Parasitology Today, vol. 6, no.

I0, 1990

323

Anisakis - f r o m the Platter to the Microfuge

J.A. Sakanari
Anisakiasis is a disease caused by the ingestion of anisakid nematodes in raw or improperly prepared fish dishes. Invading larvae penetrate the mucosa and submucosa of the gastrointestinal tract and produce lesions characterized by a marked inflammatory response.Judy Sakanari describes the biochemical and molecular studies on the proteins excreted and/trr secreted by Anisakis larvae that are currently underway to help elucidate the role these proteins may play in ti~einvasion process and the pathogenesis of anisakiasis.
Parasitic nematodes are not often regarded as media stars. Why then have articles on Anisakis appeared in The Wall Street Journal, USA Today and several California newspapers? News of this worm was aired on National Public Radio, and it has been seen wriggling around on television stations in the USA and Canada. The current rage can be traced to the 3 November 1988 issue of the New England Journal of Medicine ], which alerted clinicians and medical personnel to the disease known as anisakiasis, caused by the ingestion of larvae belonging to the family Anisakidae (Fig. 1). The letter pointed out that clinicians may be seeing an increase in the number of cases of anisakiasis as a result of the increase in the popularity of eating sushi (Fig. 2), sashimi, ceviche and lightly smoked or grilled fish. Evidently, the thought of finding worms wrapped inside a piece of beautifully prepared sushi or biting into mesquite-grilled salmon and seeing half a worm is a newsworthy item. It has been known for over six centuries that anisakid nematodes infect fish, and lesions in the stomachs of marine mammals were reported in the 1800s 6. However, it was not until the early 1960s that the 'codworm' (Pseudoterranova decipiens = Phocanema decipiens) and 'herringworm' (Anisakis spp) were regarded as potential health hazards, particularly in countries such as the Netherlands and Japan where raw fish dishe.; are commonly eaten6-15; cases of anisakiasis continue to be reported throughout Europe, Japan, North America and Hawaii 2,4,16-20. The public health significance of anisakid nematodes has prompted research on the life history, biology, morphology, epidemiology and pathology of Anisakis (for reviews see Refs 5, 10, 15, 18-20), and one area of current interest to several laboratories is
J.A. Sakanari is at the Department of Pathology, School of Medicine, University of California, San Francisco, CA 94143-0506, USA.
~) 1990,ELsevierSciencePubLishersLtd, (UK) 01694707/90/$02.00

the biochemical and molecular aspects of what Anisakis excretes and secretes. Excretory and secretory products (ES) from parasitic helminths have been studied, (1) to characterize the host's immune response during active infection, (2) as potential targets for the design of vaccines, (3) as possible serodiagnostic reagents, and (4) to understand their role in the pathogenesis of infection 2~'e2. ES products from Anisakis may play a role in the initial phases of infection by helping the worm to penetrate host tissues and once the parasite has invaded, ES products induce a cellular and humoral immune response. Invasion of host tissues Anisakis larvae can penetrate the mucosa and submucosa of the human stomach and intestine (Fig. 3)

~,

~-

Fig. I. Life cycle ofAnisakis, a parasitic nematode that belongs to the order Ascaridata (family Anisakidae). It is closely related to the large roundworms of humans (Ascaris lumbricoides) and the dog roundworm (Toxocara canis). However, unlike these terrestrial species, Anisakis completes its life history in the marine environment, infecting a wide variety of aquatic vertebrate and invertebrate hosts. Although the life cycles of Anisakis and other anisakid nematodes have not been completely described, it is believed that the life cycle goes through the following stages. (a) Adult worms are found in the stomachs of marine mammals (seals, dolphins and whales). (b) Eggs are released from female worms and pass out with the feces into the ocean. (c) First-stage larvae develop inside the egg and a second-stage larva hatches out of the egg and is (d) eaten by the first intermediate host (small crustaceans such as krill. (e) The infected hosts are eaten by fish and squid, which become infected with the third-stage larvae. The life cycle is completed when marine mammals eat the infected fish or squid; (f) humans are accidental hosts.

