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Cullen Taplin June 11, 2008
School of Aquatic and Fishery Sciences Box 355020 University of Washington Seattle, WA 98195
Abstract The black abalone (Haliotis cracherodii), is a commercially valuable single shelled gastropod inhabiting the intertidal zone along the west coast of North America. In recent decades, 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 to identify immune-related genes in the black abalone, expressed sequence tags from other species of 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 goal of this research was to identify other TLR pathway genes in black abalone and to characterize the TOLLIP gene by obtaining sequence information, examining gene expression from two populations, and correlating that expression to bacterial loads. Isolated PCR products were sequenced and identified using sequence comparison techniques (i.e. BLAST). Quantitative realtime PCR (qPCR) was performed on digestive gland and gill tissues of RLO infected and control abalone. Bacterial loads were also quantified using qPCR methods and then correlated to gene expression. 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 shown to have high similarity to other Haliotis species. No significant differences were revealed in gene expression experiments or bacterial comparisons; however, interesting patterns were discovered that warrant future study.
Introduction Black abalone (Haliotis cracherodii) are single shelled gastropods inhabiting the intertidal zone at depths of 0 – 5 m (Lindberg, 1992). Their distribution is confined to the west coast 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 fisheries landings (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 in California 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 preserving lumber 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 to considerably stress abalone and increase susceptibility to bacterial disease (Lee et al., 2001). El
Niño events in 1957, 1982, and 1997 caused significant black abalone mortalities along the California coast due to the presence of warmer ocean water (Moore et al., 2000). The largest component to black abalone mortalities along the California coast is hypothesized to be a bacterial pathogen that causes withering syndrome (WS). The bacterium has been identified as a Rickettsiales-like organism called Candidatus Xenohaliotis californiensis (RLO). Initially it was thought that RLO infects the tissue lining the gastrointestinal tract, interfering with enzymes in the gut. However, recently it was shown that RLO infects the postesophagus. Then, through mechanisms that have yet to be described, it causes a change in tissue morphology of the digestive gland (Friedman, personal communication). The bacterium prevents normal digestion of nutrients and forces the animal to rely on glycogen stores from its relatively large foot muscle. Extensive exposure to RLO causes the shrinking of the foot muscle, inability to adhere to the substrate, and ultimately death (Gardner et al., 1995). The signs of WS were noted in 1984 from commercial divers and recognized by scientists in 1987 from populations along Santa Cruz Island (Culver and Richards, 1992). Since the discovery of WS, mass mortalities exceeding 99% have been recorded along the California Channel Islands (Figure 1) (Haaker et al., 1992). WS has been found at seven of the eight island populations (Vanblaricom et al., 1993). The large mortalities of black abalone due to RLO infection have formed populations that are more resistant to the bacterium. Abalones from San Nicolas Island, an island in southern California, are more resistant to RLO through natural selective pressures than abalones in areas of northern California, like Carmel and Monterey Bay. Selective pressure due to long-term exposure to the disease is the causes of differing resistance among populations (Johnson, 2007).
Figure 1: Map of the California coast. Blue circles indicate the two sample locations. Map adapted from Friedman et al., 1997.
The current understanding of black abalones’ immune response to RLO is not well understood (Hooper et al., 2006). However, increased stress has been shown to cause a decrease in immune function, indicating that environmental contaminates and increased water temperature make abalone more susceptible to bacterial infections (Hooper et al., 2006). Abalones, like all invertebrates, only have an innate immune system and therefore have no ‘memory’ of previous infections (Arancibia, et al., 2007). Several immune components are conserved among invertebrates, but amount that different invertebrates use on the particular components can vary. Some common components include toll-like receptors, lectins in non-self recognition, and the prophenoloxidase system (Hooper et al., 2006). Recently, I used comparative bioinformatic techniques in attempts to identify immune related genes in black abalone. Several genes were found including toll-interacting protein (TOLLIP), catalase, and plancitoxin. TOLLIP is a key component in the toll-like receptor pathway (Figure 2). These receptors, which are well conserved in the animal kingdom, recognize
molecules and patterns produced exclusively by pathogens and bacteria (Singh et al., 2003). The patterns and molecules detected are critical to the survival of the microbes, which prevents resistance due to mutations. Lipopolysaccharides, peptidoglycan, and lipoteichoic acid are a few examples of recognizable molecules by toll-like receptors (Singh et al., 2003). Animals have several toll-like receptors; eleven have been identified in humans, nine in Drosophila. The main function of toll-like receptors is to signal the presence of a pathogen and production of proinflammatory cytokines (Arancibia, et al., 2007).
Figure 2: The current understanding of the toll-like receptor pathway and the individual receptor specificity with TOLLIP highlighted. Figure from Singh et al., 2003.
