C. Taplin Capstone Paper

Characterization of a toll-interacting protein gene in black abalone (

Haliotis cracherodii

)

Cullen TaplinJune 11, 2008

School of Aquatic and Fishery SciencesBox 355020University of WashingtonSeattle, WA 98195

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Abstract

The black abalone (

Haliotis cracherodii

), is a commercially valuable single shelledgastropod inhabiting the intertidal zone along the west coast of North America. In recentdecades, populations along the California coast began to experience significant declines. A bacterial infection has been found to be the major contributor to black abalone mortalities. The pathogen, a Rickettsiales-like organism (RLO), has caused mortalities exceeding 99% in some populations. There is also limited information on pathogen-host immune response. In order toidentify immune-related genes in the black abalone, expressed sequence tags from other speciesof

Haliotis

were functionally annotated and used to design primers for polymerase chain reaction(PCR) amplification. One novel gene identified was a toll-interacting protein (TOLLIP)transcript (GenBank Accession EU408785). This protein is involved in the toll-like receptor signaling (TLR) pathway, a conserved pathway producing proinflammatory cytokines. The goalof this research was to identify other TLR pathway genes in black abalone and to characterize theTOLLIP gene by obtaining sequence information, examining gene expression from two populations, and correlating that expression to bacterial loads. Isolated PCR products weresequenced and identified using sequence comparison techniques (i.e. BLAST). Quantitative real-time PCR (qPCR) was performed on digestive gland and gill tissues of RLO infected and controlabalone. Bacterial loads were also quantified using qPCR methods and then correlated to geneexpression. Results showed several other TLR genes are present in black abalone indicating the presence of the TLR pathway. Sequence comparison of TOLLIP from black abalone was shownto have high similarity to other

Haliotis

species. No significant differences were revealed in geneexpression experiments or bacterial comparisons; however, interesting patterns were discoveredthat warrant future study.

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Introduction

Black abalone (

Haliotis cracherodii

) are single shelled gastropods inhabiting theintertidal zone at depths of 0 – 5 m (Lindberg, 1992). Their distribution is confined to the westcoast of North America, extending from Coos Bay, Oregon to Cabo San Lucas, Baja California.The adult black abalone can reach a shell size of 200 mm (Parker et al., 1992). Economically,abalone are the most commercially important species of gastropods in the aquaculture industry(Chen et al., 2005). In 2002, the United States produced 169 metric tons of cultured abalone.Worldwide, 22,677 metric tons of abalone were produced, including cultured and fisherieslandings (Gordon and Cook, 2004).In recent decades, black abalone populations began experiencing declines. Fishing pressure was one factor, as laws restricting their harvest were repealed in 1968 (Parker et al.,1992). Despite the fact that black abalone are less valuable than red (

H. rufescens

) and pink (

H. corrugata

)

abalone as their meat is considered lesser quality, harvesting of wild black abalone reached 870 metric tons in 1973 (Parker et al., 1992). Overfishing of abalone inCalifornia led to closure of the commercial fishery in 1996 (Rogers-Bennett, 2004).Environmental contaminates have also been hypothesized as a cause of abalone population declines. Experiments with pentachlorophenol, a widely used pesticide for preservinglumber and an EPA priority pollutant, and abalone show a decrease in energy production and possible weakening of immune defenses (Martello et al., 2000).Another factor causing increased black abalone mortalities is an increase in water temperature due to large El Niño events and climate change. Warmer water has been shown toconsiderably stress abalone and increase susceptibility to bacterial disease (Lee et al., 2001). El