2Nd Benthic Invertebrate Taxonomy, Metagenomics and Bioinformatics (Bitmab-2) Workshop 2018

2nd Benthic Invertebrate Taxonomy, Metagenomics and Bioinformatics (BITMaB-2) Workshop 2018

Final Report for the Gulf of Mexico Alliance, Gulf of Mexico Research Initiative (GoMRI) project 16-052

Hosted by the Harte Research Institute for Gulf of Mexico Studies Texas A&M University - Corpus Christi Corpus Christi, Texas January 15-19, 2018 Acknowledgements

The BITMaB-2 workshop was part of the Gulf of Mexico Research Initiative (GoMRI) research project “Genomic Responses to the Deepwater Horizon Event and Development of High- Throughput Biological Assays for Oil Spills”. The workshop was organized by W. Kelley Thomas (Principal Investigator, University of New Hampshire), Holly M. Bik (co-Principal Investigator, University of California, Riverside), Paul A. Montagna (co-Principal Investigator, Texas A&M University – Corpus Christi), Francesca Leasi (University of New Hampshire), Krystalynne Morris (University of New Hampshire), and Michael G. Reuscher (Texas A&M University - Corpus Christi). Paul A. Montagna and Francesca Leasi reviewed this report and provided valuable feedback. Data from this project are publicly available at the Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC), doi: R5.x272.000:0001 and R5.x272.000:0002.

i Cover photo credit

The parasitic isopod Probopyrus pandalicola Packard, 1879 with attached dwarf male, collected during the workshop. Photo taken by Michael G. Reuscher.

Suggested Citation

Reuscher, M.G. 2018. 2nd Benthic Invertebrate Taxonomy, Metagenomics and Bioinformatics (BITMaB-2) Workshop 2018. Final Report for the Gulf of Mexico Alliance, Gulf of Mexico Research Initiative (GoMRI) project 16-052. Harte Research Institute for Gulf of Mexico Studies, Corpus Christi, TX, 23 pp.

Contact

Michael G. Reuscher Harte Research Institute for Gulf of Mexico Studies Texas A&M University - Corpus Christi 6300 Ocean Drive, Unit 5869 Corpus Christi, Texas 78412-5869 United States of America

Office: (361) 825-2037 Email: [email protected]

ii Table of Contents

Background ...... 1 Sampling ...... 4 Lab Work ...... 8 Taxonomic Seminars ...... 12 Bioinformatics Seminars ...... 13 Outcome and Outlook ...... 15

iii Background

The second Benthic Invertebrate Taxonomy, Metagenomics, and Bioinformatics (BITMaB-2) workshop was hosted at the Harte Research Institute for Gulf of Mexico Studies on the campus of Texas A&M University – Corpus Christi between January 15 and 19, 2018. The workshop was part of the Gulf of Mexico Research Initiative (GoMRI) research project “Genomic Responses to the Deepwater Horizon Event and Development of High-Throughput Biological Assays for Oil Spills”.

The scientific goal of the project is to develop methodologies and infrastructure for the use of genetic markers to characterize the taxonomic composition of benthic communities via metagenomics. The benefit of this approach is that benthic assessment studies and monitoring efforts will produce quicker results at lower costs than the labor-intensive morphological identification of individual benthic specimens. This is particularly important when immediate scientific results are required for informed decisions about environmental, legal, and/or financial issues following oil spills, as it has been the case in the aftermath of the Deepwater Horizon disaster.

However, the lack of genetic sequences for many benthic meiofauna and macrofauna taxa is currently a major limitation of metagenomics techniques, which means that taxonomic resolution of metagenomics analyses are currently poor. Thus, one of the main goals of the workshop was to alleviate this problem by enhancing a reference library of genetic sequences from positively identified marine benthic organisms. For this purpose, nine national and international taxonomic specialists were invited to the workshop, in addition to the taxonomic specialists on the workshop organizing committee (Table 1). These taxonomists identified benthic meiofauna and macrofauna organisms from samples obtained during the workshop and previously collected ethanol preserved specimens to the lowest taxonomic level possible. All identified specimens were then preserved in either absolute ethanol or frozen in -80˚C for genetic sequencing at the Smithsonian Institution National Museum of Natural History.

Eight of the nine invited taxonomic experts extended their visits to the Harte Research Institute the week prior to the workshop (January 8-12). During this week, they were able to focus on sample collection, identification, and preservation of benthic organisms for DNA sequencing.

Table 1. List of workshop participants. Organizing committee Workshop participants (continued) Name Institution Name Institution W. Kelley Thomas (PI) University of New Hampshire Will Ballentine Dauphin Island Sea Lab Holly M. Bik (Co-PI) University of California, Riverside Lauren Ballou Texas A&M University at Galveston Paul A. Montagna (Co-PI) Texas A&M University-Corpus Christi Titilope Bukunmi-Omidiran Texas Southern University Francesca Leasi University of New Hampshire Francesco Cicala CICESE Krystalynne Morris University of New Hampshire Brooke Denney Texas A&M University-Corpus Christi Michael G. Reuscher Texas A&M University-Corpus Christi Jennifer Dreyer Virginia Institute of Marine Science Invited Scientists Erin Easton University of Texas Rio Grande Valley Name Institution Hannah Ehrmann Texas A&M University-Corpus Christi Richard Heard University of Southern Mississippi Doug Faircloth University of Texas Rio Grande Valley Alberto de Jesús Navarrete ECOSUR-Chetumal Rachel Hale University of Southampton Wonchoel Lee Hanyang University Ashley Hamilton Texas A&M University-Corpus Christi Fabio Moretzsohn Texas A&M University-Corpus Christi Mary Colleen Hannon Texas A&M University at Galveston Tiago Pereira University of California, Riverside Amanda Heidt Moss Landing Marine Laboratories Alessandro Prantoni Federal University of Paraná Maria Herranz Matesanz University of British Columbia Adelaide Rhodes University of Vermont I-Shuo Huang Texas A&M University-Corpus Christi Melissa Rohal Texas A&M University-Corpus Christi Erin Kiskaddon Dauphin Island Sea Lab Martin V. Sørensen Natural History Museum of Denmark Jennifer Le Scripps Institution of Oceanography

