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http://journals.tubitak.gov.tr/zoology/

Turkish Journal of Zoology Turk J Zool(2014) 38: 471-478© TÜBİTAKdoi:10.3906/zoo-1304-49

Diceratocephala boschmai (Platyhelminthes: Temnocephalida) from crayfish farms in Thailand: investigation of the topographic surface and analysis

of 18S ribosomal DNA sequences

Arin NGAMNIYOM*, Thayad SRIYAPAI, Kun SILPRASITFaculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Bangkok, Thailand

* Correspondence: arin@swu.ac.th

1. IntroductionTemnocephalid platyhelminths are turbellarian ectosymbionts common on freshwater hosts; examples include the order Decapoda of Crustacea; Hemiptera, Trichoptera, and Plecoptera of Insecta; and the family Ampullariidae of Mollusca and Chelonia among the reptiles (Cannon, 1991; Damborenea and Brusa, 2008; Volonterio, 2010). Temnocephalids have a wide distribution range throughout the tropics and in the southern hemisphere, with reports of occurrences in Australia, South America, South Asia, and Africa (Cannon, 1991; Avenant-Oldewage, 1993; Edgerton et al., 2002; Volonterio, 2007). Additionally, Oki et al. (1995) reported of Temnosewellia minor that these temnocephalids have been translocated from Australia to Japan on freshwater crayfish, Cherax tenuimanus. It was also reported that T. minor on Cherax spp. was imported into Turkey (Xylander, 1997), and T. chaeropsis on C. tenuimanus into South Africa (Avenant-Oldewage, 1993). This is the reason why temnocephalids have been used for biological experiments in symbiotic ecology, embryonic development cytogenetic studies, and molecular analyses inferred from rDNA sequences (Oki et al., 1995; Baguna et al., 2001; Younossi-Hartenstein and Hartenstein, 2001; Amato et al., 2007; Du Preez and Smit, 2013; Garcés et al., 2013). Diceratocephala boschmai Baer, 1953, belonging to

the family Diceratocephalidae, was first reported in ectosymbiotic association with aquatic crayfish: for example, on Cherax boschmai, C. communis, C. pallidus, C. lorentzi, and C. longipes from Irian Jaya (Indonesia) and from C. quadricarinatus from Australia and South America (Baer, 1953; Jones and Lester, 1992 and 1996; Volonterio, 2009). Crayfish of the genus Cherax are important species in commercial aquaculture for human food consumption and as pets in many countries (Allinson et al., 2000; Nguyen et al., 2005). Herbert (1987) suggested that D. boschmai might exhibit parasitism among crayfish by consuming the contents of eggs. In spite of this possibility, investigations of D. boschmai have been scarce, and its relationships with closely related taxa, as assessable from rDNA sequences, remain unknown.

In this study, the surface topography and the molecular phylogeny of adult D. boschmai isolated from C. destructor from crayfish farms in Thailand are examined by using scanning electron microscopy (SEM) and 18S rDNA, respectively.

2. Materials and methods Fifteen adult D. boschmai and their eggs were collected from infected C. destructor from commercial crayfish farms in Bangkok, Thailand, and its metropolitan area (Figure 1A). Flatworms and eggs were maintained separately in

Abstract: In commercial astaciculture, Diceratocephala boschmai is known to be an ectosymbiont temnocephalid that is widely distributed on Cherax spp., freshwater crayfish native to Australia. This study makes the first report of D. boschmai in samples collected from C. destructor from crayfish farms in Thailand. A description of its internal anatomy is given, and its topographic surface is described using scanning electron microscopy. The phylogeny of D. boschmai in the Rhabdocoela was analyzed for the first time based on 18S ribosomal DNA (rDNA) sequences. Based on the 18S rDNA sequences, temnocephalids, including D. boschmai, are monophyletic within Rhabdocoela. The congruence between molecular data and morphological information is discussed in the text. The findings reported in the present study add to the body of knowledge on temnocephalids.

