INTRODUCTION
The opening of the Suez Canal in 1869 initiated a process of biological invasions from the Red Sea into the Mediterranean, an event commonly known as Lessepsian migration (Por, 1971; Golani 2010). This influx of marine organisms has greatly impacted the local communities in ecological, evolutionary (Sax et al. 2007), and economical terms (Arim et al. 2006). Lessepsian fishes comprise a significant percentage of all recorded invasive species in the Mediterranean Sea (Zenetos et al. 2012) and may be causing several indigenous species displacements (Golani 2010). Lessepsian migration, having both direct and indirect human-driven origins, is a phenomenon that offers a unique opportunity for studying fast evolutionary change (Palumbi 2001).
One of the most successful Lessepsian migrant species is the silver-cheeked toadfish, Lagocephalus sceleratus (Gmelin 1789), a member of the Tetraodontidae family (called puffers), widely distributed throughout the Indian and Pacific Oceans (Akyol et al. 2005). The first record of L. sceleratus invasion in the Mediterranean Sea was reported in 2003 in the Gökova Bay, in the south-eastern Aegean Sea coast of Turkey (Filiz and Er, 2004) and two years later in the North Cretan Sea (Kasapidis et al. 2007). Since then, is has spread rapidly throughout the entire Levant, Aegean and Ionian Seas (Akyol & Ünal, 2017; Kalogirou, 2013).
The survival and dispersal rate of alien species into novel habitats are dependent on multiple factors including encountering previously unknown pathogens. Thus, the invasion success may be affected by the effectiveness of the immune response (Lee and Klasing, 2004 Kolar and Logde, 2001) rendering the gene families involved in the adaptive immune response potentially under positive selection.
Lagocephalus sceleratus is a highly toxic species, which along with other pufferfishes, contains in different tissues, a toxic organic compound, tetrodotoxin (TTX) (Chua and Chew, 2009; Kosker et al., 2016). TTX is accumulated in high concentration in ovary, liver and intestine of L. sceleratus individuals, while lower amounts have been detected in skin and muscle tissues (Akbora et al., 2019). TTX is the metabolic product of TTX-producing bacteria and is retained in the fish tissues. However, the mechanisms of absorption and the differential concentration in the tissues and the organs of pupperfishes remain unclear (Nagashima and Arakawa, 2016). TTX toxicity is due to its binding to the outer pore of the NaV1.4 (SCN4) channel, blocking the transport of sodium ions across the pore (Hanifin 2010). Multiple pufferfishes have been shown to deploy TTX resistance through mutations in specific domains of NaV1.4, (Venkatesh et al., 2005, Soong and Venkatesh 2006, Jost et al., 2008).
In this study, we provide and analyse the first high-quality genome assembly of L. sceleratus . To that end, we combine Illumina and Oxford Nanopore Technology (ONT) reads to construct a highly contiguous assembly, which allows us to explore the genomic landscape of this successful invader and study the genetic background of TTX-resistance in the species. This valuable and robust genome resource will facilitate future studies on the ecology, evolution and potential exploitation of this invasive species.
MATERIALS & METHODS
Sample collection & sequencing
Animal care and handling were carried out following well established guidelines [Guidelines for the treatment of animals in behavioral research and teaching. Anim. Behav. 53, 229–234 (1997)].
One female fish (58 cm in length) was caught alive in Agios Georgios (Hersonissos), Crete, Greece (35°20’07.50”N 25°23’11.30”E) at the pre-spawning/spawning stage (as determined by stereoscopic investigation of the oocytes) and was anesthetized using clove oil. In total, 10 mL of blood was collected using a sterilized syringe and stored in tubes that contained ~1/10 of volume heparin for subsequent DNA extraction.
DNA extraction for the purpose of ONT sequencing was conducted on the day of sampling, from 2 μl of the freshly collected blood, using Qiagen Genomic tip (20G) and following ONTs protocol for DNA extraction from chicken blood. The final elution was made with 50 μl AE buffer providing 90,4 ng/μl (Qubit measurement) of high purity, high molecular weight DNA (Nanodrop ratios: 260/280 = 1,87 & 260/230 = 2,12). DNA integrity was assessed by electrophoresis in 0.4 % w/v Bio-Rad Megabase agarose gel. We constructed four ligation libraries (SQK-LSK109) following the manufacturer’s instructions. Approximately 1.2 μg of unsheared DNA was used for each library. Two of the prepared libraries were divided into two aliquots. Each library was run for approximately 24 hours on a MinION sequencer at HCMR, after which the ONT nuclease flush protocol was performed, and a fresh library or library aliquot was loaded onto the same R9.4.1 flow cell. The total run time was ~130 hours. Basecalling was done with Guppy v3.2.4 (https://community.nanoporetech.com/posts/guppy-3-2-4-release”) in High Accuracy Mode with minimum quality score 7.
For Illumina sequencing, we proceeded with two-days old, refrigerated blood samples using the same DNA extraction protocol as above. We used 4 μl of blood eluted in 100 μl AE buffer which resulted in 79,2 ng/μl (Qubit measurement) of pure DNA (260/280 = 1,85 & 260/230 = 2,21).
Template DNA for Illumina sequencing was sheared by ultrasonication in a Covaris S220 instrument. A PCR-free library was prepared with the Kapa Hyper Prep DNA kit with TruSeq Unique Dual Indexing. Paired end 2x150 bp sequencing was performed on an Illumina Hiseq4000 platform.
Tissue samples from brain, gonad, skin, liver, spleen and muscle of the same female individual, were grounded and powdered using pestle and mortar under liquid nitrogen, and except the spleen tissue were all homogenized in TRIzol® reagent (Invitrogen) assisting the homogenization procedure using needle and syringe. Total RNA was extracted from the TRIzol® homogenate according to the manufacturer’s instructions. In the case of spleen, due its nature and the inability to clean properly the RNA from “dirt” even after five washes with ethanol, the spleen RNA was finally extracted using the commercial kit NucleoSpin® RNA. The quantity of the isolated RNA was measured spectrophotometrically with NanoDrop® ND-1000 (Thermo Scientific), while its quality was tested on an agarose gel (electrophoresis in 1.5% w/v). Finally, all samples were used for mRNA paired-end library construction with the Illumina TruSeqTM RNA Sample Preparation Kits v2 following the manufacturer’s protocol (Poly-A mRNA isolation with oligo-dT beads, mRNA fragmentation, followed by transcription into first-strand cDNA using reverse transcriptase and random hexamer primers) and sequenced as 150 bp paired reads in half a lane of a HiSeq4000® following the protocols of Illumina Inc. (San Diego, CA).