Morphological and genetic characteristics of the

Parasitol Res (2014) 113:3419–3425 DOI 10.1007/s00436-014-4007-5

ORIGINAL PAPER

Morphological and genetic characteristics of the anisakid nematode Raphidascaris acus from the southwest Caspian Sea: evidence for the existence of sibling species within a species complex Mikhak Jahantab & Mohammad Haseli & Zivar Salehi

Received: 20 March 2014 / Accepted: 18 June 2014 / Published online: 2 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Recently, it has been shown that many nematode species are in fact species complex, using exact morphological and genetic studies. In this case, there are no such studies related to the genus Raphidascaris Railliet & Henry, 1915. Herein, the morphological and genetic variations among the Iranian population of the species Raphidascaris acus (Bloch, 1779) Railliet & Henry, 1915 and the other allopatric populations with morphological and genetic information were compared to show whether this species can be considered as a species complex. R. acus is an anisakid species and has been frequently reported from different host species from the Caspian Sea. Nonetheless, there are no morphological and genetic information for this species from the region. In the present study, a total of 20 specimens of R. acus were collected from Esox lucius Linnaeus, and the morphology of the Caspian population of this species was surveyed for the first time using both light and scanning electron microscopy. Meanwhile, some parts of ribosomal DNA (rDNA) including internal transcribed spacer 1 (ITS1), 5.8 s, and ITS2 were sequenced and presented as the genetic marker for this species. To understand whether R. acus can be considered as a species complex, the Caspian population of this species was compared morphologically with the allopatric populations of Czech and Canada and genetically with the allopatric population of Poland (Vistula lagoon). Morphologically, there was no difference between the Caspian and Czech populations, but the Caspian and Canadian populations differed in the length of ejaculatory duct and the presence of small triangular elevation between the bases of subventral lips. The nucleotide difference between the Caspian and Polish populations was 4.48 %. M. Jahantab : M. Haseli (*) : Z. Salehi Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran e-mail: [email protected]

In comparison with the interspecific genetic distances in the genus Raphidascaris, this value is notable. In conclusion, based on morphological and genetic differences among the allopatric populations of R. acus, this species is probably a species complex. Nonetheless, the definitive taxonomic decision in recognizing R. acus as a species complex and the description of its sibling species depend on surveying other allopatric populations morphologically and genetically accompanied by an evaluation of reproductive isolation among them. Keywords Raphidascaris acus . Caspian Sea . Species complex

Introduction A genetic marker is a conserved DNA sequence with low ratio of intraspecific polymorphism, which can distinguish species from one another (Hebert et al. 2003). Recently, it has been shown that the ribosomal DNA (rDNA) region comprising the internal transcribed spacer 1 (ITS1), 5.8 s, and ITS2 can act as a genetic marker for the identification of anisakids at the species level (Chilton et al. 1995; Zhu et al. 1998, 2000, 2001; D’Amelio et al. 2000; Zhang et al. 2007; Fang et al. 2010). Hence, recently in a publication of new anisakid species, both morphological description and genetic marker are presented (Li et al. 2012a, b; Shamsi et al. 2008). Using these genetic markers, it has also been known that some cosmopolitan anisakid morphospecies are in fact species complex (e.g., Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) sensu lato (s. l.), Pseudoterranova decipiens (Krabbe, 1878) s. l., Contracaecum osculatum (Rudolphi, 1802) s. l., Contracaecum ogmorhini Johnston & Mawson, 1941 s. l.,

