Sources of evidence for salmon in the diet of seals

Fisheries Research, 10 ( 1 9 9 0 ) 1 3 7 - 1 5 0

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Elsevier Science Publishers B.V., A m s t e r d a m

Sources of evidence for salmon in the diet of seals P.R. Boyle, G.J. Pierce and J.S.W. Diack Department of Zoology, University of Aberdeen, Aberdeen AB9 2TN (Gt. Britain)

ABSTRACT Boyle, P.R., Pierce, G.J. and Diack, J.S.W., 1990. Sources of evidence for salmon in the diet of seals. Fish. Res., 10: 137-150. Feeding trials in which salmon was fed to captive seals are described and sources of evidence for the presence of salmon in the diet of seals are evaluated. In faecal samples, the recovery rate of salmon otoliths is too low and bony remains are too fragmented to be useful. Protein extracts from the faeces of salmon-fed captive seals will react with anti-salmon antisera, but the reaction is not strong enough for the methods to be presently applied to field samples. In the digestive tract samples from seals, the use of bony remains for the identification of salmon significantly increases the probability of recognising this species. Protein extracts from digestive tract contents will react positively with anti-salmon antisera and this shows that serological methods can provide evidence for the presence of Salmonidae in the diet of seals, in the absence of solid remains.

INTRODUCTION

The incidence of the North Atlantic salmon, Salmo salar L., in the diet of phocid seals around the Scottish coast has been the subject of much attention from fishing interests (Anon., 1975; Stansfeld, 1984; King-Webster, 1985; McDonald, 1988 ). In particular, the grey seal (Halichoerus grypus) has been considered by fishermen to have a significant impact on coastal migrating salmon, and the consequent availability of salmon to commercial fishing. This view stems largely from the well publicised predatory attacks by seals on fixed nets for salmon, caged salmon at fish farms and the incidence of seal damage to fish (Rae, 1960; Rae and Shearer, 1965). Between the years 1958 and 1971, the stomach contents of a total of 609 grey seals (H. grypus) and 253 common seals (Phoca vitulina) were examined by the Department of Agriculture and Fisheries for Scotland (DAFS) (Rae, 1968, 1973 ). These extensive investigations stressed the significance of commercially important fish in the diet of seals and estimated that Salmonidae comprised ~ 30% of the diet of grey seals, most of this being S. salar. These investigations formed the basis for the calculation by Parrish and Shearer (1977) that the grey and common seal stocks breeding in Scottish 0165-7836/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.

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waters took ~ 130 000 tonnes of commercially exploited fish species. A potential defect of the procedures was that many of the seals used in the analysis had been caught or killed around various types of fishing activity, particularly salmon nets. Clearly, the provenance of these samples could introduce bias into the results of diet analysis. Subsequent investigations by the Sea Mammal Research Unit (McConnell et al., 1984; Prime and Hammond, 1985b) made extensive use of faecal remains collected from seal haul-out sites. When washed through sieves, a selection of hard structures was recovered, consisting mainly of fish otoliths, bone fragments and other remains such as cephalopod beaks. As fish otoliths can be identified usually to genus or species level (Harkonen, 1986), the incidence of various fish in the diet could be estimated. Coupled with experimental feeding trials to estimate the loss of size of otoliths during passage through the digestive tract, measurements of otolith size were also used to make a quantitative estimation of fish consumption by grey seals (Prime and Hammond, 1985a, b). In contrast to the earlier DAFS study, almost no evidence was obtained for the presence of salmon in the diet. Two major problems were encountered in the interpretation of both of these approaches to seal diet. Firstly, the degree to which the provenance of the samples determined the findings. This difficulty is inherent in any study on seals as the available digestive tract and faecal samples are very unlikely to be representative of the population as a whole. The second difficulty is the recognition of some species of fish prey. Cartilaginous fish lack otoliths and a bony skeleton, and in some fish, e.g. salmon, the skeletal structures, including otoliths, are relatively soft and friable (Casteel, 1976) and, therefore, may not endure in faecal samples as well as those of other fish. Indeed, Jobling ( 1987 ) has pointed out that the differential vulnerability of fish otoliths to the digestive processes of mammals may introduce major sources of error into prey consumption rates estimated on this basis. In recognition of these difficulties, we have begun to extend the range of methods applicable to the investigation of seal diet in order to increase the reliability of the identifications which can be made, and to increase the amount and quality of the information which can be retrieved from both digestive tract and faecal samples. Based on feeding trials with captive grey seals, and on the examination of field samples, we have evaluated the use of three lines of evidence available for the recognition of fish remains: (a) the recognition of otoliths and their persistence through the digestive tract; (b) the use of other skeletal structures; (c) the identification of protein residues from the diet in stomach and faecal samples. In this paper, we present results applicable to the recognition of salmon and compare these methods used separately and in combination. The collection and identification of otoliths is in general use for diet analysis, but since there are only two otoliths per fish which will be ingested only