Key words: Diceratocephala boschmai, Cherax destructor, topographic surface, 18S rDNA, Thailand

Received: 30.04.2013 Accepted: 28.01.2014 Published Online: 20.05.2014 Printed: 19.06.2014

Research Article

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Figure 1. A) C. destructor harboring adult D. boschmai and eggs of flatworm; B) dorsal view of an extending body; C) diagram of organ structures in dorsal view; D) diagram of reproductive complex; E, F) photomicrograph and diagram of penial stylet, respectively; G) unhatched and hatched eggs. ad, adhesive disc; at, atrium; cv, contractile vesicle; ds, dorsal side; es, ejaculatory sac; ey, eye; ev, excretory vesicle; fi, filament; in, intertentacular flange; ine, intestine; int, introvert; mo, mount; ov, ovary; pe, peduncle; pf, plane of fracture; ph, pharynx; pn, subepidermal pigment network; ps, penial stylet; rv, resorbens vesicle; s, stalk; se, seminal vesicle; sr, seminal receptacle; sp, sclerotized papillae; tc, tentacle; te, testis; tg, tentacular gland; ve, vasa efferentia; vg, vagina; vi, vitellaria; vs, ventral side.

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petri dishes containing aquarium water at approximately 26 °C. They were stained with acetic carmine and fast green (Lester and Larry, 1991) and observed under a light microscope. In order to make histological sections of the worms to confirm and interpret the location of organs, D. boschmai specimens were fixed by Bouin’s fixative solution for 12 h and were stored in 70% ethanol. Those specimens were dehydrated through a graded ethanol series and embedded in paraffin. Transversal serial sections with 6-µm thickness were prepared by using a Leica RM2125 microtome. Sections were stained with hematoxylin and eosin.

For the SEM investigation, 10 adult flatworms were immediately fixed in 2.5% glutaraldehyde fixative in 0.1 M sodium cacodylate buffer, pH 7.4, at 4 °C for 6 h. The worms were washed 3 times at 4 °C for 5 min with 0.05 M sodium cacodylate buffer, and were then postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h. They were then washed 3 times with distilled water and dehydrated through a series of ethanol washes. After dehydration, the specimens were dried in a Hitachi HCP-2 critical point drying machine using liquid carbon dioxide. The specimens were mounted on aluminum stubs, coated with gold at 20 nm of thickness in an ion-sputtering apparatus (SPI-Model sputter coater) for 1 min, and examined with a JEOL JSM-5400 electron microscope operating at 15 kV.

The organ structures were described following the terminology and abbreviations of Joffe et al. (1995), Amato et al. (2007), Volonterio (2009), and Panyarachun et al. (2010). The measurement data of the 15 individuals are given in micrometers as mean ± standard deviation.

For the molecular phylogenetic analysis, D. boschmai genomic DNA was extracted from fresh samples using a DNeasy Tissue kit (QIAGEN) according to the manufacturer’s instructions. The 18S rDNA gene was amplified by Taq DNA polymerase (Takara) using the primer pair TimA (5’-AMCTGGTTGATCCTGCCAG-3’) and TimB (5’-TGATCCATCTGCAGGTTCACCT-3’) (Noren and Jondelius, 1999). DNA amplification was carried out at thermal cycling conditions of an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 10 min. The PCR products, which were of approximately 1800 bp, were electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator. DNA from the PCR products was purified using the QIAquick Gel Extraction Kit (QIAGEN) with spin columns and stored at 4 °C.

DNA sequencing was performed by Macrogen DNA Sequencing Service, Korea. The partial sequence of the D. boschmai 18S rDNA gene was deposited in GenBank

(http://www.ncbi.nlm.nih.gov) under the accession number KC517073. The DNA sequencing results were analyzed for regions of local similarity using the online program BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The multiple sequence alignments were carried out using the ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). For the phylogenetic examination of flatworm species (Table), a bootstrap test with 1000 replications was performed to estimate the support of the neighbor-joining tree by using MEGA software, version 5.10 for Windows (Tamura et al., 2011).

