Characterization of a toll-

interacting protein gene in black

abalone (Haliotis cracherodii)

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 real-

time 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.

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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

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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 post-

esophagus. 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).

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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

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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

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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).

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 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 High-

Throughput 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.

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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 Sequence

806F 5'-GCTGGCGAAGAACTATGGTC-3'
806R 5'-ACAAAGGTCCTCGTTGCTGT-3'
866F 5'-GATGATGGAGGACGAGAGGA-3'
Table 2: Primers used to isolate a fragment of TOLLIP designed from the pacific abalone.

cDNA PCR Reaction Mix gDNA PCR Reaction Mix

12.5uL 2x GoTaq 2.5uL Buffer
1uL F Primer 0.5uL F Primer
1uL R Primer 0.5uL R Primer
1uL cDNA 1uL cDNA
9.5uL H2 O 4uL dNTPs
0.25uL genomic pol
Table 3: Reaction mixtures for PCRs. gDNA reactions utilized Advantage® HD genomic polymerase.

cDNA PCR Conditions

Denature Annealing Extension
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
gDNA PCR Conditions
Denature Annealing Extension
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.

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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 BPs Acce ssi on Numbe r Top Bl ast Hi t E Val ue

gi|82858510|gb|CX727245.1|CX727245 729 NP _031828.2 cat hepsin K [Mus musculus] 5.00E-55
gi|82858071|gb|CX726806.1|CX726806 737 CAB58121.1 T OLLIP prot ein [Mus musculus] 2.00E-54
gi|118127317|gb|DY402904.1|DY402904 534 NP _001034090.1 T NF recept or-associat ed protein ... 2.00E-33
gi|82857458|gb|CX726193.1|CX726193 741 NP _002760.1 protease, serine, 1 preproprot ein [H... 3.00E-25
gi|118127355|gb|DY402947.1|DY402947 774 ACA42558.1 deat h-associated prot ein kinase 2/CD... 4.00E-21
gi|82858044|gb|CX726779.1|CX726779 584 XP _396644.3 PREDICT ED: similar t o Myd88 CG2078... 2.00E-13
gi|118127250|gb|DW986508.1|DW986508 614 EAT 32752.1 t oll [Aedes aegypt i] 8.00E-09
gi|82858500|gb|CX727235.1|CX727235 628 BAG14263.1 GNBP1 [T enebrio molitor] 2.00E-08
gi|118127056|gb|DW986441.1|DW986441 923 ABH10823.1 recept or-int eract ing prot ein 1 [Dani... 1.00E-07
gi|82857427|gb|CX726162.1|CX726162 682 CAL50408.1 protein kinase, put at ive (ISS) [Ost ... 2.00E-07
gi|118127113|gb|DW986229.1|DW986229 697 XP _001171340.1 PREDICT ED: t oll-like recept or 9... 4.00E-05
Table 5: Related TLR genes from a Haliotis spp. database identified through PANTHER.

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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.

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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.

Treatment Comparison Statistics

Exposure
Tissue
F-stat F-crit MS d.f. p-value
Digestive Gland 0.097 241.42 1 0.76
Gill 1.12674 5.31766 14.635 1 0.31946
Location
Tissue
F-stat F-crit MS d.f. p-value
Digestive Gland 2.136 5300.57 1 0.168
Gill 1.48084 5.31766 19.2344 1 0.25832
Exposure & Location
Tissue
F-stat F-crit MS d.f. p-value
Digestive Gland 0.123 304.065 1 0.732
Gill 0.03589 5.31766 0.46614 1 0.85446
Table 6: Statistics from two-way ANOVAs on each tissue.

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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 Comparison Statistics

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Tissue R value P-value
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.

Discussion

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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

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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.

Conclusions

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 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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References

Arancibia, Sergio A., Caroll J. Beltrán, Isabel M. Aguirre, Paulina Silva, Alexis L. Peralta, Frano

Malinarich, and Marcela A. Hermoso. 2007. Toll-like Receptors Are Key Participants In

Innate Immune Responses. Biological Research. 40: 97 – 112.

Chen, Hong, Kangsen Mai*, Wenbing Zhang, Zhiguo Liufu,Wei Xu, and Beiping Tan. 2005.

Effects of dietary pyridoxine on immune responses in abalone, Haliotis discus hannai

Ino. Fish and Shellfish Immunology. 19: 241 – 252.

Culver, Carrie S., and John B. Richards. 1992. Black Abalone Mortality: Establishing a

Research Agenda. A Publication of the California Sea Grant College.

