First Record of Sepioteuthis lessoniana from the Maltese Archipelago, with Further Notes on Its Occurrence in the Central Mediterranean Sea

Abstract

The occurrence of the bigfin reef squid Sepioteuthis lessoniana, a Lessepsian migrant, is documented for the first time from the coastal waters off Malta, based on the collection of a single specimen reported through citizen science. The presence of this species in the broader Strait of Sicily region is further confirmed by a morphometric assessment and genetic analysis of another individual captured off the Pelagian island of Lampedusa. Molecular identification using mitochondrial COI and 16S rRNA markers corroborates the taxonomic identification exercise as it aligns both specimens with Indo-Pacific clades of S. lessoniana. These records represent the first genetically verified occurrences of the species in both Maltese and Italian waters. The findings extend the known Mediterranean distribution of S. lessoniana, emphasizing the Strait of Sicily as an important monitoring region for Lessepsian migrants and highlighting the combined role of integrative taxonomy and citizen science in tracking non-indigenous species introductions.

1. Introduction

In recent times, the Mediterranean Sea has been colonized by an increasing number of non-indigenous species (NIS), whose expansion is favored by the general increase in temperatures and climate change but also by anthropogenic factors (e.g., the widening of the Suez Canal) [1]. The establishment of some of these species appears to be successful in part due to their adaptive capacity and ability to compete with native species. It is therefore important to follow the evolution of their distribution over time, in order to identify which non-indigenous species are invasive and in order to understand the impacts they may cause on marine ecosystems, individual species, fisheries resources, etc.

The identity, occurrence, spread, and establishment of non-indigenous cephalopods of various origin reported in the Mediterranean Sea were exhaustively reviewed by [2]. Seven non-indigenous cephalopod species of Indo-Pacific/Red Sea origin have been recorded in the Mediterranean Sea to date [2,3,4], belonging to the following families: (i) Sepiidae, Sepia dollfusi Adam, 1941 and Sepia pharaonis Ehrenberg, 1831; (ii) Loliginidae, Sepioteuthis lessoniana R.P. Lesson, 1831; (iii) Octopodidae, Octopus cyanea Gray, 1849, Amphioctopus aegina (Gray, 1849) and Uroteuthis (Photololigo) arabica (Ehrenberg, 1831); and (iv) Tremoctopodidae, Tremoctopus gracilis (Eydoux & Souleyet, 1852). For the first five species, an autonomous migration through the Suez Canal (Lessepsian migration) has been hypothesized, while the introduction pathway for T. gracilis is still under discussion: for instance, the initial entrance into the basin could have happened through a human-mediated transfer or through Lessepsian migration, although the species could have been misidentified with the native T. violaceus Delle Chiaje, 1830 [2,3,4,5]. Finally, U. arabica, first recorded from Turkish waters, has been very recently added to the list of Mediterranean NIS cephalopods, with the species having an Indo-Pacific native range [6].

In the present study, the occurrence of the Lessepsian migrant S. lessoniana within the Strait of Sicily is confirmed through the direct examination of two specimens caught off the islands of Lampedusa (Pelagian archipelago) and of Malta (Maltese archipelago), as well as through an additional citizen science report from Malta. Sepioteuthis lessoniana is a loliginid squid occurring in coastal waters on sea grass beds, coral reefs, and sandy bottoms, up to 100 m of depth. Sepioteuthis lessoniana can reach a maximum weight of 2 kg, growing up to about 40 cm in mantle length, with females being smaller than males. It is widely distributed in the Indo-West Pacific region [7], but recently, it has established a stable population in the eastern Mediterranean Sea [2]. In fact, after its first record in Turkey (2002) [8], this species has rapidly expanded its distribution from the Suez Canal to the eastern African coasts until Tunisia (2011), spreading in most of Aegean Sea, and also reaching the Adriatic Sea in Montenegro (2015) [2]. Its rapid expansion and population increase have already fueled the commercial exploitation of this cephalopod in some eastern Mediterranean areas [9,10]. In this study, we also provide a summary of previous Mediterranean records of the species, with the aim of following the geographical expansion of this species across Mediterranean waters.