324

Parasitology Today, vol. 6, no. I O, 1990

Fig. 2. Safe sushi-just say no? A platter of sushi prepared by a Japanese sushi chef. According to Oshima 2, the sushi bar restaurants are not responsible for the increased number of cases of anisakiasis. Instead, he feels that home-prepared fish dishes are most often implicated in the transmission of the parasite. The increased number of cases has been attributed to increased reportage from the use of endoscopy 2, an increased population of marine mammalfl , increased worm infections in edible fish3'4 and the increased frequency of eating raw or lightly cooked fish I. Thorough cooking offish fillets (internal temperature of 60C for I0 min) and adequate freezing (-20C for three days) are easy, preventative measures against infection by anisakid nematodes. At room temperature, Anisakis larvae can survive for up to five days in 10% ethyl alcohol, one day in saturated NaC I , 51 days in vinegar, 65 days in soy sauce and one day in Worcester sauce 5.

contortus to molt during the exsheathment process 43, and to assist penetration of tissues by larval Strongyloides stercoralis44, Ancylostoma caninum41, Onchocerca spp 35 and Toxocara canis 36. Proteases in ES from Anisakis may also be required by the larvae to invade host tissues. ES products from living Anisakis larvae degrade macromolecular components of the extracellular matrix (trypsin-labile glycoproteins and collagen) in vitro 32, suggesting that they are capable of digesting host material. The proteins that are degraded in this assay are similar to those normally found in the mucosa and submucosa of gastrointestinal tissues. The proteases identified from Anisakis ES products hydrolyse gelatin (denatured collagen), hemoglobin and albumin at neutral pH and have relative molecular masses of 23.4, 46.1 and 54.3 kDa on gelatin substrate gels 3~'49. Biochemical analyses using inhibitors of the four classes of proteases (serine, cysteine, metallo and aspartyl), along with various synthetic peptide substrates, showed that a protease contained in Anisakis ES has serine protease activity and hydrolyses Z-ValLeu-Arg-aminomethylcoumarin, which is generally cleaved by trypsin-like enzymes 32. In addition, an immunoblot of Anisakis larvae suggested that this protease shared antigenic epitopes with rat trypsin, a mammalian serine protease. To understand further the function of the Anisakis serine protease and compare its structure with that of

and migrate to tissues of the omentum, pancreas, liver and lungs 7'23'24. Invasion of the intestinal or stomach walls commonly leads to clinical symptoms, mimicking acute appendicitis or peptic ulcers 25 but the mechanism by which these larvae penetrate tissues is not known. It has been hypothesized that ES products from Anisakis larvae are involved in the invasion process and in 1927, Mueller 26 proposed that secretory granules in the esophageal gland of Anisakis might be responsible for tissue degradation. Observations of the anterior of Anisakis from the stomach wall of a walrus and morphological studies of the esophageal gland further suggested that secreted substances functioned in extra-intestinal digestion 27'z8. Histochemical studies of the excretory organ of Anisakis larvae have revealed the presence of several enzymes including phosphatases, oxidative enzymes, phosphorylases, an aminopeptidase and an esterase, and it has been postulated that some of these enzymes might be involved in histolysis 29. Proteolytic enzymes from a number of parasitic helminths have been identified (Table 1) and some have been shown to play a role in the pathogenesis of infection. These enzymes function, for example, to prevent the coagulation of host blood, which facilitates hookworm feeding 56, to help larval Haemonchus

Fig. 3. Scanning electron micrograph of Anisakis larva penetrating the mucosa of human stomach in vitro.

Parasitology Today, vol. 6, no. I0, 1990

325
Table I. Proteases identified from parasitic nematodes Class of protease Parasitic nematode Refs 30-32 33, J. Deneris and J. Sakanari, unpublished 34 35 35 35 36

