Having a BLAST Digging into Panopea generosa Larvae: generating resources to study Pacific geoduck clam development
Jessica Blanchette and Steven Roberts
University of Washington
School of Aquatic and Fishery Sciences, Seattle, WA 98105
Abstract
Delving into the physiology of commercially significant animals can provide insight into maximizing harvest and understanding the creature as a whole. While studies typically focus on how to create optimal growth for adult organisms, less is known about the larval stages to facilitate recruitment. Observations of high rates of mortality during metamorphosis from a pediveliger to a juvenile geoduck have led to an unanswered question in the benthic invertebrate world. Molecular information can provide insight into explaining this highly stressful time of development and how other external influences may affect growth. To learn more about the physiology of geoduck clam larvae, baseline genomic resources are required. After annotating sequences of a related clam order, Veneroida, using a BLASTx algorithm and joining with a Gene Ontology information, primers pertinent for developmental processes were selected and tested via PCR and qPCR. Annotation of 1,088,642 ESTs and mRNA sequences was filtered into the GoSlim bin “development processes” and primers were selected. Of the 66,217 mRNA sequences pertaining to development, 10 primers were tested; in the end, 1 primer set was applied to the larval tissue. The single gene analyzed in this study was for actin-2—a gene coding for the actin protein, crucial for growth, reproduction, cytokinesis, and ATPase activity. The qPCR assay was applied to larval stages to detect varied expression throughout development. This study is a stepping stone to developing genomic resources for Pacific geoduck clams. With further expansion of this research with building on molecular techniques such as cloning and sequencing, full genomic resources can be created. Then, application of those resources can answer questions about mortality events in larval stages and effects of environmental change.
Introduction
Commercially important aquaculture species are widely studied to maximize harvest. Studies have largely focused on developing ideal conditions for adult yield, thus optimizing harvest and broodstock health. However, there are gaps in our understanding when it pertains to maximizing larval development to reach adult stages and this is exemplified in the Pacific geoduck clam, Panopea generosa. The Pacific geoduck clam is a commercially significant species, bringing in $60 million in Washington state in 2012, according to the Washington Department of Fish and Wildlife and the Pacific Coast Shellfish Growers Association. These clams are a fruitful industry; partially due to their being one of the largest burrowing clams and the rapid growth they undergo. While geoduck clams are one of the longest lived species, with a maximum age between 148-168 years old, most of their growth is in the first 10 years of life (Feldman et al 2004). These early years of growth are crucial in the life of geoduck and to obtain insight into the physiological processes occurring to accomplish this growth, molecular data is required. Though progress has been made in scallops and oysters, genomic resources for the Pacific geoduck clam are still extremely limited. Larval stages of the Pacific geoduck are known but still shrouded in mystery when it comes to molecular mechanisms. Understanding the physiology of P. generosa can provide insight to optimal growth and harvest and effects of external influence on larval development.
One factor affecting larval recruitment of benthic invertebrates is high risk of mortality during metamorphosis (Gosselin & Qian 1991). After developing from a trochophore to a veliger to a pediveliger, geoducks experience a metamorphosis into a settled juvenile. It is during this extremely energetically demanding stage that observations of mortality events occur frequently. Explaining this phenomenon and other potential sources of larval stress relies heavily on knowledge of physiological processes.
To begin to look into sources of larval distress, genomic resources are vital. Only with a window into the molecular mechanisms driving development can causes of damage and potential catalysts for growth be identified. The main objective of this study are to identify and annotate potential genes given sequences from a closely related taxa and to analyze how expression of genes pertinent to development change as larval stages progress.
Methods and Materials
Gene Annotation and Primer Selection
As there are currently no genomic resources for geoduck clams, expressed sequence tags (ESTs) and mRNA sequences for the clam order Veneroida, taxonomic ID #6580 were subjected to BLASTx analysis in order to annotate these undescribed sequences. Specifically the sequences were compared to the UniProtKB/Swiss-Prot database. Details of Blastx analysis available via IPython notebooks, mRNA-- http://goo.gl/JrJDzl ; ESTs-- http://goo.gl/WWQXmA. Gene Ontology information was added using gene ontology annotation files in SQLShare. Specifically the joining of tables was accomplished with the following language.