2. Material and Methods

One specimen of S. lessoniana (specimen A) was collected on 1 December 2024 by a recreational fisherman, at about 8 pm, at Guitgia (35°29′46.7″ N, 12°35′43.0″ E), on the south coast of the island of Lampedusa, within the Pelagian archipelago (Figure 1).

It was captured at a depth of 3–4 m, from the beach, using an eging rod and an artificial lure, specifically used for cephalopods, known as an ‘egi’. Subsequently, photos and video footage of the same specimen were uploaded on social media by the fisherman, which in turn triggered the rapid publication of a short note documenting this capture, based exclusively on an assessment of the digital material uploaded by the fisherman [6].

The specimen in question was concomitantly acquired by the co-authors and was photographed and examined in detail at the Wilderness Studi Ambientali facilities, in Palermo, where it is deposited in a frozen state. The species was identified at the laboratory following the keys provided by Jereb and Roper [7]. The need to directly examine the specimen in question and the need for molecular analysis were felt given that cephalopods in general are notoriously difficult to identify taxonomically through the exclusive examination of photos and video footage.

In December 2024, one of us (AD) received an anecdotal report through the Spot the Alien citizen science campaign (campaigns.com.mt) of a (possibly) additional S. lessoniana individual caught off the southeast coast of the island of Malta. Unfortunately, this claim could not be confirmed, given that the same specimen was sold and consumed prior to the report being lodged. However, the credibility of the report in question was considered high given that the citizen scientist lodging the same report is a seasoned fisher and fish hawker. In fact, the occurrence of the species in the waters of Malta was subsequently confirmed through the capture of specimen B described in the present study.

On 15 July 2025, one specimen of S. lessoniana (specimen B) was collected through spearfishing by a recreational fisher (Matthew Gatt) at Mellieha, along the northeastern coast of the island of Malta (35°58′4.8″ N, 14°22′0.6″ E) (Figure 2), from a nearshore location with a depth of just half a meter. One of us (AD) was informed of such a catch through social media. The specimen was acquired by the University of Malta and identified as above for specimen A.

2.1. Molecular Analysis

Genomic DNA was extracted from approximately 300 mg of muscle tissue of specimen A using an E.Z.N.A.^® Tissue DNA Isolation Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions; two independent extractions were performed from tissue subsamples of the same specimen, as replicates. For each extract, the cytochrome c oxidase subunit I (COI) and the 16S ribosomal RNA (16S rRNA) were amplified by polymerase chain reaction (PCR).

The COI gene (~600 bp) was amplified using the primer pair dgLCO (5′-GGT CAA CAA ATC ATA AAG AYA TYG G-3′) and dgHCO (5′-TAA ACT TCA GGG TGA CCA AAR AAY CA-3′) [11]. The 16S rRNA gene (212–244 bp) was amplified using the primers CephMLSf1 (5′-TGC GGT ATT WTA ACT GTA CT-3′) and CephMLSr1 (5′-TTA TTC CTT RAT CAC CC-3′) [12].

PCR reactions were carried out in a total volume of 24 µL, containing 1 µL of DNA, 0.5 µL of each primer (10 µM), 12.5 µL of MyTaq HS Mix 2×, and Invitrogen Ultrapure water (ThermoFisher, Waltham, MA, USA) to reach the final volume.

Thermal cycling conditions were optimized for each primer set. For COI, the cycling profile included an initial denaturation at 95 °C for 2 min, followed by 43 cycles at 95 °C for 30 s, annealing at 45 °C for 30 s, and extension at 72 °C for 45 s, with a final step at 72 °C for 5 min.

For 16S rRNA, amplification began at 94 °C for 5 min, followed by 35 cycles of 1 min at 94 °C, 30 s at 54 °C, and 1 min at 72 °C, with a final extension at 72 °C for 10 min.

