Field and Laboratory Methods for DNA Studies on Deep-sea Isopod Crustaceans

Abstract

Field and laboratory protocols that originally led to the success of published stud− ies have previously been only briefly laid out in the methods sections of scientific publica− tions. For the sake of repeatability, we regard the details of the methodology that allowed broad−range DNA studies on deep−sea isopods too valuable to be neglected. Here, a com− prehensive summary of protocols for the retrieval of the samples, fixation on board research vessels, PCR amplification and cycle sequencing of altogether six loci (three mitochondrial and three nuclear) is provided. These were adapted from previous protocols and developed especially for asellote Isopoda from deep−sea samples but have been successfully used in some other peracarids as well. In total, about 2300 specimens of isopods, 100 amphipods and 300 tanaids were sequenced mainly for COI and 16S and partly for the other markers. Although we did not set up an experimental design, we were able to analyze amplification and sequencing success of different methods on 16S and compare success rates for COI and 16S. The primer pair 16S SF/SR was generally reliable and led to better results than univer− sal primers in all studied Janiroidea, except Munnopsidae and Dendrotionidae. The widely applied universal primers for the barcoding region of COI are problematic to use in deep−sea isopods with a success rate of 45-79% varying with family. To improve this, we recommend the development of taxon−specific primers.

A Multi-gene Analysis Reveals Multiple Highly Divergent Lineages of The Isopod Chelator Insignis (Hansen, 1916) South of Iceland

“…1) with R/V Meteor (M85/3) and R/V Poseidon (POS456) in 2011 and 2013 using two types of epibenthic sleds (EBS, Rothlisberg and Pearcy 1977;Brenke 2005) and a large box corer. All samples were fixed in precooled (−20°C) 96% undenatured ethanol and treated as described in Riehl et al (2014). The specimens from the EBS were sorted on board from subsamples.…”

Discovery of Swimming Males of Paratanaoidea (Tanaidacea)

“…-Extraction of DNA from identified female and unidentified male specimens was performed at the Smithsonian Institution in Washington, D.C. ac− cording to the protocols described in Riehl et al (2014). PCRs employed two primer sets: jgLCO (5'−TIT CIA AYC AYA ARG AYA TTG G−3') and jgHCO (5'−TAI ACY TCI GGR TGI CCR AAR AAY CA−3'), or LCO−1498(Folmer et al 1994 and Nancy (5'−CCC GGT AAA ATT AAA ATA TAA ACT TC−3').…”

“…For the first 24 hours, preserved samples were gently mixed ev− ery few hours to ensure thorough preservation, and the ethanol was changed after 24 hours. A subsample of approximately 100 mL of material was sorted on board during the cruise; subsequent sorting was conducted upon return to land (see Riehl et al (2014) for sampling, preservation, and sorting details). For the RP sled (Rothlisberg and Pearcy1977), collected material was gently elutriated through a series of sieves from 1mm to 300 mm, with each fraction preserved separately.…”

The Community Structure of Deep-Sea Macrofauna Associated with Polymetallic Nodules in the Eastern Part of the Clarion-Clipperton Fracture Zone

“…The sieve residue was immediately bulk-fixed in pre-cooled (−20 • C) 96% absolute EtOH and stored at −20 • C. Every 3 to 5 h, the sample containers were carefully shaken to guarantee penetration of the EtOH through the sediment and prevent the water inside the samples from freezing (Riehl et al, 2014). After 24 h, the EtOH was decanted and replaced by new pre-cooled 96% absolute EtOH, to ensure a high EtOH concentration (Riehl et al, 2014). Subsequently, the samples were kept frozen (−20 • C) until further analysis.…”