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if the head is eaten, their absence from samples leaves a high degree of uncertainty. In the case of skeletal remains, monographs are available describing the skeletons for many North Atlantic fish species. Bone fragments are relatively more durable than otoliths, but their use in diet analysis is limited at present by the lack of a systematic approach to the diagnostic characteristics of various bones (Hansel et al., 1988 ). Serological methods (the use of antisera to recognise protein extracts) have been applied widely to questions of trophic relationships in other fields (Boreham and Ohiagu, 1978) and have recently been used to investigate predator-prey interactions in the marine environment (Feller et al., 1979; Boyle et al., 1986; Grisley and Boyle, 1988 ). Although the mammalian digestive system would be expected to break down prey proteins very efficiently, protein residues of salmon in both stomach and faecal samples from seals are potentially recognisable using serological methods. METHODS

Feeding trials using salmon were conducted with seals held on behalf of the Sea Mammals Research Unit (SMRU) at the Research Institute for Nature Management, Texel, during two separate visits to The Netherlands. Similar procedures were followed on both occasions, in the first case using groups of grey and common seals. On the second visit, an adult male grey seal was isolated in an individual space by partitions of bars across the main tank. The remaining portions of the tank held three female greys which were due to pup and four juveniles from previous years, and another adult male grey seal. The seal occupied a tank area of ~ 6 × 8 m, with an equivalent area of haulout space. When filled, the water in the tanks was 2 m deep and the surface was level with the haul-out platform. The entire tank could be emptied and refilled with water direct from the sea. Emptying took less than 1 h and filling took ~ 90 min. When the tank was drained, there was a slight bottom current. To avoid faecal material being carried through the partitions towards the drain, a filter screen of l-cm 2 aperture wire mesh, 40 cm high, was fixed to the partitions. At the floor and sides of the tank, the edges of the mesh were folded "upstream" and the whole screen fixed firmly to the bottom and to the partitions. The normal diet of the Texel seals is mackerel (Scomber scombrus). The colony of nine seals was being fed 20 kg of frozen (thawed) mackerel each morning and evening. Salmon (S. salar), whiting (Merlangius merlangus) and herring ( Clupea harengus), supplied by DAFS, Aberdeen, were taken to The Netherlands for feeding trials. Approximately 70 kg of salmon, as whole fish or large steaks, were transported and stored frozen. Fish for feeding trials was thawed and gutted before presentation. Weighed quantities of fish were

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fed individually to the isolated male seal. The remaining non-experimental group continued with an ad libitum diet of mackerel with some salmon being fed to the second male. Before trials commenced, the experimental seal had not been fed for 36 h and the tanks were drained and cleaned. The feeding regimen followed the pattern to which the seals were accustomed. Feeding took place at ~ 15:30 h and again at 08:30 h. Throughout the experimental period, the tank was drained and cleaned out each day at 10:00 h. An "experimental day" thus consisted of an evening feed followed by one the next morning, and the subsequent sample collection and cleaning. When the tank was drained each day, a thorough examination was made of the tank floor and mesh filter screens for faecal samples. Discrete faecal masses or close accumulations of faeces were numbered as individual samples. They were labelled, sealed in polythene bags and frozen ( - 20 ° ) within 1 h of collection. Any uneaten remains of the previous two meals were also collected and weighed. The weight of the "recovered" fish was then subtracted from the weight of fish fed in the previous two meals to obtain an estimate of the daily food intake. Particular attention was paid to whether the recovered fish remains contained otoliths. After each collection, the tank floor was thoroughly washed with a high-pressure water hose. To facilitate the identification of otoliths and of bone material from digestive tract and faecal remains from seals, a new reference collection has been established of northeastern Atlantic fish species. The material in this collection has then been used as the basis for identification of hard parts from salmon and other fish species from both stomach and faecal samples, together with the published keys and guides to otoliths (Scott, 1905; Brodeur, 1979; Breiby, 1985; Harkonen, 1986). The use of serological methods for identification of the diet of marine predators has been recently described by Boyle et al. (1986) and Grisley and Boyle ( 1988 ). Full details of the methods evaluated for the recognition offish species in the marine m a m m a l diet will be presented elsewhere (Pierce et al., 1990a, b ). Briefly, the methods are as follows. Protein extracts of a portion of salmon muscle were made by adding twice its weight of distilled water, or 5 m M TES (N-tris [hydroxymethyl ] methyl2-aminoethanesulphonic acid) saline adjusted to pH 7.3 with NaOH. After maceration, the mixture was centrifuged, dialysed against distilled water, and the supernatants freeze-dried and stored at - 2 0 ° C. Dutch or New Zealand rabbits were inocculated intramuscularly or subcutaneously with an initial injection of 0.5 ml of extract (protein concentration 50 mg m l - l ) with 0.5 ml Freunds' complete adjuvant. A total of 1-3 subsequent booster inoculations were given with Freunds' incomplete adjuvant. After a test bleed from the marginal ear vein, the rabbits were bled-out by cardiac puncture. After clotting overnight at 4 ° C, the blood was centrifuged; the serum was decanted off and stored at - 20°C. The titre and specificity of the various batches of salmon antisera were tested

