Experimental studies on the migration of Anisakis sp. larvae (Nematoda: ascaridida) into the flesh of herring, Clupea harengus L.

In two experiments, a batch of 38 herring from Shetland was gutted immediately on capture (0 h) and further batches were gutted 14 and 37 h after capture and storage on ice. The numbers of Anisakis larvae in the body-cavity and in the flesh (detected by pepsin-HCl digest) were then counted. The proportion of fish with worms in the flesh was higher at 14 and 37 h than at 0 h. The percentage of the total worm burden in the flesh increased between cash each time interval, which suggests that a large-scale migration of larvae into the flesh occurs in ungutted fish. There was a positive association between the numbers of larvae in the viscera and the numbers in the flesh. Most larvae occurred in the hypaxial muscles. The results are discussed in relation to the observations of earlier workers.     

Anisakid parasites of the pouting (Trisopterus luscus) from the Cantabrian Sea coast, Bay of Biscay, Spain

An epidemiological survey was undertaken of anisakids in 139 specimens (length: 13.2-24.5 cm) of pouting or bib (Trisopterus luscus) captured off the coast of northern Spain in the Cantabrian Sea. Third-stage larvae of two species of nematodes, Anisakis larvae type I and Hysterothylacium aduncum, were isolated. One adult female H. aduncum was also detected in the intestine of one pouting. Total prevalence of anisakids was 88.5%. Hysterothylacium aduncum and Anisakis showed, respectively, prevalence of 87.8% and 22.3%, mean intensity of 19.7 and 3.5, and mean abundance of 17.3 and 0.8. Analysis of infection parameters as a function of host length revealed a much higher prevalence in pouting specimens with length < 20 cm (94.4% for H. aduncum; 28.0% for Anisakis) than in those with length > or = 20 cm (65.6% for H. aduncum; 3.1% for Anisakis). The high mean intensity of Anisakis in muscle of parasitized pouting (5.9) may pose human health risks, although these are minimized by eating only thoroughly cooked pouting, as is the custom in Spain.     

Life cycle of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae)

Natural infections with Hysterothylacium haze in the Japanese common goby, Acanthogobius flavimanus, were observed in detail. In gobies in which no worm eggs were deposited, second-stage larvae were found in the digestive tract wall, and third-stage larvae occurred in the digestive tract wall, mesentery, and body cavity, whereas fourth-stage larvae and adults were found in the body cavity. This stage-habitat relationship demonstrates the infectivity of second-stage larvae to the goby and the larval migration. In heavily infected gobies, eggs and all worm stages, from hatched second-stage larvae to adults, often were found together in the body cavity of one individual host, suggesting that hatched second-stage larvae can develop in the body cavity. It was shown experimentally that H. haze develops to the second stage in the egg and does not hatch spontaneously. When a goby was fed the viscera of heavily infected gobies containing eggs and various stages of worms or artificially incubated eggs containing second-stage larvae, second- and third-stage larvae were recovered from the digestive tract wall, and fourth-stage larvae and adults were found in the body cavity. When polychaetes or crustaceans were placed in contact with infected goby viscera or incubated eggs, only second-stage larvae were recovered from the body cavity of the invertebrates. The experimental results were consistent with observations on natural infections and indicate that the direct life cycle of H. haze may involve invertebrates as transport hosts.     

Fish eggs and larvae in the diet of herring Clupea harengus membras Linnaeus, 1758 and the sprat Sprattus sprattus balticus (Schneider, 1904) (Clupeidae) in the Southeastern Baltic Sea

The qualitative and quantitative composition of fish eggs and larvae in the stomach contents of Baltic herring and sprat was examined. The occurrence of fish eggs and larvae in the diet of herring was markedly higher, compared to sprat. Eggs of four and larvae of nine fish species were found in the herring stomachs. The prey species composition of the herring and sprat diets was related to the ichthyoplankton composition and to the degree of overlapping of the prey and predator distributions. Sprat eggs were the predominant prey in the herring diet in May and sprat larvae prevailed in August. The daily ration estimates indicate that the sprat larvae consumption by herring in August exceeded the sprat egg consumption in May by many times. Cod eggs in the herring stomachs were only found at the optimal oxygen content and salinity for the cod eggs in the near-bottom water layer.     

