*Article* **An Introduction to the Study of Gastrotricha, with a Taxonomic Key to Families and Genera of the Group**

**M. Antonio Todaro 1,\*, Je**ff**rey Alejandro Sibaja-Cordero 2,3, Oscar A. Segura-Bermúdez 2, Génesis Coto-Delgado 2, Nathalie Goebel-Otárola 2, Juan D. Barquero 2, Mariana Cullell-Delgado <sup>2</sup> and Matteo Dal Zotto 1,4**


Received: 27 June 2019; Accepted: 17 July 2019; Published: 23 July 2019

**Abstract:** Gastrotricha is a group of meiofaunal-sized, free-living invertebrates present in all aquatic ecosystems. The phylum includes over 860 species globally, of which 505 nominal species have been recorded in marine sandy sediments; another 355 taxa inhabit the freshwater environments, where they are recurrent members of the periphyton and epibenthos, and, to a lesser degree, of the plankton and interstitial fauna. Gastrotrichs are part of the permanent meiofauna and, in general, they rank among the top five groups for abundance within meiobenthic assemblages. The diversity, abundance, and ubiquity of Gastrotricha allow us to suppose an important role for these animals in aquatic ecosystems; however, ecological studies to prove this idea have been comparatively very few. This is mainly because the small size and transparency of their bodies make gastrotrichs difficult to discover in benthic samples; moreover, their contractility and fragility make their handling and morphological survey of the specimens rather difficult. Here we offer an overview, describe the basic techniques used to study these animals, and provide a key to known genera in an attempt to promote easy identification and to increase the number of researchers who may be interested in conducting studies on this understudied ecological group of microscopic organisms.

**Keywords:** benthos; biodiversity; key; meiofauna; taxonomy

#### **1. Introduction**

Gastrotrichs are minute (from 60 μm to 3.5 mm in total length) vermiform, acoelomate invertebrates; they inhabit the aquatic ecosystems of the world as part of the meiofaunal communities. In freshwater habitats, gastrotrichs are members of the benthos and periphyton and, to a limited degree, also of the plankton and psammon. In marine settings, these tiny animals inhabit (mostly) the interstice of the sandy habitats and are usually the third group in density among the interstitial meiofaunal taxa, behind the nematodes and the harpacticoid copepods (e.g., abundance up to 364 ind./10 cm2) [1]; however, several studies have found them to be the second or the first most abundant meiobenthic group [2–5]. In inland waters, the group usually figures among the top five most abundant taxa, and populations may attain a density of 2600 ind./10 cm<sup>2</sup> [6]. In marine as well as in freshwater systems, the ecological role of Gastrotricha is accomplished within the detritivorous, microphagous benthic assemblage. Gastrotrichs feed on bacteria, microscopic algae, and small protists; food is ingested by aspiration thanks to the powerful, triradiated, myoepithelial pharynx. In turn, they represent prey for small

macrofauna, carnivorous ciliates, and free-living flat worms. The gastrotrichs' ecological disparity is coupled with an ample morphological diversity which may seem amazing when comparing the large and vermiform marine representatives with the tiny and tenpin-shaped freshwater forms. Despite their variety, gastrotrichs are considered to constitute a monophyletic group (phylum) based on the following synapomorphies: (1) cuticle made up of two layers, with the external layer (epicuticle) consisting of one or more plasma-membrane-like sheets (lamellar layer); (2) epicuticle covering the entire body, including the locomotor and sensorial cilia; (3) a "duo-gland system" adhesive apparatus lacking an anchor cell; and 4) peculiar helicoidal muscles enwrapping the anterior portion of the alimentary canal [7,8]. The phylum has a worldwide distribution with some 860 nominal species (as of July 2019) distributed into two orders: Chaetonotida, including 483 tenpin- or bottle-shaped species, two-thirds of which are found in inland ecosystems, and Macrodasyida, grouping 377 vermiform species, the vast majority of which are marine or, more rarely, estuarine. Only four macrodasyidan species, belonging to the genera *Marinellina* (1 sp.) and *Redudasys* (3 spp.), have been reported from freshwater habitats to date. The current classification sees the order Chaetonotida divided into 8 families and 32 genera, whereas the order Macrodasyida counts 10 families and 36 genera. The continuous description of new taxa (species, genera) and the ongoing process of re-systematization suggest that we should consider the statistics reported above as being highly conservative.