serine proteases fromL other organisms, a molecular approach was taken to clone the serine protease gene. A technique has been developed to isolate protease gene fragments not only from Anisakis but also from organisms from diverse phylogenetic groups 57. Serine protease gene fragments have been isolated from Anisakis genomic DNA using degenerate oligonucleotide primers, the design of which was based upon the consensus sequences flanking the active sites of eukaryotic serine proteases. Using low annealing temperatures (25C) during the polymerase chain reaction, four bands were amplified, each of which was subcloned and sequenced; one of the fragments (470 bp) appears to have a close homology with rat trypsin (Fig. 4). This gene fragment will be used to isolate the full-length sequence from Anisakis and can already be used as a probe to isolate serine protease genes from other parasitic nematodes. In addition to the serine protease, a metalloprotease (an aminopeptidase) has also been partially characterized from Anisakis ES products 32. However, it is not known what role this enzyme plays in the pathogenesis of anisakiasis or the biology of the parasite. Other metalloproteases have been identified from parasitic nematodes: one purified from the exsheathment fluids of Haemonchus contortus is responsible for the ecdysis of the larvae. Aminopeptidases have been associated with the molting process of the anisakid nematode Pseudoterranova decipiens 52, Haemonchus contortus5~ and Brugia pahangi (X. Hong et al., unpublished). Perhaps the Anisakis metalloprotease is also involved in the molting of the larvae from the third stage to the fourth stage in the mammalian host. Research on proteases from transformed cells and B 16 melanoma cells suggests that the process of invasion and metastasis involves a combination of metallo- and serine proteases 58'59. The serine protease functions to activate the metalloprotease, which in turn functions in the degradation of extracellular matrices. A similar protease activation cascade has been observed in the fertilization processes of Chlamydomonas 6. Further characterization of the proteases from Anisakis ES may reveal similar mechanisms in this species as well as in other infectious', organisms such as bacteria, fungi and protozoa.
Immune response to ES

Serine

Anisakis simplex Ascaris suum Dirofilaria immitis Onchocerca cervicalis O. cervipedis O. lienalis Toxocara canis

Cysteine

Angiostrongylus cantonensis Ascaris suum Dirofilaria immitis Haemonchus contortus Strongyloides ransomi Strongyloides ratti Ancylostoma caninum Anisakis simplex Ascaris suum Brugia malayi Brugia pahangi Haemonchus contortus Necator americanus

37 37 37 38 39 40 41 32 33, S.R. Morris and K.R. Kazacos, unpublished 42 X. Hang et al., unpublished 43 P.J. Hotez et al., unpublished 35

Metallo

Onchocerca cervicalis O. cervipedis Strongyloides stercoralis 44

Aspartyl

Angiostrongylus cantonensis Ascaris suum Dirofilaria immitis Ancylostoma caninum Ancylostoma ceylanicum A. duodenale Ascaris suum Dirofilaria immitis Haemonchus contortus Pseudoterranova

37 33, 37 37, 45 46 47 47 48, 49 50,J.K. Richer et al., unpublished 51

52 53, 54 55

Not determined

decipiens Strongyloides ratti Strongyloides fullebomi Trichinella pseudospiralis

G. Stewart and J.A. Sakanari, unpublished

After Anisakis larvae invade host tissues, they continue to release ES products into the surrounding tissues. HistochemicaJ[ and immunofluorescent antibody staining of Anisakis larvae embedded in human and animal tissues suggest that ES products are released from the excretory pore and form a 'cap' around the anterior section of the worm 61. The most striking histological feature of anisakiasis is massive infiltration of eosinophils around the worm, and it appears that the ES products from the larvae are inducing this cellular response. Larval Anisakis extract is chemotactic for eosinophils but

not neutrophils 62 and when in contact with the worm the eosinophils degranulate, releasing their granules onto the surface of Anisakis. Although the eosinophilic granules contain cytotoxic components including major basic protein, eosinophil-derived neurotoxin and eosinophil cationic protein, these do not appear to damage the cuticle (K. Hamann, pers. commun.). However, deposition of the eosinophilic proteins may contribute to the tissue damage surrounding the parasite, as is suspected in cases of infection by the related nematode, Balisascaris procyonis 63. Anisakis ES products have also been reported to cause mast cell degranulation 64 and to inhibit

326
Anisakis serine protease fragment

Parasitolo~ ~ y ,

vol. 6, no. lO, 1990

WVVTAAHCVYDFQNAAYWQVDLGRYNKDG~DAGVQRIRVAQIIIHPNYNPTTIVSDIALMQLT WVVSAAHC---Y--KSRIQVRLGEm~IDVVEGGEQFIDA&KIIRHPSYNANTFDNDIMLIKLN

Rat trypsin

Anisakis serine protease fragment

QPANLATGYVRPASVVTNVNQGSSLVANTQCIIAGWGDTRNTGSN--SVLRQASVPVIDY
S:oS : ..Z I~Z .S.~...~.: ..Z.:

:.:.

..:~..

