View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ZENODO Metabarcoding and Metagenomics 3: 1–19 DOI 10.3897/mbmg.3.33835 Primer Validation Development of a new set of PCR primers for eDNA metabarcoding decapod crustaceans Tomoyuki Komai1, Ryo O. Gotoh2, Tetsuya Sado2, Masaki Miya2 1 Department of Zoology, Natural History Museum and Institute, Chiba, Chiba 260-8682, Japan. 2 Department of Ecology and Environmental Sciences, Natural History Museum and Institute, Chiba, Chiba 260-8682, Japan. Corresponding author: Tomoyuki Komai ([email protected]) Academic editor: Vasco Elbrecht | Received 13 February 2019 | Accepted 3 April 2019 | Published 24 April 2019 Abstract The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and in- cluding many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Key Words mitochondrial 16S rRNA gene, biodiversity monitoring, MiDeca, natural sea water Introduction sample (multi-species approach). The latter approach has been developed with rapidly developed high-throughput Classical methods of biodiversity monitoring have been next-generation sequencing (NGS) (e.g. Taberlet et al. primarily based on the collection of specimens and subse- 2012, Thomsen et al. 2012, Miya et al. 2015, Valentini et quent morphology-based identification. Such biodiversi- al. 2016). Application of eDNA is now quite wide-rang- ty monitoring is costly and time-consuming and requires ing in studies of biodiversity, aquatic ecology and conser- considerable expertise for various taxonomic groups. vation biology (Bohmann et al. 2014, Díaz-Ferguson and Recent technological developments in molecular ecolo- Moyer 2014). gy have provided a novel tool for species detection using In particular, with regard to aquatic environments, the DNA present in aquatic or terrestrial environments (envi- multi-specific assessment and monitoring of the fauna ronmental DNA or eDNA; Taberlet et al. 2012). using eDNA have focused mainly on vertebrates (e.g. There are two major approaches to applying eDNA Thomsen et al. 2012, Kelly et al. 2014, Miya et al. 2015, analysis: “eDNA barcoding”, which aims at detecting a Andruszkiewicz et al. 2017, Ushio et al. 2017, 2018), single species in the environment (species-specific ap- which are known to release abundant DNA derived from proach); and “eDNA metabarcoding”, which simulta- faeces, body mucus, blood and sloughed tissue or scales neously detects multiple species from an environmental (Bohmann et al. 2014). The applicability of the eDNA Copyright Tomoyuki Komai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Tomoyuki Komai et al.: Development of a new set of PCR primers for... metabarcoding for aquatic invertebrates, notably those After removing problematic sequences (i.e. 18 se- with an exoskeleton (Crustacea), has received less atten- quences that could not be aligned with other sequenc- tion (Thomsen et al. 2012, Rees et al. 2014). The vast es), the remaining 249 sequences from 203 species and majority of previous studies on crustaceans yielded a five sequences of unidentified species of Cherax (Astac- “species-specific” approach detecting invasive species idea: Parastacidae) (Table 1) were subjected to multiple (e.g. Tréguier et al. 2014, Dougherty et al. 2016, Larson alignment using MUSCLE (Edgar 2004) implemented in et al. 2017) or monitoring seasonal migrations of particu- MEGA7 (Kumar et al. 2016) with a default set of param- lar species (Wu et al. 2018). eters. The aligned sequences were imported into MEGA7 The Decapoda is the largest order of the crustacean for visual inspection of the conserved and hyper-variable class Malacostraca (Arthropoda: Pancrustacea), com- regions. The visual search for a short hyper-variable re- prising more than 14,000 extant species worldwide (De gion (up to 200 bp for paired-end sequencing using the Grave et al. 2009, Ahyong et al. 2011), with continuing Illumina MiSeq) flanked by two conservative regions discovery of new species. The great majority of decapod (ca. 20–30 bp) was performed on the entire set of the two crustaceans are marine, but other environments have also aligned 12S rRNA and 16S rRNA genes. Primers were been colonised, such as lowland freshwaters, mountain designed using Primer3 (Rozen et al. 2000) accounting rivers, estuaries and even land. Decapods are also highly for G/C contents (40–60%) and melting temperature diverse in