Introduction
The phylum Tardigrada comprises microscopic animals <1 mm in length with a complex anatomy (Arakawa et al., Reference Arakawa, Yoshida and Tomita2016; Halberg et al., Reference Halberg, Persson, Møbjerg, Wanninger and Kristensen2009) such as segmented body, eight legs with claws or discs, have nervous system, digestive system, and more and is divided into three classes: Eutardigrada, Heterotardigrada, and Mesotardigrada. Mesotardigrades have not been resampled since their original description from a hot spring in Japan (Rahm, Reference Rahm1937). The eutardigrades are predominantly semi-terrestrial and limnic with a few marine species while the heterotardigrades, particularly the order Arthrotardigrada, are primarily marine (Møbjerg et al., Reference Møbjerg, Halberg, Jørgensen, Persson, Bjørn, Ramløv and Kristensen2011, Reference Møbjerg, Jørgensen, Eibye-Jacobsen, Agerlin Halberg, Persson and Møbjerg Kristensen2007). This order currently has 336 families, 160 genera, 1488 species, and 17 additional subspecies included in the Actual Checklist of Tardigrada species (Degma & Guidetti, Guidetti et al., Reference Guidetti, McInnes and Kristensen2024).
The genus Batillipes was established by Richters in 1909 with the type species B. mirus, originally described from Kiel Bay in the Baltic Sea. To date, this genus has the highest number of species recorded among other genera in the arthrotardigrades with a total of 39 species listed in WoRMS (Guidetti et al., Reference Guidetti, McInnes and Kristensen2024) and 40 species in the Actual Checklist of Tardigrada species (Guidetti et al., Reference Guidetti, McInnes and Kristensen2024), recorded from different marine environments (Guidetti and Bertolani, Reference Guidetti and Bertolani2005; Menechella et al., Reference Menechella, Bulnes and Cazzaniga2017). Identification of species in this genus is presently based on characters such as body shape (i.e. presence or absence of lateral projections), variation in caudal terminations, shape of clava and toe discs, shape of femur or leg IV and its accessory appendages (Ramazzotti and Maucci, Reference Ramazzotti and Maucci1989). In general, one of the more reliable taxonomic characters of this species is the toe arrangement pattern, especially on feet of leg IV that was introduced by Pollock (Reference Pollock1970a) and modified by Kristensen and Mackness (Reference Kristensen and Mackness2000). They proposed four patterns of toe lengths on the fourth feet. Later that, it was modified once more by Santos et al. (2018), as used posteriorly in this manuscript. However, intraspecific variation during the life cycle has also been described in several studies (Grimaldi De Zio and D’Addabbo Gallo, Reference Grimaldi De Zio and D’Addabbo Gallo1975a; Chang & Rho, 1997; Kristensen and Mackness, Reference Kristensen and Mackness2000).
In Malaysia, the terrestrial and freshwater tardigrade fauna was poorly investigated with only five species discovered (Famelobiotus scalicii Pilato, Binda & Lisi, 2004, Bryodelphax arenosus Gąsiorek, 2018, Echiniscus masculinus Gąsiorek, Vončina & Michalczyk, 2020,Insulobius orientalis Gąsiorek & Michalczyk, 2020 and Paramacrobiotus filipi Dudziak, Stec et al., Reference Stec, Dudziak and Michalczyk2020) from the Bornean part of the country, collected from moss samples (Kayastha et al., Reference Kayastha, Stec, Sługocki, Gawlak, Mioduchowska and Kaczmarek2023; Stec et al., Reference Stec, Dudziak and Michalczyk2020). There was no report of tardigrades species from Peninsular Malaysia until the study of Zieritz et al. (Reference Zieritz, Lee, Eng, Lim, Sing, Chan, Loo, Mahadzir, Ng, Yeo, Gan, Gan, Gibbins, Zoqratt and Wilson2022) who employed DNA metabarcoding to comprehensively assess the benthic freshwater invertebrate fauna of the Perak River basin, Malaysia. Although their study confirmed the presence of tardigrades, it could not determine their specific classification.
