First report of the pygmy slipper lobster Biarctus sordidus (Crustacea, Decapoda) in the Red Sea following the finding of its phyllosoma larva

Abstract

The advent of integrative taxonomy in plankton research, employing molecular and morphology-based identification, promotes the discovery of new biodiversity records, especially of larval stages. The slipper lobster family Scyllaridae consists of planktonic phyllosoma larvae, persisting weeks to many months in the water column. High interspecific larval similarities and inconsistent delineation of stages have hindered the identification of scyllarid phyllosomata to the species level using morphological characteristics. Here we report the first record of the pygmy slipper lobster, Biarctus sordidus, in the Red Sea following the finding of its phyllosoma larva, extending its known distribution from the Persian Gulf to Australia and southern China. We identified the phyllosoma collected from the Northern Gulf of Aqaba as B. sordidus using the mitochondrial 16S and 18S rRNA genes, and described its morphology to determine the larval stage. We further discuss the potential factors contributing to the delayed detection of this species.

Introduction

Slipper lobsters of the family Scyllaridae are widespread in shallow, warm temperate and tropical habitats. Their life cycle includes several planktonic phyllosoma stages, lasting weeks to months, and a final phyllosoma instar that metamorphoses into nisto, a nektonic postlarva that transforms to the benthic adult (Lavalli and Spanier, 2007). Among scyllarids, the pygmy slipper lobster, Biarctus sordidus (Stimpson, 1860), is one of the smallest species. Its distribution spans across the Indian Ocean, from Australia, Indonesia, Borneo, Philippines, Singapore, Malaysia, Thailand, South China and the Persian Gulf, at shallow depths of 2.7 to 73 m (Holthuis, 2002). Phyllosoma larvae of B. sordidus were reported from Australia (Barnett, 1989; Mcwilliam et al., 1995), Japan (Sekiguchi, 1997), Java (Tampi and George, 1975), and India (George and Thomas, 1997; Sankolli and Shenoy, 1973). However, neither adults nor larvae of B. sordidus were previously recorded in the Red Sea.

Identifying scyllarid phyllosomata to species based on their morphology is difficult due to incomplete descriptions or assignments of developmental stages, as well as disagreements among authors regarding the distinguishing characteristics of these stages (Pagliarino et al., 2013). Employing integrative taxonomy by combining DNA barcoding and morphological characterization is therefore essential for establishing the phyllosoma larval stages of scyllarid species (Guy-Haim et al., 2024). Here we used integrative taxonomy to describe an early stage phyllosoma of B. sordidus, recorded for the first time in the Red Sea.

Materials and methods

A plankton sample was collected in 6 March 2024 at 6:00 AM using a plankton net (200-μm mesh size, 50 cm opening diameter, Aquatic Research Instruments, USA), towed horizontally for 10 min at a depth of 0.5–1 m in Station A, located at the northern Gulf of Aqaba, Eilat, bottom depth ca. 700 m (29°28′N, 34°55′E, Figure 1A). Fieldwork was conducted under INPA permit no. 2024-43522.

Figure 1. A. The distribution map of Biarctus sordidus. Occurrences are presented in blue circles and the new record in a red star. Occurrences were downloaded from https://www.gbif.org/ and OBIS https://obis.org/ on 17 April 2024. Additional occurrences were added from Holthuis (2002). B–H. B. sordidus phyllosoma stage III/IV. B. ventral view. C. Antenna (a2). D. Antennule (a1). E. Mouth (mo) and maxillipeds. F. Pereiopod 4 (p4). G. Abdomen (abd). H. Exopod of pereiopod 3 (p3exp). Scale bars: B – 500 μm, C – 100 μm, D – 100 μm, E – 500 μm, F – 200 μm, G – 100 μm, H – 100 μm.

The plankton sample was examined under a stereomicroscope (SZX16, Olympus, Japan). A phyllosoma larva was identified morphologically and the developmental stage was determined following Sankolli and Shenoy (1973) and Ritz (1977). One pereiopod was cut for molecular identification, and the specimen was stored in 70% ethanol and deposited in the zooplankton collection, the National Natural History Collections, Hebrew University of Jerusalem (NNHC, HUJI).

Total genomic DNA was extracted from the pereiopod using the InviSorb Spin Tissue Mini Kit (Invitek Diagnostics, Germany) according to the manufacturer's specifications. Following the DNA extraction, the 18S rRNA gene was amplified using the primers 18S-3F and 18S-9R following Giribet et al. (1999) and the mitochondrial 16S gene was amplified using the primers 16Sar-16Sbr following Palumbi (1996). Reaction conditions were as follows: 94°C for 2 min, followed by 34 cycles of 94°C for 15 s, 49°C for 30 s, and 72°C for 1 min, and a final elongation step of 72°C for 7 min. Obtained PCR products were purified and sequenced by Hylabs (Rehovot, Israel).

A total of twenty-one 18S rRNA sequences of Scyllaridae were analysed, including one sequence of the phyllosoma of B. sordidus obtained in this study, one sequence of adult B. sordidus from Thailand (JN701622), one sequence of B. vitiensis from Guam (JN701621), and additional 18 scyllarid sequences obtained from GenBank (accession numbers and collection locality are indicated in Figure 2). Ibacus peronii and I. chacei were used as an outgroup.

