Ascaridida: Anisakidae), Parasites of Squids in Ne Atlantic

ELECTROPHORETIC IDENTIFICATION OF L3 LARVAE OF ANISAKIS SIMPLEX (ASCARIDIDA: ANISAKIDAE), PARASITES OF SQUIDS IN NE ATLANTIC

S. PASCUALI, C. ARIASI & A. GUERRA2

ILaboratorio de Parasitologia, Facti/lad de Ciencias del Mar, Universidad de Vigo, Ap. 874, 36200 Vigo, Spain 2/nsliltllO de lnvestigaciones Marinas, Cl Eduardo Cabello 6,36208, Vigo, Spain

Received 30 October 1995; accepted 27 November 1996

REFERENCE:PASCUAL(S.), ARIAS(C) & GUERRA(A.), 1995.- Electrophoretic identification of L3 larvae of Anisakis simplex (Ascaridida:Anisaki- dae), parasites of squids in NE Atlantic. Research and Reviews in Parasitology, 55 (4): 239-241. ABSTRACT:The genetic identification of the larvae of a species of Anisakis collected from North-East Atlantic squids was investigatedby electrophoretic analysis of 17enzyme loci. The correspondence of type I larvae with the sibling species A. simplex B is confirmed. Both I//ex coindetii and Todaropsis eblanae squids represent new host records for sibling B. KEYWORDS:Multilocus enzyme electrophoresis, Anisakis simplex B, squids, NE Atlantic.

INTRODUCTION Electrophoretic analyses: For the electrophoretic tests, homoge- nates were obtained from single individuals crushed in distilled water. These were absorbed in 5 by 5 mm chromatography paper Electrophoretic analysis of genetically determined (Whatman 3MM) and inserted in 10% starch gel trays. Standard allozyme polymorphisms has become a useful taxono- horizontal electrophoresis was carried out at 7-9 V cm' for 3-6 h at mic tool for explaining genetic variations between the 5° C. Gels were then sliced in two, and each part stained for a spe- natural populations of invertebrate organisms (THORPE cific enzyme. The following enzymes were studied: sorbitol dehy- & SOLE-CAVA, 1994). For example, this procedure is drogenase (SOH, E.C 1.1.1.14), malate dehydrogenase (MOH, used in the specific identification of larval stages and the E.C 1.1.1.37), isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), 6- correspondence between these forms and the adult, and phosphogluconate dehydrogenase (6-PGOH, E.C 1.1.1.43), nucle- oside phosphorylase (NP, E.c. 2.4.2.1), glutamate-oxaloacetate likewise in assessing the range of various controversial taxa of marine anisakid nematodes (BULLlNI et al., 1981; Species Life-history n Hosts Geographic origin PAGGI NASCETTI ORECCHIA et al., 1991; et al., 1993; et stage al., 1994). The most important application of genetic analysis to modern taxonomic procedures is in the detec- A. simplex A Adult 10 Phocaena North Sea, Bell tion of biological species, particularly when speciation phocaena Rock: 56°25'N processes occur with no morphological differentiation 02°IO'E (<