To date, only one publication has documented the presence of marine tardigrades in Malaysia. In 1967, Renaud-Mornant & Serène provided a brief description of Batillipes mirus from several beaches between Kuantan and Terengganu, but no further studies have been conducted in the past five decades. This study is the first to address the taxonomy of marine tardigrades in Malaysia, reporting both a new species, Batillipes malaysianus sp. nov., and a new record, Batillipes rotundiculus Rho, Min & Chang, 1999.
Materials and methods
Intertidal sediment collection was conducted on 29 November 2023 at Pantai Pancur Hitam, Labuan, Malaysia (5°22′27.9834″ N, 115°13ʹ29.496″ E). A revisited sampling was carried out on 16 September 2024 to collect additional sediment samples aiming to obtain more tardigrade samples. The sampling site was a calcareous sandy beach. The sediment sample was collected using a Perspex corer from the surface down to a maximum of 5 cm during the first sampling trip in November 2023. However, additional sediment samples were collected during the second sampling with six replicates of 0.5 m × 0.5 m quadrate area to cover more surface area. Samples were pooled into one Ziploc bag for transfer to the laboratory at Borneo Marine Research Institute, University Malaysia Sabah.
In the laboratory, collected sediments were sieved through a 500 μm mesh and decanted through a 25 μm mesh (Retch, ASTM E11) with tap water to remove macrofauna and microfauna prior to preservation for future extraction (Ng et al., Reference Ng, Chen, Mustafa, Soo, Liao and Shih2022). Sediments were washed into sample bottles and then preserved with 5% formalin and Rose Bengal (0.5 g/L) until further use. Tardigrades were sorted under a stereomicroscope (Olympus VMZ-4F) and a gridded petri dish. Tardigrades were picked out using a wire loop and underwent a series of ethanol-glycerol solutions for dehydration as proposed by Seinhorst (Reference Seinhorst1959) to make permanent mounts of tardigrades. Microscope slides of Batillipes malaysianus and Batillipes rotundiculus are deposited in the collection of tardigrades in the Borneo Marine Research Institute, Universiti Malaysia Sabah with the reference number of IPMB-Pf 13.00001 and IPMB-Pf 13.00002 respectively.
For identification, tardigrades were observed and measured. All measurements were given in micrometres (μm) and photomicrographed under 1000× magnification, using an Olympus BX53 Microscope, 80× using FSX100 Inverted Microscope (Olympus, Tokyo, Japan) with phase contrast microscope and bright field at Universiti Malaysia Sabah. Photomicrographs were also taken at Universiti Malaysia Sarawak, using an IX51 inverted microscope (Olympus, Tokyo, Japan) and captured by an INFINITY-3 digital camera (Teledyne Lumenera, Ottawa, Canada).
Results
A total of six Batillipes individuals were obtained during the two sampling trips. In November 2023, one individual was collected and identified as a new species, Batillipes malaysianus sp. nov. During the second sampling attempt in September 2024 and despite extensive efforts, only five individuals were observed. Four were shrunken, preventing a thorough identification; however, they lacked the distinct morphological characteristics of Batillipes malaysianus sp. nov., particularly the unique cuticular features. The fifth specimen was identified as Batillipes rotundiculus, representing a new record for marine tardigrades in Malaysia. Due to the exceptionally low density of marine tardigrades in the area, a single holotype was designated for the new species while a specimen was recorded for the new species occurrence.