Bioinformatics team Jessica Lunt Smithsonian Marine Station 2 Name Institution Adam Martens Environment and Climate Change Canada Taruna Schuelke University of California, Riverside Michael McGee Louisiana State University Joseph Sevigny University of New Hampshire Marta Miatta Memorial University of Newfoundland Devin Thomas University of New Hampshire Andre Giovani Morales-Núñez University of Maryland Eastern Shore Field work and lab technicians Lilian A. Palomino Alvarez UNAM, UMDI-Sisal Name Institution Phillip Randsø Natural History Museum of Denmark Noe Barrera Texas A&M University-Corpus Christi Patrick Rayle Louisiana State University Ag Center Larry Hyde Texas A&M University-Corpus Christi Michelle Stephens Smithsonian Marine Station Rick Kalke Texas A&M University-Corpus Christi Diana Marlen Ugalde Garcia UNAM, UMDI-Sisal Elani Morgan Texas A&M University-Corpus Christi Katie Vaccaro-Garska University of West Florida Terry Palmer Texas A&M University-Corpus Christi Niels Van Steenkiste University of British Columbia Jamie Smith Texas A&M University-Corpus Christi Nichelle Van Tassel University of West Florida Workshop participants Rebecca Varney University of Alabama Name Institution Colleen Walsh Louisiana State University Ag Center Nestor Ardila ECOMAR Rachel Weisend Texas A&M University-Corpus Christi Amy Baco-Taylor Florida State University Tom Wilding Scottish Association for Marine Science

This additional week was added to increase the workshop productivity towards the scientific goals of the project.

The second major objective of the BITMaB-2 workshop was training students, staff researchers, and faculty in techniques pertinent to the methodology involved in the metagenomics analyses of benthic communities. The workshop offered insights into sample processing techniques, taxonomy and identification of marine meiofauna and macrofauna organisms, proper preservation and micrography techniques, background information on metagenomics and next-generation- sequencing, and bioinformatics tools used for DNA sequence analyses. Forty workshop participants from a pool of more than 100 national and international applicants attended. The participants consisted of two undergraduate students, 17 graduate students, five postdoctoral researchers, nine staff scientists, and three faculty. Four invitees did not attend.

Overall, 60 scientists attended at the BITMaB workshop, including the aforementioned organizing committee, invited taxonomists and invited participants, as well as a team of bioinformatics specialists and a field work crew (Fig. 1). Travel expenses for members of the organizing committee, invited scientists, and the bioinformatics team were funded by the GoMRI research grant. The invited workshop participants were supported through travel funding from the NSF Research Coordination Network EukHiTS project grant, awarded to Kelley Thomas and Holly Bik.

BITMaB-2 was hosted almost exactly one year after the first workshop in 2017. The scope and the general procedures remained the same in 2018. However, several improvements were made to enhance the scientific and educational goals of the second workshop, including increased diversity and spatial extent of samples. Samples were obtained from deeper areas of the local bays, in contrast to the first workshop in 2017 when samples were collected exclusively from shore. Samples were included from the northern Gulf of Mexico deep-sea, a Louisiana marsh, and Kauai (Hawaii). A different group of taxonomists was invited to BITMaB-2, in order to increase the taxonomic diversity of organisms that would be identified over the course of the two workshops. Ludox extraction to isolate meiofauna organisms from the sediment for metagenomics analyses was performed.

Sampling

Samples were collected during the workshop, as well as the week prior to the workshop, in different South Texas bays and estuaries (Table 2, Fig. 2), including Alazan Bay, Aransas River, Baffin Bay, Copano Bay, Corpus Christi Bay, Laguna Madre, Matagorda Bay, Nueces Bay, Oso Bay, Redfish Bay, Rincon Bayou, and San Antonio Bay, as well as from the shore of the Gulf of Mexico. Most samples were collected from a small watercraft, while others were collected from shore. Sampling gear used during the field trips included push cores, shovels, and Yabby pumps. Additionally, a piece of a serpulid reef and an algal fouling community were collected. Several taxonomists accompanied the fieldwork team on the sampling trips to give advice about preferred sediment types and salinity ranges of their taxa. Sufficient amounts of marine or brackish water from the specific sampling locations were added to each container to prevent the sediment from drying out.

Figure 1. Attendants of the 2018 BITMaB-2 workshop. (Photo credit: Larry Hyde)

Table 2. List of locations sampled during the workshop Date Time Location Station Latitude Longitude Comment 1/5/18 10:15 Corpus Christi Bay NC-E 27.79722 -97.15083 15 cores in separate jars collected 1/5/18 12:53 Corpus Christi Bay NC-MK60 27.74579 -97.21583 Cores collected and pooled in bucket 1/5/18 14:00 Nueces Bay NC-B 27.85708 -97.41025 10 cores in separate jars collected 1/5/18 15:00 Nueces Bay NC-A 27.86069 -97.47358 Cores collected and pooled in bucket 1/9/18 12:35 San Antonio Bay GE-D 28.30210 -96.68435 Cores collected and pooled in bucket 1/9/18 13:35 San Antonio Bay GE-C 28.24934 -96.75837 Cores collected and pooled in bucket 1/9/18 14:15 San Antonio Bay GE-278/"Lost Island" 28.36606 -96.76080 Bucket of sediment collected 1/9/18 14:15 San Antonio Bay GE-B 28.34672 -96.74419 Bucket of sediment collected 1/9/18 15:10 San Antonio Bay GE-A 28.39352 -96.77240 Bucket of sediment collected No hydro measurements taken; collected Bob Hall Pier/Padre Island 1/9/18 15:00 Gulf of Mexico 27.583231 -97.218959 Callianassids and other benthos with Yabby Beach pump/shovel/bucket in intertidal zone No hydro measurements taken; collected benthos 1/9/18 15:30 Corpus Christi Pass Access Rd. 3 27.6493 -97.1931 with kick net/shovel/bucket 1/10/18 ? Gulf of Mexico Galveston Island, East Beach 29.310376 -94.77219 No hydro measurements taken