Phylogenetic relationships of the Gastrotricha have been questioned for a long time. By virtue of their morphological traits, many researchers have considered Gastrotricha to be close relatives of Nematoda, Rotifera, Gnathostomulida, or Kinorhyncha, within large assemblages such as the Aschelminthes, Pseudocoelomates, etc. However, phylogenetic analyses of the "Aschelminthes", grounded on genetic traits (e.g., 18S rRNA gene) showed such groupings to be polyphyletic and Gastrotricha as part of the Lophotrochozoa but with unstable alliances within the clade [9]. Recent phylogenomic studies have also dismissed the Platyzoa clade, within which Gastrotricha has been allocated for some time, and have convincingly shown Gastrotricha together with the Platyhelminthes allied in a clade named Rouphozoa as a subset within the protostomian Spiralia [10,11]. Parts of the in-group phylogenetic relationships remain unclear, e.g., the evolutionary relationships between the representatives of the two orders or within the clearly paraphyletic family Chaetonotidae. Fortunately, relationships among taxa belonging to several families, especially of the Macrodasyida, are becoming less obscure [9,12–19].

Recent overviews of the gastrotrichs' biology and morphology have been offered by several authors [20–22]. Updated information regarding, e.g., classification, distribution, literature, etc., can be found at the dedicated Gastrotricha World Portal [23] and through the World Register of Marine Species (WoRMS) [24].

#### **2. Materials and Methods**

#### *2.1. Sampling*

Sampling procedures in freshwater environments and marine ecosystems are usually analogous; qualitative studies implicate the gathering of sediment by mean of a scoop, spoon, plastic jar, or a hand-held planktonic net, while quantitative research typically uses corers of clear plastic or Plexiglas (2–5 cm inner diameter, 10–20 cm long). The sea-dweller taxa are typically interstitial, inhabiting preferentially clean, fine to medium sands, with some occurring in muddy substrata (e.g., *Musellifer* spp.) and a few that are tolerant of high sulphide or organic loads [25–30].

Qualitative intertidal sampling is typically carried out at low tide; pits are dug in the beach, and the sand from the bottom and the wall of the pits is then removed with a spoon or scoop and transferred to plastic jars (Supplementary Material Figure S1); subtidal material for qualitative studies can be taken directly by scooping up the upper 10 cm sediment surface with a plastic container (e.g., a 500 mL jar), which is immediately closed off underwater (Supplementary Material Figure S2). Jars filled with sand are then transported to the laboratory and allowed to rest for some time (1 h to

overnight) at a suitable temperature. Over the hours, the fauna move upward and will enrich the top layers of the sand, facilitating the following extraction process (see below). The horizontal distribution of Gastrotricha is patchy; consequently, the collection of several small samples is more illustrative of the taxonomic assemblage of a location than a sole big sample. Interstitial forms of freshwater habitats may be collected using similar techniques. Freshwater gastrotrichs that live on the surfaces of rooted aquatic plants, along with benthic, periphytic, and semiplanktonic taxa, are qualitatively sampled by gathering bunches of vegetation together with the bottom deposits and filtering the water through a net or a sieve with mesh of appropriate size (e.g., 25–30 μm) (Supplementary Material Figure S3). The gastrotrich-enriched sample is placed in buckets and rapidly transported to the laboratory where it is subsequently moved to small aquaria, kept at a suitable temperature, and moderately oxygenated with an air stone (Supplementary Material Figure S4).

#### *2.2. Extraction*

Freshwater and marine samples should both be processed within 5–6 days to obtain the living specimens, which are normally better suited than preserved animals for taxonomic purposes (e.g., identification) since fixation generally causes artifacts that alter and/or obscure the diagnostic characteristics. For freshwater samples only, additional checks 2–4 weeks after sampling are advisable, since taxa initially absent may be found later due to the hatching of resting eggs.

Interstitial fauna can efficiently be separated (extracted) from the sand by narcotization and decantation, using a solution of MgCl2 (7% marine sample or 1% freshwater sample) as a narcotic. For this purpose, 1–2 spoons of the fauna-enriched top layer of sand (see above) are placed into a small vessel with a sufficient amount of added narcotic solution to cover the sand. The material is then swirled and allowed to sit for 5 minutes, after which it is gently swirled again and the liquid decanted into small Petri dishes (5.5 cm). At this stage, a small amount of either seawater (marine samples) or freshwater (freshwater sample) is added to each Petri dish, which is then scanned for gastrotrichs using a binocular microscope at 40–50× magnification, preferably in transmitted light (Supplementary Material Figures S5,S6).

The freshwater, non-sandy samples placed in the small aquaria, as reported above, may be processed for gastrotrichs by sucking up with a large pipet a small amount of the detritus and the overlying water and by transferring the sucked material to a large Petri dish (9.5–12 cm); the dish is then scanned for active (motile) gastrotrichs under a dissecting microscope as described above (Supplementary Material Figure S4). Alternatively, material collected with the large pipet may be transferred to a glass flask, with an equal quantity of 2% MgCl2 solution added, aliquoted into Petri dishes of suitable diameter, and thence analyzed for narcotized gastrotrichs under a dissecting microscope.

