Morphology, morpho-taxometric and molecular characterization of the invasive alien species Caribbean leatherleaf slug Sarasinula plebeia (Gastropoda: Veronicellidae): a first record in southern Philippines

Abstract Herein, we first report the comprehensive description of the terrestrial slug, Sarasinula plebeia (Gastropoda: Veronicellidae) by employing morphology, morpho-taxometrics and molecular analysis. A rapid survey on terrestrial slug invasive alien species (IAS) was conducted in La Dicha, Malangas, Zamboanga Sibugay, the Philippines. Obtained COI gene sequences shared 100% similarities to S. plebeia from Brazil (JX532107, KM489378), Dominica (KM489500) and Vietnam (KM489367) and further supported using Bayesian analysis thus designated as S. plebeia isolate LDZS. Notably, the first reported S. plebeia in 2013 from Batan island, Batanes, northern Philippines, characterized through COI gene markers (JQ582277, JQ582278, JQ582279) showed 100% sequence similarities to a closely related veronicellid slug, Laevecaulis alte isolates (LC636101, LC636102, LC636103, and LC636104) from Japan. Taken this into account, our S. plebeia LDZS isolated from an agricultural field is the first report in the Philippines with combined diagnostic tools for the taxon.


INTRODUCTION
Terrestrial gastropods have a geographic distribution from temperate to tropical regions comprising almost 35,000 species (Barker, 2001), occupying a diverse niche, and developing different life history strategies of survival (Klussmann-Kolb & Dinapoli, 2006).Most of these terrestrial gastropod species are free-living and take part in the nutrient cycle by feeding on living and organic plant matter and releasing nutrients back to the soil in the form of feces (Theenhaus & Scheau, 1996).However, several notable species of agriculturally important pests cause economic loss on agricultural crops.
As globalization steadily progresses allowing free movement of people and goods across the world, the possible infiltration and unintentional introduction of species to new habitats are irrefutable.Such species are usually non-native to the area and were introduced through various ways.
These were labeled as invasive alien species (IAS) which pose a much greater threat to agriculture since they have no natural predator as population regulator.These IAS, of which gastropods are no exceptions, are threats to sustainable agriculture worldwide that account to agricultural loss resulting in economic loss in severe cases of infestation (Willis et al., 2006), disruption in commerce, changing the natural environment and posing risk to human health (Staples & Cowie, 2001).
One of the IAS terrestrial gastropods is Sarasinula plebeia (Fischer, 1868) (Gastropoda: Veronicellidae), commonly known as Caribbean leatherleaf slug with neotropical range (Solem, 1964;Cowie, 1998;Robinson, 1999).This species is native to Central America and has now a worldwide distribution from the Americas, Pacific regions including the Philippine islands, and is considered a serious IAS (Barker et al., 2005).In 2003, the United States Department of Agriculture (USDA) first intercepted S. plebeia (KM489380; Gomes et al., unpublished) from the Philippines.The first record of S. plebeia in the country was in Batan island, Batanes, northern Philippines, identified using cytochrome oxidase subunit I (COI) gene markers (Batomalaque et al., 2013) and another report was also in northern Philippines, from Masungi Georeserve in Tanay, Rizal with identification using morphological data (Valdez et al., 2021).
Studies on gastropods are abundant, especially on invasive terrestrial gastropods.However, supplemental work is needed given the diversity of this class.For an increased resolution and reliability of species identification, integration of different diagnostic tools is highly suggested (Robinson & Fields, 2010).Thus, this study is conducted to identify IAS terrestrial slugs in the southern part of the Philippines by means of morphological, morpho-taxometrical, and molecular analyses.We have collected a total of six slug samples from selected agricultural areas in La Dicha, Malangas, Zamboanga Sibugay, however, assessment of severity of damage to the crops was not included in the present study.Our S. plebeia LDZS isolate (MZ598573) had 100% COI gene sequence similarities to S. plebeia from Brazil (JX532107, KM489378), Dominica (KM489500) and Vietnam (KM489367) and further supported using Bayesian analysis.