5 Galveston Island, Stewart 1/10/18 ? Gulf of Mexico 29.329797 -94.734924 No hydro measurements taken Beach 1/10/18 ? Barataria Bay Fleeger Site MD3* 29.45848167 -89.94180617 Collected along edge of Spartina marsh 1/10/18 ? Barataria Bay Fleeger Site MD3* 29.45848167 -89.94180617 Collected at sheared bank edge 1/10/18 ? Barataria Bay Fleeger Site MD1* 29.46037667 -89.9208625 Collected along edge of Spartina marsh 1/10/18 ? Barataria Bay Fleeger Site MD1* 29.46037667 -89.9208625 Collected 1 m in from the bank 1/10/18 ? Barataria Bay Fleeger Ref. Site 1* 29.464091 -89.918415 Collected along edge of Spartina marsh 1/10/18 ? Barataria Bay Fleeger Ref. Site 1* 29.464091 -89.918415 Collected 1 m in from the bank 1/10/18 ? Barataria Bay Dragon Island 29.441588 -89.9057485 Along edge of Spartina marsh 1/10/18 ? Barataria Bay Dragon Island 29.441588 -89.9057485 Collected 1 m in from the bank 1/10/18 ? Barataria Bay Dragon Island 29.44142117 -89.90262667 Along edge of Spartina marsh 1/10/18 ? Barataria Bay Dragon Island 29.44142117 -89.90262667 Collected 1 m in from the bank 1/10/18 11:55 Nueces Bay Nueces Bay Boat Launch 27.8373364 -97.3810593 Bucket collected next to Marinaville Bait Stand 1/10/18 12:20 Corpus Christi Bay Indian Point 27.851054 -97.355140 Bucket of sediment collected 1/10/18 14:40 Aransas River Aransas River 28.122282 -97.309965 Bucket of sediment collected 1/10/18 15:30 Copano Bay Bayside 28.072764 -97.217279 Bucket collected near Egery Point area * Sites refer to a study by Fleeger et al. (2015): Marine Ecology Progress Series 536: 39-54.

Table 2. Continued. Date Time Location Station Latitude Longitude Comment No hydro measurements taken; 2 buckets collected 1/11/18 12:00 Oso Bay Oso Bridge 27.718032 -97.3312581 on Oso Bay side of Oso Bridge next to TAMUCC- 1 close to bridge, 1 closer to vegetation Bucket collected along Southbound lane of Upper Laguna 1/12/18 10:05 Kennedy Causeway 27.638876 -97.246760 Kennedy Causeway Access Rd. just past last exit Madre before N. Padre Island 1/12/18 10:50 Corpus Christi Pass Access Rd. 3 27.6493 -97.1931 Same sampling location as on 1/9/2018 Collected bucket next to dock at northernmost boat 1/12/18 13:00 Redfish Bay Conn Brown Harbor 27.913087 -97.133051 ramp in harbor Bob Hall Pier/Padre Island Same sampling location as on 1/9/2018; no hydro 1/13/18 ? Gulf of Mexico 27.583231 -97.218959 Beach measurements taken; 1/15/18 8:25 Baffin Bay WZ3 27.27725 -97.624867 Top 5 cm of sediment collected 1/15/18 9:10 Alazan Bay WZ5 27.3527 -97.5154 Top 5 cm of sediment collected Serpulid reef taken from channel marker 9, 1/15/18 9:47 Baffin Bay Marker 9 27.27316 -97.45328 approx. 1.0 m deep 1/15/18 11:10 Rincon Bayou RB-H 27.895472 -97.628561 Collected shovel scoops into one bucket

1/15/18 12:50 Rincon Bayou RB-A 27.907363 -97.598945 Collected one bucket with kick net

6 Collected kicknet samples into bucket - water very 1/15/18 13:30 Rincon Bayou RB-G bayou 27.88895 -97.56971 low Collected sediment with shovel into bucket - water 1/15/18 13:30 Rincon Bayou RB-G lake 27.889613 -97.569688 very low Upper Laguna Very cold and windy (TAMUCC closed); 1/16/18 10:20 Kennedy Causeway 27.638876 -97.246760 Madre collected two buckets of kick net samples 1/18/18 11:50 Redfish Bay Sta 1 27.83969 -97.13255 ~8 cores collected into bucket 1/18/18 12:05 Redfish Bay Sta 2 27.84416 -97.13088 ~8 cores collected into bucket 1/18/18 12:20 Redfish Bay Sta 3 27.84998 -97.13010 2 buckets of ~8 cores each

Figure 2. Maps of BITMaB-2 sampling locations. A. Stations in and near Corpus Christi. B. Stations north of Corpus Christi. Maps do not include additional samples provided to us from different sources. Station details can be found in Tables 2-4.

In addition to the benthic samples, hydrographic data, including temperature, salinity, conductivity, dissolved oxygen, oxygen saturation, and pH were collected with a sonde of the model YSI 6800 (Table 3). Latitude and longitude were recorded from GPS readings.

The research group of Lane Foil (Louisiana State University) collected marsh samples in Louisiana during the week prior to the workshop and sent them to us (Table 2). Kelley Thomas collected additional samples from Galveston Bay (Table 2). Ethanol preserved deep-sea samples were collected by Melissa Rohal during two C-IMAGE research cruises (Table 4). Melissa Rohal also provided ethanol preserved meiofauna samples from Rincon Bayou (Table 4). Additionally, Fabio Moretzsohn brought mollusk specimens from his personal collection (Table 4).

Lab Work

Multiple smaller sampling jars were kept in a refrigerator. Larger jars and buckets were kept in the wet lab at room temperature and aerated with multiple aquarium air stones. Different extraction techniques, tailored to the specific taxa of interest, were employed (Fig. 3). Most of the organisms were still alive after extraction. Isotonic magnesium chloride solutions were used to anesthetize the organisms and facilitate their identification. Each taxonomist had their Figure 3. Martin Sørensen and Rachel Hale processing own lab station equipped with compound a mud sample in the wet lab. (Photo credit: Larry Hyde) and stereo microscope for the identification of the specimens. Workshop participants were encouraged to work with the taxonomic experts to learn more about morphology and taxonomy of the various different benthic taxa (Fig. 4). Specimens identified to the lowest taxonomic level possible were transferred into vials with undenatured, anhydrous ethanol for subsequent DNA extraction at the Smithsonian National Museum of Natural History and subsequent genomic sequencing and assembly at UNH.

