(Stsm) Scientific Report
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SHORT TERM SCIENTIFIC MISSION (STSM) SCIENTIFIC REPORT This report is submitted for approval by the STSM applicant to the STSM coordinator Action number: CA15219-45333 STSM title: Free-living marine nematodes from the eastern Mediterranean deep sea - connecting COI and 18S rRNA barcodes to structure and function STSM start and end date: 06/02/2020 to 18/3/2020 (short than the planned two months due to the Co-Vid 19 virus pandemic) Grantee name: Zoya Garbuzov PURPOSE OF THE STSM: My Ph.D. thesis is devoted to the population ecology of free-living nematodes inhabiting deep-sea soft substrates of the Mediterranean Levantine Basin. The success of the study largely depends on my ability to accurately identify collected nematodes at the species level, essential for appropriate environmental analysis. Morphological identification of nematodes at the species level is fraught with difficulties, mainly because of their relatively simple body shape and the absence of distinctive morphological characters. Therefore, a combination of morphological identification to genus level and the use of molecular markers to reach species identification is assumed to provide a better distinction of species in this difficult to identify group. My STSM host, Dr. Nikolaos Lampadariou, is an experienced taxonomist and nematode ecologist. In addition, I will have access to the molecular laboratory of Dr. Panagiotis Kasapidis. Both researchers are based at the Hellenic Center for Marine Research (HCMR) in Crete and this STSM is aimed at combining morphological taxonomy, under the supervision of Dr. Lampadariou, with my recently acquired experience in nematode molecular taxonomy for relating molecular identifiers to nematode morphology. Two newly-designed nematodes highly universal SSU rRNA (18S) primers are planned to be used to amplify species-specific barcodes from single morphologically identified individuals sampled from the Israeli deep-sea. DESCRIPTION OF WORK CARRIED OUT DURING THE STSMS Preparation of permanent and temporary nematode slides – Aimed at establishing a collection of morphologically identified individuals as a reference for future identification. Several nematodes were picked up using a special needle and transferred into a drop of glycerol encircled with a paraffin ring, on a microscopy slide covered with a coverslip. Mildly heating of the slide melted the paraffin and glued the coverslip to the slide to create a permanent slide. A map of the individual’s location on each slide was prepared. 11 slides were prepared which contained in total 124 nematodes. Ninety-five temporary individual nematode slides served for identification and the specimens were or will be used later for the characterization of species-specific molecular markers (see below). Morphological identification Nematodes were identified with a Nomarski interference contrast (NIC) microscope under a X100 magnification considering the following morphological features: 1. Type of cuticle (smooth, annulated by punctuation or dots); 2. The shape and characteristics of the buccal cavity (large, minute, unarmed or armed tooth or jaw); 3. The number and position of the head sensilla or seta on the head; 4. The form of amphids (pocket, loop-shaped, round, spiral, etc.); 5. Spicular apparatus for the male; 6. The shape of the tail (short and round, conical, clavate, elongated or filiform); 7. The presence or absence of the preclocal supplements and their form; 8. The a, b and c De Mans morphometrical ratios. The Illustrated keys of Platt and Warwick (1983, 1988), Warwick et al. (1998), relevant taxonomic literrature from the Mediterranean and the NeMys web database were used for identification. A unique determined assembly of the above traits served for the identification of each genus. Both permanent and temporary slides were used for the identification. Molecular identification Two tasks were performed at the molecular laboratory of Dr. Panagiotis Kasapidis. 1. Extraction of DNA from single individuals using in-house prepared lysis buffer composed of 50 mM KCl, 10 mM Tris, pH 8.3, 2.5 mM MgCl2, 0.45% NP-40 (tergitol) and 0.45% Tween 20. Genomic DNA of individual nematodes was extracted in 20 μl lysis buffer reinforced with proteinase K [10 mg/ml]. The extraction solution was heated to 65°C for 60 minutes followed by 2 reaction termination at 95°C for 10 minutes, cooling to room temperature and spinning down of residuals at maximum speed for 1 min. The lysates were stored at -20°C and used later as PCR DNA templates. 