SELECT * FROM [jpb23@washington.edu].[txid6580_EST_uniprot_sprot2]blast
Left Join [sr320@washington.edu].[uniprot-reviewed_wGO_010714]unp
ON blast.Column7 = unp.Entry
Left Join [sr320@washington.edu].[SPID and GO Numbers]go
ON unp.Entry = go.SPID
Left Join [sr320@washington.edu].[GO_to_GOslim]slim
ON slim.GO_id = go.GOID
SELECT * FROM [jpb23@washington.edu].[txid6580_mRNA_uniprot_sprot2]blast
Left Join [sr320@washington.edu].[uniprot-reviewed_wGO_010714]unp
ON blast.Column7 = unp.Entry
Left Join [sr320@washington.edu].[SPID and GO Numbers]go
ON unp.Entry = go.SPID
Left Join [sr320@washington.edu].[GO_to_GOslim]slim
ON slim.GO_id = go.GOID
Sequences associated with developmental processes in were filtered using a threshold of 1e-5. Of those filtered, 10 genes were chosen for their best fit of conserved regions (1e-10 threshold) and pertinence to developmental processes (Table 1). Preliminary testing of the selected primers included polymerase chain reaction (PCR) analyzed via gel electrophoresis through a 1.5% agarose gel set to 120V for 30 minutes. Using Promega GoTaq Green Master Mix instruction and thermal cycling conditions, PCR with juvenile geoduck siphon tissue was compared to a Hyperladder II ladder to ensure correct product sizes.
Table 1. Primers were selected from BLAST annotation after determining pertinence to development.
Fit of primers was also determined by quantitative PCR of 4 of the 10 primer choices. After determining the appropriate primers by a visual scan, 10µl Bio-Rad 2X Sso Fast EvaGreen Supermix was utilized in a 20µl reaction volume per the manufacturers instruction; 0.5µl of 10µM forward and reverse primer, 2µl cDNA, and 7µl water was used in each qPCR reaction. Reactions were then placed in a thermalcycler for 30 seconds at 95° C, then underwent 40 cycles of 95° C for 5 seconds and 55° C for 10 seconds. Final extension was for 10 seconds at 55° C.
Sampling
Pacific geoduck, Panopea generosa, trochophores, early-veligers, late-veligers, early-metamorphosed, and settlers were collected from Taylor Shellfish hatchery after a spawning event started February 11, 2014. Initial sampling began with next-day stages of trochophores until settlement, February 28th. All stages were taken in duplicate, except the single juvenile, and analyzed in technical duplicate. Samples of these stages were extracted from the tank, rinsed, and stored in RNAlater until further use.
Gene Expression
Extraction of RNA started by homogenizing all individuals of each stage as a single sample. Each tube containing P. generosa tissue was centrifuged and supernatant RNAlater pipetted off. For the settlers, the interphase between RNAlater and gravel substrate was used as tissue for RNA extraction. Total RNA was extracted from homogenized samples using TriReagent (Molecular Research Center, Inc., Cincinnati OH) following the manufacturer’s instructions. The final RNA pellet was resolubilized in 200µl DEPC water and stored in -80°C.