Table 3. Hydrographic variables measured at the different sampling stations. Station details are provided in Table 1. Date Station Sonde depth (m) Temperature (˚C) Conductivity (mS/cm) Salinity (ppt) DO (%) DO (mg/l) pH 0.10 9.35 45.81 29.49 100.90 9.57 8.22 1/5/18 NC-E 2.90 9.36 45.93 29.57 100.70 9.54 8.24 0.10 9.23 46.39 29.90 99.80 9.46 8.37 1/5/18 NC-MK60 3.73 8.95 46.41 29.89 100.80 9.63 8.37 0.10 8.53 44.46 28.47 103.30 10.06 8.34 1/5/18 NC-B 0.90 8.50 44.47 28.48 105.20 10.25 8.34 0.10 8.56 39.37 24.89 104.60 10.40 8.35 1/5/18 NC-A 1.00 8.20 39.80 25.21 109.40 11.06 8.38 0.10 12.56 33.53 21.00 119.70 11.19 8.47 1/9/18 GE-D 1.20 12.28 43.50 28.06 112.10 10.02 8.40 0.10 12.39 36.47 23.05 119.80 11.09 8.54 1/9/18 GE-C 1.70 11.85 40.33 25.76 155.60 14.31 8.52 1/9/18 GE-278/"Lost Island" 0.10 12.99 26.64 16.34 143.80 13.76 8.88

9 0.10 13.42 28.60 17.67 168.50 15.72 8.93

1/9/18 GE-B 1.50 11.66 43.12 27.77 148.50 13.53 8.61 0.10 13.04 24.90 15.20 143.60 13.76 8.91 1/9/18 GE-A 1.00 12.31 35.91 22.60 172.70 16.13 8.77 1/10/18 Nueces Bay Boat Launch 0.10 13.77 45.14 29.21 103.40 8.97 8.15 1/10/18 Indian Point 0.10 17.77 45.97 29.88 125.90 10.74 8.16 1/10/18 Aransas River 0.10 16.28 2.24 1.15 100.60 9.81 8.85 1/10/18 Bayside 0.10 16.20 20.86 12.52 93.20 7.71 8.34 1/12/18 Kennedy Causeway 0.10 7.99 48.27 31.17 96.40 9.36 7.85 1/12/18 Access Rd. 3 0.10 10.06 47.02 30.40 101.00 9.38 8.00 1/12/18 Conn Brown Harbor 0.10 13.47 43.83 28.28 107.60 9.39 8.10 0.10 12.50 55.778 50.18 111.40 8.66 8.18 0.50 12.50 55.774 50.18 98.20 7.70 8.20 1/15/18 Baffin Bay 1.00 12.50 55.768 50.19 97.80 7.62 8.21 1.50 12.50 55.795 50.21 101.20 7.88 8.21 2.00 12.60 55.300 50.36 83.80 6.48 8.21

Table 3. Continued Date Station Sonde depth (m) Temperature (˚C) Conductivity (mS/cm) Salinity (ppt) DO (%) DO (mg/l) pH 0.10 13.10 57.367 50.99 111.20 8.50 8.20 0.50 13.20 57.374 50.98 104.40 7.98 8.20 1/15/18 Alazan Bay 1.00 13.10 57.345 50.99 99.80 7.63 8.21 1.50 13.20 57.349 51.00 97.50 7.45 8.21 0.10 13.00 53.155 46.90 112.00 8.80 8.24 0.50 13.00 53.149 46.92 109.20 8.59 8.24 1/15/18 Baffin Bay 1.00 13.00 53.155 46.89 104.80 8.24 8.25 1.50 13.00 53.310 47.11 101.50 7.97 8.24 2.00 12.90 53.270 49.24 99.70 7.75 8.22 0.10 13.06 3.90 2.09 105.60 10.64 8.00 1/15/18 RB-H 1.00 13.14 4.34 2.30 109.40 11.34 8.10 1/15/18 RB-A 0.10 18.53 31.97 19.95 127.90 10.65 8.11 1/15/18 RB-G bayou 0.10 17.93 34.87 22.00 136.30 11.30 8.54

1/15/18 RB-G lake 0.10 17.93 34.87 22.00 136.30 11.30 8.54

10 1/16/18 Kennedy Causeway 0.10 6.12 47.78 30.61 92.40 9.38 7.80 0.10 6.57 42.84 27.18 101.00 10.33 7.62 1/18/18 Redfish Bay Sta 1 2.40 8.35 46.36 29.83 101.70 9.80 7.72 0.10 6.04 42.00 26.56 103.50 10.79 7.89 1/18/18 Redfish Bay Sta 2 3.00 6.48 45.69 29.20 101.60 10.27 7.87 0.10 5.81 42.11 26.60 100.70 10.55 7.97 1/18/18 Redfish Bay Sta 3 3.00 8.05 45.46 29.18 101.00 9.79 7.94

Table 4. Locations of other samples examined during the workshop Date Time Location Station Latitude Longitude Comment 7/11/81 ? Corpus Christi Bay Jetty at McGee Beach 27.782029 -97.393043 Collected by M.C. Castiglione 9/12/90 ? Corpus Christi Bay Indian Point 27.850285 -97.354920 Collected by J.W. Tunnell 9/16/91 ? Gulf of Mexico Port Aransas Jetties 27.833957 -97.043701 Collected by J.W. Tunnell Shallow, low salinity Nueces River delta; coll. By 11/3/15 ? Rincon Bayou C 27.89878 -97.60417 M. Rohal 12/30/16 ? Kauai (Hawaii) Salt Pond Beach 21.90014 -159.60776 Collected by F. Moretzsohn 12/30/16 ? Kauai (Hawaii) Salt Pond Beach 21.900195 -159.609096 Collected by F. Moretzsohn 12/30/16 ? Kauai (Hawaii) Salt Pond Beach 21.900259 -159.609123 Collected by F. Moretzsohn 4/13/17 ? Gulf of Mexico Port Aransas 27.794651 -97.083228 Collected by F. Moretzsohn 6/16/17 ? Corpus Christi Bay N/A 27.824958 -97.319641 Collected by F. Moretzsohn 7/23/17 19:02 Gulf of Mexico PCB06 (C-IMAGE) 29.12283 -87.26572 C-Image sample from 995 m, coll. by M. Rohal 7/28/17 0:18 Gulf of Mexico DSH08 (C-IMAGE) 29.12175 -87.86792 C-Image sample from 1120 m, coll. by M. Rohal 7/29/17 20:26 Gulf of Mexico GP03 (C-IMAGE) 28.74727 -89.19283 C-Image sample; collected by M. Rohal 7/30/17 ? Gulf of Mexico SL16150 (C-IMAGE) 28.62718 -89.98500 C-Image sample from 242 m, coll. by M. Rohal 12/6/17 23:52 Gulf of Mexico SW01 (C-IMAGE) 28.23900 -89.12097 C-Image sample from 1125 m, coll. by M. Rohal 11 12/7/17 ? Corpus Christi Bay University Beach 27.715434 -97.321834 Collected by F. Moretzsohn