Friedman, Carolyn S., Marilyn Thomson, Calvin Chun, Peter L. Haaker, and Ronald P. Hendrick.

1997. Withering Syndrome of the Black Abalone, Haliotis cracherodii (Leach): Water

Temperature, Food Availability, and Parasites as Possible Causes. Journal of Shellfish

Research. Vol. 16, No. 2, Pgs. 403 – 411.

Garnder, George R., John C. Harshbarger, James L. Lake, Thomas K. Sawyer, Kathy L. Price,

Mark D. Stephenson, Peter L. Haaker, and Heidi A. Togstad. 1995. Association of

Prokaryotes With Symptomatic Appearance of Withering Syndrome in Black Abalone

Haliotis cracherodii. Journal of Invertebrate Pathology. 66: 111 – 120.

Gordon, Roy H. and Peter A. Cook. 2004. World Abalone Fisheries and Aquaculture Update:

Supply and Market Dynamics. Journal of Shellfish Research. Vol. 23 No. 4

Pgs. 935 – 939.

Haaker, Peter L., David O. Parker, Heidi Togstad, Daniel V. Richards, Gary E. Davis, and

Carolyn S. Friedman. 1992. Mass Mortality and Withering Syndrome in Black Abalone,

Haliotis cracherodii. In: S. A. Shephard, M. J. Tegner, and S. A. Guzmán del Próo,

2
 editors. Abalone of the World: Biology, Fisheries, and Culture. Oxford: Blackwell

Scientific. pp 214 – 224.

Hooper, Celia, Rob Day, Ron Slocombe, Judith Handlinger, and Kristen Benkendorff. 2007.

Stress and Immune Responses in Abalone: Limitations in Current Knowledge and

Investigative Methods Based On Other Models. Fish and Shellfish Immunology.

22: 363 – 379.

Johnson, Christina S. 2007. Disease-Resistant Black Abalone Discovered. California Sea Grant

News Release.

Lee, K.-K., P.-C. Liu, Y.-C. Chen, and C.-Y. Huang. 2001. The Implication of Ambient

Temperature With the Outbreak of Vibriosis in Cultured Small Abalone Haliotis

diversicolor Supertexta Lischke. Journal of Thermal Biology. 26: 585 – 587.

Lindberg, David L. 1992. Evolution, Distribution, and Systematics of Haliotidae. In: S. A.

Shephard, M. J. Tegner, and S. A. Guzmán del Próo, editors. Abalone of the World:

Biology, Fisheries, and Culture. Oxford: Blackwell Scientific. pp 3 – 18.

Martello, Linda B., Carolyn S. Friedman, and Ronald S. Tjeerdema. 2000. Combined Effects of

Pentachlorophenol and Salinity Stress on Phagocytic and Chemotactic Function in Two

Species of Abalone. Aquatic Toxicology. 49: 213 – 225.

Moore, James D., Thea T. Robbins, and Carolyn S. Friedman. 2000. Withering Syndrome in

Farmed Red Abalone Haliotis rufescens: Thermal Induction and Association with a

Gastrointestinal Rickettsiales-like Prokaryote. Journal of Aquatic Animal Health.

12: 26 – 34.

Parker, David O., Peter L. Haaker, and Heidi A. Togstad. 1992. Case Histories for Three Species

of Colifornia Abalone, Haliotis corrugata, H. fulgens and H. cracherodii. In: S. A.

2
 Shephard, M. J. Tegner, and S. A. Guzmán del Próo, editors. Abalone of the World:

Biology, Fisheries, and Culture. Oxford: Blackwell Scientific. pp 384 – 394.

Rogers-Bennett, Laura, Brain L. Allen, and Gary E. Davis. 2004. Measuring Abalone

Recruitment in California to Examine Recruitment Overfishing and Recovery Criteria.

Journal of Shellfish Research. Vol. 23 No. 4 Pgs 1201 – 1207.

Singh, Bhanu P., R. S. Chauhan, and Kokesh K. Singhal. 2003. Toll-like Receptors And Their

Role In Innate Immunity. Current Science. vol. 85, no. 8, pgs. 1156 – 1164.

Vanblaricom, G. R., J. L. Ruediger, C. S. Friedman, D. D. Woodard, and R. P. Hedrick. 1993.

Discovery of Withering Syndrome Among Black Abalone Haliotis cracherodii Leach,

1814, Populations at San Nicolas Island, California. Journal of Shellfish Research. Vol.

12 No. 2 Pgs. 185 – 188.