Indian Journal of Geo-Marine Sciences Vol. 45(3), March 2016, pp. 453-458

Morphological and histophathological studies of dirus infection in internal organ of Caranx ignobilis

Alagarsamy Sakthivel, Periyasamy Selvakumar & Ayyaru Gopalakrishnan* Center of Advanced Study in Marine Biology Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamilnadu, India *[E-mail: [email protected]]

Received 26 December 2014; revised 28 January 2015

Adult worms of possessed a proboscis which is long, cylindrical with a uniform width measuring up to 0.42±0.02 (0.37-0.48) mm in length and 0.13±0.02 (0.07-0.16) mm in width. Scanning electron microscopy [SEM] photographs were revealed that the proboscis hooks large, uniform size (15 rows of 13 hooks each) with a row of longer hooks at the base. Histopathological study revealed that the fish intestinal tissues totally destroyed the ultrastructure of the infected intestinal tissues, such as mucosal epithelium, stratum granulosum, lamina propria, muscular and serosa of the wall of the intestine forming a tunnel surrounded with collagenous fibers and granulocytes. Inflammation, granular cells formation, connective tissue proliferation was associated with the host immune madulation of the infected intestine.

[Keywords: Acanthocephalus dirus, Histopathology, Internal organs, SEM, Caranx ignobilis.]

Introduction of direct attachment between the proboscis and Fish parasite infection constitutes one of the host intestine 8. The affected fish intestine were most important problems and challenges in irreversible mechanical damage caused by the throghout world. Numerous reports are available attachment of the armed proboscis affects the on the harmful effects of many acanthocephalan architecture of the intestinal tissues leading to parasites on the digestive tract and associated pathological changes. Since the damege of the organs of different fish species1-6 but, no more intestinal villi, formation of the granular tissues information is not available due to and capsule formation associated with host Acanthocephalus dirus infections in Caranx immune responses which seriously affect the ignobilis. Therefore the present study was digestive and absorptive efficiency of the 9. undertaken to determine the pathological changes Moreover in the heavy infections they can cause in the intestine of naturally infected Caranx occlusion of the gut and invasion/migration of ignobilis with Acanthocephalus dirus as well as parasites into uncommon locations 10. Similarly, in morphology of the parasite using the scanning this study aslo described intestinal damage in elactrone microscope. caranx ignobilis by acanthocephalan parasites, Acanthocephalan is frequently seen in the histologycal as wll as scanning electron intestines of wild fresh water and marine fishes. microscopy (SEM) study was undertaken. Recntly Sakthivel et al., (2014) have been repoted Parasitization of the intestinal tract with even a acanthocephalan (Echinorhynchus sp.) infection of few acanthocephalids leads to acute inflammation yellowfin tuna (Thunnus albacares) from and apparently is capable of influencing the Nagapattinam, south east coast of India7. growth rate of small fishes 11. Roubal (1993) Moreover, acanthocephalans attach to the host studied the comparative histopathology of intestinal wall using its hooked proboscis, causing Longicollum () infecting the extensive tissue damage and potential fatality in alimentary tract and spleen of a fish. He observed various vertebrates. The pathological effects of L. that the neck and proboscis had penetrated the thecatus can be seen microscopically at the point entire gut wall 12. A layer of compact, rounded 454 INDIAN J. MAR. SCI., VOL. 45, NO. 3 MARCH 2016