The goal of this research was to both confirm the presence of the TLR pathway and characterize the TOLLIP gene in black abalone. To confirm the TLR pathway was present in black abalone, related genes were discovered through functional relationships. Characterization
of TOLLIP was conducted by obtaining genetic sequence, comparing differential TOLLIP gene expression between resistant and susceptible populations, and correlating bacterial loads to TOLLIP gene expression. For gene expression experiments, gill and digestive gland tissue were examined. From this research, abalone farms, breeding programs, restoration efforts and monitoring programs of currently threatened black abalone populations could benefit. Methods and Materials Black abalone samples were provided by Dr. Carolyn Friedman’s lab. Her lab maintained black abalone in control and regularly RLO exposed treatments. Furthermore, her lab had two populations of black abalone; both split into the control and exposed groups. One population was from San Nicolas Island, CA (the resistant population) and the other was from Carmel, CA (the susceptible population). There were approximately 40 abalone total between the four treatment groups. Discovering Related TLR Genes To discover TLR pathway genes in black abalone, a system called PANTHER (Protein Analysis Through Evolutionary Relationships) was utilized (www.pantherdb.org). This system classifies genes based on their function and is able to predict the function of un-described genes based on nucleotide relationships. Related genes were identified by searching within the TLR pathway and using a database of Haliotis spp. genes. Sequencing the TOLLIP Gene Gill tissue was collected from an abalone in a control group. Total RNA was isolated using an MO Bio PowerSoilTM RNA isolation kit (MO Bio Laboratories, Inc.). The kit used various centrifuge steps, RNA capture columns and included a chloroform extraction. The extracted RNA was reverse transcribed into cDNA (Table 1).
The coding sequence of TOLLIP was first investigated. Primers were designed from the TOLLIP EST (gene accession number CX726806) of the closely related pacific abalone (H. discus) (Figure 3). The first primers designed were 806F and 806R. After successful sequencing of the partial gene using these two primers, 866F was designed to capture more upstream sequence (Table 2). Polymerase chain reaction (PCR) on cDNA was conducted to amplify the gene (Table 3 and 4). Purification of the band was done by cutting it out from the gel and using Ultrafree®-DA spin columns (Millipore Corporation). Samples were sequenced through HighThroughput Sequencing Solutions at the University of Washington. Genomic sequencing of TOLLIP was carried out by extracting total genomic DNA and PCR was conducted using Advantage® HD Polymerase Mix to amplify the large gene fragment (Clontech Laboratories, Inc.) (Tables 3 and 4). Again, primers 806F and 806R were used.
Reverse Transcription Protocol 1. Get 0.25 ug of mRNA or 25 ug total RNA 2. Add RNase free water up to 5 ul in a 0.6 ml tube 3. Mix 4. Spot spin 5. Heat at 75C for 5 min in thermocycler 6. Put directly on ice for 5 min or longer 7. Make Master Mix: PER RXN 4 ul 5x Buffer (AMV RT Buffer) 8 ul dNTPs (10 mM total) 1 ul AMV RTranscriptase 1 ul Oligo dT Primer 1 ul RNase free water Total = 15 ul 8. Add MM to tube with diluted mRNA in it (total volume now 20 ul) 9. Vortex 10. Spot spin 11. Incubate at RT for 10 min 12. Incubate at 37C for 1 hr in thermocycler 13. Heat inactivate @ 95C for 3 min 14. Spot spin 15. Leave on ice or store at –20C
Table 1: Method used for cDNA formation from RNA.
Figure 3: Map of the TOLLIP EST from the pacific abalone, a closely related species. Boxes indicate primers: 866F in red, 806F in green, and 806R in blue. Punitive introns are labeled with blue “I”.
Primer Name 806F 806R 866F
Sequence 5'-GCTGGCGAAGAACTATGGTC-3' 5'-ACAAAGGTCCTCGTTGCTGT-3' 5'-GATGATGGAGGACGAGAGGA-3'
Table 2: Primers used to isolate a fragment of TOLLIP designed from the pacific abalone.
cDNA PCR Reaction Mix 12.5uL 1uL 1uL 1uL 9.5uL 2x GoTaq F Primer R Primer cDNA H2 O
gDNA PCR Reaction Mix 2.5uL 0.5uL 0.5uL 1uL 4uL 0.25uL Buffer F Primer R Primer cDNA dNTPs genomic pol
Table 3: Reaction mixtures for PCRs. gDNA reactions utilized Advantage® HD genomic polymerase.
cDNA PCR Conditions
Denature 95°C for 5 min Cycles 1-40: 95°C for 1 min Cycles 1-40: 50°C for 1 min Cycles 1-40: 72°C for 1 min 72°C for 10 min Annealing Extension
gDNA PCR Conditions
Denature 94°C for 5 min Cycles 1-30: 98°C for 10 sec Cycles 1-30: 68°C for 12 min 72°C for 10 min
Table 4: PCR temperature conditions used. gDNA annealing/extension cycle called for 1 min per kilobase.