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and Contracaecum rudolphii (Hartwich, 1964) s. l.), comprising several sibling species (Nascetti et al. 1993; Abollo et al. 2003; Mattiucci et al. 2003; Shamsi et al. 2009). The genus Raphidascaris Railliet & Henry, 1915 consists of three subgenera namely Ichthyascaris Wu, 1949, Sprentascaris Petter & Cassone, 1984, and Raphidascaris Railliet & Henry, 1915 (Moravec et al. 1990; Moravec and Nagasawa 2002). Despite a relatively high species diversity in this genus, the ITS1, 5.8 s, and ITS2 regions of only three species belonging to the subgenus Ichthyascaris, including Raphidascaris lophii (Wu, 1949), Raphidascaris trichiuri (Yin & Zhang, 1983), and Raphidascaris longispicula Li et al., 2012, are completely available (Damin and Heqing 2001; Li et al. 2012b; Xu et al. 2012). Except for Raphidascaris acus (Bloch, 1779) Railliet & Henry, 1915 (see Kijewska et al. 2008), there is no information about the ITS and 5.8 s sequences of two subgenera Sprentascaris and Raphidascaris. To date, no study has been published on whether there are species complexes within the genus Raphidascaris. R. acus is a cosmopolitan species reported from different host species (see Smith 1984; Moravec 1994; Nagasawa et al. 2007). The Caspian Sea and its drainage basin are also the regions from where this nematode species has frequently been reported (Khara et al. 2011; Pazouki et al. 2011; Rahanande et al. 2011; Sattari et al. 2001, 2005). Nonetheless, the morphological and genetic characteristics of R. acus are unknown in this water body. In the present study, the morphological and genetic characteristics of R. acus are presented from the Caspian Sea for the first time, and in order to evaluate R. acus as a species complex, the Caspian population is compared morphologically and genetically to the allopatric populations for which the related information is available.

Materials and methods Sampling and microscopic examination In May 2013, a total of 32 specimens of Esox lucius Linnaeus (13 males and 19 females; body weight 0.2–0.750 kg) were caught by local fishermen from the Anzali Lagoon, southwest of the Caspian Sea. The anisakid worms were isolated from the intestines and washed in physiological saline. For each nematode, a small piece of the mid-body was removed and stored in 98 % ethanol for molecular analyses and the rest of the body was stored in 70 % for morphological studies. The adult nematodes were cleared in glycerin, studied under light microscope, and identified using published keys (Moravec 1994; Gibbons 2010). Measurements are in micrometers and were taken using an ocular micrometer and presented in the text as the range followed by the mean and standard deviation in parentheses. The number of nematodes examined (N) and

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the total number of measurements (n) in cases when more than one measurement was taken per worm are also presented. Drawings were made using a drawing tube attached to an Iranian hp NP-21 microscope. For examination with scanning electron microscopy (SEM), the nematodes were dehydrated in an ethanol series and dried in hexamethyldisilazane, mounted on stubs, coated with gold, and examined in a Vega ІІ Tescan-LMU. The morphology of the Caspian population of R. acus was compared respectively to the Czech (the Bystrice River, the Danube River basin; Moravec 1971, 1994) and Canadian (Smoky hollow lake, Ontario; Smith 1984) populations. Voucher specimens and molecular vouchers have been deposited in the Museum d’Histoire Naturelle, Geneva, Switzerland (MHNG) (morphological vouchers (20 specimens): MHNG-INVE-88872–88891; molecular vouchers (7 specimens): MHNG-INVE-F2031– F2037). Molecular analyses Seven adult specimens were randomly selected for molecular analyses. Genomic DNA was extracted using a Column Genomic DNA Extraction mini Kit (Takapouzist Co., Iran) according to the instructions of the manufacturer. Polymerase chain reaction (PCR) was performed to amplify the ITS1, 5.8 s, and ITS2 regions of rDNA. Each PCR (25 μl) contained 2.5 μl 10× PCR buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl), 1.5 μl MgCl2 (50 mM), 1 μl dNTP mix (10 mM of each dNTP), 1 μl (=10 pmol) of each primer, 0.5 μl Taq DNA polymerase (2.5 units/μl, Takapouzist Co., Iran), and 4 μl DNA template. The ITS1, 5.8 s, and ITS2 were amplified using the previously described primers NC5 (5′-GTAGGTGAACCTGCGGAAGG ATCATT -3′; Zhu et al. 1998) and NC2 (5′- TTAGTTTCTTTT CCTCCGCT -3′; Zhu et al. 1998) under the following thermocycling profile: 5 min of initial denaturation at 95 °C; 40 cycles of 45 s at 95 °C (denaturation), 45 s at 55 °C (annealing), 45 s at 72 °C (extension), and 5 min of terminal extension at 72 °C. The amplicons were purified by the AccuPrep PCR Purification Kit (Takapouzist Co., Iran). Takapouzist Co., Iran, carried out the sequencing bidirectionally using the primers NC5 and NC2. The sequences were edited manually by Chromas Pro v. 1.4.1 (Technelysium Pty Ltd, Australia) and aligned using online ClustalW2 program (http:// www.ebi.ac.uk/Tools/msa/clustalw2). The ITS and 5.8 s sequences of the Caspian population of R. acus were deposited in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov) under the accession number KM047505 and compared with the relevant partial sequence of the Polish population (Vistula Lagoon; Kijewska et al. 2008) as well as with the relevant sequences of the other species of the genus Raphidascaris formerly deposited in the GenBank database. The pairwise comparison of sequence differences was calculated using the formula presented by Chilton et al. (1995).