The effects of salinity and temperature on the development and survival of fish parasites

In brackish water the variety of marine and freshwater parasite species is considerably reduced. The distribution in brackish water of most marine endoparasites is restricted by the salinity tolerance of their hosts, most of the parasite species are more tolerant than their hosts. The influence of salinity and temperature on nine species has been examined; first stage larvae of Contracaecum aduncum develop in 0-32‰ salinity; Cryptocotyle lingua proved to be infective at salinities down to 4‰. The greatest resistance was found in Anisakis larvae from herring Clupea harengus , which survived for more than half a year. Parasites in the fish intestines appear to be unaffected by changing water salinities, as the osmolarity in the intestines stays nearly constant. Marine ectoparasites ( Acanthochondria depressa, Lepeophtheirus pectoralis ) survive about three times longer than freshwater species ( Piscicola geometra, Argulus foliaceus ) when salinity is 16‰. High temperature increases the effects of adverse salinities on parasites. There is evidence that none of these ecto-parasitic species can develop within the range of 7-20‰ salinity.     

Population biology and behavior of larval Eustrongylides tubifex (Nematoda: Dioctophymatida) in poikilothermous hosts

A total of 178 channel catfish (Ictalurus punctatus), 162 freshwater drum (Aplodinotus grunniens), 60 smallmouth bass (Micropterus dolomieui) and 2,488 yellow perch (Perca flavescens) were examined for larval Eustrongylides tubifex (Nitzsch 1819) J?gerskiold 1909 during 1973 and 1974. Older, large fish exhibited higher prevalence, larger mean numbers of larvae in infected fish, larger mean numbers of larvae in all fish examined, and wider range of intensity of infection. Infections were most extensive in yellow perch. The majority of larvae were removed from mesenteric cysts. Under experimental conditions, larvae migrated from cysts through the body wall when environmental temperatures reached or exceeded 40 C. Fourth-stage larvae removed from yellow perch successfully reinfected a variety of poikilothermous experimental hosts. Both young-of-the-year and I+ age/size class yellow perch exhibited increasing monthly larval worm burdens during portions of the sampling period. Older (II+) age/size class yellow perch did not exhibit significantly increasing worm burdens. No significant differences were noted in numbers of male and female larvae or in worm burdens in male and female yellow perch.     

A study of the assimilation of fluorescent pigments of microalgae Isochrysis galbana by the early larval stages of turbot and herring

The appearance of large supranuclear vacuoles in the enterocytes of 1- and 4-days post-hatch larvae of turbot and herring, respectively, revealed by the pinocytotic uptake of a fluorescent marker (FITCdextran), indicates a potential for the absorption of dissolved nutrients by the endotrophic stages of marine fish larvae. Ingestion of algal cells by turbot larvae was observed soon after hatching, but low level pinocytotic absorption of algal material was first seen during the second day. More extensive lysis of algal cells and pinocytotic absorption occurred 24 h later. Although lysis of I. galbana was shown to occur at low external osmolarities, it is unlikely that sufficiently low osmolarities present in the hind gut of turbot larvae explained the observed rupture of algae. Other mechanisms for digestion are discussed. In newly hatched herring larvae, algal cells were unable to pass beyond the constriction in the mid-gut caused by the yolk sac. When algal cells were eventually seen to pass into the hind gut, there was no evidence of algal digestion or absorption throughout the remaining endotrophic and early exotrophic stages of herring larvae, although pinocytosis was observed to occur mid-way through the endotrophic stage.     

