Integrative species delimitation and community structure of nematodes in three species of Australian flathead fishes (Scorpaeniformes: Platycephalidae).

This study aimed to determine the integrative characterisation of nematodes from three species of edible flathead fishes (Scorpaeniformes: Platycephalidae) in New South Wales, Australia, and describe nematode communities within three species of flatheads. Tiger (Platycephalus richardsoni (Castelnau); n = 20) and sand flatheads (Platycephalus bassensis (Cuvier); n = 20), sourced from the Nelson Bay area, and dusky flathead (Platycephalus fuscus (Cuvier); n = 20) from the Manning River, Taree, were examined for the presence of nematodes. The nematodes were initially classified morphologically as 12 different morphotypes belonging to the families Anisakidae (Anisakis types I, II, and III, Contracaecum type II, Terranova types I and II), Raphidascarididae (Hysterothylacium types IV, VI, VIII, and H. zhoushanense larva), and Gnathostomatidae (Echinocephalus sp. larva), Capillariidae (Capillaria sp.), followed by genetic identification through sequencing of the internal transcribed spacer (ITS-1, 5.8S, ITS-2) regions. Phylogenetic analyses revealed the evolutionary relationship between the identified larval specimens in the present study with available GenBank larval and adult nematodes. Sand flathead was 90% infected with nematodes followed by tiger flathead at 85% and dusky flathead at 15%. Nematodes infecting estuarine dusky and oceanic sand and tiger flatheads contrasted markedly. The analysis of similarities (ANOSIM) showed significant differences (p < 0.001) in the composition of taxa within nematode communities between the three species of flatheads (global R = 0.208) with the highest difference being between sand and dusky flatheads (R = 0.308, p < 0.001). The findings of the present study provide a foundation for future investigations of the community composition, life cycles, and distribution of nematode populations in edible fish in Australia and explore and clarify their significance to public health.

New insights into the coexistence of Contracaecum rudolphii A and Contracaecum rudolphii B (Nematoda: Anisakidae) in Phalacrocorax carbo sinensis from Sardinia: genetic variability and phylogenetic analysis.

Contracaecum sp. nematodes are important parasites of fish eating birds that can cause animal health problems. In the present study, specimens of Contracaecum rudolphii sensu lato, from the great cormorant Phalacrocorax carbo sinensis from Sardinia, were characterized based on morphological and molecular data. The morphological analysis allowed to identify all the fourth stage larvae (n = 1918) as Contracaecum sp., and adults, male (n = 5845) and female (n = 8312), as C. rudolphii sensu lato. Population genetics and phylogenetic relationships were inferred based on mitochondrial and nuclear markers. Multiple sequence alignment of the ribosomal internal transcribed spacer showed the coexistence of C. rudolphii A (n = 157), C. rudolphii B (n = 22) and a rare heterozygote of these species. Moreover, mitochondrial markers, namely NADH dehydrogenase subunits I (nad1), cytochrome c oxidase subunit (cox1 and cox2) and small subunit of rRNA (rrnS), showed that the studied C. rudolphii A populations had undergone bottleneck, or founder effect event, subsequent to a rapid population growth and expansion. The observed heterozygote is with a mitochondrial pattern of C. rudolphii B. Although, both Contracaecum species showed high genetic diversity, no genetic structure between localities was detected. Phylogenetic reconstructions supported the paraphyly of the avian Contracaecum species including C. ogmorhini (parasite of otariids).

Morphological and molecular identification of Hysterothylacium larvae (Nematoda: Raphidascarididae) in marine fish from Tunisian Mediterranean coasts.

The taxonomy of Hysterothylacium genus in Mediterranean waters remains incomplete and unresolved. The aim of the current study was to investigate the morphological and molecular identification of selected species of Hysterothylacium larvae in marine fish from the Tunisian Mediterranean coasts. A total of 192 marine fish samples were examined. In total, thirty-seven third-stage larvae of Hysterothylacium were morphologically identified as Hysterothylacium type V. In the present study, representatives of this type from the Mediterranean Sea were genetically characterized for the first time by sequencing the rDNA ITS (ITS1-5.8S-ITS2) regions and mtDNA cox2 gene. This study represents the first report of Hysterothylacium type V from the Mediterranean Sea. We also report Mullus barbatus, M. surmuletus, and Pagellus erythrinus as new hosts for this larval type. Based upon molecular and phylogenetic analyses considering the rDNA ITS regions, the Hysterothylacium type V described here was classified as a new genotype, named Genotype B. The valid genetic data of the described Hysterothylacium type V in the present study can be used to establish the phylogenetic relationships among Hysterothylacium species from the Mediterranean Sea and worldwide for future research.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

A new species of Raphidascaris (Nematoda: Raphidascarididae) infecting the fish Gymnogeophagus balzanii (Cichlidae) from the Pantanal wetlands, Brazil and a taxonomic update of the subgenera of Raphidascaris based on molecular phylogeny and morphology.