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Results Morphological examination of R. acus from the Caspian Sea Description (Figs. 1 and 2) General: Medium-sized worms, whitish nematodes, cuticle with transverse striations. Three anterior lips with prominent lateral flanges (Fig. 2a, b). Anterior part of each lip with two lobes. Two lateral double papillae on dorsal lip; each subventral lip with one lateral amphid, one single papilla, and one double papilla (Figs. 2a, b). Lateral alae distinct, starting from the region between the bases of subventral and dorsal lips and extending posteriorly to mid part of tail (Figs. 1a, e and 2a). Interlabia absent (Fig. 2a, b). Esophagus muscular, broader posteriorly than anteriorly, representing 8.5–13.31 % of body length. Excretory pore ventral, posterior

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to nerve ring (Fig. 1c). Ventriculus glandular, approximately as long as wide. Ventricular appendix relatively long. Intestinal caecum absent (Fig. 1c). Tail conical in both sexes, curved ventrally in male (Fig. 1e, f). Male (based on nine adult specimens): Length of body 19,512–36,585 (27,967±7,352; N=6), maximum width 366–731 (545±156, N=6). Dorsal lip 72–93 (86±9, N=6) long, maximum width 75–105 (85.5±17, N=3); subventral lips 70–98 (82±8, N=9, n=14) long, maximum width 70– 105 (88±10, N=9, n=10). Esophagus 2,352–3,528 (2,967± 419, N=9) long, representing 8.95–13.31 % of body length. Nerve ring 461–784 (605±139, N=4) from anterior extremity. Ventriculus 108–225 (162±39.5, N=8) long, 98–196 (158±32, N=8) wide; ventricular appendix 1,098–1,715 (1,278 ± 202, N = 8) long. Ejaculatory duct 686–1,666 (1,210±361, N=7) long. Spicules subequal, 688–863 (750 ±62.5, N=8) long, representing 48.44–114.41 % of length of ejaculatory duct and 2.01–3.71 % of body length. Posterior papillae subventral, arranged in two rows, 16–19 pairs of precloacal, 1 pair of adcloacal, and 4 pairs of postcloacal papillae. Medioventral papilla anterior to cloaca present. Lateral phasmids present. Tail curved, 117–196 (172±27, N=7) long. Female (based on 11 adult specimens; two specimens examined for SEM): Length of body 20,244–44,634 (29,823± 8,234; N=11), maximum width 415–1,097.5 (728±199.5, N= 11). Dorsal lip 72–98 (87.5±10, N=8) long, maximum width 79 (N=1); subventral lips 68–105 (82±12, N=11, n=15) long, maximum width 70–100 (81±10, N=5, n=11). Esophagus 2,156–4,606 (3,078±681, N=11) long, representing 8.5– 12.83 % of body length. Nerve ring 441–588 (527±62, N= 4) from anterior extremity. Ventriculus 98–265 (165±49, N= 10) long, 98–274 (164±52, N=10) wide; ventricular appendix 539–1,616 (1,079 ± 332, N = 10) long. Vulva preequatorial, 11,956–6,517 from anterior end, representing 25.35–29.08 % of body length. Vagina muscular, posterior to vulva. Phasmids present. Tail 225–470 (341±100.5, N=4) long. Remarks