Figure 4. Alberto de Jesús Navarrete teaching Diana Marlen Ugalde Garcia, Lilian Abigaid Palomino Álvarez, and Will Ballentine about nematodes. (Photo credit: Larry Hyde)

Nematodes were transferred to vials with deionized water and frozen at -80˚C. Stereo and compound microscopes with attached cameras were used to produce voucher images (Fig. 5).

Francesca Leasi performed Ludox extraction of benthic meiofauna (Fig. 6). This technique facilitates the quick separation of sediment from organisms. The extracted organisms were then stored for metagenomics analyses. Francesca would frequently explain and demonstrate the different steps of the extraction method to workshop participants.

Taxonomic Seminars

The taxonomists provided comprehensive overviews over the taxa of their expertise during twenty- minute long presentations (Fig. 7). The covered taxa included Bivalvia (Fabio Moretzsohn), Cumacea (Richard Heard and Adelaide Rhodes), Gastropoda (Fabio Moretzsohn), Gnathostomulida (Martin Sørensen), Harpacticoida (Wonchoel Lee), Kinorhyncha (Martin

Figure 6. Francesca Leasi demonstrating Figure 5. Alessandro Prantoni taking Ludox extraction to Hannah Ehrmann and micrographic pictures. (Phot credit: Larry Doug Faircloth (Photo credit: Lilian Abigaid Hyde) Palomino Álvarez)

Sørensen), Nematoda (Alberto de Jesús Navarrete and Tiago Pereira), Oligochaeta (Alessandro Prantoni), Polychaeta (Michael Reuscher), and Tanaidacea (Richard Heard and Adelaide Rhodes) (Table 5). The speakers elaborated on morphology, current taxonomic status and systematic classification, ecology, and natural history of the different taxa. Francesca Leasi gave a lecture introducing background, goals, and methods of the GoMRI project “Genomic Responses to the Deepwater Horizon Event and Development of High-Throughput Biological Assays for Oil Spills”.

Bioinformatics Workshops

The bioinformatics workshops were a combination of lectures, practical computer exercises, and discussions, led by the bioinformatics teams of the University of New Hampshire (Kelley Thomas, Joseph Sevigny, and Devin Thomas) and the University of California, Riverside (Holly Bik, Taruna Schuelke). The workshop participants were introduced to metagenomics, metabarcoding,

Table 5. List of presentations of the daily taxonomic seminars. Presenter Presentation title Richard Heard Cumacea and Tanaidacea Alberto de Jesús Navarrete The ecology of marine nematodes Genomic Responses to the Deepwater Horizon event and development of high- Francesca Leasi throughput biological assays for oil spills Wonchoel Lee Diversity of Copepoda Fabio Moretzsohn Bivalvia and Gastropoda of the Gulf of Mexico The Phylum Nematoda: phylogeny, systematics, and taxonomy with an Tiago Pereira emphasis on marine nematodes Alessandro Prantoni Overview of marine clitellates / oligochaetes Michael G. Reuscher Polychaeta Biodiversity of Tanaidacea (Crustacea: Peracarida) in the Northern Gulf of Adelaide Rhodes Mexico Martin V. Sørensen Gnathostomulida and Kinorhyncha

Figure 7. Alessandro Prantoni presenting on Oligochaeta. (Photo credit: Larry Hyde)

high-throughput sequencing, genome assembly and annotation, and whole metagenome shotgun analysis (Fig. 8, Table 6). Workshop participants were able to accomplish practical exercises on their own computers, to acquire and practice skills in the Unix command language BASH and the QIIME 2 pipeline, both essential open-source software tools for the analyses of genomic sequence data.

After the daily plenary seminars, workshop participants split into smaller groups to further practice, discuss, and troubleshoot the covered topics among themselves and with the bioinformatics experts.

Figure 8. Workshop participants during the daily bioinformatics seminar. (Phot credit: Larry Hyde)

Outcome and Outlook

A total of 290 benthic organisms were identified and preserved for genomic sequencing at the Smithsonian Institution National Museum of Natural History (Table 7). They belong to at least 97 different taxa and this number is likely to increase as the identification of the oligochaetes is

Table 6. Overview of topics covered during the bioinformatics workshop Activity Type Activity details Facilitators Introduction to the principles of metabarcoding and metagenomics; Introduction to Genomics, Bioinformatics: Kelley Thomas Lecture Generating data via high-throughput sequencing; data structures and DNA sequencing and Assembly of Genomes and Holly Bik conventions Anatomy of a Command; Finding Help; Pathways and Directories; Introduction to the command line Lecture/Practical Moving About; Move, Copy and Delete; Odds and Ends; Using Nano; UNH team environment and the world of BASH Viewing Files (less is more). Bash (Continued) Grep; Redirecting Standard Out; Wildcards and Bash and Command line tricks Lecture/Practical Globbing; Variables and parameter substitution; .Bashrc; File UNH team permissions; Shell Scripts; Nohup removing adaptors and quality filtering with Trimmomatic: Data quality Genome assembly and annotation Lecture/Practical control; Assembling a genome with SPADeS; generating assembly UNH team metrics with QUAST; annotating a genome with Prokka Discussion and Q&A session about Genome Discussion Kelley Thomas Assembly results and data analysis Overview of changes in QIIME 1 vs QIIME 2 pipelines, introduction to Dr. Holly Bik Intro to the QIIME 2 pipeline for microbial Lecture/Practical data QA/QC – dada2 quality filtering, read merging, etc., taxonomy and UCR &