Raphidascaris (Sprentascaris) andersoni n. sp. (Nematoda: Raphidascarididae) collected in the intestine of the humphead cichlid Gymnogeophagus balzanii (Perugia) from the Pantanal wetlands, State of Mato Grosso do Sul (Brazil) is described and genetically characterized. The new species differs from its congeners mainly by having a conspicuous papilla-like formation slightly anterior to the cloacal aperture. Furthermore, males of R. (S.) lanfrediae and R. (S.) mahnerti have caudal alae, and R. (S.) hypostomi and R. (S.) pimelodi lack lateral alae, whereas in the new species caudal alae are absent and lateral alae present. The remaining congeners, namely, R. (S.) marano and R. (S.) saltaensis differ from Raphidascaris (Sprentascaris) andersoni n. sp. mainly because males have three pairs of postcloacal papillae (vs five pairs). In the phylogenetic reconstructions, using three nuclear genetic markers (18S, ITS1-5.8S-ITS2 and 28S rDNA) and one mitochondrial (cox1 mtDNA), the new species was separated from other representatives of Raphidascarididae, and the absence of monophyly in Hysterothylacium and Raphidascaroides was confirmed. Moreover, the subgenera Sprentascaris and Ichthyascaris appeared to be monophyletic. Therefore, even though Raphidascaris (Raphidascaris) was apparently not monophyletic, the subgenera of Raphidascaris should be re-erected as valid genera. The updated diagnoses of Ichthyascaris, Raphidascaris and Sprentascaris are given. The present study represents the first parasitological survey in G. balzanii.

Hysterothylacium fabri (Nematoda: Raphidascarididae) in Mullus surmuletus (Perciformes: Mullidae) and Uranoscopus scaber (Perciformes: Uranoscopidae) from the Mediterranean.

Raphidascarididae are among the most abundant and widespread parasitic nematodes in the marine environment. The life-cycle of most raphidascaridid species is poorly known and information about their distribution and host range is lacking in many geographical areas, as is the taxonomy of several species. A study of larval and adult stages of Hysterothylacium fabri (Rudolphi, 1819) Deardorff and Overstreet, 1980 (Nematoda: Raphidascarididae) infecting the striped goatfish Mullus surmuletus Linnaeus, 1758 (Mullidae) and the Mediterranean stargazer Uranoscopus scaber Linnaeus, 1759 (Uranoscopidae) from the Ionian Sea (central Mediterranean) has been carried out by combining light and scanning electron microscopy observations and molecular analyses through polymerase chain reaction-restriction fragment length polymorphism and sequencing of the ITS rDNA gene. Results indicate that U. scaber and M. surmuletus represent suitable definitive and intermediate/paratenic hosts of H. fabri, respectively, in the Mediterranean and highlight the importance of combining genetic and morphological data to study the taxonomy and epidemiology of parasites widely distributed in different fish species and aquatic ecosystems.

Similar yet different: co-analysis of the genetic diversity and structure of an invasive nematode parasite and its invasive mammalian host.

Animal parasitic nematodes can cause serious diseases and their emergence in new areas can be an issue of major concern for biodiversity conservation and human health. Their ability to adapt to new environments and hosts is likely to be affected by their degree of genetic diversity, with gene flow between distinct populations counteracting genetic drift and increasing effective population size. The raccoon roundworm (Baylisascaris procyonis), a gastrointestinal parasite of the raccoon (Procyon lotor), has increased its global geographic range after being translocated with its host. The raccoon has been introduced multiple times to Germany, but not all its populations are infected with the parasite. While fewer introduced individuals may have led to reduced diversity in the parasite, admixture between different founder populations may have counteracted genetic drift and bottlenecks. Here, we analyse the population genetic structure of the roundworm and its raccoon host at the intersection of distinct raccoon populations infected with B. procyonis. We found evidence for two parasite clusters resulting from independent introductions. Both clusters exhibited an extremely low genetic diversity, suggesting small founding populations subjected to inbreeding and genetic drift with no, or very limited, genetic influx from population admixture. Comparison of the population genetic structures of both host and parasite suggested that the parasite spread to an uninfected raccoon founder population. On the other hand, an almost perfect match between cluster boundaries also suggested that the population genetic structure of B. procyonis has remained stable since its introduction, mirroring that of its raccoon host.