3. Results 3.1. Morphological observations In adult D. boschmai specimens, the average size of the body is 5 mm long and 2 mm wide. The bodies of the adults are tongue-shaped and bear 2 tentacles at the right and left sides of the anterior end. A smooth intertentacular flange (640 ± 121.2) is positioned at the anterior end, and a bluntly rounded adhesive disc (415 ± 72.6 in basal width) is at the posterior end. There is a stalk between the worm body and the adhesive disc (Figure 1B). A large pharynx (586 ± 122.1 in length and 611 ± 142.6 in width) is positioned medially, close to the anterior end (posteriorly to the eyes) and covered by subepidermal pigment networks. These pigments are distributed anteriorly from the eyes to the tentacles and the intertentacular flange and posteriorly to the intestinal area at the beginning of the posterior part. Two excretory vesicles are lateral to the pharynx in the anterior area. The saccate intestine lacks septae (Figure 1C).

In the reproductive complex, vitellaria are located dorsal to the intestine (Figure 1C). Tentacular glands are dispersed laterally, anteriorly to the testes (Figure 1C).

Ovary is ovoid (96 ± 9.9 in length and 49.9 ± 5.9 in width), located subterminally on the right side of the intestine, presenting a short oviduct that enters the cylindrical vagina (129 ± 18.4 in length and 125 ± 17.7 in width). A large resorbens vesicle (151 ± 19.3 in length and 122 ± 14.4 in width) is on the left side of the anterior area of the ovary and opens into the ootype. Four sclerotized papillae with irregular lobes are situated in the posterior part of the vagina. Single rod-shaped seminal receptacles were seen (22 ± 2.6 in length) just below the left side of the resorbens vesicle opening into the vagina through the ootype (Figure 1D). Two irregularly shaped testes (291 ± 40.2 in length and 152 ± 20.8 in width) are situated posterolaterally. The vasa efferentia, of which the right duct is longer than the left, extend from the testes to the claviform seminal vesicle (51 ± 11.2 in length and 26 ± 7.2 in width). Ejaculatory sac seems to be pyriform (47 ± 9.4 in length and 21 ± 8.3 in width). A contractile vesicle (90 ± 16.3 in length and 97 ± 13.9 in width) with

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a rounded aspect is located intermediately between the seminal vesicle and the penial stylet. The curved penial stylet (219 ± 10.6 in length and 68.8 ± 6.1 in basal width), which originates from the contractile vesicle, curves up into the atrium. Introvert (51 ± 6.3 in length and 31 ± 4.1 in width) with 19–20 crowns of spines, positioned at the distal portion of the stylet (Figures 1E and 1F).

Unhatched eggs (667 ± 34 in length and 326 ± 19.2 in width) are clavate and light yellowish-brown in color.

Filaments are present on the apical end of the eggshells. Embryo eyes were seen subterminally from the apical end. Hatched eggs show a horizontal plane of fracture, and the peduncle of the eggs adheres to the shrimp shell (Figure 1G).3.2. SEM investigationUnhatched and hatched eggs are scattered on shrimp shells, attached by the thick peduncles and additional filaments (Figures 2A and 2B). Short filaments are placed subpolarly

Table. Species used in the phylogenetic analysis, with GenBank accession numbers of 18S rDNA and their references.