After RNA was extracted, each sample was treated with a Promega TURBO DNA-free kit. Each 5 ng sample of RNA was combined with “DNA-free” buffer, 0.5 ml DNase reagent and water to 50 µl , then incubated for 30 min at 37 °C. Next, another 0.5 ml DNase reagent was added and the solution incubated for 30 min at 37°C again. About 0.1 of the total volume-worth of DNase Inactivation Reagent was added to the total mix and the tube rested for 2 minutes at room temperature. After centrifuging the tube at 10,000g for 1.5 min and transferring the liquid to a new tube, the RNA was reverse transcribed to cDNA. Reverse transcription proceeded with approximately 1 µg RNA per reaction in 25 total reaction volumes. The RNA was combined with 0.5µl Promega oligo dT primer per 1 ug RNA and incubated at 70C for 5 minutes then stored on ice. Then, a 6.75µl combination of 5µl Promega M-MLV Buffer, 1.25µl 10 mL dNTP, and 0.5µl M-MLV RT reagent were added to each reaction. This solution incubated for 1 hour in 42°C then a final rest of 95°C for 3 minutes. All resulting cDNA was then stored in -20°C until qPCR could be performed. Quantitative PCR followed Bio-Rad instruction as previously stated. Gene expression was then calculation using a crude expression formula: expression=10^(-(0.3012*Ct)+11.434
Results
Joining BLAST results with the Uniprot/Swiss-prot database resulted in 17,818 annotated expressed sequence tags (ESTs) relevant to developmental processes—filtered out via GoSlim bin. Of those annotated, 10,912 (61%) ESTs have a 1.0E-10 e-value threshold. Swiss-prot joining of the BLAST for mRNA (taxonomic ID #6580) lead to 66,217 annotated genes pertaining to developmental processes. Of the annotated development genes, 23,170 (35%) have a threshold e-value of 1.0E-10; this filter was used as the lowest subset for primer selection. From the PCR analyzing the 10 selected primers, the primer amplifying the gene for “actin-2” was determined to both have the best fit for geoduck cDNA and presence in all larval tissues (Fig.1).Quantitative PCR confirmed that the amplification was of a single product.
Figure 1. Results of BLAST joining to annotate Veneroida ESTs is divided into the above three functions. Primers applied to larval stages were extracted from the “developmental process” GoSlim bin.
When the qPCR assay was applied to the trochophores, veligers, settlers, and one juvenile with a single primer set for actin-2, expression was noted to be high in trochophores and less in settlers and juveniles. The veliger sample appeared to have skewed results due to differences in initial RNA concentration and will be excluded from analysis. Though there was a subtle difference between settler and juvenile stages there is a conspicuous increase in expression of the actin-2 gene in trochophores (Table 2, Fig 2).
d
Discussion
Results from this study act as a baseline for geoduck clam genomic resources. The BLAST results from annotating the Veneroida sequences are applicable to future clam studies.
The primers selected here are in a very finite scope of available genes from the BLAST and thus represent a limited portion of the genome. However, from even the limited selection, several primers were viable and one was successfully found to be pertinent to developmental processes and was expressed in all developmental stages. This gene (actin-2) is for the production of actin, functioning in ATPase activity and assists in growth, reproduction, embryo development, actin cytoskeleton organization, and cytokinesis (NCBI). All of these processes are important in the life of a larval geoduck and this particular sequence is now a known gene within geoduck clams. This is supported by the expression results; the abundance of expression in trochophores indicates rapid growth and energy expenditure. Unfortunately, without the inclusion of veliger data, a clear picture of how this gene expression changes throughout each stage is not achieved.
For future study, repeating the assay with adjustments for veliger complications would resolve one issue found here. Beyond these findings, the addition of more molecular techniques such as cloning and sequencing would be beneficial. Developing genomic resources for geoduck clams is a vital first step in learning more about their physiology and optimizing their growth. There are numerous factors that play a role in growth and development, they all act in tandem to contribute to mortality events and stress to hinder reaching adulthood (Gosselin & Qian 1997). Fully understanding how these aspects relate to an animal’s growth and development and maximizing harvest for commercial needs require further exploration of geoduck physiology.
Acknowledgements
Many thanks to Taylor Shellfish Hatchery, Quilcene for providing larval samples and to Sara Wyckoff and Molly Jackson for collection of the samples. Additional thanks to Sam White for assistance in lab techniques and RNA extraction, Brent Vadopalas for his knowledge of geoduck life history, and to Gregory Jensen, Claire Olsen, Mackenzie Gavery, Emma Timmins-Schiffman, Jake Heare, Hannah Wear, Charles Deuber, and Katie Jackson. This study would not have been possible without funding from the University of Washington School of Aquatic and Fishery Sciences.
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