ecology 16 assignment steps, downstream alpha/beta diversity analyses UNH teams Discussion and Q&A session about QIIME Discussion Holly Bik results and data analysis Metabarcoding (amplicon) and workflows, analyzing eukaryotic data (e.g. 18S), visualizing data and interpreting QIIME results, expanded UCR & UNH QIIME 2 analysis continued Lecture/Practical community analyses and visualization tools in R (phyloseq, metacoder, teams etc.) Mapping DNA sequence reads to reference UNH team and genomes to investigate genetic diversity or Lecture/Practical Kelley Thomas profiling gene expression Genome visualizations using UCSC Genome UNH team and Lecture/Practical Browser Kelley Thomas Introduction to Whole Metagenome Shotgun Kelley Thomas Lecture Analysis and Holly Bik

currently still in progress. Specimens belonged to the higher taxa Amphipoda, Bivalvia, Branchiura, Decapoda, Gastropoda, Gnathostomulida, Harpacticoida, Isopoda, Kinorhyncha, Nematoda, Oligochaeta, Platyhelminthes, Polychaeta, and Tanaidacea. The majority of the identified taxa will be sequenced for the first time and as such, they will be important additions to the benthic metagenomics reference library.

Compared to the first BITMaB workshop, the number of identified specimens more than doubled and the number of different taxa increased considerably. One of the main reasons for the increased productivity was the addition of another sampling week prior to the workshop. While most of the taxonomists attended the Bioinformatics seminars and interacted with workshop participants during the workshop, they were able to focus entirely on sample collection and identification of benthic organisms in the week prior. Additionally, samples during BITMaB-2 were more diverse than the year before. In 2017, all samples were collected from shore with shovels and buckets. In 2018, more bays were sampled and we were able to collect samples from deeper areas of the bays from a boat. The deep-sea and marsh samples, as well as the Fabio Moretzsohn’s mollusks from miscellaneous locations added further to sample diversity.

Workshop participants received training in all the techniques necessary to perform metagenomics analysis. This included sample processing in the wet lab, such as meiofauna and macrofauna extraction from the sediment through stacked sieve sample washing. In contrast to the first BITMaB workshop, there was a lab station dedicated to Ludox extraction, which sparked lots of interest from workshop participants. Taxonomic insights into some of the most important marine benthic taxa were provided in the lab, where workshop participants were given the opportunity to interact with the taxonomic experts, as well as through the daily taxonomic seminars. The bioinformatics seminars conveyed crucial background information and for the analysis of next- generation sequencing and metagenomics data, as well as programming skills for the BASH and QIIME 2 platforms.

BITMaB-2 concludes the workshop series of the GoMRI project “Genomic Responses to the Deepwater Horizon Event and Development of High-Throughput Biological Assays for Oil Spills”. However, we hope that we can continue to offer these workshops in the future to advance methodologies and infrastructure of benthic metagenomics analyses, and to educate the new generation of scientists in all necessary techniques.

Table 7. List of identified specimens for genetic analysis. Taxon Species ID Count Notes Taxonomist Sample Site Date (Y-M-D) Amphipoda Caprellidae gen. sp. 3 Rhodes Nueces Boat Launch 2018-01-10 Amphipoda Cymadusa compta 3 Heard Kennedy Causeway 2018-01-12 Amphipoda Deutella meerta 2 Heard Kennedy Causeway 2018-01-12 Amphipoda Grandidierella bonnieroides 1 Heard Kennedy Causeway 2018-01-12 Amphipoda Haustoriidae gen. sp. 1 Heard ? ? Amphipoda Melphidipidae gen. sp. 1 Rhodes PCB06 2017-07-23 Amphipoda Paracaprella sp. 1 Genus ID uncertain Heard Nueces Boat Launch 2018-01-10 Aplacophora Prochaetodermatidae gen. sp. 4 Moretzsohn DSH08 2017-07-28 Bivalvia Amygdalum sp. 1 Juvenile, genus ID uncertain Moretzsohn Kennedy Causeway 2018-01-16 Bivalvia Brachidontes domingensis 2 Cracked the shell Moretzsohn Kennedy Causeway 2018-01-12 Bivalvia Crassostrea virginica 3 Tissue clip for DNA extraction Moretzsohn Kennedy Causeway 2018-01-12 Bivalvia Cyrtopleura costata 1 Tissue clip for DNA extraction Moretzsohn TAMUCC Beach 2017-12-07

Bivalvia Donax variabilis 3 Heard Bob Hall Pier 2018-01-13

18 Bivalvia Unidentified 1 Moretzsohn PCB06 2017-07-23 Branchiura Argulus sp. 1 Heard ? ? Decapoda Austinixa behreae 1 Heard ? ? Decapoda Callichirus islagrande 2 Heard Bob Hall Pier 2018-01-13 Decapoda Emerita cf. talpoida 1 Heard ? ? Decapoda Palaemonetes sp. 3 Juvenile Heard Kennedy Causeway 2018-01-12 Gastropoda Astyris lunata 1 Moretzsohn Kennedy Causeway 2018-01-16 Gastropoda Batillaria minima 3 Heard Kennedy Causeway 2018-01-12 Gastropoda Batillaria minima 1 Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Batillaria minima 1 Moretzsohn Kennedy Causeway 2018-01-16 Gastropoda Batillaria minima 2 Juvenile Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Batillaria minima 1 Tubeworm on the shell Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Berghia sp. 1 Moretzsohn Kennedy Causeway 2018-01-16 Gastropoda Bittiolum varium 3 Heard Kennedy Causeway 2018-01-12

Table 7. Continued Taxon Species ID Count Notes Taxonomist Sample Site Date (Y-M-D) Gastropoda Boonea impressa 3 Cracked the shell Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Cerithium eburneum 5 Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Crepidula convexa 3 Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Crepidula cf. fornicata 1 Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Crepidula fornicata 2 Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Echinolittorina hawaiiensis 1 Moretzsohn Salt Pond Beach 2016-12-30 Gastropoda Echinolittorina placida 1 Moretzsohn Indian Point 1990-09-12 Gastropoda Echinolittorina placida 1 Moretzsohn McGee Beach jetties 1981-07-11 Gastropoda Echinolittorina placida 1 Moretzsohn Port Aransas jetties 1991-09-16 Gastropoda Janthina globosa 1 Moretzsohn Port Aransas 2014-04-13 Gastropoda Janthina janthina 1 Moretzsohn Port Aransas 2014-04-13 Gastropoda Littoraria pintado 1 Moretzsohn Salt Pond Beach 2016-12-30

