Microscopie 20.qxp_Layout 1 10/10/17 08:38 Pagina 31 CONTRIBUTI SCIENTIFICI Scanning electron microscopy in the taxonomical study of free-living marine nematodes Lucia Cesaroni, Loretta Guidi, Maria Balsamo, Federica Semprucci Department of Biomolecular Sciences (DiSB), University of Urbino, loc. Crocicchia, 61029 Urbino, Italy Corresponding author: Federica Semprucci Dipartimento di Scienze Biomolecolari (DiSB), University of Urbino, loc. Crocicchia, 61029 Urbino, Italy Tel. +39 0722304248 E-mail: [email protected] Summary Free-living marine nematodes are microinvertebrates composing one of the most diversified groups of the marine biota, with more than 7000 species. This means that only the 20% of the species is currently known. Several morphological features can help their taxonomical identification such as cephalic, cervical and body setae, amphids, cuticle, spicules and tail that may also have a func- tional role. Given the small size of these organisms, they differ in minute characters that can be detected more effectively by scan- ning electron microscopy (SEM). This study presents an overview of the use of SEM on some nematode species collected in the Maldivian archipelago, and highlights the importance of this technique in the taxonomical study of nematodes as well as its poten- tialities in the functional investigation of some of their structures. Key words: nematodes, taxonomic identification, morphological characters, adaptations, scanning electron microscopy. Introduction body, but mainly present on the head region. Arrangement, shape, position and size of sensillae Nematodes are cylindrical elongated worms with and lateral organs (amphids) are species-specific and free-living, symbiotic or parasite life style. In particu- consequently are diagnostic characters for the taxo- lar, free-living species have a body length of about 1- nomical identification. Among them, the amphid is 2 mm and are one of the most abundant component the largest and most complex sensorial organ of the of the meiofaunal assemblage: to date about 7000 nematode cephalic region (Decraemer et al., 2014). species have been described, but this number has Nematodes contribute to the benthic energy flows been estimated as only a small part of the real diver- in different ways (Giere, 2009) and have a central sity of the phylum (Appeltans et al., 2012). role in the trophic chains of marine habitats Nematodes show different body shapes, from the (Danovaro et al., 2008; Semprucci and Balsamo, most common cylindrical form to a short or thick 2012; Semprucci et al., 2015). They have numerous trophic styles that may also be recognized by the dif- body (i.e. order Desmoscolecida) or to a “S” and “ε”- shaped body (i.e. families Draconematidae and ferent morphology of the buccal cavity (Wieser, Epsilonematidae). Other nematodes are completely 1953). covered with bacterial ectosymbionts arranged in Habitats show a nematode fauna of different com- multiple layers (i.e. sub-family Stilbonematinae) or position in species that are characterized by specific may present close relations with ectocommensals morphological and physiological adaptations. It is such as Suctoria (Semprucci and Balsamo, 2012; well known that both the general shape of nematode Ansari et al., 2017). bodies and the structure of their organs may be cor- These organisms have a pseudocoelic body cavity related to environmental factors prevailing in the filled with a high-pressure fluid where the reproduc- habitat for which they are adapted (Wieser, 1959). tive system lies (Platt and Warwick, 1983). Their Due to the small body size, light microscopy has body wall is formed by an external thick cuticular some limits in effectively discerning minute morpho- layer made of collagen. The cuticle usually bears sen- logical details. Several publications focused on the sory structures (sensillae) distributed along the taxonomy and systematics of nematodes have applied ultrastructural techniques, but the latter are 31 Settembre 2017 microscopie Microscopie 20.qxp_Layout 1 10/10/17 08:38 Pagina 32 CONTRIBUTI SCIENTIFICI still underemployed. This paper is a review on the is a major character reported in all taxonomical great variety of morphological features of nematodes descriptions of the species and is used also as an ele- that can be analyzed by scanning electron ment of phylogenetic discussion (e.g. Jensen, 1979; microscopy (SEM), like cephalic, cervical and body Lorenzen, 1994; Leduc, 2013; Semprucci, 2015); how- setae, amphids, cuticle, spicules and tail, and high- ever, this character is valid only in adults because lights the importance of this technique in the taxo- setae may be subject to changes from juvenile to nomical study of nematode species. adults stage. In the head