Early development and life cycle of Contracaecum multipapillatum s.l. from a brown pelican Pelecanus occidentalis in the Gulf of California, Mexico.

The initial developmental stages of Contracaecum multipapillatum (von Drasche, 1882) Lucker, 1941 sensu lato were studied using eggs obtained from the uteri of female nematodes (genetically identified) found in a brown pelican Pelecanus occidentalis from Bahía de La Paz (Gulf of California, Mexico). Optical microscopy revealed a smooth or slightly rough surface to the eggs. Egg dimensions were approximately 53 × 43 µm, although after the larvae had developed inside, egg size increased to 66 × 55 µm. Hatching and survival of the larvae were greater at 15°C than at 24°C, and increased salinity resulted in a slight increase in hatching but seemed to reduce survival at 24°C, but not at 15°C. The recently hatched larvae measured 261 × 16 µm within their sheath. When placed in culture medium, the larvae grew within their sheath, and a small percentage (~2%) exsheathed completely (314 × 19 µm). The larvae continued to grow and develop once they had exsheathed, attaining mean dimensions of 333 × 22 µm. Although they did not moult during culture, optical microscopy revealed a morphology typical of third-stage larvae. Finally, the genetic identity between the larval parasites collected from mullet Mugil curema and adult female parasites collected from the brown pelican suggests a life cycle of C. multipapillatum in which the mullet are involved as intermediate/paratenic hosts and the brown pelicans as final hosts in the geographical area of Bahía de La Paz.

A model for the development and growth of the parasitic stages of Parascaris spp. in the horse.

Literature documenting the growth and development of Parascaris spp. infections was used to develop a model describing worm dynamics in the young horse. The model incorporates four main variables; the rate at which larvae migrate through host tissues to return to the small intestine, the proportion of migrating larvae which succeed in returning to the small intestine, the rate of growth in size of maturing and adult worms and the survival rate of maturing and adult worms. In addition, the number of eggs laid each day by adult female worms is calculated as a function of worm size (length) and is used to calculate faecal egg output of the foal. Published data describing the rate of migration through host tissues, and the growth of worms following their return to the small intestine, was used to derive relationships describing these processes. However, only limited data exists relating the survival of migrating larvae and mature worms in the intestine to host age and experience of infection. Therefore, relationships and coefficients describing these variables were modified so that output aligned with published experimental results. As a consequence, the model has not yet been evaluated against an independent data set, and so remains as the best 'current hypothesis' for the dynamics of this parasite. Hopefully, future experiments designed to test specific assumptions and outputs of the model will lead to a better understanding of the biology of this important parasite. For example, the most influential variable in determining model output is the survival rate of worms in the small intestine. In the model, worm survival declines in response to both the increasing age of the horse and the increasing cumulative length of worms in the intestine (used as a proxy for crowding). Given the importance of this variable to model behaviour and the paucity of experimental data on this topic this would seem a priority for future study. Initial experiments using the model suggest that a single anthelmintic treatment, administered soon after patency of initial infection, may effectively control environmental contamination with Parascaris spp. eggs while allowing a small 'refugia' of susceptibility to delay the emergence of anthelmintic resistance. Further evaluations of the practicality of this approach may be worthwhile.

Baylisascaris procyonis Demography and Egg Production in a California Raccoon Population.

California has more reported human raccoon roundworm cases than any other state due to large and overlapping human and raccoon populations. Infection by this parasite, Baylisascaris procyonis , is relatively benign in raccoons but can cause severe pathology in other species. Disease risk is driven by environmental egg contamination which increases with raccoon density, worm intensity, and worm prevalence. To improve knowledge about risk to humans and other species, 189 raccoons from southern California were examined to investigate how host age and season affect worm abundance, demography, and fecundity. Adult worms were present in animals as young as 10 wk and 100% of 4-mo-old raccoons were infected. Although 80% of sampled raccoons hosted adult B. procyonis , prevalence and abundance were lower in older animals. There were more worms in juvenile than in adult raccoons, resulting in a convex age-intensity profile. Coupled with raccoon demography, this drove fall peaks in parasite abundance and egg production. Eggs per-gram feces averaged 4,606 ± 661 (SE), and this output increased with worm intensity, with no evidence that crowding reduced parasite size or fecundity. High parasite egg outputs from hosts in this California raccoon population increase human exposure risk, and this risk could be reduced by management strategies that target heavily infected juvenile raccoons.

