Dataset: Caribbean PaV1 survey
Deployment: Butler_Caribbean

From February 2006- February 2007 and from September 2010 through October 2011, adult Panulirus argus tissues were obtained directly from fishermen as fresh or recently frozen tissue samples (fifth walking leg) from locations throughout the Caribbean. In most cases, approximately 100 lobsters were sampled from each country or sample location and carapace length and sex were taken for each lobster. Samples consisted of a 1 – 2 cm piece of a walking leg preserved in clear rum (40% ethanol, 80 proof), shipped to the Virginia Institute of Marine Science and transferred upon receipt into 95% ethanol for long-term storage in preparation for genetic analysis.

Molecular diagnostics and DNA sequencing
Genomic DNA was extracted using Chelex resin (Biorad, Hercules, CA). Using sterile methods, aliquots of ~15mg of tissue or 150µl of hemolymph in anticoagulant, were incubated with 50µl of 10% Chelex resin (w/v) and 12µl of proteinase K (Qiagen, Valencia, CA) at 60ºC for approximately 4 hrs. Once tissue lysis was complete, samples were vortexed for 15 s, heated at 100ºC for 10 min, vortexed briefly again and then centrifuged at 13,600 rpm for 1 min. The DNA (supernatant) was removed and stored at either -20ºC or 4ºC prior to genetic analysis. For quality control, genomic DNA from each lobster was assessed by amplifying the small subunit ribosomal RNA (SSU) using ‘universal’ SSU primers. The amplified target DNA fragment was approximately 1800 bp in length.

Diagnosis of PaV1 was done using PCR primers published previously with modifications to reagent concentrations, thermocycling parameters, and reaction volume as described in Moss et al. (2012). The concentrations of the assay reagents were 1X PCR buffer (20mM Tris-HCl (pH 8.4), 50mM KCl), 0.2 mg ml-1 bovine serum albumin (BSA), 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 uM 45aF (forward primer), 0.5 uM 543aR (reverse primer) and 1.0 unit Taq polymerase (Invitrogen, Carlsbad, CA). Aliquots of 2.5 uL of lobster genomic DNA were added to 22.5 uL of PCR reagents for a final PCR volume of 25 uL. An additional control reaction containing 2.5 ?l of ddH20 plus PCR reagents in the above concentrations was included as a separate control (no DNA). The thermocycling parameters were an initial denaturation step at 94°C for 5 min followed by 40 cycles of 94°C for 45 s, 63°C for 45 s, 72°C for 1 min, all followed by a final elongation step at 72°C for 10 min. Aliquots of 10 ?L of the PaV1 PCR product or 6 ?L of the SSU product were loaded onto a 2% agarose gel (w/v), electrophoresed at 100V, stained with ethidium bromide and examined under UV light. Images were recorded using the Alpha Innotech FluorChem? (San Leandro, CA) imaging system.

Amplification products from all spiny lobsters found to be PCR positive for PaV1 were cloned and sequenced. Briefly, between 4 and 8 clones were sequenced per sample. PCR products of the correct size (~500 bp) were cloned into the plasmid pCR4-TOPO and then transformed into Escherichia coli using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) using half the manufacturer’s recommendation of vector and chemically-competent cells. Selection was based on ampicillin resistance. Transformed bacterial colonies were screened for inserts using a boil-prep method followed by PCR amplification using the M13 vector primers. Before sequencing, PCR products were treated with shrimp alkaline phosphatase (SAP) and exonuclease I (Exo I) (Amersham Biosciences, Piscataway, NJ) to remove excess oligonucleotides and dNTPs. Bidirectional sequencing was performed using the Big Dye Terminator Kit v3.1 (Applied Biosystems, Norwalk, CT) with M13 sequencing primers in 5 uL reactions with one eighth the concentration of Big Dye recommended by the manufacturer’s protocols. Sequence products were re-suspended in 20 uL of Hi-Di formamide (Applied Biosystems, Foster City, CA) and 10 uL of each were electrophoretically separated on an ABI 3130 Prism Genetic Analyzer.

Vector trimming and sequence editing were performed using CodonCode Aligner v3.7.1.1. Sequences were aligned using the ClustalW algorithm in MacVector v.12.5.1. Settings for pairwise alignment were an open gap penalty of 10.0 and an extend gap penalty of 0.1. Settings for the multiple alignment stage were an open gap penalty of 10.0 and an extend gap penalty of 0.05. Following ClustalW alignment, the resulting alignment was edited by hand. Sequences were examined for unique alleles using Collapse v.1.2. Measures of genetic distance and nucleotide diversity were performed using MEGA v.5.0.