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using crossed immunoelectrophoresis (CIE; Clarke and Freeman, 1966 ) and fused-rocket immunoelectrophoresis (FRIE). Details of these methods are given in Grisley and Boyle ( 1988 ). The antisera were not completely specific to S. salar and reactions of identity were seen with other Salmonidae, but not with fish species from other families. Seal faeces were collected at haul-out sites, in several areas of Scotland: the Moray Firth, Orkney, Summer Isles and the Isle of May. Seal carcasses were obtained from those shot at salmon nets and elsewhere by fishermen; seals taken by DAFS in the course of other studies, and seals found dead on the shore, possibly as a consequence of the phocine distemper virus. The carcasses were opened by a median ventral incision and the digestive tract, including the oesophagus, was excised after ligaturing at both ends. After freeing from associated mesenteries, it was divided into 4-8 ligatured sections, in all cases separating both the stomach and the rectum. Sections of digestive tract were stored in individually labelled polythene bags at - 2 0 °C until required for processing. Both faecal and digestive tract samples were processed by two routes. Aqueous protein extracts were made, freeze-dried and stored as dry powder for subsequent serological testing. The insoluble residue was washed through nested sieves (smallest mesh diameter 0.355 mm ) to collect otoliths and other hard fragments. RESULTS Recovery of hard remains from faeces of the captive seals was limited to fragments of unrecognisable bones, including vertebrae, fish eye lenses, some scales and an occasional otolith. The intake of fish by the Texel grey seal over a I 0-day feeding trial is shown in Fig. 1. Previously, the animal had been fed on mackerel, but no food had been given for 36 h before the start of the feeding experiment. For the first 3 days, herrings were presented at each feeding session. Initially, the seal would take the fish, but after some "mouthing" a n d chewing, most of it was rejected. Despite presentation of herring at each feeding time, almost nothing was ingested. On the fourth day, salmon steaks were offered, 2.48 kg in the afternoon and 1.15 kg at the morning feed, but only a very small quantity was actually ingested. Throughout Day 5, the salmon steaks offered were skinned and the food intake rose to 3.80 kg ( 57% ) of the 6.65 kg of fish offered. Skin was also removed from the fish on Day 6 and the intake rose to 5.5 kg (78%) of the 7.08 kg offered. Thereafter, from Days 7 to 9, the salmon steaks presented were no longer skinned, but the seal fed actively at each meal. The food was brought to the surface each time, held between the front limbs and chewed. The seal was increasingly efficient at dealing with the fish, leaving only a small amount of skin and bone material. This improved "utilisation" of the meal is

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Fig. l. Summary of the feeding regimen and food intake of a grey seal at Texel showing the weight of food presented daily (solid line) and the intake of the seal (dotted line) obtained by subtraction of the weight of food remains. Species of fish in the diet and the numbers assigned to faecal samples recovered are shown on the diagram. shown in Fig. 1 and by Day 9 the daily intake of salmon was 11.75 kg (93%) o f the 12.6 kg presented. Details o f the recovery of hard parts from the faecal samples collected from the feeding trial on the isolated seal are shown in Table 1. A total o f 24 salmon heads were presented in the salmon meals. O f these, five complete or damaged heads were recovered from the tank, leaving a net intake by the seal of 19 heads. From this intake, a maximum of 38 otoliths could have been recovered from faecal samples. As Table 1 shows, a single otolith was found and this had been seriously eroded during its passage through the digestive tract to the point where recognition in a faecal sample from the wild would have been doubtful (Fig. 2 ). There were recognisable scales in some samples and more frequent eye lenses. N o bone fragments were recognisable, except vertebrae. The occasional mollusc and crustacean remains are considered to be incidental to the feeding trials. The particular adult male seal used in these feeding trials required an initial learning period before salmon was taken readily and consumed. The other male, a much larger and older seal, took salmon steaks at once and on several occasions was seen to swallow whole heads (although only two otoliths were recovered from its faeces). Unfortunately, we have insufficient data to quantify recovery rates o f otoliths from the second animal. Examination o f wild seal faecal scats reveals very little evidence o f salmo-