Differential expression of ß-tubulin isotypes in different life stages of Parascaris spp after exposure to thiabendazole.

Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to ß-tubulin, which prevents polymerisation of microtubules. In this study, ß-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120 h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study ß-tubulin gene expression of both isotypes in parasacaris eggs after exposure to different concentrations of thiabendazole (TBZ) for five days at 25 °C. A strong pattern of differential expression of ß-tubulin and isotype 1 was observed in all stages, while isotype 2 expression was mainly found at an early phase of the embryogenesis. For isotype 1, a 5-fold increase was observed during the first 48 h, but gene expression gradually decreased after 72, 96 and 120 h. Isotype 2 was only expressed during the first 24h, followed by a 130-fold decrease at (time points) 72, 96 and 120 h. The in ovo larval developmental test, in which we exposed initially unembryonated eggs to increased concentrations of TBZ, did affect isotype 1 gene expression but not isotype 2. This assumes that each isotype has specific functions in different life stages. This is in agreement with the 'multi-tubulin' hypothesis, which states that different tubulin isotypes are required for specialised microtubule functions. Isotype 1 is the most likely drug target for BZs, as isotype 2 was only expressed at very low levels later in development. Increasing concentrations of TBZ altered ß-tubulin isotype 1 gene expression after exposure of the eggs for five days, but this was not seen for isotype 2.

Anguilla anguilla intestinal immune response to natural infection with Contracaecum rudolphii A larvae.

The European eel, Anguilla anguilla, is a major warm-water fish species cultured in North and South Europe. Seventy-one A. anguilla collected between 2010 and 2015 from the Comacchio lagoons were examined. Fish were infected and damaged by larvae (L3) of the nematode Contracaecum rudolphii A, which were encapsulated within the thickness of the intestinal wall and within the external visceral peritoneum (serosa). Conspicuous granulomas, visible at sites of infection, were arranged in a trilayer, formed by a series of concentric whorls. The cells involved in the immune response and their distribution in the granuloma layers were assessed by immunohistochemical, immunofluorescence, and ultrastructural techniques. The outer part of the granuloma contained macrophages, macrophage aggregates, and mast cells (MCs) scattered among fibroblasts. This layer was vascularized, with degranulation of MCs occurring in close proximity to the capillaries. The middle layer was rich in MCs and fibroblasts. The inner layer, closest to the parasite larva, consisted mainly of dark epithelioid cells, some of which were necrotic. Non-necrotic epithelioid cells formed desmosomes between themselves or with fibroblasts. Within the granulomas, numerous cells of different types were positive to proliferative cell nuclear antigen antibody, indicating a high degree of cellular proliferation around the larvae.

First record of larvae of Hysterothylacium (Nematoda: Anisakidae) with zoonotic potential in the pirarucu Arapaima gigas (Osteichthyes: Arapaimidae) from South America.

Third-stage larvae (L3) of Hysterothylacium sp. were collected by the first time in juveniles of pirarucu Arapaima gigas farmed in the Rio Preto da Eva, Amazonas state. Ninety-eight (98) out of 100 examined fish showed to be parasitized. Five hundred and ninety larvae of Hysterothylacium sp. were collected from the intestines, stomach and pyloric caeca. The mean intensity of parasite indexes was 6.02 (±5.75) ranging from 1 to 40 larvae per host and the mean abundance was 5.9 (±5.76). The A. gigas is the new host record for larvae of Hysterothylacium sp. in Brazil, and this is the first record of larvae of Hysterothylacium (Nematoda: Anisakidae) with zoonotic potential in the pirarucu from South America.

First record of larvae of Hysterothylacium (Nematoda: Anisakidae) with zoonotic potential in the pirarucu Arapaima gigas (Osteichthyes: Arapaimidae) from South America / Primeiro registro de larvas de Hysterothylacium sp.(Nematoda: Anisakidae) com potencial zoonótico em pirarucu Arapaima gigas (Osteichthyes: Arapaimidae) na América do Sul

Abstract Third-stage larvae (L3) of Hysterothylacium sp. were collected by the first time in juveniles of pirarucu Arapaima gigas farmed in the Rio Preto da Eva, Amazonas state. Ninety-eight (98) out of 100 examined fish showed to be parasitized. Five hundred and ninety larvae of Hysterothylacium sp. were collected from the intestines, stomach and pyloric caeca. The mean intensity of parasite indexes was 6.02 (±5.75) ranging from 1 to 40 larvae per host and the mean abundance was 5.9 (±5.76). The A. gigas is the new host record for larvae of Hysterothylacium sp. in Brazil, and this is the first record of larvae of Hysterothylacium (Nematoda: Anisakidae) with zoonotic potential in the pirarucu from South America.

