Patterns of abundance and maturity among three species of parasitic nematodes ( Pseudoterranova decipiens , Contracaecum osculatum , Anisakis simplex ) co-existing in Sable Island grey seals ( Halichoerus grypus )

The abundance and maturity of three species of anisakine nematode ( Pseudoterranova decipiens , Contracaecum osculatum , Anisakis simplex ) that co-occurred in the stomachs of Sable Island grey seals were examined in relation to seal growth and seasonal considerations. Sealworm ( P. decipiens), the predominant nematode in these seals, typically reached numbers of 400 to 2000 worms per stomach. C. osculatumand A. simplexwere usually found in much smaller numbers of 40 to 100 and 20 to 60 worms, respectively, per stomach. All three species initially increased in abundance as the seals grew, but after most of a seals’ growth had been attained P. d cipiensabundance continued to increase with age, A. simplexnumbers either continued to increase or were simply maintained, while C. osculatumabundance declined. Numbers of both P. decipiensand A. simplexdeclined during winter breeding/pupping and summer moulting fasts or partial fasts, and rose during the regular feeding periods between the fasts. Conversely, numbers of C. osculatumrose during the breeding period, and also during the moulting period in younger seals. We believe this could be attributed to some degree of feeding on prey species in the immediate vicinity of Sable Island that were not preferred during focused feeding periods, and that the inclination to feed during fasting periods decreased as seals grew. An inverse relationship between worm abundance and worm maturity, attributable to the seasonal changes in rates of ingestion of immature worms, was more pronounced for C. osculatumthan P. decipiens . C. osculatumwas usually represented by much higher proportions of mature worms than P. decipiens . This could be entirely related to the longer periods of time dedicated to feeding than spent breeding or moulting, but higher mortality rates of immature C. osculatumor greater longevity of mature C. osculatumcould also have occurred. A simplex, generally associated with cetacean species as final hosts, rarely matured in grey seals. We have doubts that the grey seal could be considered a final host for A. simplex. Fowler, G.M. and Stobo, W.T. 2001. Patterns of abundance and maturity among three species of parasitic nematodes ( Pseudoterranova decipiens, Contracaecum osculatum, Anisakis simplex ) coexisting in Sable Island grey seals ( Halichoerus grypus ). NAMMCO Sci. Publ. 3: 149-160. 149 NAMMCO Scientific Publications, Volume 3 cilitate direct comparisons with results of an earlier study (Stobo et al. 1990). In this paper we have applied generalized linear analysis of deviance (McCullagh and Nelder 1983) to logtransformed nematode abundances, using the statistical utilities in S_Plus (Chambers and Hastie 1992). For statistical hypothesis testing, probability levels <.05 were considered significant. Estimation of the number of polynomial parameters to reflect curvature of the relationships between worm abundance and explanatory variates, and the order of entry of effects into models, were determined using the S-Plus step function, which iteratively applies a χ test of significance for entry or removal of a term in successive models. Seven sampling trips were made in each of 1983 and 1989, with the later 1989 study scheduled to correspond temporally with 1983 sampling. As in Stobo and Fowler (2001), we designated the January-February trip as month 2, and the March trip (very end of month) as month 4, to more reasonably scale the magnitudes of time passed between trips for the modelling process. The seal samples were partitioned into 3 life history groups (corresponding approximately to pups, juveniles, and adults) for descriptive and analytical purposes, and each life history stage was modelled separately. The pups were treated as a separate group since they do not develop nematode infections until they begin independent feeding; for this reason, no pups were taken during the January trips. However yearlings taken in January 1983 and 1989 (12 to 14 months old) were included in the pup life stage group to represent the infections carried by the pups at the end of the first year of life. The 2 older life stages were determined from preliminary analyses of deviance as ages 1 to 6 (designated as juveniles) and ages 7 and older (designated as adults). This partitioning of the ages was considered optimal due to the absence of significant interactions between age and other explanatory variables in modelling P. decipiensabundance (Stobo and Fowler 2001). As such these stages reflected phases of infestation that were generally, but loosely, associated with the stages of maturity of the seals. With respect to C. osculatumand A. simplexabundance in the current analysis, these stages presented one instance of a significant interaction, pertaining to the model of C. osculatuminfections in pups (Stobo and Fowler 2001). But since this interaction occurred between age in months of pups and study year (1983 versus 1989 sampling), we saw no reason to change our maturity classification. The analysis of P. decipiensinfections was reproduced from the earlier paper (Stobo and Fowler 2001), and the same approach was applied to C. osculatumand A. simplexin this paper. The original inclusion of C. osculatumas a covariate in the P. decipiensmodel is our only instance of treating the abundance of a co-occurring nematode species as a variate. We knew from exploratory analyses that A. simplexabundance exhibited serial correlation with P. decipiens abundance, and the C. osculatumand A. simplexdata were too dispersed to resolve covariance with other species in their own models. RESULTS AND DISCUSSION P. decipienswas the most abundant nematode species in the stomachs of grey seals, although C. osculatumattained appreciable numbers in the fall and winter, especially in pups (Table 1). A. simplexusually occurred only in small numbers. The results of modelling (Table 2) indicated that growth (age or length) and season (trip) were the major significant effects determining the abundance of the three species of nematode in grey seals, with sex and study year attaining significance as lesser effects in some models. Age or length of seals were relevant to the abundances of all three nematodes in every life history stage with the possible exception of A. simplexin adults (but see below). Seasonal effects on nematode abundances were evident in juvenile and adult life history stages, but could not be investigated with pups. During the first year of life the pups acquire worms for the first time as they learn to feed, no minima or maxima in worm abundances yet established to correlate with seasons. The assumption of normality of the distributions of log-transformed nematode counts appears reasonable for all models (Fig. 1). The abundances of all three parasite species increase as seals grow older or larger (Fig. 2). 151 NAMMCO Scientific Publications, Volume 3 INTRODUCTION During investigations into the relationship between the sealworm Pseudoterranova decipiens , and the grey seal Halichoerus grypus , the stomachs of 553 grey seals were sampled over the course of two years (1983 and 1989) to elucidate seasonal and longterm changes in sealworm abundance (Stobo et al. 1990, Stobo and Fowler 2001). All animals were taken at Sable Island, a major North Atlantic haulout and pupping site approximately 160 km off the east coast of Nova Scotia, Canada. The primary focus of this field work was the sealworm, but specimens of two other prevalent species of anisakine nematodes, Contracaecum osculatum and Anisakis simplex , were also counted and staged for maturity in the same fashion as P. decipiens . Subsequent analysis of the data showed a relationship between P. decipiensand C. osculatumabundances (Stobo and Fowler 2001). In this paper, the abundance and maturity of the 3 most common species of stomach nematodes found in grey seals ( P. decipiens, C. osculatum, and A. simplex) are examined in relation to the age, length and sex of the seal host, as well as season and year of sampling. All three anisakine nematode species in this study are characterized by life cycles that include fish species as intermediate hosts (Scott and Martin 1957, Young 1972, Platt 1975, Margolis and Arthur 1979). The majority of these hosts are demersal (McClelland et al. 1990, Marcogliese 1995), and include most of the groundfish species of interest to fisheries in the northwest Atlantic. Capelin ( Mallotus villosus) and cod ( Gadus morhua ) are known to be major intermediate hosts in the Gulf of St Lawrence (McClelland et al. 1985, Boily and Marcogliese 1995, Marcogliese 1995). In the vicinity of Sable Island, only large cod and white hake ( Urophycis tenuis ) have been found to host C. osculatum, and only in small numbers (McClelland et al. 1990). However, capelin in this area have not been examined for nematode infestations. The grey seal ( Halichoerus grypus ) appears to be the main final host of P. decipiensin the North Atlantic (Scott and Fisher 1958, Young 1972, Mansfield and Beck 1977, McClelland 1980, Bjørge MS 1984), and a major final host of C. osculatumin the Gulf of St Lawrence (Marcogliese et al. 1996). Grey seals have not been regarded as an important final host of A. simplex, rather cetaceans are considered the u ual final hosts of this nematode (van Thiel 1966). Experimental work with captive seals (McClelland 1980) indicated that P. decipiens often survived 6 weeks in grey seals, during which time they completed their life cycle (produced eggs). We lack similar data related to longevities of sexually viable C. osculatumand A. simplexin the grey seal. Grey seal pups begin to acquire stomach nematodes shortly after they commence independent f eding (Stobo et al. 1990, Stobo and Fowler 2001). Grey seals of all subsequent ages are infected, worm abundance increasing as the seals grow larger (Bjørge MS 1984, Stobo and Beck 1985, Wiig 1988, Stobo et al. 1990, Stobo and Fowler 2001). Due to sexual dimorphism in grey seals (ma