Species GenBank accession number References

IngroupTemnosewellia minor AY157183 Lockyer et al. (2003)Temnocephala sp. 1 AJ012520 Littlewood et al. (1999)Temnocephala sp. 2 AF051332 Baguna et al. (2001)Diceratocephala boschmai KC517073 This studyDidymorchis sp. AY157182 Lockyer et al. (2003)Castrella truncata AY775777 Willems et al. (2006)Microdalyellia rossi AJ012515 Littlewood et al. (1999)Mesostoma lingua AY775759 Willems et al. (2006)Strongylostoma elongatum AY775771 Willems et al. (2006)Castrada viridis AY775753 Willems et al. (2006)Phaenocora unipunctata AY775762 Willems et al. (2006)Olisthanella truncula AY775761 Willems et al. (2006)Gyratrix hermaphroditus AY775739 Willems et al. (2006)Polycystis naegelii AY775743 Willems et al. (2006)Pseudomonocelis ophiocephala AY775735 Willems et al. (2006)Diascorhynchus rubrus AJ012508 Littlewood et al. (1999)Acrorhynchides robustus AY775737 Willems et al. (2006)Cirrifera sopottehlersae AY775733 Willems et al. (2006)Coelogynopora axi AY775734 Willems et al. (2006)Paromalostomum fusculum AJ012531 Littlewood et al. (1999)Haplopharynx rostratus AJ012511 Littlewood et al. (1999)Geocentrophora wagini AJ012509 Littlewood et al. (1999)Schizochilus caecus AJ775747 Willems et al. (2006)Geocentrophora baltica AF065417 Noren and Jondelius (1999)Nematoplana coelogynoporoides AJ012516 Littlewood et al. (1999)Polystylophora novaehollandiae AJ270161 Littlewood et al. (2000)Provortex tubiferus AJ312269 Noren and Jondelius (2002)Pterastericola australis AJ012518 Littlewood et al. (1999)Cheliplana cf. orthocirra AJ012507 Littlewood et al. (1999)OutgroupCaenorhabditis elegans EU196001 Kiontke et al. (2007)

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Figure 2. Surface topography of D. boschmai. A–C) Unhatched and hatched eggs; D) ventral view of a specimen; E) mouth with protruding pharynx; F) thread-like filaments adhered to the pharynx; G, H) a higher magnification of write-dot boxes in 2E; I, J) a higher magnification of write-dot boxes in 2D; K) ventral view of a specimen at posterior end; L) dorsal view of a specimen; M) a higher magnification of write-dot box in Figure L. ad, adhesive disc; af, adhered filaments; ci, ciliated cell; ds, double spines; fi, filament; gr, groove; gv, gravel-like units; in, intertentacular flange; mo, mouth; op, opercular plate; pe, peduncle; pi, pit; sp, single spine; tb, trabecular meshwork; tc, tentacle; th, thread-like filaments; tr, trunk; vi, villi..

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on the clavate eggs. The opercular plates are conspicuous and perpendicularly arranged to the longitudinal axis of the eggs (Figure 2C).

In the observed adult flatworm, the thick trunk of the pharynx was seen protruding from the mouth (Figure 2D). Thread-like filaments with single and double spines were seen embedded into the lumpy surface of the pharynx (Figures 2E and 2F), but these are exogenous. Cilia are present on the surface surrounding the pharynx and are dense close to the mouth (Figures 2G and 2H). Villi connected into a network widely cover the 2 anterolateral tentacles (Figure 2I). A similar network was seen from the middle of the body to the beginning of the posterior region on the ventral side, interrupted at intervals by grooves on the body wall that are the result of the contraction of the body (Figure 2J). In the posterior area, surrounding and on the adhesive disc area, the surface is characterized by gravel-like units (Figure 2K). An irregular surface extends throughout the dorsal surface, where a few empty pits were also seen (Figures 2L and 2M). 3.3. Phylogenetic testThe phylogenetic relationship of D. boschmai to 29 other species was analyzed by examining the 18S rDNA gene. In the relationships with closely related taxa, the phylogenetic result showed a common ancestry for species belonging to Typhloplanoida, Dalyellioida, and Temnocephalida, but not Kalyptorhynchia or Proseriata. Among the Temnocephalida, D. boschmai, Didymorchis sp., Temnosewellia minor, and Temnocephala sp. were monophyletic. Diceratocephala boschmai was a sister group to 2 Temnocephala species. The clade of the Temnocephalida was the sister group to the clade containing the Typhloplanoida species Castrella sp. and Microdalyellia sp. The 18S rDNA sequences clearly isolated D. boschmai from 5 species of Proseriata (Pseudomonocelis ophiocephala, Cirrifera sopottehlersae, Coelogynopora axi, Nematoplana coelogynoporoides, and Polystylophora novaehollandiae), 2 species of Lecithoepitheliata (Geocentrophora wagini and Geocentrophora baltica), and 1 species each of Macrostomida (Paromalostomum fusculum) and Haplopharyngida (Haplopharynx rostratus). All species were also highly distant from the outgroup (Caenorhabditis elegans) (Figure 3).