region, the most common pattern of setae is represented by 3 circles around the mouth as Materials and Methods reported in Figure 1A, where hair-like setae are showed and arranged following the scheme 6+6+4. Specimens were obtained from sediments collect- The distinctive feature of this specimen (genus ed from the Central part of the Maldivian Desmodorella, family Desmodoridae) is the presence Archipelago, mainly from Ari, South Malé, and of rows of short setae along the whole body. Felidhoo atolls (Indian Ocean). Sediment samples Although there is no evidence that this specific fea- were taken by a Scuba diver using a plexiglass corer ture of Desmodorella is a possible adaptation to the (Ø2 cm). They were treated with a 7% MgCl2 aqueous habitat conditions, the representatives of this genus solution for narcotizing fauna, and then fixed with a are dominant in medium-coarse sands likely thanks neutralized formaldehyde solution (4% final concen- to the body stoutness provided by the combination tration). Meiofauna were separated from sediment of a thick cuticle and the presence of these short after decantation through a 42 µm mesh sieve and setae along the whole body (Semprucci et al., 2010). centrifugation in Ludox HS 30 (Pfannkuche and Another possible arrangement of the cephalic setae Thiel, 1988). All meiofaunal animals were counted is in two circles with the scheme 6+10. This pattern and sorted by taxon level (mainly phylum and order) is visible in Figure 1B that shows a specimen of the under a stereo-microscope (Leica G26). Some nema- genus Bathylaimus (family Tripyloididae) with the tode specimens especially interesting from a mor- mouth surrounded by 3 high, rounded lips deeply phological point of view were selected for an ultra- incised. The 6 labial setae are very short, while 6 structural analysis by SEM. To apply this technique, longer and 4 shorter jointed setae lie at the same the selected organisms were first rinsed in cacody- level. Jointed setae are rare, but they are a character- late buffer to remove formalin medium and possible istic feature of the family Tripyloididae. foreign material and subsequently they were dehy- Also the presence of setae in the oesophageal drated in a graded ethanol series, 5 minutes for each region (cervical setae) and their position and length solution. Lastly the specimens were transferred in are of taxonomical importance. They are generally hexamethyldisilazane (HMDS) and allowed to dry, mechanoreceptors, possibly with also a chemorecep- according to Hochberg and Litvaitis (2000). Dry tive function (Decraemer et al., 2014). specimens were mounted on aluminum stubs, and Figure 1C shows a nematode of the genus sputter-coated with gold-palladium for 5 minutes. Pseudosteineria (family Xyalidae) with very long Observations were carried out under a SEM Philips subcephalic setae that are subdivided in 8 groups at 515, at the University of Urbino, and under a SEM the same level of the amphids. As argued previously, Q200FEG, at the Agency of the Regional so developed structures could be have an important Environmental Protection of Pesaro. function as an adaptation to the interstitial habitat. The somatic setae on the general surface of the body may be arranged randomly or in a definite pat- tern. Figure 1D shows a particular example of stout Results and Discussion somatic setae of a specimen of the genus Tricoma (family Desmoscolecidae). An important role of Cephalic, cervical and body setae these somatic setae in the locomotion of Sensillae (also called setae) have generally a tactile Desmoscolecidae within the substratum has been function. For instance, long setae probably lead the documented by different authors (see Decraemer worm in the interstitial habitat, especially in exposed and Rho, 2014 for details). sediments, or may be used as adhesive organs as in Finally, setae may be present or absent in the tail some representatives of the family Epsilonematidae region, and their length and number have a relevance (Wieser, 1959; Gad, 2002). in the specific identification. A well-developed cau- The description of the number, arrangement and dal seta in a specimen of the genus Pseudosteineria length of the labial and cephalic setae in nematodes is visible in Figure 1E. microscopie Settembre 2017 32 Microscopie 20.qxp_Layout 1 10/10/17 08:38 Pagina 33 CONTRIBUTI SCIENTIFICI Amphids dorsally so that amphids appear loop-shaped, with Amphids are bilateral and generally symmetrical only one turn, or multi-spiral (with numerous turns). structures situated on the head region and are the The same group includes also amphids with other main sensory organs of the nematodes. They are gen- shapes such as circular and transverse. The non-spi- erally recognized as chemoreceptors, but