Species Sod Adk-2 Lap-I Ap-I Ap-2 PepB MATERIAL AND METHODS A. simplex A 100 lOO 100,102 100 100 100 A. simplex B 92 105 85, 90, 94, 97 92 96 70,80,90 Collection methods: Anisakis sp. type I larvae (Ascaridida: Anisa- kidae) were recovered from the digestive tract and gonad of Illex coindetii (Verany, 1839) and Todaropsis eblanae (Ball, 1841) Table 2.- Biochemical keys for the electrophoretic identification squids. Host specimens were sampled from commercial vessels of larvae and adults of Anisakis simplex A and Anisakis simplex B trawling on the continental shelf and slope off Galicia (NW Spain) (adapted from ORECCHIAet al., 1986; NASCETTIet al., 1986; between 1992 and 1994. MANTTIUCCeIt al., 1991). 240 S. PASCUAL, C. ARIAS & A.GUERRA transaminase (GOT, E.C. 2.6.1.1). adenylate kinase (ADK, E.C. Loci Alleles Loci Alleles 2.7.4.3). phosphoglucomutase (PGM, E.C. 2.7.5.1), esterase-l , 2 (EST, E.C. 3.1.1.1), tripeptide aminopeptidase (PEPB, E.C. 3.4.-.- Sdh 100 0,95 Pgm-2 100 1,00 ), aminopeptidase-I, 2 (AP, E.C. 3.4.-.-), leucine aminopeptidase- 105 0,05 19' I, 2 (LAP,E.C. 3.4.11.1), mannose phosphate isomerase (MP!, 9' ESI-I 100 1,00 E.C. 5.3.1.8), and glucose phosphate isomerase (GPI, E.C. Mdh 100 1,00 2' 5.3.1.9).PEPB has a substratum 10 mg of LEU-LEU-LEU, 10 mg 7' ESI-2 90 0,04 of O-Dianisina in 5 ml of buffer system Tris-HCI pH 8,00. For AP- Idh 93 0,37 96 0,21 I, AP-2 the following recipe has been used: 20 mg of LEU-ALA, 100 0,60 100 0,67 10 mg of O-Dianisina in 5 ml of buffer solution Tris HCI pH 8,00. 107 0,Q3 105 0,08 Details of the enzymes studied and the electrophoretic techniques 7' 4' used follow NASCETTI et al. (1986). Locus and allele designation 6-Pgdh 93 0,42 PepB 70 0,28 was as follows: isozyme loci were numbered in order of decrea- 7 0,04 80 0,64 sing mobility from the most anodal: alleles were named numeri- 100 0,54 90 0,08 cally according to their mobidity relative to the commonest one (= 11' 9' 100) in the reference population (Anisakis simplex A from the Me- Np 92 0,08 Ap-J 92 1,00 diterranean Sea) (Table I). 100 0,92 8' 7' Ap-2 96 1,00 GOI-2 93 0,79 5' RESUL TS AND DISCUSSION 97 0,01 Lap-I 90 0,76 100 0,16 94 0,21 105 0,01 97 0,03 For all the polymorphic loci, genotype frecuencies clo- 110 0,Q3 17' sely matched the expectations based on the Hardy- Wein- Lap-Z 100 1,00 berg equilibrium (HWE) model, in accordance with the 16' 17' existence of a single gene-pool within specimens collec- Adk-2 105 1,00 Mpi 100 1.00 ted from the different squid species examined. The pre- 28' 29' sence of diagnostic loci allowed the specific identifica- Gpi 100 1,00 tion of all the specimens analyzed as corresponding to 12' the sibling Anisakis simplex B (Table 2). Table 3 shows the allele frequencies observed at the loci Table 3.- Allele frequencies at 17 enzyme loci in Anisakis simplex studied. All individuals showed activity in the 17 loci tes- B larvae, parasites of IIlex coindetii and Todaropsis eblanae in ted (Table 4). Five loci (MDH, NP, PGM-2, EST-I, PGI) NW Spain. Allele frequencies were calculated after pooling alleles from all sampling data. ' = number of individuals analyzed. were monomorphic with the same alleles in both species. One group of 3 loci (6-PGDH, EST-2, LAP-2) showed allele frequencies which were either similar to other popu- BULLlNI (L.). ASCETTI (G.) & GRAPELLI (C.), 1981.- uovi dati lations of A. simplex B from the orth Atlantic waters or sulla divergenza e sulla variabilita genetica delle specie gemelle showed slight variations. A third group (SDH, MPl, IDH) Ascaris lumbricoides- A. suum e Parascaris univalens-P. equo- rum. Parassitologia, 23: 139-142. had significant differences between A. simplex A and B. HOCHBERG(F.G.), 1990.- Diseases of Mollusca: Cephalopoda. In: It is important to stress the presence of A. simplex B in Diseases of marine animals, Vol. Ill, Cephalopoda 10 Urochor- cephalopods from NW Spain and its occurrence in both data (0. Kinne edit.). Biologisches Anstalt Helgoland, Ham- ommastrephid squid species, representing new host re- burg: 47-227. cords for sibling B. The identification of our material as NASCETTI (G.), PAGGI (L.), ORECCHIA (P.), SMITH (l.W.), MAT- A. simplex 8 also agrees with the opinions held by BE- TIUCCI (S.) & BULLlNI (L.), 1986.- Electrophoretic studies on VERL Y-BURTON (1978) and ORECCHIA et al. (1986). the Anisakis simplex complex (Ascaridida: Anisakidae) from the Mediterranean and the North East Atlantic. lnternational Jour- These authors note that A. simplex A is chiefly distribu- nal for Parasitology, 16: 633-640. ted in the Mediterranean, while B is primarily A. simplex NASCETTI(G.),CiANCHI (R.), MATTIUCCI(S.), D' AMELlO(S.), OREC- distributed in North Atlantic waters. CHIA(P.), PAGGI (L.), BRATTEY(1.), BERLAND(B.), SMITH (l.W.) & BULLI I (L.), 1993.- Three sibling species within Contracae- cum osculatum ( ematoda, Ascaridida, Ascaridoidea) from the ACKNOWLEDGEME TS Atlantic Artic Boreal region: reproductive isolation and host pre- ferences. lnternational Journal for Parasitology, 23: 105-120. We specially thank Prof. Dr. Lia Paggi and eo-workers (lstituto di ORECCHIA(P.), PAGGI (L.), MATTIUCCI (S.), SMITH (l.W.), NAS- Parassitologia, Universita degli Studi di Roma, La Sapienza, Roma, CETTI (G.) & BULLlNI (L.), 1986.- Electrophoretic identification Italia) for help with electrophoretic identification of anisakids. of larvae and adults of Anisakis (Ascaridida: Anisakidae). Jour- nal of Helminthology, 60: 331-339. ORECCHIA (P.), MATTIUCCI (S.), D' AMELlO (S.), PAGGI (L.), PLOTZ REFERENCES (L), CiANCHI (R.), NASCETTI (G.), ARDUINO (P.) & BULLI I (L.), 1994.- Two new members in the Contracaecum osculatum BEVERLy-BURTO (M.), 1978.- Population genetics of Anisakis complex (Nematoda, Ascaridoidea) from the Antartic, lnterna- simplex ( ematoda: Ascaridoidea) in Atlantic salmon (Salmo tional Journal for Parasitology, 24: 367-377. salar) and their use as biological indicators of host-stocks. Envi- PAGGI (L.), NASCETTI (G.), CiANCHI (R.), ORECCHIA(P.), MAT- ronmental Biology of Fishes, 3: 369-377. T1UCCI(S.), D'AMELlO (S.), BERLA D (B.), BRATTEY (L), SMITH Anisakis simplex B in NE Atlantic squids 241