Rat trypsin

SPATL---NSRVSTVSLPRSCGSS---GTKCLVSGWGNTLSSGTNYPSLLQCLDAPVLSD

Anisakis serine protease fragment

NLC S S W Y - S T L ~ S F C A G Y Q E G G I D A C QGDSGGP

Rat trypsin

SSCKSSYPGKITSNMFCLGFLEGGKDSCQGDSGGP

Fig. 4. Amino acid translation of the Anisakis serine protease gene fragments shows a 43% identity with a mammalian serine protease, rat trypsin.

lymphocyte blastogenesis 65'66, but it is not known which components of the ES products induce these effects; however, fractionation of ES material may give greater insight into the role of excreted and/or secreted proteins in modulating the cellular immune response. A number of Anisakis ES antigens are recognized by sera from patients with a n i s a k i a s i s 49'66'67, and these proteins may be useful as potential serodiagnostic reagents. In comparing the reactivity of infected human sera to ES antigens and larval homogenates, Poggensee et al. 68 found that better sensitivity was obtained with the ES antigens although cross-reactivity with other helminth antigens was noted; for example, sera from patients with schistosomiasis and ascariasis also gave a positive reaction to the ES antigens. The most promising reagent for use in the serodiagnosis of anisakiasis is a monoclonal antibody that recognizes two ES proteins of 40 and 42 kDa (Ref. 69). This antibody reacts with the intestine, excretory gland and muscle cells of Anisakis larvae and does not cross-react with Echinococcus multilocularis, Pseudoterranova decipiens larvae, Trichinella spiralis, Dirofilaria immitis, Toxocara cati or Ascaris suum. Yagihashi et al. 7 showed that the best sensitivity to ES proteins was obtained for IgG, IgA and IgM at four and five weeks after the onset of illness when they used this monoclonal antibody in a sandwich form of an enzyme-linked immunosorbent assay. Two peaks of sensitivity for IgE were obtained at day 1 and one week after the onset of symptoms. However, there is currently no reliable serodiagnostic assay for anisakiasis, and the disease is often misdiagnosed. Cloning known ES proteins may therefore be useful in developing a specific, quantitative assay for serodiagnosis. Moreover, ES antigens in conjunction with monoclonal antibodies might be used to confirm the diagnosis of anisakiasis and also to aid in distinguishing acute versus chronic disease.

Conclusion Excretions and/or secretions from larval Anisakis are a complex mixture of proteins vital for the establishment of the parasite in its host but also responsible for the pathogenesis of this infection. Isolation of the antigens that evoke cellular and humoral immune responses may help characterize the host response to Anisakis infections and aid in the development of a serodiagnostic assay. Structural and functional analyses of the proteolytic enzymes may not only give insight into the mechanism of invasion by Anisakis and other infectious organisms, but also enhance our knowledge of the molecular evolution of the enzymes themselves.
Acknowledgements

I am grateful to J.H. McKerrow, J. Bouvier, B. Keene and J. Deneris, Department of Pathology, University of California, San Francisco, for reviewing this manuscript and giving helpful suggestions. I thank E.M. Shimazu for typing the manuscript and the California Sea Grant College Program, Academic Senate and School of Medicine, University of California, San Franciscofor providing support for the research on Anisakis.
References

1 McKerrow, J.H., Sakanari, J.A. and Deardorff, T.L. (1988) New Engl.J. Med. 319, 1228-1229 20shima, T. (1987) Parasitology Today 3, 44-48 3 Chandra, C.V. and Khan, R.A. (1988)J. Parasitol. 74,1038-1040 4 M611er, H. and Schr6der, S. (1987) Arch. Lebensmittelhygiene 38, 123-127 50shima, T. (1972) in Progress of Medical Parasitology in Japan (Morishita, K., Komiya, Y. and Matsubayashi, H., eds), Vol. IV, pp 305-393,Meguro ParasitologicalMuseum 6 Myers, B.J. (1976) Trans. Am. Microsc. Soc. 95,137-142 7 Yokogawa, M. and Yoshimura, H. (1967) Am. J. Trop. Med. Hyg. 16,723-728 8 Myers, B.J. (1970)J. Wildlife Dis. 6,266-271 9 Myers, B.J. (1975)J.MilkFoodTechnol. 38,774--782 10 Margolis,L. (1977)J. Fish. Res. Bd. Canada 34, 887-898 11 Van Thiel, P.H. and Van Houten, H. (1967) Trop. Geogr. Med. 19, 56-62 12 Ishikura, H., Hayasaka, H. and Kikuchi, Y. (1967) Sapporo Med. 32,183-196 13 Hayasaka, H., Ishikura, H. and Takayama, T. (1971) Int. Surg. 55, 8-14 14 Jackson, G.J. (1975)J. Milk Food Technol. 38,769-773