I IN NT TR RO OD DU UC CT TI IO ON N D uring investigations into the relationship between the sealworm Pseudoterranova decipiens, and the grey seal Halichoerus grypus, the stomachs of 553 grey seals were sampled over the course of two years (1983 and 1989) to elucidate seasonal and longterm changes in sealworm abundance (Stobo et al. 1990, Stobo andFowler 2001).All animals were taken at Sable Island, a major North Atlantic haulout and pupping site approximately 160 km off the east coast of Nova Scotia, Canada.The primary focus of this field work was the sealworm, but specimens of two other prevalent species of anisakine nematodes, Contracaecum osculatum and Anisakis simplex, were also counted and staged for maturity in the same fashion as P. decipiens.Subsequent analysis of the data showed a relationship between P. decipiens and C. osculatum abundances (Stobo and Fowler 2001).In this paper, the abundance and maturity of the 3 most common species of stomach nematodes found in grey seals (P.decipiens, C. osculatum, and A. simplex) are examined in relation to the age, length and sex of the seal host, as well as season and year of sampling.
All three anisakine nematode species in this study are characterized by life cycles that include fish species as intermediate hosts (Scott and Martin 1957, Young 1972, Platt 1975, Margolis and Arthur 1979).The majority of these hosts are demersal (McClelland et al. 1990, Marcogliese 1995), and include most of the groundfish species of interest to fisheries in the northwest Atlantic.Capelin (Mallotus villosus) and cod (Gadus morhua) are known to be major intermediate hosts in the Gulf of St Lawrence (McClelland et al. 1985, Boily and Marcogliese 1995, Marcogliese 1995).In the vicinity of Sable Island, only large cod and white hake (Urophycis tenuis) have been found to host C. osculatum, and only in small numbers (McClelland et al. 1990).However, capelin in this area have not been examined for nematode infestations.
The grey seal (Halichoerus grypus) appears to be the main final host of P. decipiens in the North Atlantic (Scott and Fisher 1958, Young 1972, Mansfield and Beck 1977, McClelland 1980, Bjørge MS 1984), and a major final host of C. osculatum in the Gulf of St Lawrence (Marcogliese et al. 1996).Grey seals have not been regarded as an important final host of A. simplex, rather cetaceans are considered the usual final hosts of this nematode (van Thiel 1966).Experimental work with captive seals (McClelland 1980) indicated that P. decipiens often survived 6 weeks in grey seals, during which time they completed their life cycle (produced eggs).We lack similar data related to longevities of sexually viable C. osculatum and A. simplex in the grey seal.
Grey seal pups begin to acquire stomach nematodes shortly after they commence independent feeding (Stobo et al. 1990, Stobo andFowler 2001).Grey seals of all subsequent ages are infected, worm abundance increasing as the seals grow larger (Bjørge MS 1984, Stobo and Beck 1985, Wiig 1988, Stobo et al. 1990, Stobo and Fowler 2001).Due to sexual dimorphism in grey seals (males grow faster and larger than females), males carry more worms than females of the same age (Stobo and Fowler 2001).Grey seals do not appear to completely eliminate their stomach nematodes at any time during the year but seasonal fluctuations in abundance have been observed, possibly due to the breeding fast (late December to early February) or changes in diet composition (Wiig 1988, Stobo et al. 1990, Haug et al. MS 1991, Stobo and Fowler 2001).In previous analyses, Stobo et al. (1990) and Stobo and Fowler (2001) described a seasonal cycle of abundance of P. decipiens in grey seals, suggesting declines in abundance coincident with increases in proportions of mature worms during the breeding season when the adult seals are fasting.Declines were also observed following the moulting season (roughly middle of May to middle of June) when partial fasting may occur.