fibroblasts and scattered connective tissue fibers Results were surrounding the neck and proboscis. Best of The examination of the intestine Caranx my knowedge this is the first report of caranx ignobilis was exposed the presence of heavy ignobilis infected by Acanthocephalus dirus infection by Acanthocephalus dirus (Fig. 1). parasite as well as infected fish intestine ultra- Worms were yellow to orange in colour and they structure and anatoy of the parasite also studied. were seen in attached to the posterior region of the Material and Methods intestine (Fig. 2a). The intensity of infection was The examined fish species Caranx ignobilis varied from moderate to very heavy with the were collected throughout the year (2013) from numbers varying from 8 to 13 in Caranx ignobilis. the Nagapattinam coast (10º 45' 36.25" N and 79º The worm possessed a proboscis which was long, 50' 59.54" E) Tamil Nadu, India. Total length and cylindrical with a uniform width measured up to weight of the fishes were measured and examined 0.42 ± 0.02 (0.37-0.48) mm in length and 0.13 ± for the parasitic infection in the intestine, site of 0.02 (0.07-0.16) mm in width (Fig. 2b and c). attachment; orientation numbers of parasites in Proboscis hooks observed by SEM was large, each fish were recorded. Infected fishes were kept uniform in size (15 rows of 13 hooks each) with a in the ice box and brought to the laboratory. The row of longer hooks of Acanthocephalus dirus entire intestine was dissected open longitudinally (Fig. 3b and c). Proboscis, anterior end as well as and examined for the presence of acanthocephalan posterior end is one of the most important identical parasites under a stereo zoom microscope. organs of this parasite (Fig. 3a and d). Intestine along with the parasite was placed under refrigeration for a day for dislodging the attached parasites and for extending and averting their proboscis. Parasites attached firmly to the walls/tissue of the intestine were dissected out with the aid of fine needles and forceps. The specimens were then fixed in cold 75% ethanol. For preparing whole mounts, the specimen was pressed slightly between glass slides, fixed in AFA (alcohol–formalin–acetic Fig. 1-Infected intestine showing attached Acanthocephalus acid) over night, stained with Semichon's dirus. acetocarmine stain) and counter stained with fast green. For scanning electron microscopy, parasites Histopathological studies revealed that, severel were fixed in 2.5% glutaraldehyde followed by 1% damage in the wall of the infected intestine (Fig. Osmium peroxide, dehydrated inacetone series, 4a). Intestinal folds at the sites of parasite critical point dried, gold coated and observed attachment had their tips eroded and appeared under a SEM (JEOC JMS 5610LV) at an flattened along with increased mucus production acceleration voltage of 8kv and electron (Fig. 4b). Degeneration and necrosis of the micrographs taken. Prior to stored in the ice box mucosal epithelium were evident in many areas some of the infected samples were cut and and they were totally eroded, drastically reducing preserved in the 10% buffer formalin for the absorptive area available for the digestive and histopathology study, then the samples were absorptive functions of the animal. In intestinal dehydrated in ethanol series, cleared,embedded in bilinear the site of parasite attachment, the paraffin and 6 μmthick sections were taken using a epithelial cells were found detached, capillaries Yorco Microtome. The sections were stained using and they were ruptured and clumps of RBC was Harris Haematoxylin and Eosin and examined seen lying free in the lumen (Fig. 4c). under a light microscope photomicrograph were taken.

SAKTHIVEL et al.: MORPHOLOGICAL AND HISTOPHATHOLOGICAL STUDIES OF ACANTHOCEPHALUS DIRUS 455

and it can clearly express that heavy infection of Caranx ignoblis with Acanthocephalus dirus. It may have important consequences for host- parasitized fitness. Morover overall, the detrimental effects on its monarch host as well as on the co-occurring nematode parasite caused dramatic pre-adult mortality of monarch butterflies and thereby strongly reduced Caranx ignoblis health and latter, the parasite requires the monarch to reach adulthood for its transmission. In this Fig. 2-(a) Stereo microscopic view of the Acanthocephalus dirus. (b) Proboscis of Acanthocephalus dirus arrow showing effect was harmful to the host and resembles the head with clear hooks. (c) and Bursa. protective effects for example, previous research The presoma of the parasites were found to has demonstrated that infections, pathological pierce the mucosal epithelium, lamina propria, effects were localized around the attachment of the stratum granulosum, muscular and service of the adult worm. The extent of damage is proportional wall of the intestine forming a tunnel surrounded to the depth of penetration of the proboscis 13. with collage nousfibbers and granulocytes. Moreover in the case of our present investication Inflammation, granular tissue formation, also similarly express that heavy infection and connective tissue proliferation and associated host proboscis was deeply penetrated in caranx immune reactions were evident in the tissues. ignobilis intestine. Aggregation of lymphocytes and the presence of a Bullock (1963) have been studied that generally large number of granulocytes and fibroblasts acanthocephalans cause more damage to the suggest inflammatory responses (Fig. 4d). Though intestinal tissues and induce a more complex host the parasite was enclosed in a fibrous capsule of response, mainly, due to deeper penetration in to host origin, the integument of the parasite the intestine mucosa and worm burdens14. appeared to be unaffected by the inflammatory Intestinal parasites of vertebrates can induce responses mounted by the host's immune system. inflammation of the host digestive tract, resulting in an altered gastrointestinal function, namely Discussion enhanced secretion and propulsive motility of the The current study recorded the food fishes were gut 15. affected by acanthocephalan parasite. Commonly Acanthocephalus dirus had showed strong result

Fig. 3-Scanning electron micrographic view of antereior and trunk adults Acanthocephalus dirus (a), Scanning electron micrographic view of proboscis and trunk of adults Acanthocephalus dirus (b), Close view of proboscis with details of hooks (c), Posterior end of the Acanthocephalus dirus (d). 456 INDIAN J. MAR. SCI., VOL. 45, NO. 3 MARCH 2016

Fig. 4 (a) Infected intestine showing deeper penetration of Parasite proboscis. (b) Higher magnification microphotography illustrating the parasite penetration area surrounded by abundant multinucleate inflammatory cells. (c) Histological section showing the damage caused by the Acanthocephalus dirus. (d) Inflammation granular tissue formation, connective tissue proliferation.