Figure 2. Maximum-Likelihood phylogenetic tree of Biarctus sordidus based on the 18S rRNA gene, using the K2 + G substitution model. The outgroups Ibacus peronii and I. chacei were used as a root node. The numbers in blue indicate the percentage of ML bootstrap support (1000 replicates) for nodes that received at least 60% support. The scale bar denotes the estimated number of nucleotide substitutions per site.

A total of fifteen 16S mtDNA sequences of Scyllaridae were analysed, including one sequence of the phyllosoma of B. sordidus obtained in this study, two sequences of adult B. sordidus from Thailand (JN701710) and Australia (AY583888), two sequences of B. pumilus from South Africa (ON960177- ON960178), one sequence of B. vitiensis from Guam (JN701709), and additional nine scyllarid sequences obtained from GenBank (accession numbers and collection locality are indicated in Figure 3). Ibacus chacei was used as an outgroup.

Figure 3. Maximum-Likelihood phylogenetic tree of Biarctus sordidus based on the mitochondrial 16S gene, using the HKY + G substitution model. The outgroup Ibacus chacei was used as a root node. The numbers in blue indicate the percentage of ML bootstrap support (1000 replicates) for nodes that received at least 60% support. The scale bar denotes the estimated number of nucleotide substitutions per site.

Sequence alignments were conducted using ClustalW embedded in MEGA v11.0 (Tamura et al., 2021). The best-fitting substitution models were selected according to the Bayesian Information Criterion using Maximum-likelihood (ML) model selection in MEGA. ML analyses were performed using the K2 + G (18S) and HKY + G (16S) models with 1000 bootstrapping replicates each.

Results

Morphological description

Biarctus sordidus (Stimpson, 1860): phyllosoma, stage III (following Sankolli and Shenoy, 1973) / IV (following Ritz, 1977) (Figure 1B).

Dimensions: TL (total length) 2.24 mm; CL (cephalic length) 1.52 mm; CW (cephalic width) 1.49 mm; ThW (thorax width) 0.73 mm; ThL (thorax length) 0.76 mm; A1L (antennular length) 0.63 mm; A2L (antennal length) 0.22 mm; AbdL (abdomen length) 0.21 mm.

Cephalic shield subcircular, twice wider than thorax. Eyestalks segmented. Antennule unsegmented with inner flagellum as a small bud. Right antennule damaged. Antenna short uniramous with small seta on either side of tip. Pereiopod 3 with unsegmented setose exopod. Pereiopod 4 more than twice longer than abdomen. Uropods not visible.

Molecular identification

A DNA fragment of 577 bp of the 18S rRNA gene was sequenced from the phyllosoma larva of B. sordidus and assembled from forward and reverse sequences. The sequences were deposited in NCBI GenBank under the accession numbers PP702379 (18S) and PP960534 (16S). NCBI blastn yielded 99.61% identity of the 18S sequence to B. sordidus from Guam, and 96.33% identity of the 16S sequence to B. sordidus from Thailand. Maximum likelihood analysis of 18S Scyllaridae sequences obtained from GenBank showed that the phyllosoma from the Red Sea is clustered together with B. sordidus within the Biarctus clade, with high bootstrap support.

Discussion

The Red Sea is home to seven scyllarid species: the clamkiller slipper lobster, Scyllarides tridacnophaga, the Aesop slipper lobster, S. haanii, the dark-spot locust lobster, Gibbularctus gibberosus, the flathead lobster, Thenus orientalis, the hunchback locust lobster Petrarctus rugosus, Eduarctus lewinsohni, and Biarctus pumilus (Holthuis, 1968; Holthuis, 2002). Here we report an eighth scyllarid in the Red Sea, Biarctus sordidus, following the finding of its phyllosoma larva in the northern tip of the Gulf of Aqaba, Eilat.

Using laboratory-hatched eggs of adults collected from Moreton Bay, southeast Queensland, Australia, Ritz (1977) described the first phyllosoma stage of B. sordidus, and based on plankton samples he provided keys to its eight phyllosoma stages. Sankolli and Shenoy (1973) described the first six phyllosoma stages of laboratory hatched B. sordidus collected from western India, stating that ‘each phyllosoma stage (instar) took, on an average, five days to moult to the next’. Thus, it can be estimated that the larval duration of B. sordidus is approximately 40 days. We found the stage III/IV phyllosoma of B. sordidus in the beginning of March in the offshore waters of the northern Gulf of Aqaba. We can therefore hypothesize that a potential recruitment of this species in the Red Sea may take place during April.

Similar to its congener B. pumilus, B. sordidus inhabits shallow hard sandy bottoms within its distributional range. The location and shape of the cardiac and gastric teeth, minute morphological features, differentiate between these sister taxa (Holthuis, 2002). Thus, it is plausible that former biodiversity surveys in the Red Sea have confused B. sordidus with B. pumilus. Another reason for the late detection of B. sordidus in the Red Sea might be a recent introduction by ship ballast, as was previously hypothesized to explain the introduction of the Caribbean spiny lobster Panulirus argus to Cape Verde Archipelago (Freitas and Castro, 2005). Cargo ships that arrived at the Port of Eilat in Israel and the Port of Aqaba in Jordan, both located at the Northern Gulf of Aqaba, could have been the vector of such an introduction. Nonetheless, there is no former evidence for ballast-mediated introductions of phyllosoma larvae. Further studies are needed to unveil whether the pygmy slipper lobster has established populations in the Red Sea.