19 Gastropoda Nerita picea 1 Moretzsohn Salt Pond Beach 2016-12-30

Gastropoda Neritina virginea 3 Moretzsohn Kennedy Causeway 2018-01-12 Gastropoda Neritina virginea 1 Moretzsohn Kennedy Causeway 2018-01-16 Gastropoda Peasiella tantilla 1 Tissue clip for DNA extraction Moretzsohn Salt Pond Beach 2016-12-30 Gastropoda Scyllaea pelagica 1 Moretzsohn Port Aransas 2014-04-13 Gastropoda Texadina sphinctostoma 2 Moretzsohn GE-A 2018-01-12 Gastropoda Unidentified 1 Juvenile Moretzsohn Kennedy Causeway 2018-01-12 Gnathostomulida Haplognathia ruberrima 1 Sørensen Redfish Bay Sta.1 2018-01-18 Harpacticoida Echinolaophonte armiger 14 Extracted from algae rinsing Lee Kennedy Causeway 2018-01-16 Harpacticoida Harpacticus obscurus 21 Extracted from algae rinsing Lee Kennedy Causeway 2018-01-16 Harpacticoida Metis holothuria 3 Rhodes NC-E 2018-01-05 Harpacticoida Miraciidae gen. sp. 4 Extracted from algae rinsing Lee Kennedy Causeway 2018-01-16 Harpacticoida Pseudostenhelia wellsi 7 Lee RB-C 2015-11-03 Isopoda Cymothoa exigua 1 Moretzsohn Corpus Christi Bay 2016-12-30 Isopoda Dynomella angulata 3 Rhodes Nueces Boat Launch 2018-01-10

Table 7. Continued Taxon Species ID Count Notes Taxonomist Sample Site Date (Y-M-D) Kinorhyncha Echinoderes augustae 3 Sørensen NC-MK60 2018-01-05 Kinorhyncha Echinoderes bookhouti 1 Sørensen NC-E 2018-01-05 Kinorhyncha Echinoderes spinifurca 3 Sørensen NC-E 2018-01-05 Kinorhyncha Echinoderes spinifurca 1 Sørensen Redfish Bay Sta.1 2018-01-18 Kinorhyncha Echinoderes sp. 3 Sørensen GE-B 2018-01-09 Kinorhyncha Leiacanthus langi 1 Sørensen NC-E 2018-01-05 Kinorhyncha Leiacanthus langi 1 Sørensen Redfish Bay Sta.1 2018-01-18 Kinorhyncha Leiacanthus langi 1 Sørensen Redfish Bay Sta.2 2018-01-18 Kinorhyncha Pycnophyidae gen. sp. 1 Sørensen NC-MK60 2018-01-05 Nematoda Adoncholaimus sp. 1 Navarrete Redfish Bay Sta.1 2018-01-18 Nematoda Adoncholaimus sp. 1 Navarrete DSH08 2017-12-06 Nematoda Adoncholaimus sp. 1 Photo voucher Navarrete SW01 2017-07-28

Nematoda Adoncholaimus sp. 1 Male; photo voucher Pereira East Beach 2018-01-10

20 Nematoda Adoncholaimus sp. 1 Photo voucher Pereira NC-E 2018-01-05 Nematoda Cephalanticoma sp. 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Cheironchus sp. 1 Photo voucher Pereira NC-E 2018-01-05 Nematoda Chromadorella sp. 2 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Daptonema sp. 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Daptonema sp. 1 Navarrete Nueces Boat Launch 2018-01-10 Nematoda Daptonema sp. 2 Navarrete Redfish Bay Sta.1 2018-01-18 Nematoda Daptonema sp. 3 Photo voucher Pereira East Beach 2018-01-10 Nematoda Daptonema sp. 1 Pereira East Beach 2018-01-10 Nematoda Daptonema sp. 2 Photo voucher Pereira NC-E 2018-01-05 Nematoda Daptonema sp. 1 Male; photo voucher Pereira RB-G 2018-01-15 Nematoda Daptonema sp.1 3 Navarrete Nueces Boat Launch 2018-01-10 Nematoda Daptonema sp.2 1 Navarrete Bob Hall Pier 2018-01-09 Nematoda Daptonema sp.2 1 Photo voucher Navarrete Bob Hall Pier 2018-01-09

Table 7. Continued Taxon Species ID Count Notes Taxonomist Sample Site Date (Y-M-D) Nematoda Daptonema sp.2 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Daptonema sp.2 1 Navarrete Nueces Boat Launch 2018-01-10 Nematoda Daptonema sp.3 1 Navarrete East Beach 2018-01-10 Nematoda Daptonema sp.3 1 Photo voucher Navarrete SW01 2017-12-06 Nematoda Daptonema sp.3 2 Navarrete SW01 2017-12-06 Nematoda Desmodora sp. 1 Photo voucher Pereira NC-E 2018-01-05 Nematoda Desmoscolex sp. 1 Navarrete DSH08 2017-07-28 Nematoda Desmoscolex sp. 1 Photo voucher Navarrete SW01 2017-12-06 Nematoda Desmoscolex sp. 1 Navarrete SW01 2017-12-06 Nematoda Dichromadora sp. 1 Navarrete East Beach 2018-01-10 Nematoda Dichromadora sp. 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Dichromadora sp. 1 Navarrete Nueces Boat Launch 2018-01-10