Taxonomic accounts
Phylum Tardigrada Doyère, 1840
Class Heterotardigrada Marcus, 1927
Order Arthrotardigrada Marcus, 1927
Family Batillipedidae Ramazzotti, 1962 (emended by Gallo D’Addabbo et al., Reference D’Addabbo, Sandulli and Grimaldi2005)
Genus Batillipes Richters, 1909
Batillipes malaysianus sp. nov. Chen & Ng, 2024
Diagnosis
Batillipes with tubular primary clavae. Well-developed dome-shaped secondary clavae, appearing as conspicuous ventral platelet-like circular structures. Internal cirri and median cirrus without cirrophores. Cephalic cirri with sharp distal tips. On the fourth leg, middle toes 3 and 4 of different lengths, with toe 3 shorter than toe 4 (toe arrangement pattern B1 according to Santos et al. (2018)). Four pairs of conspicuous dorso-lateral body projections are present. Semi-circular lateral body projection 1 separates the head from the body. Lateral body projections between legs III and IV are triangular with an elongated and thick blunt tip turned backward. The caudal apparatus consists of a prominent semicircular projection. Ventral cuticle uniformly punctated with presence of irregularly organized setae of different lengths.
Type locality
Pantai Pancur Hitam, Labuan, Malaysia at 5°22′27.9834″ N, 115°13ʹ29.496″ E; sandy/calcareous beach, intertidal zone.
Etymology
The name refers to the country where the species was found, Malaysia.
Material examined
Holotype (deposited at the Collection Museum of Borneo Marine Research Institute IPMB-Pf 13.00001): adult, female, collected at Pantai Pancur Hitam, Labuan, Malaysia, mounted in glycerol (Figure 1).
Batillipes malaysianus sp. nov. collected at Pantai Pancur Hitam, Labuan, Malaysia, observed and photomircrographed using Olympus FSX100 inverted phase contrast microscope (PCM). Scale bar 16 µm.
Description of holotype
Female with body length 139 µm (152 µm including the caudal apparatus) and 48 µm wide between the 2nd lateral body projection (between legs I and II) (Figure 1). Large and prominent salivary gland (3.5 µm) is observed in mounted specimens. Trapezoidal head (Figure 2) separated from the body by a neck constriction followed by lateral processes (1st body projection). Internal and median cephalic cirri inserted dorsally without cirrophores (Figure 2A, B, D). External cirri, bearing cirrophores, with sharp distal tips, inserted ventrally, below the common pedestal bearing lateral cirri A and primary clava (Figure 2C–D). Lateral cirri A, located dorsally in relation to the shorter constricted primary clavae which are tubular in shape with blunt end and black punctation (Figure 2B, D). A van der Land’s organ is present at the base of the primary clavae (Figure 2B).
Ventral-anterior portion of Batillipes malaysianus sp. nov. observed and photomircrographed using (A)–(B) Olympus FSX100 inverted microscope phase contrast microscope (PCM) and (C) IX51 inverted microscope, captured by an INFINITY-3 digital camera. (A) Pharyngeal bulb and salivary gland (black arrow); (B) white arrows shows constricted primary clava while black arrows show van der Land’s organ; (C) black arrow show the sharp distal tips of ec; (D) detailed ventral-anterior sketch of Batillipes malaysianus sp. nov. (mc – median cirrus; ic – internal cirrus; ec – external cirrus; lc – lateral cirrus; pc – primary clava; sc – secondary clava). Scale bar 16 µm.
Ventral mouth opening in a slightly protruding circular structure (Figure 2D). Buccal tube 8 μm long and 1 μm wide. Stylets (7 μm long) with strong stylet supports. Ovoid pharyngeal bulb, about 18 μm long and 15 μm wide, with placoids (about 5 μm long). Four pairs of conspicuous dorso-lateral body projections are present. Semi-circular lateral body projection 1 separates the head from the body. Lateral body projections between legs III and IV are more elongated than others lateral body projections, triangular with thick blunt tip turned backward.
Sensory spines are present on all legs. Sharply pointed and short (11.2 μm long) cirri E (cE) are located dorsally between lateral processes and leg IV, not longer than caudal apparatus (Figure 3). The caudal apparatus consists of a prominent semicircular projection. Telescopic legs with toes that have the distal stalk enlarged distally and small circular/ovoid suction discs with thin braces. Legs I–IV have six toes (toes formula 6-6-6-6), indicating that the holotype is an adult (Kristensen and Mackness, Reference Kristensen and Mackness2000). On the fourth pair of legs, toes are arranged according to the B1 pattern (Santos et al., 2018): [(Toe 3 < Toe 4) ≤ Toe 1 & Toe 6] (Figure 4).