Sampling site establishment
A survey for the occurrence of terrestrial slugs was conducted from an agricultural area in La Dicha, Malangas, Zamboanga Sibugay (07°40′8.832″N,123°00′15.048″E)(Fig. 1).A purposive sampling utilizing the transect walk method was undertaken during sampling for terrestrial slug collection.Undergrowth of bushes, grasses, below fallen trees and stones were inspected for the presence of slugs.Collected samples were washed and transferred to sterile containers for further processing.The voucher sample specimens in 95% ethanol solution are deposited in the Department of Biological Sciences, Mindanao State University -Iligan Institute of Technology, Iligan City and the University of the Philippines Los Baños, Museum of Natural History (https://mnh.uplb.edu.ph).

Morphology and morpho-taxometrics analyses
Morphology was assessed by describing key characters along their morpho-taxometric values, such as total body length in stretched condition, circumference obtained at the widest region of the body using a thread (scaled to millimeter) and live weight (g) using digital weighing scale (Das & Parida, 2015).

Molecular analysis
For molecular analysis, COI gene was used as a marker for species identification.Tissue samples obtained from the slug were cut and submerged into 95% ethanol solution.Genomic DNA (gDNA) of the slug tissue samples was extracted using PureLink® Genomic DNA Mini Kit (Invitrogen, Thermo Fisher Scientific).Gene amplification by PCR was performed with the following components: template gDNA, universal HCO (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) and LCO Figure 1.Map showing the sampling site (blue dot) in the selected area for terrestrial slug in La Dicha, Malangas, Zamboanga, Sibugay, southern Philippines.
Capillary sequencing (bidirectional) was performed at the Philippine Genome Center-DNA Core Sequencing Facility (PGC-DCSF), University of the Philippines-Diliman, Quezon City.Fluorescent-labeled chain terminator dNTPs with the reaction components viz.amplicons, primers, and ABI BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) were used.Cycling parameters on Bio-Rad T100 Thermal Cycler include: pre-hold at 4℃; 96℃ for 1 min; 25 cycles of 96℃ for 10 sec, 50℃ for 5 sec, 62℃ for 4 min and a hold at 4℃. Ethanol precipitation was carried out to remove unincorporated ddNTPs, excess primers and primer dimers.Capillary electrophoresis was done on the ABI 3730xl DNA Analyzer using a 50 cm 96-capillary array, POP7TM Polymer, and 3730xl Data Collection Software v3.1.Base calling was done on the Sequencing Analysis Software v5.4.

Sequence alignment and phylogenetic analysis
The generated COI gene sequences were assembled and trimmed using the BioEdit program (Hall, 1999).The assembled sequences were BLASTn-searched in National Centre for Biotechnology (NCBI) and compared with closely related sequences deposited.Phylogenetic analysis was done with other identified closely related species.Sequences were first aligned using MUSCLE in MEGA version 11 followed by manual trimming of poorly aligned ends, then pairwise distance analysis using Maximum Composite Likelihood model was used to determine the number of base substitutions between sequences (Tamura & Nei, 1993).Bayesian phylogenetic analysis (MrBayes 3.2.7)was employed for constructing the phylogenetic relationship inferred by obtained COI sequences and carried out using GTR+I+G model; analyses were run under 1 × 10 6 generations (4 runs) and Monte Carlo Markov chains were sampled every 100 generations and 20% of the converged runs were regarded as burnins (Huelsenbeck & Ronquist, 2001).

RESULTS
A total of six slug samples were collected from La Dicha, Malangas, Zamboanga, Sibugay.Combination of morpho-taxometric and molecular analysis showed that it is the Caribbean leatherleaf slug, S. plebeia and designated as S. plebeia isolate LDZS (MZ598573).

Morphological description
Sarasinula plebeia isolate LDZS has brownish dorsal notum without stripes (green), mantle is absent, body is flat and elongated when crawl, foot running from anterior to posterior end in a narrow line between the hyponotum.Colored arrows indicate the following characters: hyponotum (red), foot (orange), perinotum (yellow), entire notum (green) and ocular tentacle (blue) (Fig. 2).

Morpho-taxometric analysis
Sarasinula plebeia isolate LDZS has a mean body length of 48.18 mm, a circumference of 28.5 mm, and mean live weight of 1.28 g, respectively (Table 1).

