Introduction
Trichinellosis is a zoonotic disease caused by parasitic nematodes of the genus Trichinella (Gottstein et al., 2009). This disease is a public-health concern throughout the world (Pozio, 2001; Dupouy-Camet, 2000).The main sources of human infection are larvae from meat of pork, game and horse. Although human infections have not been reported for certain genotypes, all Trichinella species are considered as human pathogens (Dupouy-Camet et al., 2002). The nervous system of parasitic nematodes plays a key role in the control of all activities of the worm and is thus the main target when new anti-parasitic drugs are developed. The distribution and function of different neurotransmitters have been investigated in numerous helminth groups (Terenina & Gustafsson, 2003, 2014). Nematodes have been extensively studied particularly in Caenorhabditis elegans (Chase & Koelle, 2007). A wide range of signaling molecules, including the bioamine serotonin (5-HT), has been identified. A variety of experimental approaches, including immunocytochemical ones, have revealed 5-HT in 8 classes of neurons in C. elegans. Numerous specific functions of 5-HT have been defined. These include, feeding, locomotion, egg laying and copulatory behavior (Horvitz et al., 1982; Desai et al., 1988; Mcintire et al., 1992; Loer & Kenyon, 1993; Duerr et al., 1999; Sze et al., 2000). Some studies have been made on neurotransmitters in plant nematodes (Holden-Dye & Walker, 2011). According to Masler (2007, 2008) 5-HT influences the general motility, the egg laying process and the stylet activity of Heterodera glycines and Meloidogyne incognita. The effect of serotonin on the reproductive behavior of Heterodera schachtii has also been recorded (Jonz et al., 2001). The receptors for 5-HT and the genes encoding the enzymes of the 5-HT-synthesis have been identified in C. elegans (Loer & Kenyon, 1993; Olde & McCombie, 1997; Hamdan et al., 1999; Sze et al., 2000; Ranganathan et al., 2000; Hobson et al., 2003).
Only limited information is available regarding the function of 5-HT in parasitic nematodes. According to Brownlee et al. (1995) 5-HT has an effect on the pharyngeal musculature of the parasitic nematode Ascaris suum. The presence of 5-HT in the neurons of the pharynx and the tail area of the ventral nerve cord of male A. suum has been described by Martin and Donahue (1989), Johnson et al. (1996) and Fellowes et al. (1999, 2000). According to Rao et al. (2011), 5-HT occurs in the nervous system of the highly pathogenic nematode of small ruminants, Haemonchus contortus, where it was localized in the amphidal and pharyngeal neurons of adult female and male worms. 5-HT was also found in a ray of sensory neurons and in a few ventral cord motor neurons in adult males. According to Rao et al. (2011) 5-HT had an inhibiting effect on the movement of the worms.
Data on the nervous system and neurotransmitters of Trichinella spp. are scarce. The presence of cholinesterase activity (enzyme of cholinergic system) in Trichinella spiralis (Ramisz, 1965; De Vos & Dick, 1992) and presence of several catecholaminergic neurons in the anterior end of this nematode (Lee & Ko, 1991) have been described. Despite its medical importance there are no reports dealing with the presence and function of serotonin (5-HT) in Trichinella. In the present study the distribution of 5HT in Trichinella is investigated. This is thus the first report on the distribution of the neurotransmitter 5-HT in Trichinella.
Discussion
The nervous systems of parasitic nematodes, and in particular that of Trichinella pseudospiralis – the causative agent of human trichinellosis – are poorly described. In this study, we describe the pattern of 5-HT-IR neurons and nerve processes in adult of T. pseudospiralis and in the tail end of the male worms. In the head region, one pair of large neurons, two pairs of small neurons and an additional single neuron occur, some of which equipped with processes extending in anterior and posterior direction. Based on the present knowledge it is difficult to compare the 5-HT-IR nervous system of T. pseudospiralis with that of C. elegans. However, it is tempting to look for homologies between the two worms. In the anterior region of C. elegans, two pairs of 5-HT-IR neurons have been described in detail by Jafari et al. (2011). In C. elegans, the first pair is formed by the right and left pharyngeal neurosecretory motor neurons (NSMR and NSML). The pair of large 5-HT-IR neurons in T. pseudospiralis could be homologous to the above mentioned neurons in C. elegans. In C. elegans the second pair is formed by the right and left amphid chemosensory neurons (ADFR and ADFL), which are defined by having two amphid sensory dendrites each. The small 5-HT-IR structures observed in the tip of the head of T. pseudospiralis could be homologous to the double dendrites of ADFR and ADFL in C. elegans.
Furthermore, Jafari et al. (2011) describe three adjacent ring inter neurons, RIH (unpaired), AIMR and AIML (right and left) in C. elegans.
We suggest that one of the smaller pairs of neurons in T. pseudospiralis is of the same type as the amphidial (ADFs) in C. elegans. The other pair of neurons and the unpaired neuron in T. pseudospiralis are perhaps homologous to the ring inter neurons AI Ms and the single RIH (unpaired interneuron) in C. elegans.