Acanthocephalan parasites was attached to the mechanism, they are able to seriously disrupt the epithelial mucosa alone (Acanthogyrus and integrity of the mucosalgut layer, inducing lesion Acanthocephalus sp) and becomes extreme, with of wide degree: from shallow erosions to deep extensive granuloma and subsequent fibrosis, ulcerations with hemorrhage and perforation of the when the worm's proboscis is anchored in the gut wall. These cases usually result in peritonitis muscle layer or entirely perforates the intestinal and septicemia with the lethal outcome, which is wall Pomphorhynchus spp.16. Similarly, in the case rarely noticed in the wild, except in cases of mass of the presen study, Caranx ignobili intestine have mortalities. Another important factor in the heavy infection due to Acanthocephalu sp. Such pathogenesis of gastrointestinal helminths a as, mucosal epithelium, lamina propria, stratum reduction in the host feed intake that has negative granulosum, muscular and service of the wall of economical repercussions in the farm systems, the intestine forming a tunnel surrounded with whereas in a wild population slowly but collage nousfibbers and granulocytes. constantlyweakens the fish, making the host more Inflammation, granular tissue formation, susceptible to predation 17. connective tissue proliferation and associated host The pathogenicity caused by acanthocephalans immune reactions were evident in the tissues. also depends on the parasite/host species and site At the same time, depending on their acumen of localization 16. Extensive inflammation, SAKTHIVEL et al.: MORPHOLOGICAL AND HISTOPHATHOLOGICAL STUDIES OF ACANTHOCEPHALUS DIRUS 457

peritonitis due to perforation of the gut and Conclusion systemic clinical changes (anurhersia) will occur The Acanthocephalus dirus as observed in only with massive infections, most often in farmed Caranx ignobilis species is inconsonance with the fish 18. But in the case of current phenomenon was various finding and can be linked with many carried out the histopathology study of the ecological factors. Parasites are normally in a parasitised visceral organs of Caranx ignobili complex dynamic equilibrium with their hosts. damaged by Acanthocephalus dirus were Many parasites were responsible for high mortality undertaken. rate, particularly in younger stages. Worms did Sommerville (1982) also reported that not produce any visible ill effects on the general pronounced inflammatory response and focal health of the . However, histopathological haemorrhages accompanie spenetration and early studies revealed severe pathological changes and migration of metacercarial 19. The inflammatory the mechanical damage caused by the worms have reaction predominated by infiltrating totally destroyed the architecture of the intestinal macrophages, is particularly intense around tissues. Therefore, the authors suggest a new host unencysted migrating metacercariae and preceded record, intensity, of the parasite and complete the eventual enclosurein a fibrous capsule of the research in acanthocephalan in the intestine of fish encapsulating metacercaria 20. Similarly the and also the decline of this economically current phenomena was carried out high important native fish in Nagapattinam. possibilities could be related to infected intestine changes into microvasculature including Acknowledgement enlargement of the lymphatic vessels, which is Authors would like to thank Department of directly proportional to the lymphocytic Biotechnology, (BT/ PR14992/SPD/ 11/ infiltration observed around the parasitic infected 1332/2010). Government of India, New Delhi for region 21. Akinsanya (2007) reported degenerative the financial support and to the fisher woman and changes in gut wall, liver and pancreas of man on the Nagapattinam coast for the supply of Synodontis schall as a result of Wenyoniavirilis host fishes both on landing and also in the market. infection. In this present study also carried out acanthocephalan parasites were recovered from References Caranx ingnobilis fish species. 1. Esch G W, Huffines W J, Histopathology associated Several technical hurdles encountered in this with endoparasitic helminthes in bass, J. Parasitol, 59 (1973) 306 – 313. study was the presence of “gaps” in the 2. Hine P M, Kennedy C R, Observations on the histological sections of both and distribution, specificity and pathogenecity of the smallmouth bass intestine. These gaps were acanthocephalan Pomphorhynchus laevis (Muller), J. observed between the proboscis and the host tissue Fish Biol, 6 (1974) 521 – 535. 3. Dezfuli BS, Pironi F, Giari L, Domeneghini C, Bosi G, to which it was attached. This apparent retraction Effect of pomphorhynchus laevis (Acanthocephala) on of the proboscis from the host tissue has been putative neuromodulators in the intestine of naturally observed previously 22 and is considered an artifact infected Salmotrutt, Dis. Aquat Org, 51 (2002a) 27 – of fixation that occurs when the parasite is killed 35. at the time of preservation. That is, there would be 4. Dezfuli B S, Giari L, Simoni E, Bosi G, Manera M, Histopathology, immune-histochemistry and no gap in an actual infection of Caranx ignobilis ultrastructure of the intestine of Leuciscuscephalus (L.) in a living yellow perch or small mouth bass naturally infected with Pomphorhynchus laevis because the parasite by definition attached to the (Acanthocephala), J. Fish Dis, 25 (2002b) 7 – 14. intestinal wall with its hooks. Evidence of this is 5. Dezfuli B S, Giovinazzo G, Lui A, Giari, L, Inflammatory response to Dentitruncus truttae apparent in that the tissue surrounding the gaps (Acanthocephala) in the intestine of , Fish and damaged, presumably from contact with the Shel fish Immunol, 24 (2008) 726 – 733. proboscis. 6. Dezfuli B S, Lui A, Giovinazzo G, Boldrini P, Giari L, Intestinal inflammatory response of powan Coregonus lavaretus (Pisces) to the presence of anthocephalan infections, Parasitology, 136 (2009) 929 – 937. 458 INDIAN J. MAR. SCI., VOL. 45, NO. 3 MARCH 2016