POPULA nONS POPULA nONS

Genotyes ICN ICS TEN TES Genotypes ICN ICS TEN TES

Sdh Got-2 100/100 2 6 93/93 2 4 5 14 100/105 0 I 93/97 0 0 I 0 N 0 2 0 7 93/100 1 2 2 5 X' 0,04 93/105 0 1 0 0 93/100 0 I 0 I Mdh 100/100 1 0 0 0 lOO/lOO 2 3 4 4 N 4 8 8 20 N 2 3 4 4 X' 0,87 0,89 0,43 0,62

Idh Adk-2 93/93 1 I 2 4 105/105 6 8 10 20 93/100 0 I 6 10 N 6 8 10 20 lOO/lOO 4 7 2 4 100/107 1 0 0 0 Pgm-2 N 6 9 10 18 lOO/lOO 4 5 6 14 X' 6,07 3,24 0,4 0,22 N 4 5 6 14

6-Pgdh Est-1 93/93 0 1 4 2 100/100 4 0 6 2 93/100 I I 3 3 N 4 0 6 2 97/100 I 0 0 I 100/100 4 0 2 3 Est-2 N 6 2 9 9 90/96 0 I 0 X' 0,24 0,22 0,8 1,29 96/96 0 I 0 96/100 1 I 0 Np lOO/lOO I 2 3 92/100 0 I 0 I 100/105 2 0 0 100/100 2 2 3 3 N 4 0 5 3 N 2 3 3 4 X' 1,44 2,45 X' 0,12 0,08 Lap-1 PepB 90/90 6 4 70/70 0 0 2 2 90/94 5 70/80 0 I 3 I 90/97 1 70/90 0 0 I 0 94/94 I 80/80 2 I 2 3 N 0 6 0 II 80/90 0 0 I I X' 0,67 N 2 2 9 7 X' 0,22 0,28 3,52 Lap-2 lOO/lOO 6 II Ap-1 N 0 6 0 II 92/92 6 3 10 5 N 6 3 10 5 Mpi 100/100 6 9 10 20 Ap-2 N 6 9 10 20 96/96 4 0 6 5 N 4 0 6 5 Gpi 100/100 6 3 10 9 N 6 3 10 9

Table 4.- Genotype frequencies in Anisakis simplex B from Illex coindetii and Todaropsis eblanae in waters off Galicia. Nenumber of individuals tested; TCN=/. coindetii to the north (North Galicia: Cape Finisterre-Cape Estaca de Bares); rcs=!. coindetii to the south (West Galicia: Mifio-C. Finisterre); TEN=T. eblanae to the north (idem); TES=T. eblanae to the south (idem).

(l.W.) & BULLINI (L.), 1991.- Genetic evidence of three species kids. Bulletin of the Scandinavian Society for Parasitology, within Pseudoterranova decipiens (Nematoda, Ascaridida, As- 4(2): 25-39. caridoidea) in the North Atlantic and Norwegian and Barent THORPE (l.P.) & SOLE-CAVA (A.M.), 1994.- The use of allozyme seas. International Journal for Parasitology, 21: 195-212. electrophoresis in invertebrate systematics. Zoologica Scripta, PAGGI (L.) & BULLINI (L.), 1994.- Mollecular taxonomy in Anisa- 23: 3-18.