21 Nematoda Dichromadora sp. 1 Photo voucher Pereira Bob Hall Pier 2018-01-09

Nematoda Dichromadora sp. 1 Photo voucher Pereira East Beach 2018-01-10 Nematoda Diplopeltula sp. 1 Photo voucher Navarrete SW01 2017-12-06 Nematoda Diplopeltula sp. 1 Navarrete Redfish Bay Sta.2 2018-01-18 Nematoda Diplopeltula sp. 1 Navarrete ? ? Nematoda Dorylaimopsis sp. 1 Navarrete East Beach 2018-01-10 Nematoda Dorylaimopsis sp. 1 Navarrete Redfish Bay Sta.1 2018-01-18 Nematoda Dorylaimopsis sp. 1 Navarrete Redfish Bay Sta.2 2018-01-18 Nematoda Dorylaimopsis sp. 3 Female Navarrete Sl1650 2017-07-30 Nematoda Dorylaimopsis sp. 2 Male Navarrete Sl1650 2017-07-30 Nematoda Dorylaimopsis sp. 1 Photo voucher Pereira Redfish Bay Sta.3 2018-01-18 Nematoda Halalaimus sp. 1 Genus ID uncertain; photo voucher Pereira ? ? Nematoda Linhomoeus? 1 Genus ID uncertain Navarrete Redfish Bay Sta.1 2018-01-18 Nematoda Linhomoeus? 1 Genus ID uncertain; female Navarrete Sl1650 2017-07-30 Nematoda Longicyatholaimus sp. 1 Photo voucher Pereira NC-E 2018-01-05

Table 7. Continued Taxon Species ID Count Notes Taxonomist Sample Site Date (Y-M-D) Nematoda Mesacanthion? sp. 1 Genus ID uncertain; photo voucher Pereira Bob Hall Pier 2018-01-09 Nematoda Mesacanthion? sp. 1 Genus ID uncertain Pereira Bob Hall Pier 2018-01-09 Nematoda Metachromadora sp. 1 Photo voucher Pereira East Beach 2018-01-10 Nematoda Monhystera sp. 1 Navarrete DSH08 2017-07-28 Nematoda Neochromadora sp. 1 Navarrete Bob Hall Pier 2018-01-09 Nematoda Neochromadora sp. 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Neochromadora sp. 1 Photo voucher Pereira Bob Hall Pier 2018-01-09 Nematoda Odontophora sp. 1 Male; photo voucher Navarrete Bob Hall Pier 2018-01-09 Nematoda Oncholaimidae gen. sp. 1 Pereira Bob Hall Pier 2018-01-09 Nematoda Onyx sp. 1 Female; photo voucher Navarrete Bob Hall Pier 2018-01-09 Nematoda Paramononhystera? sp. 2 Genus ID uncertain; photo voucher Pereira East Beach 2018-01-10 Nematoda Pomponema sp. 1 Photo voucher Navarrete Bob Hall Pier 2018-01-09

Nematoda Pomponema sp. 1 Male; photo voucher Pereira Bob Hall Pier 2018-01-09

22 Nematoda Promonhystera sp. 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Nematoda Psileonema sp. 1 Photo voucher Pereira PCB06 2017-07-23 Nematoda Rhynchonema sp. 1 Photo voucher Pereira Bob Hall Pier 2018-01-09 Nematoda Sabatieria sp. 1 Photo voucher Navarrete SW01 2017-12-06 Nematoda Setosabatieria sp. 1 Navarrete Redfish Bay Sta.2 2018-01-18 Nematoda Siphonolaimus sp. 1 Female Navarrete Sl1650 2017-07-30 Nematoda Sphaerolaimus sp. 1 Female Navarrete Sl1650 2017-07-30 Nematoda Sphaerolaimus sp. 1 Photo voucher Navarrete SW01 2017-12-06 Nematoda Sphaerolaimus sp. 1 Photo voucher Pereira SW01 2017-12-06 Nematoda Sphaerolaimus sp. 2 Pereira SW01 2017-12-06 Nematoda Synodontium sp. 1 Photo voucher Pereira East Beach 2018-01-10 Nematoda Synodontium sp. 1 Pereira East Beach 2018-01-10 Nematoda Synonchiella sp. 1 Photo voucher Pereira Bob Hall Pier 2018-01-09 Nematoda Syringolaimus sp. 1 Photo voucher Pereira PCB06 2017-07-23

Table 7. Continued Taxon Species ID Count Notes Taxonomist Sample Site Date (Y-M-D) Nematoda Terschellingia sp. 2 Photo voucher Pereira East Beach 2018-01-10 Nematoda Theristus sp. 1 Photo voucher Pereira Bob Hall Pier 2018-01-09 Nematoda Theristus sp. 5 Photo voucher Pereira RB-C 2015-11-03 Nematoda Thoracostomopsidae gen. sp. 1 Photo voucher Pereira ? ? Nematoda Viscosia sp. 3 Navarrete Redfish Bay Sta.1 2018-01-18 Nematoda Viscosia sp.1 2 Photo voucher Pereira East Beach 2018-01-10 Nematoda Viscosia sp.2 2 Photo voucher Pereira East Beach 2018-01-10 Nematoda Viscosia sp.3 1 Photo voucher Navarrete Nueces Boat Launch 2018-01-10 Posterior end for DNA extraction; Oligochaeta Identification in progress 9 Prantoni Lost Island 2018-01-09 specimen kept as voucher Posterior end for DNA extraction; Oligochaeta Identification in progress 15 Prantoni Oso Bridge (CC Bay) 2018-01-11 specimen kept as voucher Posterior end for DNA extraction; Oligochaeta Identification in progress 1 Prantoni Oso Bridge (Oso Bay) 2018-01-11 specimen kept as voucher 23 Platyhelminthes Unidentified 2 Moretzsohn Kennedy Causeway 2018-01-12

Posterior end for DNA extraction; Polychaeta Alitta sp. 1 specimen kept as voucher; photo Reuscher Lost Island 2018-01-09 voucher Polychaeta Alitta sp. 1 Reuscher Lost Island 2018-01-09 Tissue from palps and parapodia Polychaeta Alitta sp. 1 for DNA extraction; specimen kept Reuscher Matagorda Bay 2018-01-11 as voucher Posterior end for DNA extraction; Polychaeta Lumbrineris sp. 1 Reuscher Bob Hall Pier 2018-01-09 specimen kept as voucher Posterior end for DNA extraction; Polychaeta Mediomastus californiensis 1 Reuscher Oso Bridge (CC Bay) 2018-01-11 specimen kept as voucher Posterior end for DNA extraction; Polychaeta Syllis sp. 1 Reuscher Oso Bridge (CC Bay) 2018-01-11 specimen kept as voucher Tanaidacea Agathotanais hanseni 1 Rhodes SW01 2017-12-06 Tanaidacea Hargeria rapax 5 Female Rhodes Baffin Bay 2018-01-15 Tanaidacea Hargeria rapax 1 Male Rhodes Baffin Bay 2018-01-15