Direct Estimation of Food Consumption of Juvenile Fish in a Shallow Inlet of the Southern Baltic

The species composition and feeding behaviour of fish larvae and juveniles was investigated in a shallow inlet of the Southern Baltic. The aim of the study was to examine if there was a predatory impact of young fish on the observed annual collapse of the copepod population. Dominant fish species were herring (Clupea harengus), perch (Perca fluviatilis) and roach (Rutilus rutilus). Roach larvae preferred shallower areas, whereas young herring and perch were found in open water. Highest consumption values were always observed in the evening. By computing complete evacuation of the digestive tract in approximately 3.5 hours, daily consumption in percent of fish fresh weight was 1.3, 4.7 and 5.5% for young herring, roach and perch, respectively. The impact of young fish on the dynamics of the population of the copepod species Eurytemora affinis is negligible.     

The mortality and fecundity of Haemonchus contortus in parasite-naive and parasite-exposed sheep following single experimental infections.

Parasite-exposed lambs and their parasite-naive controls were experimentally infected once only with 30,000 H. contortus larvae at 3, 9, 12, and 20 weeks following termination of a moderate immunizing infection of 30,000 H. contortus larvae. Previously exposed lambs, challenged at 3 weeks, had a significant reduction in the total H. contortus worm burden as compared to parasite-naive controls. No difference in the total H. contortus worm burden was found between parasite-exposed or parasite-naive lambs challenged at 9 weeks or thereafter. Female worms were found to be significantly smaller in lambs previously exposed to the parasite as compared to those found in parasite-naive lambs. The average parasite fecundity was 4700 eggs per female worm per day. Previous exposure of the lambs to the parasite had no effect on parasite fecundity. Various mathematical models were used to examine parasite fecundity. Parasite fecundity was found to increase in the initial post-challenge period reaching a constant value approximately 58 days after challenge infection. No density-dependent constraints on fecundity were observed.     

Pathogenicity of Ichthyophonus hoferi for laboratory-reared Pacific herring Clupea pallasi and its early appearance in wild Puget Sound herring

Laboratory-reared pathogen-free Pacific herring were exposed to pure cultures of Ichthyophonus hoferi, and reproduced the disease seen in naturally infected fish--thus fulfilling Koch's Postulates. Pathogen-free herring used in this study were reared from artificially spawned eggs incubated in filtered, UV-sterilized seawater, eliminating the variables associated with multiple infections, which are common in wild herring. Wild free-ranging herring were captured monthly from June through October by dip net from 'herring balls' located in the northern Puget Sound. I. hoferi infections were identified in these fish soon after metamorphoses, about 4 mo post-hatch. The prevalence increased from 5 to 6% in 0-yr fish to 24% in 1-yr-old fish to 50 to 70% in fish over 2 yr old, with no associated increase in mortality. The route of natural transmission to wild herring was not determined, but carnivorous fish became infected and died when they were experimentally fed tissues infected with the organism. In vitro culture of tissues was the most sensitive method for identifying both clinical and subclinical infections.     

Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae.

The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy. Larvae were hatched and kept in filtered seawater or in seawater with penicillin and streptomycin. The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments. Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus. The posterior region of the intestinal lumen appeared completely occluded with bacteria. The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp. dominated in larvae exposed to antibiotics. The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics. Thus, a dramatic change in the microflora resulted from incubation with antibiotics. Nonpigmented yeasts were detected in both larval groups. Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated. These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae. The possible significance for larval health and nutrition is discussed.     

Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae.

The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy. Larvae were hatched and kept in filtered seawater or in seawater with penicillin and streptomycin. The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments. Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus. The posterior region of the intestinal lumen appeared completely occluded with bacteria. The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp. dominated in larvae exposed to antibiotics. The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics. Thus, a dramatic change in the microflora resulted from incubation with antibiotics. Nonpigmented yeasts were detected in both larval groups. Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated. These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae. The possible significance for larval health and nutrition is discussed.     