Black arrows show cirri E on both sides of the holotype. They are located dorsally between lateral processes and leg IV. Observed and photomircrographed under IX51 inverted microscope, captured with an INFINITY-3 digital camera. Scale bar 5 µm.
Leg IV (left) of Batillipes malaysianus sp. nov., holotype, observed and photomircrographed using IX51 inverted microscope, captured with an INFINITY-3 digital camera. T1–T6 indicate the sequence of toes on this leg. Scale bar 5 µm.
Ventral cuticle consists of delicate punctation with no transverse cuticular folds observed. Unorganized setae of different lengths were also observed on the ventral cuticle from below the pharyngeal bulb to leg IV (Figure 5A and B). Rosette-like gonopore delimited by two anterior cuticular platelets forming a groove towards the anus (Figure 5C and D). Figure 6 shows the ventral cuticle with three focus layers under an inverted microscope. The gradual visibility of the dorsally inserted median cirrus across layers (Figure 6A–C) confirms that the setae originate from the cuticle rather than being attached debris. Table 1 shows the morphometric data of holotype (n = 1).
Ventral details of Batillipes malaysianus sp. nov., observed and photomircrographed using IX51 inverted microscope, captured by an INFINITY-3 digital camera. (A)–(B) show unorganized setae on the ventral cuticle (black arrows). (A) Setae present after pharyngeal bulb; (B) setae between legs III and IV; (C) detail of female gonopore (go) and anus (an) of holotype; (D) detailed drawing of female gonopore (go) and anus (an). Scale bar (A)–(B) 16 µm; (C)–(D) 5 µm.
Ventral observations of Batillipes malaysianus sp. nov., under IX51 inverted microscope, captured by an INFINITY-3 digital camera. (A) The punctated cuticle is clearly visible. (B) As the focus shifts deeper, the setae become more distinct. (C) The cuticle is no longer visible, but the setae appear more prominent. Scale bar 5 µm.
Morphometrics of holotype Batillipes malaysianus sp. nov.
Remarks
The specimen was mounted with the ventral side facing up, limiting the observation of the dorsal characteristics. Despite extensive efforts, including two separate sampling events and multiple replicates during each session, only a single specimen of this new species was collected. These efforts highlight the extremely low density of marine tardigrades in the study area, as observed during this initial pilot study.
Differential diagnosis
Batillipes malaysianus sp. nov. has the toe arrangement of subgroup B1 according to Santos et al. (2018): [(Toe 3 < Toe 4) ≤ Toe 1 & Toe 6]. The key characteristic that distinguishes Batillipes malaysianus sp. nov. is that its primary clavae are constricted, with a van der Land’s organ present at the base, a unique characteristic not observed in any other species within Group B1 except the latest described species B. wyedeleinorum. Additionally, the holotype displays unique morphological traits, including setae on the ventral cuticle, a feature not reported in any other species within Group B1.
Besides the key characteristics mentioned above, the sp. nov. can be easily identified from the 13 species in Group B1 by their four pairs of lateral body projections and the lack of an ala-like caudal apparatus. Of these species, only B. chandrayani, B. similis, and B. littoralis have cephalic cirri with sharp distal tips like the distal tips of the sp. nov. In addition, B. malaysianus sp. nov. can be distinguished from the three species mentioned above by the shape of the caudal apparatus. B. littoralis has a three-pointed spiky apparatus, B. similis has a single conical projection, and B. chandrayani has a triangular-shaped caudal spike, whilst the sp. nov. possesses a prominent semi-circular projection.
Batillipes rotundiculus Rho et al. (Reference Rho, Min and Chang1999)
Material examined
Adult, female, collected from Pantai Pancur Hitam, Labuan, Malaysia at 5°22ʹ27.9834″ N, 115°13ʹ29.496″ E; sandy/calcareous beach, intertidal zone. Specimen mounted in glycerol (Figure 7).