TOLLIP Gene Expression Using the methods described above, cDNA was made from two tissue sources. Digestive gland tissue was isolated from twenty abalone (five from each treatment group) five months from the start of the experiment. Gill tissue was isolated eight months after beginning the experiment and three abalone were taken from each treatment. Quantitative real-time PCR (qPCR) was carried out utilizing an Opticon-2 Continuous Fluorescence Detection System to measure expression of TOLLIP in each sample (Bio-Rad). CT values were normalized to corresponding 16S RNA values to compare relative abundance of TOLLIP. Real-time PCR miner was used to analyze qPCR data (www.miner.ewindup.info/miner/). Two-way ANOVAs and single factor ANOVAs were performed with Microsoft Excel 2007. For correlations of TOLLIP gene expression to bacterial loads, both digestive gland and gill tissues from RLO exposed abalone were examined. Bacterial loads were provided from Nate Wright, a member of Dr. Friedman’s lab, also using qPCR methods. Results Discovering Related TLR Genes Several TLR related genes were identified through PANTHER (Table 5). Those of most interest were TLR9, NFkB, and several TRAFs.
Se qID gi|82858510|gb|CX727245.1|CX727245 gi|82858071|gb|CX726806.1|CX726806 gi|118127317|gb|DY402904.1|DY402904 gi|82857458|gb|CX726193.1|CX726193 gi|118127355|gb|DY402947.1|DY402947 gi|82858044|gb|CX726779.1|CX726779 gi|118127250|gb|DW986508.1|DW986508 gi|82858500|gb|CX727235.1|CX727235 gi|118127056|gb|DW986441.1|DW986441 gi|82857427|gb|CX726162.1|CX726162 gi|118127113|gb|DW986229.1|DW986229 BPs Acce ssi on Numbe r Top Bl ast Hi t 729 NP _031828.2 737 CAB58121.1 534 NP _001034090.1 741 NP _002760.1 774 ACA42558.1 584 XP _396644.3 614 EAT 32752.1 628 BAG14263.1 923 ABH10823.1 682 CAL50408.1 697 XP _001171340.1 cat hepsin K [Mus musculus] T OLLIP prot ein [Mus musculus] T NF recept or-associat ed protein ... protease, serine, 1 preproprot ein [H... deat h-associated prot ein kinase 2/CD... E Val ue 5.00E-55 2.00E-54 2.00E-33 3.00E-25 4.00E-21
PREDICT ED: similar t o Myd88 CG2078... 2.00E-13 t oll [Aedes aegypt i] GNBP1 [T enebrio molitor] recept or-int eract ing prot ein 1 [Dani... protein kinase, put at ive (ISS) [Ost ... PREDICT ED: t oll-like recept or 9... 8.00E-09 2.00E-08 1.00E-07 2.00E-07 4.00E-05
Table 5: Related TLR genes from a Haliotis spp. database identified through PANTHER.
Obtaining TOLLIP Sequence The partial coding sequence of TOLLIP in black abalone was successfully obtained and was submitted to GenBank. When compared to the Pacific abalone’s TOLLIP gene, black abalone’s TOLLIP gene shows 97.5% similarity in the nucleotide sequence and 99.5% in the protein sequence (Figure 4). The genomic sequence of the TOLLIP gene was isolated in gel electrophoresis. However, because of its large size (>10kbp), sequencing was not carried out due to time restrictions. Lastly, comparisons were made with the sea urchin’s (Strongylocentrotus purpuratus) genomic TOLLIP sequence. Six punitive introns in the abalone TOLLIP sequence were identified (Figure 3).
Figure 4: TOLLIP coding sequence comparisons of the Pacific abalone and black abalone.
Comparing Differential TOLLIP Gene Expression Figure 5 displays TOLLIP gene expression levels and standard errors for the four treatment groups and two tissue examined. For both tissues, two-way ANOVA values for each tissue were also recorded (Table 6).
Figure 5: TOLLIP gene expression levels for two tissues from four treatment groups.