In vitro development of the fish parasite Hysterothylacium aduncum from the third larval stage recovered from a host to the third larval stage hatched from the egg

Anisakids are parasitic nematodes of fish worldwide, producing economic and human health concerns. It is thus important to ascertain their in vitro life cycle in laboratory studies. Here we describe the in vitro development of third-stage larvae (L3) of Hysterothylacium aduncum isolated from blue whiting Micromesistius poutassou, to the hatching of L3 from eggs obtained from H. aduncum worms grown in GLIT medium (a modified mixture of Yaeger's LIT [Liver Infusion Tryptose] and Grace's media) at pH 4.0, 13 degrees C and with 5% CO2 in air. Under these conditions, L3 recovered from fish developed to mature adults (3.4 to 6.2 cm in length), with oviposition starting from Day 26 in culture. Fertilized eggs (mean size 64 x 52 microm) had a thick, rugose eggshell and were larger than unfertilized ones (mean size 49 x 42 microm), whose eggshells were thin and smooth. Eggs laid during the first and second week of oviposition, and maintained in 2.8% NaCl solution at 13 degrees C, developed to L3. Under these maintenance conditions, between 20.6 and 52.5% of the eggs laid during the first week developed into larvae. Motile larvae, enclosed in a sheath, hatched from between 2 and 11% of these eggs. The larvae started to hatch 23 d after deposition. These larvae were 144 to 215 microm in length, enclosed in a 237 to 305 microm-long sheath. This GLIT culture medium may help to study the biology of this and other anisakids.     

Discrimination of Baltic herring populations with respect to Anisakis simplex larvae infection

Samples of herring Clupea harengus in the Baltic were collected during the spawning season in western, middle and eastern waters off the Polish coast. Two samples were taken per area, several morphometric characters were measured and some meristic characters (vertebrae and gill rakers) were counted. Infection with Anisakis simplex larvae, total length, age and maturity stage were recorded. Univariate and multivariate statistical tests were used to compare the samples, discriminant analysis was applied to separate herring populations and the fraction of herring populations in each area was estimated. The results obtained indicated that infected fish represented a mixed group of western and central Baltic herring. In the first samples collected from each area, the share of the western population decreased from 40% in the western to 18% in the middle and 13% in the eastern coasts. The share of western herring was higher in the second samples collected in the same area and decreased from 91% in the western coast to 14 and 32% in the middle and eastern ones, respectively.     

Temperature-driven changes in early life-history stages influence the Gulf of Riga spring spawning herring (<Emphasis Type="Italic">Clupea harengus m</Emphasis>.) recruitment abundance

Processes occurring during early life-history stages influence the year-class abundance of marine fish. We found that the abundance of 1-year-old spring spawning herring is statistically significantly determined by the number of post-flexion herring larvae in the Gulf of Riga (Baltic Sea). The abundance of consecutive developmental stages of larvae: yolk-sac, pre-flexion, flexion and post-flexion strongly correlated with each other, indicating that factors which already influence the yolk-sac stage are important in determining the abundance of post-flexion herring larvae. Winter air temperature before spawning determined the timing of maximum abundance of pre-flexion herring larvae, but not their main prey: copepod nauplii, implying that different mechanisms governing major preconditions for the formation of year-class strength. The abundance of post-flexion larvae displayed a potential dome-shaped relationship with sea surface temperature experienced after hatching. We suggest that increased summer temperatures, which exceed the physiological optimum negatively, affect the survival of post-flexion herring larvae. Overall, future climate warming poses an additional risk to larval herring survival and this may lead to a reduction in those herring stock which rely on recruitment from shallow coastal areas.     