2. Broadly universal 18S primer pair was previously designed by me, brought to Crete and was used to PCR amplify an approximately 550 bp amplicon. The applied PCR conditions were: 95°C – 2 min; 40 cycles (94°C – 30 sec, 54°C – 30 sec, 72°C – 30 sec); 72°C – 7 min. I intended also to sequence the amplicons in Crete but the Corona crisis-related shortening of the STSM prevented this task to be completed. DESCRIPTION OF THE MAIN RESULTS OBTAINED A number of 219 individual nematodes were morphologically identified from the permanent and the temporary slides to 59 genera belonging to 7 orders and 22 families. The individuals were selected to represent samples from a variety of depths covering the entire study province. The 95 individuals from the temporary slides will be used for further molecular work comparing the two nematode identification methods. Table 1 – The revealed genera Class Order Family Genus Chromadorea Chromadorida Chromadoridae Acantholaimus Chromadorea Chromadorida Chromadoridae Actionema Chromadorea Plectida Aegialoalaimidae Aegialoalaimus Chromadorea Monhysterida Xyalidae Amphimonhysrella Enoplea Enoplida Enchelidiidae Bathyeurystomina Enoplea Enoplida Enchelidiidae Belbolla Chromadorea Chromadorida Chromadoridae Chromadorella Enoplea Enoplida Phanodermatidae Crenopharynx Chromadorea Monhysterida Xyalidae Daptonema Chromadorea Desmodorida Desmodoridae Desmodora Chromadorea Desmoscolecida Desmoscolecidae Desmoscolex Chromadorea Chromadorida Chromadoridae Dichromadora Chromadorea Araeolaimida Diplopeltidae Diplopeltula Enoplea Enoplida Ironidae Dolicholaimus Chromadorea Monhysterida Sphaerolaimidae Doliolaimus 3 Chromadorea Araeolaimida Comesomatidae Dorylaimopsis Chromadorea Monhysterida Xyalidae Elzalia Chromadorea Chromadorida Chromadoridae Endeolophos Chromadorea Chromadorida Neotonchidae Filitonchus Chromadorea Chromadorida Selachinematidae Gammanema Chromadorea Monhysterida Xyalidae Gnomoxyala Enoplea Enoplida Oxystominidae Halalaimus Chromadorea Plectida Haliplectidae Haliplectus Chromadorea Chromadorida Chromadoridae Hypadontholaimus Chromadorea Araeolaimida Comesomatidae Laimella Chromadorea Monhysterida Linhomoeidae Linhomoeus Chromadorea Chromadorida Cyatholaimidae Longicyatholaimus Chromadorea Chromadorida Cyatholaimidae Marylynnia Chromadorea Desmodorida Desmodoridae Metachromadora Chromadorea Chromadorida Cyatholaimidae Metacyatholaimus Chromadorea Plectida Ceramonematidae Metadasynemella Chromadorea Monhysterida Linhomoeidae Metalinhomoeus Chromadorea Monhysterida Sphaerolaimidae Metasphaerolaimus Chromadorea Desmodorida Microlaimidae Microlaimus Chromadorea Araeolaimida Comesomatidae Minolaimus Chromadorea Desmodorida Desmodoridae Molgolaimus Chromadorea Chromadorida Neotonchidae Neotonchus Chromadorea Monhysterida Xyalidae Omicronema Enoplea Enoplida Oxystominidae Oxystomina Chromadorea Chromadorida Cyatholaimidae Paracyatholaimus Chromadorea Monhysterida Linhomoeidae Paralinhomoeus Chromadorea Plectida Paramicrolaimidae Paramicrolaimus Chromadorea Monhysterida Xyalidae Paramonhystera Enoplea Enoplida Enchelidiidae Pareurystomina Chromadorea Araeolaimida Comesomatidae Pierrickia Chromadorea Chromadorida Cyatholaimidae Pomponema 4 Chromadorea Plectida Ceramonematidae Pselionema Chromadorea Desmodorida Desmodoridae Pseudonchus Chromadorea Monhysterida Xyalidae Retrotheristus Chromadorea Araeolaimida Comesomatidae Sabatieria Chromadorea Monhysterida Xyalidae Scaptrella Chromadorea Araeolaimida Comesomatidae Setosabatieria Chromadorea Monhysterida Sphaerolaimidae Sphaerolaimus Enoplea Enoplida Rhabdolaimidae Syringolaimus Chromadorea Monhysterida Monhysteridae Thalassomonhystera Chromadorea Monhysterida Linhomoeidae Thershelingia Chromadorea Desmoscolecida Desmoscolecidae Tricoma Chromadorea Araeolaimida Comesomatidae Vasostoma Enoplea Enoplida Oncholaimidae Viscosia The most common orders were Chromadorida and Monhysterida and the prevailed families were Xyalidae, Comesomatidae and Chromadoridae. The DNA extraction buffer and the in-house designed primers were initially tested on 16 randomly selected individuals, before application on the 95 identified individuals. The DNA extracts were used to PCR amplify the ~550 bp amplicon from the 18S rRNA. Out of the 16 templates, 14 successfully amplified amplicons were detected on an agarose gel. ~550 pb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 n/c Fig. 1 - PCR amplification of the 18S amplicon. For further processing of the results, this data will be sent for sequencing by a service laboratory. The created species-specific sequences are aimed to serve as a reference molecular taxonomy library and will have to completely overlap the high throughput sequencing (HTS) of samples. 5 Therefore, all the future sequencing, including that of single individuals will be performed by the Illumina HTS method. Unfortunately, the STSM was prematurely terminated because of air-traffic closure in Greece due to the Co-Vid19 pandemic. However, a solid basis was laid for the combined morphological- molecular identification