M MA AT TE ER RI IA AL LS S A AN ND D M ME ET TH HO OD DS S
Details of the field sampling and processing of the 553 grey seals in this study, and the general analytical approach are provided in Stobo and Fowler (2001).The specific methods of analysis used here represent a subset of the methods presented in Stobo and Fowler (2001), which applied some older analytical techniques to fa-cilitate direct comparisons with results of an earlier study (Stobo et al. 1990).In this paper we have applied generalized linear analysis of deviance (McCullagh and Nelder 1983) to logtransformed nematode abundances, using the statistical utilities in S_Plus (Chambers and Hastie 1992).For statistical hypothesis testing, probability levels < .05were considered significant.Estimation of the number of polynomial parameters to reflect curvature of the relationships between worm abundance and explanatory variates, and the order of entry of effects into models, were determined using the S-Plus step function, which iteratively applies a χ 2 test of significance for entry or removal of a term in successive models.
Seven sampling trips were made in each of 1983 and 1989, with the later 1989 study scheduled to correspond temporally with 1983 sampling.As in Stobo and Fowler (2001), we designated the January-February trip as month 2, and the March trip (very end of month) as month 4, to more reasonably scale the magnitudes of time passed between trips for the modelling process.The seal samples were partitioned into 3 life history groups (corresponding approximately to pups, juveniles, and adults) for descriptive and analytical purposes, and each life history stage was modelled separately.The pups were treated as a separate group since they do not develop nematode infections until they begin independent feeding; for this reason, no pups were taken during the January trips.However yearlings taken in January 1983 and 1989 (12 to 14 months old) were included in the pup life stage group to represent the infections carried by the pups at the end of the first year of life.The 2 older life stages were determined from preliminary analyses of deviance as ages 1 to 6 (designated as juveniles) and ages 7 and older (designated as adults).This partitioning of the ages was considered optimal due to the absence of significant interactions between age and other explanatory variables in modelling P. decipiens abundance (Stobo and Fowler 2001).As such these stages reflected phases of infestation that were generally, but loosely, associated with the stages of maturity of the seals.With respect to C. osculatum and A. simplex abundance in the current analysis, these stages presented one instance of a significant interaction, per-taining to the model of C. osculatum infections in pups (Stobo and Fowler 2001).But since this interaction occurred between age in months of pups and study year (1983 versus 1989 sampling), we saw no reason to change our maturity classification.
The analysis of P. decipiens infections was reproduced from the earlier paper (Stobo and Fowler 2001), and the same approach was applied to C. osculatum and A. simplex in this paper.The original inclusion of C. osculatum as a covariate in the P. decipiens model is our only instance of treating the abundance of a co-occurring nematode species as a variate.We knew from exploratory analyses that A. simplex abundance exhibited serial correlation with P. decipiens abundance, and the C. osculatum and A. simplex data were too dispersed to resolve covariance with other species in their own models.