PCR products were visualized by agarose gel electrophoresis using a 1 kb GeneRuler DNA Ladder (ThermoFisher) as a molecular weight marker, and then they were purified using a Wizard^® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Purification and One-Shot Sanger sequencing were performed by the internal service of Stazione Zoologica Anton Dohrn (Naples, Italy). Chromas version 2.6.6 (Technelysium Ltd., South Brisbane, Queensland, Australia) was used for manual curation and quality control of the sequences.

2.2. Phylogenetic Analysis

The purified sequences underwent a homology search using the Basic Local Alignment Search Tool (BLAST) algorithm from the National Center for Biotechnology Information (NCBI) at https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed 5 June 2025). Multiple sequence alignment was conducted using the online CLUSTAL W tool [13] (https://www.genome.jp/tools-bin/clustalw; accessed 5 June 2025) with default parameters.

The best-fitting nucleotide substitution model was determined in MEGA version 12 [14] using the “Find Best DNA/Protein Models (ML)” function. Model selection was based on the Bayesian Information Criterion (BIC), which identified the Tamura 3-parameter model with a proportion of invariant sites (T92 + I) as the most suitable. Phylogenetic trees were reconstructed under this model using the Maximum Likelihood (ML) method with 1000 bootstrap replicates and partial deletion for sites with less than 90% data coverage, resulting in a final alignment of 597 positions. In addition, Bayesian inference (BI) analyses were performed in Geneious Prime^® 2025.0.3 (Biomatters; https://www.geneious.com, accessed on 15 June 2025) using the MrBayes plugin v3.2.6 [15]. As the T92 model is not implemented in MrBayes, the next more complex model, Hasegawa–Kishino–Yano with a proportion of invariant sites (HKY + I), was applied, following the recommendations of the MrBayes manual [16]. Two independent runs with four Markov chain Monte Carlo (MCMC) were executed for 1,100,000 generations, sampling every 200 generations, with the first 25% of samples discarded as burn-in. Convergence was assessed by effective sample sizes (ESS > 1000) and potential scale reduction factors (PSRF~1.0). Sequences of Sepiotheutis sepiodea and S. australis were used as outgroups, according to Cheng et al. [17]. The final tree was exported and edited in Interactive Tree of Life (iTOL) v7 [18].

3. Results

Specimens A and B were females, had a dorsal mantle length (DML) of 163 mm and 260 mm, and weighed 203 g and 815 g, respectively. The specimens had a broad mantle, were oval in outline, and their fins extended nearly the full length of the mantle (Figure 1 and Figure 2). Their arms were equipped with sucker rings with pointed teeth, whereas the tentacular clubs were long, with 4 series of suckers, and with median manal suckers enlarged. Measurements of these individuals are reported in

Table 1. Morphometric measurements (mm) and weights (g) of the female specimens of S. lessoniana caught in the waters of Lampedusa on 1 December 2024 (A) and of Malta on 15 July 2025 (B).

Measurements	Specimen A ♀	Specimen B ♀
	mm	mm
Total length with tentacles (TL1)	425	654
Total length without tentacles (TL2)	207	302
Dorsal mantle length (DML)	163	260
Ventral mantle length (VML)	145	238
Mantle width (MW)	64	73
Head length (HL)	42	54
Head width (HW)	40	62
Fin length (FL)	142	240
Fin width (FW)	23–24	57
Distance between fin tips	100	195
Funnel length (FuL)	32	31
Maximum arm length (AL)	213	365
Tentacle length (TeL)	83	165
Weight (g)	203	815

.

The upper surface of the body was covered by large, pale yellow chromatophores, less numerous along the ventral part, but absent on the fins. The fresh specimens had a brownish color with greenish tones (especially around the eyes).

Molecular Analysis

The use of the primer set CephMLSf1/CephMLSr1 provided sequences with 98–100% similarity to S. lessoniana isolate Sles21 (

Table 2. Next relative by GeneBank Alignment showing phylogenetic affiliation of S. lessoniana samples.

