RAPID COUNTING OF NEMATODA IN SALMON 

 BY PEPTIC DIGESTION 



Introduction 



A major problem in the management of 

 the international North Pacific salmon fish- 

 ery is the resolution of a line or corridor 

 in the North Pacific Ocean which equitably 

 divides high seas stocks of salmon according 

 to their continental origin. One of the 

 techniques for the characterization of the 

 salmon stocks employs the use of naturally 

 occurring parasites as indexes of racial 

 likeness. 



Among the more promising of the "tracer 

 parasites" is the larval nematode Anisakis 

 sp. Criteria for specific identification 

 of Anisakis larvae are not well established. 

 The species considered in this study may be 

 one of several reputed species from fish- 

 eating mammals. The larvae in salmon appear 

 to be morphologically identical with those 

 in herring. Furthermore, the ecological 

 background of herring and salmon have much 

 in common to support a hypothesis of iden- 

 tity between these larval parasites. Bishop 

 and Margolis (1955) considered the Anisakis 

 larvae in herring ( Clupea pallasii ) from 

 the coast of British Columbia to be the 

 intermediate stage of Anisakis simplex 

 (Rudolphi, 1809), a stomach parasite of 

 whales and seals. Whatever the taxonomic 

 status of these forms, there is no evidence 

 to suggest that more thaji one species is 

 represented in salmon. 



The worms are colorless, translucent 

 organisms which usually occur, to a greater 

 or lesser degree, tightly coiled in lentic- 

 ular cysts on serosal surfaces of visceral 

 organs and in the somatic musculature of 

 the salmon, within and between the myotomes. 

 The difficulty of detection, recovery, and 

 enumeration of these parasites in salmon 

 musculature by the routine method of dissec- 

 tion and direct observation is a deterrent 

 to a satisfactory rate of progress for 

 thorough quantitative analysis of the infec- 

 tion phenomenon. Therefore, em investiga- 

 tion weis undertaken to explore and develop 

 other suitable methods of isolation and 

 enumeration of the larval worms. 



Various methods of chemical and 

 enzymatic digestion of the host musculature 

 were studied. Of these, only peptic diges- 

 tion at relatively high temperatures proved 

 efficient and economically suitable for use 

 with large numbers of samples. 



Procedure 



The fish are filleted and skinned. 

 Each fillet is weighed to the nearest gram 

 and then broken into several smaller pieces 

 to increause initial surface area. The 

 material from an individual fillet is placed 

 in a digestion vessel (a beaker or jar of 

 approximately 4 liters' capacity). Five 

 milliliters per gram of flesh of a 0.25- 

 percent pepsin solution (2.5 grams of gran- 

 ular pepsin, 1:10,000 made to 1 liter with 

 1-percent HCl ) are added to the vessel. 

 The mixture is stirred gently, and the pH 

 is adjusted to 1.2 with concentrated HCl. 



The digestion vessel is placed in a 

 constant-temperature water bath or air oven 

 where the temperature of the digestion mix- 

 ture is raised to and maintained at the 

 temperature optimum for the most rapid di- 

 gestion (52° ± 2* C). The digestion is 

 permitted to continue for 15 minutes after 

 the optimum temperature has been attained. 

 During the heating and holding periods, 

 the mixture is stirred mechjuiically at a 

 moderately high speed using a glass stir- 

 ring rod. 



Following the holding period, the 

 mixture is permitted to stand at room tem- 

 perature for 5 minutes without agitation. 

 During this period, the peptonized tissue 

 forms a layer at the top of the solution, 

 cuid the worms settle to the bottom. The 

 layer of peptonized material is decanted. 

 The remaining liquid is agitated to suspend 

 the worms and then filtered through a 

 standard 80-mesh grading sieve. The resi- 

 due on the screen is washed several times 

 with cold water and then transferred to a 

 large Petri dish to which is added a few 

 milliliters of 10 percent formaldehyde. 