R RE ES SU UL LT TS S A AN ND D D DI IS SC CU US SS SI IO ON N
P. decipiens was the most abundant nematode species in the stomachs of grey seals, although C. osculatum attained appreciable numbers in the fall and winter, especially in pups (Table 1). A. simplex usually occurred only in small numbers.The results of modelling (Table 2) indicated that growth (age or length) and season (trip) were the major significant effects determining the abundance of the three species of nematode in grey seals, with sex and study year attaining significance as lesser effects in some models.Age or length of seals were relevant to the abundances of all three nematodes in every life history stage with the possible exception of A. simplex in adults (but see below).Seasonal effects on nematode abundances were evident in juvenile and adult life history stages, but could not be investigated with pups.During the first year of life the pups acquire worms for the first time as they learn to feed, no minima or maxima in worm abundances yet established to correlate with seasons.The assumption of normality of the distributions of log-transformed nematode counts appears reasonable for all models (Fig. 1).
The abundances of all three parasite species increase as seals grow older or larger (Fig. 2).This trend is maintained for P. decipiens throughout the life of a seal.The trend for C. osculatum is maintained until seals are nearly fully grown, after which the numbers begin to decline.The reason for the eventual decline in C. osculatum abundance remains a matter of conjecture.It may result from competitive displacement by P. decipiens or simply change in prey preference with age or size of seals, as originally speculated (Stobo and Fowler 2001) The increasing trend is only evident for A. simplex among younger animals, although the difference between sexes of adults (Table 2) may represent a growth component (male grey seals are larger than females).Similarly, the significance of sex in the pup model of P. decipiens abundance may be capturing the onset of sexual dimorphism in grey seals, a feature that the length variable is inadequate to characterise (Stobo and Fowler 2001).The animals can increase their mass, in terms of weight, at a greater rate than their length alone represents.
Two seasonal patterns in nematode abundances predominate for adult and juvenile seals (Fig. 3).The abundances of P. decipiens and A. simplex decrease during winter breeding/pupping and summer moulting periods, and increase during the normal feeding periods between these events.C. osculatum abundance, however, only parallels the other nematodes during the moulting decline and post-moult increase in adult seals.The pattern is reversed with respect to the winter and spring.C. osculatum abundances rise during breeding and drop off during the post-breeding feeding period.In juveniles the entire pattern of C. osculatum abundance is inverted relative to that of P. decipiens and A. simplex.The association between increasing C. osculatum abundance and breeding or moulting periods suggests some degree of feeding in the local area at these times, but likely on prey species not usually preferred.Mothers with pups are known to fast during the breeding period, but could become infested with C. osculatum upon arrival at the island, prior to giving birth.Male and juvenile seals may do the same, but may also be feeding to some extent during the pupping and mating period.Younger animals may be more inclined towards feeding during breeding or moulting periods than older animals.This would account for the full inversion of the C. osculatum pattern in juveniles, assuming that older seals feed less during the moult.
A portion of the C. osculatum infections apparent during the breeding period may be associated with animals that feed in the Gulf of St Lawrence, where this nematode typically predominates over P. decipiens in grey seals (Marcogliese et al. 1996)  Bank that grades into the Gully) since the mid-1980's, cooler water temperatures having made the eastern part of the Shelf suitable for this species (Frank et al. 1996).Capelin are the only known major fish host of C. osculatum (Marcogliese 1995) that might qualify as a transient candidate, and are common closer to Sable Island than the fish sampled by McClelland et al. (1990), but to the east of the waters McClelland et al. (1990) sampled.Estimates of capelin abundance were also greater in 1989 than 1983 (Frank et al. 1996).This is consistent with the higher abundances of C. osculatum carried by adult grey seals in 1989 relative to 1983.However, the prey contents of 57 grey seal stomachs, taken from Sable Island during the breeding season (Benoit and Bowen 1990), included invertebrates but no capelin.This suggests that invertebrate hosts might seem a likelier source of C. osculatum than fish around Sable Island, and could explain the large C. osculatum infections attained by pups and young seals that are probably less skilled at catching mobile fish prey than older seals.But only 6 of the 57 stomachs in the Benoit and Bowen (1990) study contained any food at all (and we do not know the ages of these 6 animals).Furthermore, the differences between study years in the pup and adult C. osculatum models do not parallel each other.The 1983 abundances in pups were usually greater than 1989 abundances (see Fig. 2), whereas for adults 1989 abundances were greater than 1983 abundances (see Fig. 3).This reversal in relative abundances of C. osculatum between years for pups and adults may denote differences in mechanisms of transmission with age.These confounding differences between sample years and age groups prevent us from determining the extent to which prior residency in the Gulf of St Lawrence might be relevant to the magnitudes of C. osculatum infections in juvenile and adult seals around Sable Island.
Maturity of P. decipiens and C. osculatum appears to be inversely related to abundance, with an offset of about a month (Fig. 4).The general pattern was discussed for P. decipiens by Stobo