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TABLE 1 Food intake and recovery of hard parts from faecal material of a grey seal trolled diet Day

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8.16

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9.69

9.48

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11.75

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10

4.65

4.65

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Recovery of hard material from faeces (0.355-mm mesh)

Crustacea, seaweed, 1 lens -

Crustacea, herring vertebrae, fragments of salmon vertebrae, 2 lenses Crustacea, fragments of salmon vertebrae, 2 lenses, bone fragments, 1 salmon otolith Fragments of salmon vertebrae, scales, 1 lens Seaweed, mollusc shell, crustacea, scales, 2 lenses, fragments of bone and vertebrae 3 lenses, fragments of vertebrae

Mollusc shell, crustacea, bone fragments, 23 lenses, mackerel vertebrae, 1 mackerel otolith

nid fish. Details of the incidence of all fish remains in the field sample will be presented elsewhere, but it is relevant to note here that only one salmon otolith was recovered from seal scats in the Moray Firth area ( n = 408 ) and one from samples elsewhere in Scotland (n = 415 ). No other salmonid skeletal material was recovered from the faeces. The presence of salmon in protein extracts from faeces of the salmon-fed seals was tested using FRIE, against antisera raised to salmon flesh. The salmon extract and control homologous extract from salmon flesh were pipetted into adjacent wells and allowed to diffuse into the surrounding gel for 3 0 min, and then electrophoresed into a gel plate containing rabbit anti-salmon antiserum. Selected results are shown in Fig. 3. The control salmon extracts

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Fig. 2. Photograph of salmon otoliths recovered from faecal samples of captive seals compared with a pair of otoliths from the reference collection extracted directly from a fish (top of picture).

give a series of precipitin peaks (concentric "rockets"), indicating reactions between a number of proteins and antibodies in the polyspecific antiserum. In contrast, the sample extracts gave only faint indications of reactions (Fig. 3a). The earlier series of controlled feeding experiments on a mixed group of seals gave slightly more definite reactions (Fig. 3b) in which, importantly, the sample peaks linked with those from the homologous extract. These "reactions of identity" do not occur with extracts of non-salmonid fish and can be interpreted as positive recognition of Salmonidae in the sample, but it is doubtful whether the reaction is sufficiently clear to be useful for the recognition of salmon in wild caught faeces. Contents of the digestive tract of seal carcasses generally provide more possibilities for the recognition of fish prey, including salmon. In Fig. 4, the hard parts recovered by sieving (mesh 0.3 5 5 m m ) from the stomach of a wild grey seal killed in a salmon net are displayed. The material is arranged in three columns: on the left, a series of gadoid bones including a cod urohyal; in the centre, an assemblage of salmonid bones including caudal skeleton and lingual plate (probably of sea trout), underneath the salmonid bones are four cod otoliths and some lumpsucker denticles; in the right hand column are lumpsucker bones. These bones are readily identifiable from the reference collection skeletons. This example clearly shows the potential of bone material for the recogni-

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Fig. 4. Display of solid materials sieved from a seal stomach. The bones are arranged in three columns showing gadoid bones on the left; salmonid bones in the middle column, with four cod otoliths and lumpsucker denticles assembled at the lower part; on the right are all lumpsucker bones.

tion of salmonids in the digestive tract since no salmonid otoliths were recovered in this instance. In a field sample of 53 seal digestive tracts (grey and common seals combined), the incidence of Salmonidae in the diet was more than doubled if evidence from fish skeletons was used in addition to otoliths. As might be expected given the degree of protein degradation due to exposure to digestive processes, protein extracts from seal stomach contents were more likely to give positive evidence of the presence of salmonids when tested serologically than were those from seal scats. The results of serological testing of protein extracts from material in the stomach of a wild grey seal killed at a salmon net are shown in Fig. 5. The linkage of lines of precipitin reaction