For obvious reasons, quantitative studies should be based on fixed material. To reduce artifacts that may hamper species identification, treatment of the freshly collected material (samples) with a solution of magnesium chloride (7% for marine or 1% for freshwater samples) for 5–10 min is very much suggested before the material is fixed. Fixation may be done using a solution of 10% borax-buffered formalin; later, some rose bengal (1%) may be added to ease sorting. Gastrotrich specimens in the quantitative samples may be extracted from the sandy substrata using the same techniques used for other meiofaunal taxa, e.g., by elutriation and multiple decantations. Extraction from samples containing fine sediment and rich in detritus can be carried out by centrifugation using the silica gel LUDOX AM (*d* = 1.210) to create a gradient [31]. The supernatant should be filtered using a 20–30 μm mesh sieve to concentrate the gastrotrichs.

#### *2.3. Morphological Analysis*

Morphometric data should be acquired on living, relaxed specimens mounted on a microscope slide and covered with a square coverslip (15–18 mm). As the mounting of a gastrotrich may be tricky, the following practice carried out routinely at the first author laboratory may facilitate the task. To mount the specimen of interest, a drop of the same medium the specimen is extracted from is put on a clean microscope slide and a single gastrotrich is transferred to it by using a micropipette (mouth or hand held). In the case of freshwater medium, to relax/anesthetize the specimen, a small amount of 1% magnesium chloride solution can be added to the liquid containing the gastrotrich; alternatively, small crystals of alkaloids such as novocaine or procaine are put at the edge of the water so they dissolve gradually in the water, anesthetizing the animal. Thereafter, a clean coverslip (cover glass) is carefully put on the water. To avoid excessive animal compression, the coverslip should not be used as it is; instead, small modelling clay posts are attached beneath its corners before it is put in place (Supplementary Material Figures S7,S8). As deep morphological survey requires the use of oil immersion optics (e.g., 60×, 100×) it is important the specimen be positioned far from the sides of the coverslip. Proper positioning of the specimen at (or near) the center of the slide and its dorso-ventral orientation may be attained by adding a tiny amount of the liquid medium to the cover glass sides or by absorbing the liquid with a piece of blotting paper. Animals gently compressed between the slide and the coverslip are then observed under an upright biological microscope, preferably using DIC (differential interference contrast) lenses (Supplementary Material Figure S9). Fine anatomical traits may necessitate SEM observation; for this purpose, specimens are opportunely prepared (e.g., by hesamethildysilazane or the CPD (critical point drying) technique) [32,33].

Identification of formalin-fixed gastrotrichs from a location may be facilitated by a preliminary identification of the local fauna based on living specimens. Regardless, identification of preserved material can be executed on animals included in watery solutions, or better, established on (semi)permanent mounts. The latter can be set by including the gastrotrichs in a solution of glycerol–formalin (1:3), and the coverslip is then sealed with nail polish or Glyceel. Alternatively, specimens may be mounted in absolute glycerol on an H-S slide after immersion in a 10% glycerol–ethanol solution, which is allowed to evaporate in an oven at 40 ◦C for 2–4 days [34]. However, in many cases, permanent mounts—even in the case of uncontracted, well-oriented specimens—do not permit a full identification as many of the diagnostic traits deteriorate over time. Consequently, for taxonomic purposes, photos or high-resolution video sequences of living, relaxed specimens may deliver superior long-lasting records of the anatomical characteristics of a species compared with specimens mounted on microscope slides.

## *2.4. Taxonomic Key*

The following key, modified from [35], encompasses the valid families and genera of Gastrotricha (Figures 1–20) described to date [24]. Two families (Redudasyidae and Hummondasyidae, belonging to Macrodasyida) and five genera (*Bifidochaetus* and *Cephalionotus* belonging to Chaetonotida and *Anandrodasys*, *Hummondasys*, *Thaidasys*, and *Kryptodasys* belonging to Macrodasyida) included herein have been established since the publication of the previous keys [12–15,36,37]. For the inclusion of *Megadasys* among the Planodasyidae (order Macrodasyida), see [18].

The key is designed to be used by researchers and students who have a general knowledge on how to identify animals but may not have much expertise on Gastrotricha; it is practical in style and is grounded on relevant discriminatory traits as they appear in relaxed mature animals. In most cases, anatomical traits are those which are easily visible using differential interference contrast optics and which are countable. However, to facilitate the assignment, it is imperative that the mounted specimen to be identified is oriented in a dorso-ventral fashion. The following abbreviations are used in the key: PhIJ, pharyngo-intestinal junction; TbA, adhesive tubes of the anterior series; TbD, adhesive tubes of the dorsal series; TbP, adhesive tubes of the posterior series; TbV, adhesive tubes of the ventral series.