DISCUSSION
Sarasinula plebeia is already recorded in the Philippines with introduction to a critical ecosystem such as in Masungi Georeserve in Tanay, Rizal (Valdez et al., 2021), identified based on the morphological key characters for the species.Prior to this, S. plebeia was initially recorded from Batan island, Batanes, northern Philippines (Batomalaque et al., 2013), identified using COI gene marker with sequences submitted to NCBI as S. plebeia voucher BSD1 (JQ582277), S. plebeia voucher BSD2 (JQ582278), and S. plebeia voucher BSD3 (JQ582279).However, we found a discrepancy in the designation of the said terrestrial slug from Batan island.The COI gene sequences of this first reported S. plebeia in 2013 (JQ582277, JQ582278, and JQ582279) were BLASTn-searched and found to be closely related to another veronicellid slug, Laevicaulis alte from Japan (Hirano et al., 2022).Percent similarities between the misidentified L. alte as S. plebeia from Batan island with the L. alte isolates from Japan is 100%, showing 0.00 number of base substitutions using pairwise distance analysis and forming one clade in the phylogenetic tree.For this particular group, the use of only one tool to identify species is not sufficient and robust.Solely depending on either COI gene marker or morphological key characters may result in the misidentification of the species.
For instance, the group Systellommatophora: Veronicilloidea have degree 2 limacization (Simone, 2018), with almost indistinguishable morphological characters among the taxa.The high intraspecific morphological variation among veronicellid slugs (Kim et al., 2016) renders identification of the species based on morphology alone difficult, requiring supplemental molecular analysis (Robinson & Fields, 2010).We therefore propose a re-description of the terrestrial slug species found in the Batan island (identification using COI gene marker only) and in the Masungi Georeserve (identification using morphology only), pending on the availability of voucher specimens.We also recommend dissecting for the reproductive part of our slug, which is an important key character to increase resolution of species identification, and also including DNA barcoding of more S. plebeia samples in the area.Aside from being a successful IAS, another risk that S. plebeia poses is on human health; S. plebeia is sometimes found in areas near human settlements (Garcia et al., 2007).This terrestrial slug is known to be the vector for rat lungworm nematode species like Angiostrongylus cantonensis and A. costaricensis which are causative agents of meningoencephalitis and abdominal angiostrongyliasis (Laitano et al., 2001).This association with a parasitic nematode coupled with its preference close to the human population, poses serious risk in health with successful infection sometimes leading to death if left untreated.Like other veronicellid slugs, S. plebeia is a hermaphroditic herbivore that feeds on a wide variety of plants.It devastates agricultural, horticultural fields as well in suburban areas worldwide due to its non-discriminate feeding habit as a polyphagous species (Hata et al., 1997;Robinson & Hollingsworth, 2004).Generally, this species is found in moist areas such as below leaf litters and decaying matters to avoid desiccation, they are also known to burrow in soil with depths ranging from 25 cm to 1 m during dry season.They rapidly reproduce during the wet season with, at its peak, juvenile maturity reaching at ~ 2.5 months (Garcia et al., 2007).
Here, we recovered S. plebeia partially buried underground, from an orchard of mango trees beside several plots of rice paddies; however, the severity of damage caused by this species to agricultural crops was not assessed and is an excellent research outlook.This terrestrial slug, along with other gastropods, are pests in agriculture causing reduced leaf surface area, stagnant growth, contaminating crops with their feces (Port & Port, 1986, South, 1992;Glen & Moens, 2002;Port & Ester, 2002) and consuming seeds, stems and meristems through voracious feeding habits, which results to reduction of growth and loss of plant vigor (South, 1992).Though they have relatively limited individual mobility over a large span of area and distances, trade in food, horticulture commodities, and people facilitated their spread (Howlet, 2012).

CONCLUSION
This study showed that one of the top IAS worldwide is established in the Philippines, wherein S. plebeia were identified using morphology, morpho-taxometric and molecular analysis with COI gene as marker.To our knowledge, this is the first recorded S. plebeia in southern Philippines.

Figure 3 .
Figure 3. Phylogenetic relationship of Sarasinula plebeia isolate LDZS (bold) and related sequences inferred by the COI sequences through Bayesian analysis using GTR+I+G model showed a strong relation with posterior probability value of 1. Position of S. plebeia (JQ582279, JQ582278, JQ582277) also showed strong relation with L. alte (PP value of 1).Scale bar represents the estimated substitution per site.

Table 2 .
Pairwise distance analysis using Maximum Composite Likelihood model, showing number of base substitutions (below diagonal) per site from between sequences using COI gene sequences of Sarasinula plebeia isolate LDZS (bold) and related taxa.Standard error estimates (above diagonal) are shown.