Fig. 1 Line drawings of Raphidascaris acus from the Caspian Sea: a, b cephalic end; c anterior part; d region of vulva; e posterior part of female; f posterior part of male. Scale bars: a, b 100 μm; c–f 200 μm

The morphological comparison among the Caspian specimens and two other allopatric populations are given in Table 1. In general, there was no morphological difference between the two Caspian and Czech populations, and the quantitative characters overlapped. For most of the characters, the two Caspian and Canadian populations quantitatively overlapped except for the length of the ejaculatory duct (see Table 1). In addition, there is a small triangular elevation between the bases of subventral lips in Canadian specimens (see Smith 1984), but this case is absent in the Caspian specimens. Moravec (1971, 1994) did not also report this elevation in the Czech population of R. acus.

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Fig. 2 Scanning electron micrographs of Raphidascaris acus from the Caspian Sea: a lateral ala between the bases of dorsal and subventral lips and b lacking triangular elevation between subventral lips

Genetic characteristics

Discussion

There was no sequence variation among the Caspian specimens of R. acus. The ITS and 5.8 s sequences of the Caspian population were compared with the Polish sequences (GenBank accession number AY603537.1) at 891 nucleotide positions and differed in 40 positions. The sequence variation between the two Caspian and Polish populations was 4.48 % (Fig. 3). Meanwhile, the interspecific sequence variations of the genus Raphidascaris are given in Table 2.

The present study is the first case in which the allopatric populations of one species of the genus Raphidascaris were compared to discover sibling species in a species complex. Therefore, the data of this investigation cannot be compared with a similar study in the genus Raphidascaris and must be compared with similar studies related to the family Anisakidae in which the genetic distances have also been calculated based on the formula of Chilton et al. (1995). One of the anisakid

Table 1 Morphological comparison among allopatric populations of R. acus Caspian specimens Male

Female

Canadian specimens

Czech specimens

Male

Male

Female

Female

Body length

19,512–36,585

20,224–44,634

31,500–39,500

40,500–49,200 10,000–44,000

16,000–43,900

Body width Posterior lip length Posterior lip width Subventral lip length Subventral lips width Nerve ring from anterior extremity Esophagus length Ventriculus length Ventriculus width Ventricular appendix length Tail length Distance of genital pore from anterior end Spicule length Number of preannal papillae Number of postannal papillae Length of ejaculatory duct Ratio of esophagus length to body length Ratio of spicule length to body length

366–731 72–93 75–105 70–98 70–105 461–784 2,352–3,528 108–225 98–196 1,098–1,715 117–196 – 688–863 16–19 4 686–1,666 8.95–13.31 % 2.01–3.71 %

415–1,097.5 72–98 79 67.67–105 70–100.33 441–588 2,156–4,606 98–265 98–274 539–1,616 225–470 6,517–11,956 – – – – 8.5–12.83 % –

630–830 92–118 – 92–118 – 630–830 3,450–4,110 – 170–240 1,440–2,070 235–291 – 544–802 16–21 4 1,700–2,570 1–10 % 1–2 %

810–1,070 93–132 – 93–132 – 600–740 3,510–4,620 – 215–276 1,370–2,230 500–730 9,830–11,650 – – – – 8–9 % –

120–1,560 78–180 98–228 78–182 100–218 462–737 1,600–4,580 133–372 108–332 800–2,110 108–239 – 440–1,200 17–20 5 – 10–16 % 2–4 %

470–931 107–198 201–240 100–224 226–260 665–798 2,510–5,150 199–332 149–332 226–2,660 258–490 4,080–9,970 – – – – 12–16 % –

