In vitro Culture of Larval Anisakid Parasites of the Chilean Hake Merluccius gayi

Abstract

The present note reports the results of the first attempt to cultivate anisakid larvae from the Southern Hemisphere. Larval anisakids were obtained from the viscera and muscles of the hake, Merluccius gayi, caught in the Pacific Ocean off San Antonio, Chile (33?35'S, 71?37'W). The culture medium was based on that described by Van Banning (1971, J. Cons. Int. Explor. Mer 34: 84-88) for Anisakis larvae type I; fresh bovine liver was homogenized in 500 ml of 0.9% NaCl solution, the pH was adjusted to 2. To culture Phocanema larvae, two drops of heparinized human blood were added to 1 ml of medium. The culture was carried out in a water bath at 34 C. The medium was changed every day and any change or molt noted. At the desired stages of development, the worms were fixed in 1% glutaraldehyde in 0.25 M buffer cacodylate, pH 7.4, and processed for scanning electron microscopy. Eighty-nine (42%) of 210 Anisakis larvae type I reached the adult stage; all the worms were identified as Anisakis simplex. Fifteen (25%) of 61 Phocanema larvae survived to develop into adults which were all identified as Phocanema decipiens. Our results confirmed those of Grabda (1976, Acta Ichthyol. Piscat. 6: 119-141) regarding the in vitro culture of A. simplex. On the other hand, reports of in vitro culture of third stage larvae of P. decipiens have described the occurrence of only one molt before reaching the adult stage (Townsley et al., 1963, J. Fish. Res. Bd. Can. 20: 743-747; McClelland and Ronald, 1974, Can. J. Zool. 52: 471-479). However, we found that P. decipiens from the Chilean hake molted twice. The first molt occurred 4 to 7 days and the second 20 to 25 days after starting the culture. These observations are in good agreement with those reported by McClelland (1980, Exp. Parasitol. 49: 128-136) who experimentally infected harbor and gray seals with P. decipiens larvae from the Atlantic cod. However, the time required for in vitro molting se ms to be slightly longer as compared with in vivo molting. The morphology of larval and adult stages of P. decipiens has already been described by McClelland (1980, loc cit.) and that of A. simplex by Grabda (1976, loc. cit.). However, we believe that the oral region of both species and the tail region of P. decipiens have not been sufficiently illustrated. In our observations, we found that the heads of A. simplex and P. decipiens may be distinguished by lip size and denticle distribution. In A. simplex, the dorsal lip is larger than that of P. decipiens. The central portion of each lip is divided into two lobes defined by a ridge of small denticles, these lobes being proportionally larger in P. decipiens than in A. simplex. The form and disposition of the denticles are different in both species. In P. decipiens, the dentigerous ridges extend up the lateral margins of the lobes, viewed on face they outline a well-defined W, while the ridges of A. simplex end more medially, and are straighter with a small projection between the lobes of the lip (Figs. 1, 2). The denticles of A. simplex are longer and sharper than those of P. decipiens (Figs. 3, 4). The tail region of P. decipiens is characterized by the presence of three postanal plates of spined ridges; it is possible to note in the males that one spicule is shorter than the other, but both of them are of the same thickness (Fig. 5). There are also in this region 3 pairs of postanal papillae on each side (Fig. 6). The Chilean hake, Merluccius gayi, harbored a mixed infection of Anisakis and Phocanema species (Carvajal et al., 1979, J. Fish Biol. 15: 671-677) and the morphological