ecology and include a number of commercially (Tm: 50–60 °C). important species (such as shrimp, prawn, lobster, cray- The new universal primers for decapod eDNA were fish, king crab, snow crab etc.), attracting much scientific designed on the 16S rRNA gene (for details, see Results and economic interests. and Discussion) and were named MiDeca-F/R (F and R The importance of marker selection in eDNA me- represent forward and reverse, respectively). tabarcoding has recently been emphasised (Coissac et al. 2012, Deagle et al. 2014). As there is no ideal uni- In silico evaluation of variation in the target region versal metabarcode (Riaz et al. 2011), marker selection could be specific to the target taxonomic group. The aim Interspecific differences within the amplified DNA se- of this study was to develop new universal PCR primers quences are required for accurate taxonomic assign- for eDNA metabarcoding of decapod crustaceans, which ments. To computationally evaluate levels of interspe- enable detection of multiple species for biodiversity as- cific variations within the target region (hereafter called sessment and monitoring. The performance of newly “MiDeca sequence”) across different taxonomic groups developed primers (herein named “MiDeca”) was tested of decapods, 254 whole mitogenome sequences used for using tissue-derived DNA extracts from 250 species and the primer development were subjected to calculation of an eDNA sample from natural seawater collected from pairwise edit distances (intra-species, inter-species, in- the near-shore environment. ter-genus and inter-family levels, respectively). The edit distance is defined as the minimum number of single-nu- cleotide substitutions, insertions or deletions that are re- Materials and methods quired to transform one sequence into the other. (Jones and Pevzner 2004). Primer development Test of primers with tissue extracted DNA Mitochondrial rRNA genes have been recommended for identification of animal taxa because they have a similar In order to examine the performance of MiDeca primers, taxonomic resolution to the COI marker and they present extracted DNA from a single individual of each 250 spe- conserved regions that flank variable regions, which al- cies across the suborder Dendrobranchiata and 10 of 11 lows the design of primers with high-resolving power for infraorders of the suborder Pleocyemata was used to am- the target taxonomic group (Deagle et al. 2014). In order plify MiDeca sequences (Table 2). Total genomic DNA to identify a suitable region in the mitogenome for species was extracted from each tissue (thoracic or pleon muscle identification based on eDNA, 267 whole mitogenome or pereopod or pleopod muscle), which was preserved in sequences of Decapoda registered in the databases have 70–99% ethanol for one to more than 10 years, using a been downloaded from NCBI as of 17 October 2017. DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) Three requirements were considered for designing with an elution volume of 100 µl. the new primers: 1) a target amplicon consisting of few- DNA concentrations were measured and recorded with er than 200 bp is desirable because the eDNA will often a NanoDrop Lite spectrophotometer (Thermo Fisher Sci- be degraded; 2) the amplified regions include sufficient entific, DE, USA). PCR was carried out with 30 cycles of interspecific differences for all target species; and 3) con- a 8.0 µl reaction volume (divided from the original PCR served regions (20–30 bp) across all target species are mastermix) containing 2.2 µl sterile distilled water, 3.8 µl located at both ends of the short hyper-variable region to 2 × Gflex PCR buffer (Takara, Otsu, Japan), 0.4 µl of each simultaneously amplify the target sequences (Riaz et al. primer (5 µM), 0.2 µl Tks Gflex DNA polymerase (Taka- 2011, Miya et al. 2015, Valentini et al. 2016). ra, Otsu, Japan) and 1.0 µl template. The thermal cycle https://mbmg.pensoft.net Metabarcoding and Metagenomics 3: e33835 3 profile after an initial 1 min denaturation at 94 °C was as (8 litre polyethylene bucket) was thoroughly decontami- follows: denaturation at 98 °C for 10 s, annealing at 50 °C nated with a 10% bleach solution before use. Surface wa- for 10 s and extension at 68 °C for 10 s with the final ter was collected using 10 casts of the bucket fastened to a extension at the same temperature for 7 min.