Drinking and water absorption by the larvae of herring (Clupea harengus) and turbot (Scophthalmus maximus)

The efficiencies of water absorption from the guts of the larvae of herring ( Clupea harengus L.) and turbot ( Scophthalmus maximus L.) were estimated by two methods, The first method was based on the differences in the rates of accumulation, by drinking, and clearance from the gut of radiolabelled inert markers. The second method used the equilibrium level of radioactivity in the larvae to measure the concentration of the markers in the gut above background as water is absorbed from the gut. Water absorption efficiencies for herring larvae drinking sea water were estimated to be 77% using both methods. When external salinity was reduced to 50% sea water, drinking rates and water absorption efficiency in herring larvae fell substantially.
Estimates of water absorption efficiency of seawater-adapted turbot larvae were similar (71–84%) to that of herring using both methods. Although temperature had a marked effect on both the rate of drinking and water absorption, there was no significant thermal effect on the efficiency of absorption from the guts of turbot larvae. The limitations of the techniques and the implications of the estimates in terms of water balance in fish larvae are discussed.     

Intact antigen uptake in intestinal epithelial cells of marine fish larvae

Endocytosis of bacteria by epithelial cells in the hindgut of 4- to 6-day-old cod ( Gadus morhua L. ) larvae and of 10- to 12-day-old herring ( Clupea harengus L. ) larvae was demonstrated by fluorescence microscopy and transmission electron microscopy. Fixed sheep erythrocytes and bacteria-sized latex particles were not ingested by these cells. By immunohistochemical methods intact bacterial antigens were observed in columnar epithelial cells in the foregut of 4-day-old yolk-sac larvae of cod. In these cells a preferential uptake of Vibrio fischeri and Vibrio salmonicida cells as compared to Flavobacterium sp. was found. The possible role of these findings in the establishment of an indigenous microflora, defence against pathogens or nutrient absorption in fish larvae is discussed.     

Regulation of Haemonchus contortus populations in sheep exposed to continuous infection

Groups of sheep were infected three times weekly for 15 weeks with infective larvae of Haemonchus contortus at four rates ranging from 600 to 4800 larvae per week. At 3-weekly intervals, sheep from each group were killed for total worm counts after receiving a dose of radiolabelled larvae which enabled a direct measurement of establishment of incoming larvae during the continuing infection. Peak H. contortus burdens were reached between 6 and 9 weeks of infection, and were related to infection rate. In the groups receiving 2400 and 4800 larvae per week, worm numbers then declined rapidly and by the end of the experiment were lower than those in the groups given 600 or 1200 larvae per week. Establishment of incoming larvae was not influenced by infection rate, and declined from 45% in the first 4 weeks of infection to insignificant levels during the final 6 weeks. The proportion of incoming larvae arrested in their development increased as the infection progressed. It was concluded that H. contortus numbers were regulated by development of resistance to infection and by a loss of established worms which was related to the current rate of larval intake, and to the host's previous experience of infection.     

Effects of alternative prey on predation intensity from herring Clupea harengus and sandeel Ammodytes marinus on capelin Mallotus villosus larvae in the Barents Sea

Stomach contents from lesser sandeel Ammodytes marinus and herring Clupea harengus caught at one and three stations, respectively, were analysed to investigate predation intensity on capelin Mallotus villosus larvae. Most capelin larvae were found in the anterior sections of the stomachs close to the oesophagus, indicating that rapid digestion rates of larvae reduced the abundance in the posterior sections. The anterior sections of the stomachs had alternating layers of either copepods or capelin larvae and krill. This shows that the individual predators switched between feeding on either copepods or on krill and capelin larvae. A total of 549 capelin larvae was found in 440 fish stomachs. Capelin larvae were found in 20% of the sandeel stomachs, and 24, 34 and 62% of the stomachs from the three herring stations, respectively. Many of the predator stomachs contained more than five larvae, and up to 28 larvae were found in one herring stomach. The herring appeared to prey selectively on the largest capelin larvae, and the number of larvae per stomach was much higher in stomachs where krill had recently been eaten than where copepods were dominant. The predation intensity measured in this study is much higher than reported in earlier studies, and it is suggested that the predators were feeding using a searching image, and that the concentrations of alternative prey (copepods and krill) varied around a switching threshold.