Ventrum of Batillipes rotundiculus observed and photomircrographed with an IX51 inverted microscope (Olympus, Tokyo, Japan), captured with an INFINITY-3 digital camera (Teledyne Lumenera, Ottawa, Canada). Scale bar 25 µm.
Description
Body length 102 µm measured from anterior margin to caudal extreme region. Body slightly wider posteriorly with width 28 µm. Ventral cuticle transparent with fairly regular punctations (Figure 8H). Head 23 µm wide, measured distance between bases of lateral cephalic cirri (Figure 8A). All cephalic sensory cirri forming a pedunculate base distinctly and not frayed at tips. The length of median cephalic cirrus is 6 µm; internal cirri are 11 µm, external cirri are 3 µm; and lateral cirri are 14.5 µm inserted ventrally, originating together with primary clavae from the same enlarged pedestal on each side of head (Figure 8A-B). Cheek’s region is smoothly rounded.
Detailed drawing of B. rotundiculus specimen with labels: mc – median cirrus; ic – internal cirrus; lc – lateral cirrus; ec – external cirrus; pc – primary clava; lp – lateral projection; so – sensory organ; mo – mouth; pb – pharyngeal bulb; st – stylet; ce – cirrus E. (A) Ventro-anterior region of B. rotundiculus (black arrow: stylet); (B) median cirrus; (C) pharyngeal bulb; (D) mouth; (E) detail of female gonopore (go) and anus (an); (F) leg IV; (G) cirrus E; (H) punctation of ventral cuticle; (I) sensory organ on leg II. Scale bar 5 µm.
Pharyngeal bulb (Figure 8C) relatively small and ovoid (length, 13.5 µm, wide, 11.5 µm); stylet (7.5 µm); stylet support and placoids not confirmed clearly (Figure 8A) but mouth protrusion clearly visible (Figure 8D). Body with distinct neck constriction (21 µm); prominent lateral projection presents between head and leg I; blunt lateral projections present between legs I and II, and legs II and III, while legs III and IV have triangular lateral projections with thick blunt tip turned backwards. Cirrus E (cE) thin and relatively short (15 µm) located dorso-laterally (Figure 8G), just posterior to the last body projection. Caudal apparatus prominent, forming a semi-circle.
Legs telescopic and simply cylindrical. Sensory spines are present on all legs (Figure 7I). Each leg with six toes with formula of 6-6-6-6 with fourth leg toe arrangement of group A: middle toes (3 and 4) on feet of leg IV equal in length (considering toe 1 as the most cephalically) according to Santos et al. (2018) (Figure 7F). Female gonopore rosette-like, between fourth leg pair (Figure 7E).
Distribution
Intertidal and shallow sublittoral sand bottom, around Korean coast (Rho et al., Reference Rho, Min and Chang1999); Ría do Barqueiro (Veiga, Reference Veiga2002) and Labuan, Malaysia (current study).
Remarks
The specimen in the current study is similar to the holotype described by Rho et al. (Reference Rho, Min and Chang1999) except for the lateral projections between legs III and IV. Rho et al. (Reference Rho, Min and Chang1999) described them as blister-like, yet the current study observed that they are more similar to the specimen described in the dissertation of Veiga (Reference Veiga2002), which are triangular with thick blunt tip turned backwards.
A dichotomous key to the identification of species of Batillipes
The latest key was proposed by Santos et al. (2018), which uses toe arrangement patterns, shape of primary clava, ventral body projections between legs III and IV, and details of cuticular sculpture as main taxonomic characters, allowing an easier identification based on observations under light microscopy. In this study, the key has been modified and updated to include new species described between 2018 and 2024 including Batillipes malaysianus sp. nov. (Table 2). Global distribution and habitat characteristics of each Batillipes species were also summarized in Table 3.