Sample	Next Relative by GenBank Alignment (AN **, Organism)	Hom § (%)	AN
S11_16S	S. lessoniana isolate Sles21 16S KF854049.1	98	PV789287
S12_16S	S. lessoniana isolate Sles21 16S KF854049.1	100	PV789288
Sl1_COI	S. lessoniana isolate Sles20 (COI) gene KF854086.1	99	PV785481
Sl2_COI	S. lessoniana isolate Sles20 (COI) gene KF854086.1	100	PV785482

** Accession number; ^§ homology.

), obtained from a specimen collected from waters off Durban, South Africa [19].

Similarly, sequences amplified using the primer set dgLCO/dgHCO showed the highest similarity, 99% and 100%, to S. lessoniana isolate Sles20, also collected from Durban, South Africa [19]. Our sequences have been deposited in the GenBank database under accession numbers S11_61S PV789287, S12_16S PV789288, Sl1_COI PV785481, and Sl2_COIPV785482.

The ML (Figure 3) and the Bayesian (Figure 4) trees were constructed using the higher-quality COI sequence obtained from our specimen. Based on BLAST analysis, seven of the closest matching sequences from GenBank were selected and included in the alignment. To minimize redundancy and ensure geographic representation, one representative sequence per sampling site was retained. The resulting trees show that our sample clusters within a supported clade (bootstrap value and posterior probability = 0.8) comprising S. lessoniana sequences from South Africa, Indonesia, and Taiwan. Within this group, our sequence forms a distinct sub-cluster with S. lessoniana from South Africa (KF854086.1), also supported by a bootstrap value and posterior probability of 1.0. Sepioteuthis sepioidea (KF854085.1) and S. australis (MK185986.1) highlighted their divergence from S. lessoniana specimens.

4. Discussion

The bigfin reef squid S. lessoniana, a Lessepsian migrant, was recorded for the first time in the Mediterranean Sea from Iskenderun Bay, Turkey [6,8]. It is today widely distributed within eastern and central parts of the basin (Figure 5), and it is common in those Mediterranean localities, where it has acquired some commercial importance, including Egypt [20], Libya [21], Turkey [22], and the Greek waters of the south Aegean Sea [9,23] and Syria [24].

The record from Lampedusa represents the first well-documented evidence of the occurrence of S. lessoniana in Italian waters. Recently (January 2022), another adult individual belonging to the same species was caught from the same location (Lampedusa Island) by a recreational fisher, and preserved frozen, but unfortunately, it was lost during shipping to the Stazione Zoologica Dohrn (SZN) laboratory and, as a result, it was impossible to document this event well or provide useful data.

As observed for other Lessepsian species, the occurrence of S. lessoniana around the islands of Lampedusa and Malta is particularly noteworthy, not merely because of its presence, but due to the pivotal role these islands play as biogeographical sentinels. Located along the Strait of Sicily—a recognized transition zone between the western and eastern Mediterranean basins—they represent natural ‘gateways’ where newly arriving species can first be detected before potentially expanding westward and vice versa. Previous studies have highlighted that these islands consistently act as early detection hotspots for non-indigenous species (NIS), owing to their strategic position at the interface of distinct faunal assemblages and major current systems [25,26]. Thus, the documentation of S. lessoniana at Lampedusa further reinforces the importance of these islands as priority sites for long-term monitoring of NIS dispersal pathways in the Mediterranean. Moreover, the first report of this potentially invasive species in the Maltese archipelago is of crucial importance, given that each country addresses alien species outbreaks differently, introducing targeted management actions and outreach interventions that take into account the specific socio-economic context of each nation.