Parasitology Today, vol. 6, no. I O, 1990

15 Smith, J.W. and Wooten, R. (1978) in Advances in Parasitology (Lumsden, W.H.R., MullLer,R. and Baker, J.R., eds), Vol. 16, pp 93-163, Academic Press 16 Oshima, T. and Kliks. M. (1987)Int.J. Parasitol. 17,415-421 17 Hubert, B., Bacou, J. and Belveze, H. (1989) Am. J. Trap. Med. Hyg. 40, 301-303 18 Bier, J.W. et al. (1987) BaiUiere's Clin. Trap. Med. Comm. Dis. 2, 723-733 19 Sakanari, J.A. and McKerrow, J.H. (1989) Clin. Microbial. Rev. 2, 278-284 20 Ishikura, H. and Namiki:, M. (1989) Gastric Anisakiasis in Japan, Springer-Verlag 21 Lightowlers, M.W. and Rickard, M.D. (1988) Parasitology 96, 5123-5166 22 Maizels, R.M. and Selkirl%M.E. (1988) in TheBiology of Parasitism (Englund, P.T. and Sher, A., eds), pp 285-308, Alan R. Liss 23 Rushovich, A.M. etal. (1983)Am.J. Clin. Pathol. 80, 517-520 24 Kobayashi, A., Tsuji, M. and Wilbur, D.L. (1985) Am. J. Trap. Med. Hyg. 34, 310-313 25 Ohtaki, H. and Ohtaki, R. (1989) in Gastric Anisakiasis in Japan (Ishikura, H. and Namiki, M., eds), pp 37-46, Springer-Verlag 26 Mueller, J.F. (1927)Z. Zellforsch. 5,495-504 27 Hoeppli, R. (1933)LingnamSci.J. 12(Suppl.), 1-11 28 Hsu, H.F. (1933)Chin. Med.J. 47, 1247-1288 29 Ruitenberg, E.J. and Loendersloot, H.J. (1971)J. Parasitol. 57, 1149-1150 30 Matthews, B.E. (1982)J.l~relminthol. 56,177-183 31 Matthews, B.E. (1984)J. tIelminthol. 58,175-185 32 Sakanari, J.A. and McKerrow, J.H.J. Parasitol. (in press) 33 Knox, D.P. and Kennedy, M.W. (1988)Mol. Biochem. Parasitol. 28, 207-216 34 Tamashiro, W.K., Rao, Ni. and Scott, A.L. (1987)J. Parasitol. 73, 149-154 35 Lackey, A. et al. (1989)Exp. Parasitol. 68,176-185 36 Robertson, B.D. etal. (1989)Exp. Parasitol. 69, 30-36 37 Maid, J. and Yanagisawa,T. (1986)J. Helminthol. 60, 31-37 38 Cox, G.N. et al. Mol. Biochem. Parasitol. (in press) 39 Dresden, M.H., Rege, A.A. and Murrell, K.D. (1985) Exp. Parasitol. 59,257-263 40 Rege, A.A. and Dresden, M.H. (1987)Exp. Parasitol. 64,275-280 41 Hotez, P.J. etal. (1985)J.Biol. Chem. 260, 7343-7348

