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).