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NAMMCO Scientific Publications, Volume 3 and Fowler ( 2001), who attributed fluctuations in the proportions of mature worms to lack of recruitment during full or partial fasts (existing worms maturing) and recruitment of immature worms during more dedicated feeding periods.
It was also noted that the proportions of mature P. decipiens were higher in 1983 than 1989 during four of the seven trip pairs (no difference between years for the other three trip pairs).We speculated that this could be due to colder water temperatures in the late 1980's slowing development in intermediate hosts, or that gradual reduction in size of intermediate fish hosts over time (due to commercial fishing) resulted in progressively smaller worms being ingested by grey seals.In either case the worms would require more time to mature in the final host.
There was no suggestion of any significant differences in C. osculatum maturity between 1983 and 1989 studies in this analysis (Table 3).This might lend greater weight to the smallerprey-size/smaller-worm-size hypothesis over the colder water hypothesis as the explanation for the lesser proportion of mature  3).Much or all of this difference could be attributable to the single dominant recruitment pulse, but does not rule out the possibilities that C. osculatum experiences higher juvenile mortality rates, or simply lives longer than P. decipiens.
A. simplex rarely matures in adult grey seals, and then only during the breeding period when mature A. simplex typically represented 4-8% of total numbers of infected animals.This suggests that the grey seal is not truly a final host of A. simplex, but that the worm can persist for some time post-ingestion.While a few mature individuals were found, we question whether they were reproductively viable.

A AC CK KN NO OW WL LE ED DG GE EM ME EN NT TS S
We are grateful to the several people who assisted in the collection of these samples, Brian Beck, Andrew Wynn, and John Horne in particular.We are indebted to Gerry Forbes, officerin-charge and other staff of the Canadian Atmospheric Environment Service on Sable Island for ground support and their unfailing willingness to assist us in staying operational at all times.Dr. Gary McClelland, Dr. Gwyneth Jones and John Martell sorted and identified the nematode specimens.

Table 1 .
. Mean abundances of nematodes, by life history stage, in the stomachs of grey seals during each trip period on Sable Island.
152Sealworms in the North Atlantic: Ecology and Population Dynamics *These are 14 month old animals, not newborns.

Table 2 .
Analyses of deviance of log worm abundance in grey seals.Terms in italics not retained in final model.The preceder "poly" defines the term as a polynomial (term,degrees of freedom).The C.o. term denotes log Contracaecum osculatum abundance.Insignificant interactions are not shown.The age variable denotes months in pups, and years in juveniles and adults.

Table 3 .
Comparison of the mean proportion of mature Pseudoterranova decipiens and Contracaecum osculatum in the stomachs of adult grey seals by trip between 1983 and 1989.
* t-test for samples with unequal variances used