327 42 Petralanda, I., Yarzabal, L. and Piessens, W.F. (1986) Mol. Biochem. Parasitol. 19, 51-59 43 Gamble, H.R., Purcell, J.P. and Fetterer, R.H. (1989) Mol. Biochem. Parasitol. 33, 49-58 44 McKerrow, J.H. et al. (1990) Exp. Parasitol. 70,134-143 45 Swamy, K.H.S. and Jaffe, J.J. (1983) Mol. Biochem. Parasitol. 9, 1-14 46 Oya,H. and Noguchi, I. (1977)J. Parasitol. 26,307-313 47 Pritchard, D.I., McKean, P.G. and Schad, G.A. (1990) Parasitology Today 6, 154-156 48 Juhasz, S. and Nemeth, I. (1970) Acta Vet. Acad. Sci. Hung. 27, 217-224 49 Kennedy, M.W. etal. (1988)Mol. Biochem. Parasitol. 31, 35-56 50 Sato, K., Takahashi, J. and Sawada, T. (1976)Jpn.J. Parasitol. 25, 8-9 51 Rogers, W.P. (1982)Int.J. Parasitol. 12,495-502 52 Davey, K.G. and Sominerville, R.I. (1974) Int. J. Parasitol. 4, 241-259 53 Lewert, R.M. and Lee, C-L. (1956)J. Infect. Dis. 99, 1-14 54 Wertheim, G. etal. (1983)J. Helminthol. 57,241-246 55 Matthews, B.E. (1975)Parasitology 70, 25-38 56 Hotez, P.J. and Cerami, A. (1983)J. Exp. Med. 157, 1594-1603 57 Sakanari,J.A. et al. (1989) Proc. Natl Acad. Sci. USA 86, 4863-4867 58 Mignatti, P., Robbins, E. and Rifkin, D.B. (1986)Cell47,487-498 59 Chen, J-M. and Chen, W-T. (1987)Cell48, 193-203 60 Snell, W.J., Eskue, W.A. and Buchanan, M.J. (1989)J. Cell Biol. 109, 1689-1694 61 Bier,J.W. and Raybourne, R.B. (1988)Proc. Helminthol. Sac. Wash. 55, 91-94 62 Tanaka, J. and Torisu, T. (1978)J. Immunol. 120, 745-749 63 Hamann, K.J. etal. (1989)Am.J. Trap. Med. Hyg. 40, 180-196 64 Kobayashi, A., Kumada, M. and Ishizaki, T. (1972)Jpn. J. Med. Sci. Biol. 25,335-344 65 Raybourne, R. et al. (1983) Exp. Parasitol. 55,289-298 66 Raybourne, R., Deardorff, T.L. and Bier, J.W. (1986) Exp. Parasitol. 62, 92-97 67 Sakanari,J.A. et al. (1988) Am.J. Clin. Pathol. 90, 107-113 68 Poggensee,U. et al. (1989)Zbl. Bakt. Hyg. A 270, 503-510 69 Takahashi, S., Sato, N. and Ishikura, H. (1986)J. Parasitol. 72, 960-962 70 Yagihashi,A. etal. (1990)J. Infect. Dis. 161,995-998

A Comparison of Trans-RNA Splicing in Trypanosomes and Nematodes

J.E. Donelson and W. Zeng
Many aspects of the biology of kinetoplastids are unique, so it is surprising that they share with nematodes an unusual post-transcriptional process called trans-splicing. During this process, a small conserved R N A sequence is added to the 5' non-translated ends of transcribed R N A s of protein-encoding genes. Trypanosomes and nematodes are the only organisms to date in which these sequences have been described, and the biological significance of trans-splicing remains a mystery but may be of wider occurrence in invertebrates. In this review, John Donelson and Wenlin Zeng compare the process in nematodes and trypanosomes and speculate on its raison d'fitre. Parasitologists have ta:aditionally been divided into two groups, those who study protozoa and those who investigate helminths. This separation reflects the
John E. Donelson and Wenlin Zeng are at the Department of Biochemistry and Howard Hughes Medical Institute, University of Iowa, Iowa City, IO 52242, USA.
~) 1990,EtsevierSciencePublishersLtd, (UK) 01694707/90/$0200

extensive biological differences between these two groups of parasites. Protozoan parasites are microscopic, unicellular eukaryotes with short generation times and unique structural and metabolic features. Helminths, in contrast, are macroscopic, multicellular organisms with much longer generation times and many properties more similar to their mammalian hosts than to protozoa. A consequence of these striking differences is that investigators studying the two groups of parasites often have few interests in common except a shared fascination with the way parasitism is achieved and how host defense mechanisms are thwarted. Recently however, a common feature of trypanosomes and nematodes has emerged unexpectedly from examinations of their RNA processing mechanisms. Trypanosomes and nematodes have been found to share a novel post-transcriptional process called trans-splicing. In this process an RNA sequence of 39 nucleotides in trypanosomes and 22 nucleotides in nematodes, called the spliced leader