The specimen of S. lessoniana from Malta constitutes the first record of the species for the country, and it is also the first NIS cephalopod recorded from the island, whilst the specimen of S. lessoniana reported from Lampedusa represents the second Lessepsian cephalopod found in Italian waters, following various records of T. gracilis from the Tyrrhenian Sea, the Strait of Messina, and the Adriatic Sea [2,3,27,28]. The fact that the Italian sample recorded in this study formed a distinct sub-cluster with the South African sample, coupled with the current known distribution of the species within the Mediterranean (which is mainly restricted to the eastern basin), suggests a possible Lessepsian migration of the species into the Mediterranean, rather than a possible entry of the species into the basin through the Atlantic route.

It is to be remarked that the species named S. lessoniana represents a species complex [7,29], and even the species found in the Mediterranean should be more correctly reported as S. cf lessoniana complex. To date, in fact, the taxonomic identity of the Mediterranean specimens generally assigned to S. lessoniana has not been validated through genetic analysis [2,30]. An accurate identification of S. lessoniana is challenging, and consequently, researchers have turned to molecular tools, particularly mitochondrial DNA markers such as the Cytochrome Oxidase I (COI) gene and 16S ribosomal RNA (16S rRNA), to ensure precise species recognition and trace its geographic origin. Therefore, DNA-based identification methods have become increasingly important, especially when dealing with early-stage specimens, degraded samples, or newly introduced populations. In this study, the COI gene, considered the main reference of DNA barcoding for animals, and the mitochondrial 16S rRNA gene, widely used for phylogenetic analyses and complementary species identification, were used for phylogenetic identification of the species. The COI + 16S approach provided a double validation of the species identity. This helped to overcome cases of ambiguous COI results or poor-quality samples. It also further confirmed the approach as the standard one for reliable species-level diagnosis, particularly for invasive taxa with limited baseline data.

Further studies should investigate the genetic structure of different geographic populations of the species, including the collection of morphometric data to reveal any potentially overlooked macroscopic differences in the species complex.

It also is very important to trace the movements and expansion of this invasive species inside the Mediterranean Sea, in order to understand if there is a risk that it could further colonize central areas and the western basin, establishing stable populations in these areas, as well as to assess its impact on native marine ecosystems (e.g., through competition with native species) [31,32]. The success of this cephalopod is probably linked to a flexible reproductive strategy, which consists of prolonged or multiple spawning events during its life span [7].

Citizen science initiatives represent an increasingly valuable tool for the monitoring of non-indigenous species (NIS), particularly in marine systems where sustained scientific surveys are logistically demanding and costly [33]. In Malta, the ongoing Spot the Alien campaign [34] exemplifies how citizen science projects can generate reliable data, especially through the involvement of both recreational and professional fishers who spend extensive time at sea and are therefore pivotal in detecting changes in species occurrence. Ultimately, such initiatives provide early-warning signals of new introductions, extend the spatial and temporal coverage of monitoring efforts, and foster public engagement with marine biodiversity issues. However, citizen science data also carry limitations, including variability in data quality, uneven sampling effort, and the need for expert validation to avoid misidentifications. In this context, citizen science is expected to play an increasingly important role, particularly since the bigfin reef squid S. lessoniana could represent an opportunity and resource for both professional and recreational fishers, given the current overexploitation of several native fishery resources in the Mediterranean [9,10]. The establishment of S. lessoniana in the Mediterranean underscores the need for early detection systems and genetic surveillance networks. Future key actions in the monitoring and management of the species should employ the use of environmental DNA (eDNA) to detect the presence of squid in water samples on a broader scale, while promoting the consumption and commercialization of such a species in the local community. The development of region-specific reference libraries for different genetic marker sequences is both challenging and essential. Such libraries require extensive and validated sampling across geographic areas, combined with accurate taxonomic identification and high-quality sequencing. They are necessary because populations of the same species may show regional genetic variation, and without reference data specific to a given area, molecular identification can be uncertain or misleading. Region-specific libraries therefore play a key role in confirming future records and in clarifying phylogenetic relationships between populations. Furthermore, by contributing to global databases such as BOLD and GenBank, they enhance the accuracy and reliability of large-scale marine biodiversity monitoring and invasive species tracking.