4. DiscussionIn this study, by examining material found on the crayfish Cherax destructor from Thailand, we describe the internal anatomy of the temnocephalid D. boschmai as observed in whole mounts and histological sections. These results are similar to a recent report by Volonterio (2009), in which D. boschmai was found on the surface of C. quadricarinatus from an Uruguayan astaciculture farm and was used to provide a detailed description of the

species. The specimens found in Thailand are similar to the Uruguayan D. boschmai: a similar anatomy was found in the reproductive complex and in the shape and size of the penial stylet; the penial stylets of Thai D. boschmai also approach the atrium along a curved path.

This work presents the first description of the eggs of D. boschmai using SEM. According to our observations, the egg plane of fracture and polar filaments are similar in structure to those reported by Volonterio (2009).

Epidermal surfaces of D. boschmai have not been studied by SEM in previous studies. The observation of large ciliated areas on the ventral surface is in agreement with the observations made with light microscopy by Baer (1953) and subsequent authors. The forms and positions of posttentacular syncytia were described in D. boschmai and other temnocephalids such as T. dendyi, Craspedella sp., and Achenella sathonota (Joffe et al., 1995, 1996). Unfortunately, the specimens used for SEM herein were not relaxed during the fixative process; as a result of the contraction, artifacts were obtained that precluded the observation of the plate pattern.

In the 18S rDNA analysis, D. boschmai was grouped within the Temnocephalida as a sister group to Temnocephala spp. This result confirms the molecular point of view in which Temnocephalida is considered to be monophyletic within the Rhabdocoela, and it is in agreement with a report by Willems et al. (2006), in which Rhabdocoela was monophyletic based on the 18S rDNA sequences of 62 rhabdocoels, including Temnocephala spp. Therefore, this result fills a gap, placing D. boschmai into the scheme of the phylogenetic relationships within the Rhabdocoela as classified by 18S rDNA sequences. Furthermore, it is congruent to the recent study of Van Steenkiste et al. (2013), which clarified the dalytyphloplanid phylogeny based on complete 18S rDNA and partial 28S rDNA, in which C. truncate and M. rossi were positioned within Dalyellioida. However, G. buccinicala and Provortex spp. were separated from Dalyellioida into Neodalyellioida. Two dalytyphloplanids were so closely related with Temnocephalid Temnocephala spp. and Temnosewellia minor that both orders were classified in Neotyphloplanoida.

To our knowledge, this is the first study to report the occurrence of the ectosymbiotic D. boschmai on C. destructor from Thailand. Hebert (1987) and Volonterio (2009) found that temnocephalids have been translocated with Cherax quadricarinatus from Australia to other countries with warm water and tropical climates. Therefore, our findings suggest that D. boschmai may also have been translocated to Thailand by associating with Cherax destructor.

In summary, the present study is the first to describe the internal anatomy and surface morphology of D. boschmai

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from crayfish farms in Thailand. The 18S rDNA sequences used for the phylogenetic assessment of the relationships of D. boschmai within the Rhabdocoela place the species

among the closely related Temnocephalida, although detailed morphological and accurate phylogenetic analyses using rDNA sequences remain to be performed.

Figure 3. The neighbor-joining phylogenetic tree based on the 18S rDNA gene, showing the relationships of D. boschmai with 29 other turbellarian species.

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