147

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Fig. 5. Protein extracts of the fluid content from a seal digestive tract and control extracts of salmon flesh electrophoresed into rabbit anti-salmon antiserum (fused rocket immunoelectrophoresis). The material shown as a dark line above the oesophagus (o) and stomach (s) wells, linked to the peaks in the control rockets indicates reaction of identity. Material from the intestine (i) in three wells gave no reaction, between samples from the oesophagus and the stomach with peaks in the adj acent standard salmon extract provides evidence o f reactions o f identity and is sufficient to establish the presence o f Salmonidae. Serological screening o f the gut contents of wild grey and c o m m o n seals (n = 5 3 ) has produced evidence o f salmonids in the diet where no hard parts were present. Conversely, there are cases where evidence from hard parts suggests that salmonids were present, but serological screening proved negative. DISCUSSION The results from experimental feeding clearly suggest that evidence for salmon in the diet of seals is very difficult to obtain.

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In faecal samples, the recovery of otoliths was completely unrepresentative of the quantity of food intake and it must be concluded that recognition of otoliths alone cannot be used to estimate the incidence of salmonids in the seal diet. Recognisable bony material was also very infrequent and the application of this approach to the field sample did not alter the incidence of Salmonidae. This result is in contrast to that for most other fish species where recognition in the faeces can be markedly improved by the use of skeletal remains as well as otoliths (Pierce et al., 1990c ). The serological methods for salmon applied to seal faeces clearly have some potential for the recognition of the fish in the diet. However, the antisera we have produced are not of sufficient strength and specificity to propose their application to field samples. There is scope for the technical improvement of these methods for diet determination from faeces, but their routine application is more probable for fish species other than salmon (Pierce et al., 1990a). In the digestive tract, even when salmon otoliths are not present, evidence from both skeletal material and serological testing of protein extracts can be reliably used. Applied to field samples, these methods used in combination can considerably improve the recovery of information. Samples of gut contents from fresh seal carcasses are not likely to be readily available and the use of a more complex approach to diet investigation to maximise the return of information is fully justified. It is possible that these methods would be applicable to stomach contents of seals obtained by lavaging, especially as protein extracts are made from the fluid content of the stomach and do not require the evidence of solid material. In combination with programmes of study on seal biology which involve the routine catching and release of seals, serological testing of the stomach contents obtained in this way could provide wholly new information on the diet of marine mammals. ACKNOWLEDGEMENTS

This work forms part of a joint project between the DAFS, Aberdeen, and the University of Aberdeen, funded by the Scottish office over the period 1 June-30 September 1989. Paul Thompson, David Miller (University of Aberdeen), John Hislop and Bill MacDonald (DAFS) provided continuous help with the field samples; Maxine Grisley provided scientific advice on serological methods and, with Jill Robertson and Val Pratt, provided essential support in laboratory and animal house methods. We are also grateful to A1 Miller, Ishbel Clark and Andy Lucas for their practical help on many aspects of the project. Facilities and hospitality at the Research Institute for Nature Management, Texel, The Netherlands, were provided by courtesy of Peter Reijenders, and John Harwood kindly allowed access to the grey seals held there on behalf of the Sea Mammals Research Unit. Antisera were raised under Home

SALMON IN SEAL DIETS

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Office licence and collections made on the Isle of May under a permit from the Nature Conservancy Council.

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sea Resources. Sea Mammal Research Unit, Natural Environment Research Council, Cambridge, pp. 59-83. Prime, J.H. and Hammond, P.S., 1985b. The diet of grey seals in the North Sea assessed from faecal analysis. In: Sea Mammal Research Unit Report on The impact of grey seals on North sea Resources. Sea Mammal Research Unit, Natural Environment Research Council, Cambridge, pp. 84-99. Rae, B.B., 1960. Seals and the Scottish fisheries. Scott. Dep. Agric. Fish. Mar. Res., 1960: 1-39. Rae, B.B., 1968. The food of seals in Scottish waters. Scott. Dep. Agric. Fish. Mar. Res., 1968: 1-23. Rae, B.B., 1973. Further observations on the food of seals. J. Zool. London, 169: 287-297. Rae, B.B. and Shearer, W.M., 1965. Seal damage to salmon fisheries. Scott. Dep. Agric. Fish. Mar. Res., 1965: 1-39. Scott, T., 1905. Observations on the otoliths of some teleostean fishes. Annu. Rep. Fish. Board Scot., 24: 48-82. Stansfeld, J., 1984. Scotland's grey seals. Salmon Net, XVII (August): 72-74.