametes. The temperature-mediated lethal and sublethal effects on red abalone reproduction described here, combined with temperatures known impacts on abalone growth, kelp abundance, and disease status, clearly demonstrate population-level consequences. We suggest that temperature needs to be explicitly incorporated into red abalone recovery and management planning, because Californias ocean has warmed and is predicted to warm in the future.

THE INTRODUCED SABELLID POLYCHAETE TEREBRASABELLA HETEROUNCINATA IN CALIFORNIA: TRANSMISSION, METHODS OF CONTROL AND SURVEY FOR PRESENCE IN NATIVE GASTROPOD POPULATIONS

Abstract The sabellid polychaete Terebrasabella heterouncinata (Fitzhugh & Rouse 1999) has a unique life history in which larvae settle on the edge of gastropod shells and rely on shell deposition to create a tube with an opening to the exterior. This worm was accidentally imported to California, USA on abalone from South Africa in the 1980s and spread with abalone shipments to most culture facilities and some public aquaria throughout the state. Its ability to infest Californias native gastropods has sparked concern regarding potential establishment in intertidal habitats adjacent to facilities that held sabellid-positive abalone. We examined the ability of T. heterouncinata to transmit between individual turban snails, Tegula funebralis. We found that transmission between T. funebralis did occur, but at a significantly slower rate than that between red abalone Haliotis rufescens. During 2002 to 2006 native gastropods (turban snails and limpets) were collected at most sabellid-exposed sites and no T. heterouncinata were detected; it thus appears that this species has not become established in California. Freshwater exposure was examined as a method to kill T. heterouncinata in shell fragments that may remain after abalone are removed from production or display units. Freshwater immersion for up to 8 hours but not 16 or 32 h resulted in survival of adults and/or larvae resident in brood chambers. In a similar study, motile T. heterouncinata larvae were found to survive up to 32 sec of freshwater exposure, whereas none survived a 64-sec exposure. These data can be used by abalone culture and display facilities to establish reliable sanitization procedures to prevent T. heterouncinata transmission or reinfestation.

Implementing a Restoration Program for the Endangered White Abalone (Haliotis sorenseni) in California

ABSTRACT A restoration program including wild population surveys, captive breeding, health monitoring, recovery site preparation, and recovery modeling has been implemented to restore white abalone (Haliotis sorenseni) populations in California. White abalone once supported a lucrative fishery and are now endangered, nearing extinction at less than 1% of baseline abundances. Recent deep water surveys indicate that populations continue to decline with no signs of recruitment, despite the closure of the fishery in 1996. Four sites with artificial reefs (n=12/site) in optimal white abalone habitat were established. No wild white abalone have been found at these sites. Captive abalone were spawned in the spring of each year from 2012 to 2015. Each year, the production of 1-y-old abalone has increased in the captive breeding program from approximately 20 in 2012, to 150 in 2013 and an estimated 2,000 in 2014. In 2015, the breeding program reached two milestones: (1) most successful spawning season to date and (2) the hatchery distributed 200 captive-reared abalone to 4 partner institutions within the White Abalone Recovery Consortium (WARC). The WARC is made up of federal and state agencies, universities, public aquaria, and aquaculture organizations, all committed to white abalone restoration. The next steps for the program include expanding the captive breeding program to increase production, monitoring abalone health and genetic diversity, and conducting stocking studies to enhance growth and survival in the ocean. The goal of the stocking program is to create a reproductive population in the wild to bring white abalone back from the brink of extinction.

Denman Island disease (causative agent Mikrocytos mackini) in a new host, Kumamoto oysters Crassostrea sikamea.

Mikrocytos mackini, causative agent of Denman Island disease in Pacific oysters Crassostrea gigas and other oyster species, was found in 2011 in a previously unreported host, the Kumamoto oyster C. sikamea, in Humboldt Bay, California, USA. The detection was also the first reported finding of M. mackini in California. Prevalence was estimated as high as approximately 27% from pooled samples analyzed by PCR. Higher prevalence appeared related to longer residence time in the bay and somewhat colder than typical winter seawater temperatures. No M. mackini was detected in Humboldt Bay juvenile Kumamoto oysters or Pacific oyster brood or seed stock in 2011 or 2012.

Monomorphic pathogens: The case of Candidatus Xenohaliotis californiensis from abalone in California, USA and Baja California, Mexico

Withering syndrome (WS) is a chronic wasting disease affecting abalone species attributed to the pathogen Candidatus Xenohaliotis californiensis (CXc). Wild populations of blue (Haliotis fulgens) and yellow (H. corrugata) abalone have experienced unusual mortality rates since 2009 off the peninsula of Baja California and WS has been hypothesized as a possible cause. Currently, little information is available about the genetic diversity of CXc and particularly the possible existence of strains differing in pathogenicity. In a recent phylogenetic analysis, we characterized five coding genes from this rickettsial pathogen. Here, we analyze those genes and two additional intergenic non-coding regions following multi-locus sequence typing (MLST) and multi-spacer typing (MST) approaches to assess the genetic variability of CXc and its relationship with blue, yellow and red (H. rufescens) abalone. Moreover, we used 16S rRNA pyrosequencing reads from gut microbiomes of blue and yellow abalone to complete the genetic characterization of this prokaryote. The presence of CXc was investigated in more than 150 abalone of the three species; furthermore, a total of 385 DNA sequences and 7117 16S rRNA reads from Candidatus Xenohaliotis californiensis were used to evaluate its population genetic structure. Our findings suggest the absence of polymorphism in the DNA sequences of analyzed loci and the presence of a single lineage of CXc infecting abalone from California (USA) and Baja California (Mexico). We posit that the absence of genetic variably in this marine rickettsia may be the result of evolutionary and ecological processes.

Differing responses of red abalone (Haliotis rufescens) and white abalone (H. sorenseni) to infection with phage-associated Candidatus Xenohaliotis californiensis

The Rickettsiales-like prokaryote and causative agent of Withering Syndrome (WS)—Candidatus Xenohaliotis californiensis (Ca. Xc)—decimated black abalone populations along the Pacific coast of North America. White abalone—Haliotis sorenseni—are also susceptible to WS and have become nearly extinct in the wild due to overfishing in the 1970s. Candidatus Xenohaliotis californiensis proliferates within epithelial cells of the abalone gastrointestinal tract and causes clinical signs of starvation. In 2012, evidence of a putative bacteriophage associated with Ca. Xc in red abalone—Haliotis rufescens—was described. Recently, histologic examination of animals with Ca. Xc infection in California abalone populations universally appear to have the phage-containing inclusions. In this study, we investigated the current virulence of Ca. Xc in red abalone and white abalone at different environmental temperatures. Using a comparative experimental design, we observed differences over time between the two abalone species in mortality, body condition, and bacterial load by quantitative real time PCR (qPCR). By day 251, all white abalone exposed to the current variant of Ca. Xc held in the warm water (18.5 °C) treatment died, while red abalone exposed to the same conditions had a mortality rate of only 10%, despite a relatively heavy bacterial burden as determined by qPCR of posterior esophagus tissue and histological assessment at the termination of the experiment. These data support the current status of Ca. Xc as less virulent in red abalone, and may provide correlative evidence of a protective phage interaction. However, white abalone appear to remain highly susceptible to this disease. These findings have important implications for implementation of a white abalone recovery program, particularly with respect to the thermal regimes of locations where captively-reared individuals will be outplanted.