Ratio of spicule length to ejaculatory duct length

48.44–114.41 %

–

31–32 %

–

–

–

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Fig. 3 Alignment of the first internal transcript spacer (ITS1), 5.8 s, and second internal transcript spacer (ITS2) for the Caspian and Polish (AY603537) Raphidascaris acus. The numbers refer to the alignment position, and asterisks indicate positions with the same nucleotides

Table 2 Pairwise comparison of ITS and 5.8 sequences among the species of Raphidascaris R. trichiuri R. lophii R. longispicula

R. trichiuri (%)

R. lophii (%)

R. longispicula (%)

R. acus (Caspian Sea) (%)

– 0.44 3.85

0.44 – 3.74

3.85 3.74 –

24.84 25.41 24.41

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genera is Contracaecum Railliet & Henry, 1912 for which several sibling species were recently presented (Shamsi et al. 2008, 2009). Based on morphological and genetic examinations of specimens of C. rudolphii (Hartwich, 1964) isolated from two species of cormorants in Australia, Shamsi et al. (2009) recognized two sibling species within the C. rudolphii species complex. The most significant morphological differences based on which two morphotypes were recognized as two valid species included differences in the diameter of the labial region, the distance of the deirids from the anterior end, the distance from cloaca to the posterior end, the length of body, lips, esophagus, intestinal caecum, and ventricular appendix. Base-pair nucleotide differences of 0.9 % and 1.8– 3.2 % for ITS1 and ITS2, respectively, were detected between two morphotypes. In another case, Shamsi et al. (2008) described Contracaecum pyripapillatum which resembles Contracaecum multipapillatum. The validity of this species was only on the basis of the difference in the shape of the preanal papillae, being pyriform in C. pyripapillatum and circular in C. multipapillatum. Despite this insignificant morphological difference, the sequence variation in ITS1 and ITS2 between them was respectively 3.4–3.8 % and 6 %. There are only three species of the genus Raphidascaris for which the ITS and 5.8 s sequences are available (see Li et al. 2012a). These three species namely R. trichiuri, R. lophii, and R. longispicula belong to the subgenus Ichthyascaris. Herein, the sequence variation among these three species was calculated using the formula of Chilton et al. (1995), because in the case of the genetic distance between the two Caspian and Polish populations, it is more logical to consider the sequence variations among the congeners. The sequence variations were 3.74, 3.85, and 0.44 %, respectively, between R. lophii and R. longispicula, R. trichiuri and R. longispicula, and finally R. lophii and R. trichiuri. The value calculated for the sequence variation between the Caspian and Polish populations was 4.48 % that is considerable compared with the interspecific values of the genus. It is worth mentioning that the ITS sequences of the Polish population are partial (GenBank accession number AY603537.1), and the sequence variation value may be increased if the complete sequences are provided. Because of lacking morphological information about the Polish nematodes, a comparison between this population and other allopatric populations is impossible. The lack of morphological difference between the Caspian specimens and those based on which Moravec (1971, 1994) described R. acus from Czech Republic does not indicate the genetic similarity between Czech and Caspian or even between Czech and Polish populations because genetic and morphological divergence may unsimultaneously happen (Nascetti et al. 1993). Certainly, a significant sequence variation between the two Caspian and Polish populations is strong evidence on the lacking gene flow between the Caspian Sea and Vistula Lagoon.

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Given the morphological and genetic criterions based on which anisakid species were validated as discussed in the above paragraphs, the presence of the triangular elevation between the bases of the subventral lips only in the Canadian specimens, the difference in the length of ejaculatory duct between Canadian and Caspian populations, and a considerable genetic distance between the Caspian and Polish populations are so significant that R. acus can be considered as a species complex. The present study revealed the morphological and genetic divergence among allopatric populations of R. acus for the first time, but recognizing allopatric morphtypes and genotypes as sibling species depends on the knowledge about the reproductive isolation among allopatric populations provided by surveying their hybrids.

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