Treatm Com ent parison S tatistics
E xpos ure F-stat F-crit MS d.f. Digestive Gland 0.097 241.42 Gill 1.12674 5.31766 14.635 L a oc tion T s is ue F-stat F-crit MS d.f. Digestive Gland 2.136 5300.57 Gill 1.48084 5.31766 19.2344 E xpos ure & L a oc tion T s is ue F-stat F-crit MS d.f. Digestive Gland 0.123 304.065 Gill 0.03589 5.31766 0.46614 T s is ue
Table 6: Statistics from two-way ANOVAs on each tissue.
p-value 1 0.76 1 0.31946 p-value 1 0.168 1 0.25832 p-value 1 0.732 1 0.85446
Correlating Bacterial Loads to TOLLIP Gene Expression Correlations of Bacterial loads to TOLLIP expression in both gill and digestive gland tissues are presented in Figure 6. Each tissue was first examined with all exposed samples and then examined for each location. Linear regressions were also calculated (Table 7).
Bacterial Com parison S tatistics
T s is ue R v lue a P lue -va Digestive Gland 0.002698544 0.91193011 Gill 0.19311379 0.38326094
Table 7: Linear regression statistics for each tissue of bacterial load to TOLLIP gene expression correlations.
Figure 6: Correlations of bacterial loads to TOLLIP gene expression.
Discovering Related TLR Genes As expected, gene identification through PANTHER indicates that the TLR pathway is present in black abalone. Furthermore, these genes indicate an immune function for the TLR pathway. Several kinases and proteases, a toll like receptor 9, MyD88, and TNF receptors support this conclusion. These results are encouraging, as gene identification in black abalone has received little attention. Each of the genes identified here could be of interest for further study on the TLR pathway in black abalone. Obtaining TOLLIP Sequence The high similarity for a partial coding region of the TOLLIP gene compared to other abalone species also matched expectations. Animals within the same genus are expected to have similar genomes. However, another reason for the high similarity could be that the TOLLIP primers used were designed from an expressed sequence tag (EST) from a pacific abalone. By their nature, ESTs are typically conserved regions of a gene. Nonetheless, the isolated sequence of TOLLIP from black abalone is novel as little sequence information is available for the species. Comparing Differential TOLLIP Gene Expression As presented in Table 6, the differences in TOLLIP expression isolated from gill and digestive gland tissues among the four treatments were not significant. For both tissues, the lack of statistical power from small sample sizes among treatment groups could explain this result. Another explanation for non-significant results in digestive gland tissue, especially the San Nicolas Island population, could be it is an unrelated tissue as far as RLO infection is concerned. The digestive gland was chosen for study because originally it was believed to be the target of RLO infection. However, only recently was it discovered that RLO actually infects the post-esophagus and causes a change in tissue morphology of the digestive gland. Therefore, an
immune response in the digestive gland is not expected. Furthermore, digestive gland was harvested five months after the beginning of the experiment. Assuming RLO infection occurred soon thereafter, it may be that the TLR pathway is useless for the animal five months later. The TLR pathway is a first line of defense by signaling the presence of a pathogen. Once the abalone has been infected, it may revert to other immune response systems to fight the infection. Lastly, abalone from San Nicolas Island may have developed or rely more heavily on a separate method to fight RLO infection. While gene expression was not significant, a pattern in gill tissue seems to emerge that could be of interest. TOLLIP gene expression in control samples appears to be higher in control samples than RLO exposed samples for both populations. While this was opposite of expectations, one possible explanation is that the TLR pathway in RLO exposed abalone treatments is more active and transcribing gene transcripts more quickly. Quantitative PCR measures gene transcripts, not proteins produced. Further studies are needed to support this explanation. Correlating Bacterial Loads to TOLLIP Gene Expression As discussed above, a similar pattern of lower TOLLIP transcript at higher bacterial loads can be seen in gill tissue. With digestive gland tissue, there is no detectable pattern, further supporting the possibility that RLO infection is not associated with digestive gland. When examined by population, no consistent pattern could be detected. Again, low sample sizes are definitely affecting statistical power.
The study provided insight into the toll-interacting protein in black abalone and function of the toll-like receptor pathway. Partial coding sequence and genomic DNA for the TOLLIP gene was isolated – a first for this species of abalone. Several TLR pathway molecules were identified, confirming the presence of the pathway in black abalone. Lastly, data from gene expression studies suggests that the TLR pathway is active in the gill tissue. With these results, future research on black abalone’s immune response to RLO infection could examine TOLLIP expression in other tissues (notably the post-esophagus), explore other molecules in the TLR pathway, and compare laboratory gene expression results to expression found in wild populations. Acknowledgements I would like to thank Dr. Carolyn Friedman and her lab staff for providing abalone tissue samples, giving insight into RLO infection, and for allowing the use of figures for this paper and a previous oral presentation. Thanks goes to Sam White for guidance on experiment protocols and Dr. Steven Roberts for being my faculty mentor throughout the research. Lastly, acknowledgements go to Sea Grant California and NOAA for providing partial funding.
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