7. Alagarsamy Sakthivel, Periyasamy Selvakumar, acanthocephalan infections, J. Morphol, 112 (1963) 23 Ayyaru Gopalakrishnan, Acanthocephalan – 44. (Echinorhynchus sp.) infection of yellowfin tuna 15. Palmer J M, Greenwood-Van Meerveld B, Integrative (Thunnus albacares) from Nagapattinam, south east neuroimmuno modulation of gastrointestinal function coast of India, Journal of Coastal Life Medicine, 2 during entericparasitism, Journal of Parasitology, 87 (2014) 596 – 600. (2001) 483 – 504. 8. Nickol BB, Crompton DWT, Biology of the 16. Mcdonough J, Gleason LN, Histopathology in the Acanthocephala, Cambridge University Press, rainbow darter, Etheostomaaeruleum, resulting from Cambridge, England (1985). infections with the Acanthocephalans, 9. Ali Othman Al Ghamdi, Description of Pomphorhynchus bulbocolli and acanthocephalusdirus, rhadinorhynchus dorsoventrospinosus (acanthocephala: J. Parasitol, 67 (1981) 403 – 409. rhadinorhynchidae) from the red spotemperor 17. Mercer J G, Mitchell P I, Moar K M, Bisse A, Geissler lethrinuslentjan with new host and localityrecords in S, Bruce K, Chappell L H, Anorexia in rats infected Saudi Arabia, Journal of the Egyptian Society of with nematode, Nippostron gylusbrasiliensis: Parasitology, 43 (2013) 209-214. experimental manipulations, Parasitology, 120 (2000) 10. Nickol B B, Fish Diseases and Disorders. Phylum 641 – 647. Acanthocephala, In: Protozoan and Metazoan 18. Bauer O N, The ecology of freshwater Infections, Woo, P.T.K. (ed.), 2nd Ed. Vol. I. CAB fish.Inves.Gosud. NauchIssled. Inst. Ozer. Rech. Ryb, International, Walling ford (2006). Khoz, 49: 5-206 (In Russian, English transl. Israel Prog, 11. Ahangar M A, Occurrence of pomphorhynchus sp. Sci. Trans Cat, (1959) 622, 3 – 215. (acanthocephala) in the liver of schizothoraxesosinus in 19. Sommerville C. The pathology of Haplorchis pumilio jhelum river, Kashmir, Trends in parasitology (Loos, 1896) infection in cultured tilapias. J Fish Dis Research, 2 (2012) 2319 – 3158. 1982; 5. 12. Roubal, Frank R, Comparative histopathology of 20. Yekutiel D. Metacercaria infections of cichlid fry in Longicollum (Acanthocephalata; Pomphorhyncliridae) Lake Kinneret. M. Sc. Thesis, Hebrew University of infection in the alimentary tract and spleen of Jerusalem (Hebrew text, English summary). 1985. Acanthoporgnu sanstrites, Int. J. Parasitol, 23 (1993) 21. Akinsanya Bamidele, Histopathological study on the 391 – 397. parasitised visceral organsof some fishes of Lekki 13. Mercer J G, Mitchell P I, Moar K M, Bisse A, Geissler Lagon, Lagos, Nigeria, Egypt, Soc, Parasitol 43 (2007) S, Bruce K, Chappell L H, Anorexia in rats infected 209 – 214. with nematode, Nippostron gylusbrasiliensis: 22. Venard C E, Warfel J H, Some effects of two species of experimental manipulations, Parasitology, 120 (2000) Acanthocephala on the alimentary canal of the 641 – 647. largemouth bass, Journal of Parasitology, 39 (1953) 187 14. Bullock W L, Intestinal histology of some salmonid – 190. fishes with particular reference to the histopathology of