Of the 5-HT-IR pairs of neurons in the anterior end of the model worm, C. elegans, two pairs have been shown to be serotonin producing. Members of one pair have been defined as NSMR and NSML (right and left pharyngeal neurosecretory motoneurons). The members of the other pair are noted as, ADFR and ADFL (right and left amphid chemosensory neurons) and are defined as having two amphid sensory dendrites each. The serotonin produced by these two cell pairs has been shown to diffuse into extracellular spaces (Jafari et al., 2011). On the other hand the authors reported three adjacent ring inter neurons, RIH (unpaired), AIMR and AIML (right and left), instead, to transport serotonin (noted as serotonin reuptake transporters, SERT) controlling in this way a behavioral response to food deprivation. In the present study it was found difficult to actually pinpoint the identity of the neurons observed although, because of the surprising similarity among nematode structure, the serotonin reacting neurons found in our study are likely to be homologues of the neurons defined in C. elegans.
In Haemonchus contortus, which infects small ruminants, four 5-HT-IR neurons situated within the pharynx have been found (Rao et al., 2011). The authors suggest that the pair of brightly stained neurons, located posteriorly to the nerve ring, is homologous to the neurosecretory motor neurons (NSM) in C. elegans (Albertson & Thompson, 1976) and Ascaris suum (Johnson et al., 1996). However, in H. contortus these cell bodies are located posterior to the nerve ring, whereas in C. elegans they are situated anterior to the nerve ring. The difference in the location of these cell bodies in the two species has been discussed by Rao et al. (2011). In H. contortus, the NSM cell bodies are located in the anterior mid region of the pharynx (Rao et al. 2011). In A. suum, the same cells have been shown to be located at the very posterior end of the pharynx (Johnson et al. 1996). We suggest that the pair of large 5-HT-IR neurons in the head region of T. pseudospiralis is homologous to the NSM in A. suum and H. contortus (Johnson et al., 1996; Rao et al., 2011). In H.contortus, the two neurons, located anterior to the NSM, are supposed to be amphidal neurons (Rao et al., 2011).
Small 5-HT IR structures are visible in the tip of the head of T. pseudospiralis. We assume that they are related to the cephalic sense organs described by transmission and scanning electron microscopy in T. spiralis, T. pseudospiralis and other Trichinella species (Kim & Ledbetter, 1980; Hulinská & Shaikenov, 1984; Li-chtenfels et al., 1983). Our immunocytochemical data suggest that the small 5-HT-IR structures in the tip of the head apparently are localized to ADFL and ADFR dendrites in the lateral amphids and that the serotoninergic nervous system takes part in the function of head sense structures in T. pseudospiralis.
In the vulval region of T. pseudospiralis, abundant innervation by serotoninergic neurons was observed. Intensely stained 5-HT-IR nerve elements formed a complex network comprising neurons and nerve fibers. We suggest that the serotoninergic nervous system participates in the egg laying of T. pseudospiralis.
When comparing the 5-HT innervation of the vulval region of different nematodes differences appear. In T. pseudospiralis, the innervation of the vulval region is more extensive than in free living nematodes, where only single 5-HT-IR neurons have been observed (Loer & Rivard, 2007).
An interesting observation in our study was the presence of 5-HT-IR structures located along the whole body of T. pseudospiralis. Nematodes of the order Trichinellida (Ley & Blaxter, 2002) are characterized by having bacillary bands (rows of hypodermal gland cells) present also in Dioctophymatida (e.g. Eustrongylides). The ultrastructure of the of hypodermal gland cells of trichinellid nematodes studied earlier indicated that the region contains glandular and non-glandular cells (Wright, 1963, 1968; Sheffield, 1963; Jenkins, 1969; Bruce, 1970;McDarby et al., 1987) and, occasionally, neurons and sensory receptors (Wright, 1968; Jenkins, 1969; Wright & Chan 1973). According to Zdjarska and Nebesarov (2000) the sense receptors in Capillaria pterophylli (trichinellid nematode of Capillariidae) occur in association with the hypodermal gland cells in a limited part of the nematode body, i.e. in the proximal region between the level of the nerve ring and the end of stichosome.
Studies of the surface morphology of Trichinella spp. (T. spiralis, T. nativa, T. pseudospiralis) by scanning electron microscopy have shown the presence of small pores in the hypodermal gland cells running along the nematode body (Kim & Ledbetter, 1980; Lichtenfels et al., 1983). Lichtenfels et al. (1983) found rows of hypodermal gland cells to initiate 50 μm from the anterior end in Trichinella spp. and to reach 25 μm from the posterior end. While mainly single, pores of the hypodermal gland cells were found to be organized in double subventral rows in the more central regions of the worms. In newborn larvae, muscle larvae, intestinal larvae, and adults of Trichinella spiralis, the presence of hypodermal gland cells with pores was demonstrated using light, scanning and transmission electron microscopy by Kozek (2005).
The proposed functions of the hypodermal gland cells are osmoregulatory and absorptive (Wright, 1963; Wright & Chan, 1973), and secretory (Reichels 1955; Bruce 1974). A sensory function has been suggested by the presence of nerve endings (Wright & Chan, 1973; Kozek, unpublished observations) (See Kozek, 2005). It is evident that 5-HT-IR in the structures located along the body of T. pseudospiralis is connected with the hypodermal gland cells of the worm. The function of serotonin in these structures is unknown. Further studies are needed.
In conclusion, our results present the first report on the identification and localization of the neurotransmitter serotonin in T. pseudospiralis. The localization of the 5-HT-IR structures in the nematode are concentrated to the nervous system of the anterior end and the vulval regions as well as to stained structures running along the trichinella body, which probably correspond to structures of the hypodermal glands previously described in Trichinella species. Further studies are needed to clarify the function of serotonin in T. pseudospiralis.