A dichotomous key to the identification of species of Batillipes
Global distribution and habitat characteristics of Batillipes species (Tardigrada: Heterotardigrada)
Discussion
According to Kaczmarek et al. (Reference Kaczmarek, Bartels, Roszkowska and Nelson2015), marine tardigrades such as Batillipes mirus and Parastygarctus higginsi were reported from Malaysia as early as 1967. However, no subsequent studies on marine tardigrades in the region have been conducted since then. This study provides an update on marine tardigrade species in Malaysia for the first time in over half a century. There are 39 Batillipes species listed in the World Register of Marine Species (Guidetti et al., Reference Guidetti, McInnes and Kristensen2024; WORMS, 2024) and 40 species in the Actual Checklist of Tardigrada species (Guidetti et al., Reference Guidetti, McInnes and Kristensen2024). However, WoRMS and the Actual Checklist of Tardigrada have not yet updated their records to include the most recently described species, Batillipes chandrayaani Vishnudattan, Rubal, Nandan, 2024 from the mid littoral region of Mandapam Mini Coral beach, Tamil Nadu, Southeastern Indian coast and Batillipes wyedeleinorum Bartels, Fontoura, Nelson, Kaczmarek, 2024 from the British Virgin Islands. In fact, there should be 42 species: in WoRMS list, Batillipes kalami Vishnudattan, Rubal & Bijoy Nandan, 2023 is missing and in both lists, B. caudatus Hay, 1917 should be considered as a separated species from B. mirus, which according to Kristensen and Mackness (Reference Kristensen and Mackness2000) and Jørgensen et al. (Reference Jørgensen, Boesgaard, Møbjerg and Kristensen2014) was unjustifiably synonymized with the type species by Marcus (1929). With the addition of B. malaysianus sp. nov. described in this study, the total number of Batillipes species increases to 43.
The observations made on the holotype in this study were unfortunately limited, as no additional individuals of the same species were collected. Nevertheless, the holotype is considered a new species due to its remarkable and unique morphological features. The toes arrangement – Group B1 and the constricted primary clavae is a trait unique to this species and Batillipes wyedeleinorum, yet, unlike B. wyedeleinorum, cephalic cirri of B. malaysianus sp. nov. have sharp distal tips. Additionally, the presence of scattered setae on the ventral cuticle, a feature absents in all known species of the genus, provides strong support for its designation as B. malaysianus sp. nov. (Bartels et al., Reference Bartels, Fontoura, Nelson, Orozco-Cubero, Mioduchowska, Gawlak, Kaczmarek and Cortés2021; Kristensen and Mackness, Reference Kristensen and Mackness2000; Santos et al., Reference Santos, da Rocha, Gomes and Fontoura2017; Vishnudattan et al., Reference Vishnudattan, Rubal and Nandan2024).
Given that Malaysia is one of the world’s megadiverse countries, ranked 12th in the world according to the National Diversity Index (https://www.cbd.int/countries/profile?country=my), it is expected that more species of marine tardigrades will be discovered by improving future sampling efforts. Additionally, the integration of molecular techniques, such as environmental DNA analysis and metabarcoding, will be crucial for advancing our understanding of tardigrade diversity. Future research incorporating these molecular tools will provide a more accurate and comprehensive assessment of species diversity, as well as insights into intraspecific morphological and molecular variations. Such advances will contribute to a growing understanding of tardigrade ecology and their role within marine ecosystems.
Data availability statement
The holotype of Batillipes malaysianus in this study is available at the Collection Museum of Borneo Marine Research Institute with deposition code IPMB-Pf 13.00001.
Authors contributions
Chen, C.A., conceptualization, validation, investigation, resources, writing – review and editing, supervision, project administration, funding; Ng, W.L, methodology, formal analysis, investigation, data curation, writing – original draft, visualization; Teng, S.T., visualization, writing – review and editing; Kajita, T., validation, writing – review and editing.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The author(s) declare none.
Ethical standards
This study did not involve any study subjects that require ethical approval, nor did it involve endangered or protected species. All sampling and laboratory procedures adhered to standard methodological protocols.