Resumo Larvas de terceiro estágio (L3) de Hysterothylacium sp. foram coletadas pela primeira vez em juvenis de pirarucu Arapaima gigas cultivados no Rio Preto da Eva, Estado do Amazonas. Noventa e oito (98) dos 100 peixes examinados estavam parasitados. Quinhentos e noventa larvas de Hysterothylaciumsp.foram coletados no intestino, estômago e cecos pilóricos. O índice parasitário de intensidade média foi de 6,02 (±5,75) variando de 1 a 40 larvas por hospedeiro e o de abundância média foi de 5,9 (±5,76). A. Gigas é um novo registro de hospedeiro para larvas de Hysterothylacium sp. no Brasil, também sendo, o primeiro registro de larvas de Hysterothylacium sp. com potencial zoonótico em pirarucu da América do Sul.

Analysis of the effect of soil saprophytic fungi on the eggs of Baylisascaris procyonis.

Baylisascaris procyonis is a soil-transmitted helminth mainly found in raccoons (Procyon lotor) which can also affect other domestic and sylvatic animals, as well as humans, when the eggs released in the feces of parasitized raccoons are accidentally ingested. Three assays have been conducted to assess the effect of three saprophytic fungi, Mucor circinelloides, Paecilomyces lilacinus, and Verticillium sp., on the eggs of B. procyonis. Firstly, their ovicidal effect was in vitro ascertained by placing 1 mL with 2 × 10(6) spores of each fungus in Petri plates with water-agar (2 %) and simultaneously adding 200 eggs of Baylisascaris/plate. Two in vivo probes were carried out, by spraying the fungal spores (3 mL containing about 2 × 10(6) spores/mL) on the feces of raccoons and coatis (Nasua narica) passing eggs of B. procyonis in a zoological park; the other assay consisted of evaluating the activity of the fungi after adding sand to fecal samples from raccoons. An ovicidal type 3 activity characterized by morphological damage of the eggshell with hyphal penetration, internal egg colonization, and embryo alteration was observed for all the tested fungi. In the plate assays, viability of Baylisascaris eggs reduced significantly by 53-69 % with Mucor, 45-62 % with Paecilomyces, and 52-67 % with Verticillium. A similar ovicidal effect was detected in the feces with sand. These results demonstrate the usefulness of spraying spores of M. circinelloides, Pa. lilacinus, or Verticillium sp. on the feces of animals infected by Baylisascaris to decrease the numbers of viable eggs and, thus, the risk of infection.

Baylisascaris potosis n. sp., a new ascarid nematode isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana.

We describe a new nematode species, Baylisascaris potosis n. sp., isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana. The nematode was found in fecal specimens, identified morphologically, and confirmed genetically. The new species is similar to Baylisascaris procyonis, Baylisascaris columnaris, and other Baylisascaris species, but is distinguished by the position of the male phasmidial pole. Nuclear and mitochondrial DNA sequence analyses confirmed that the new species is phylogenetically distinct from all the members of the genus Baylisascaris, and groups with B. procyonis and B. columnaris. This nematode is the 10th species assigned to the genus Baylisascaris.

Effects of urbanization on prevalence of Baylisascaris procyonis in intermediate host populations.

Baylisascaris procyonis is an intestinal parasite of raccoons (Procyon lotor) that can also infect humans and a wide range of wildlife species. Prevalence of B. procyonis in raccoon populations appears to decrease as the landscape urbanizes, but less is known about prevalence in the small-mammal intermediate hosts of the parasite. We measured prevalence of B. procyonis in populations of mice (Peromyscus spp.) in forest preserves along a gradient of urbanization in Illinois. Prevalence in the mouse intermediate host exhibited a trend opposite raccoons: prevalence increased as surrounding human population density increased. This counterintuitive result may be due to higher overall environmental loads of B. procyonis in urban areas due to higher raccoon densities. Our results emphasize the need to understand fully the transmission dynamics of B. procyonis in its definitive and intermediate hosts in order to design and implement effective strategies to mitigate zoonotic risks to humans.

Frequency of deposition and location of Baylisascaris procyonis eggs in raccoon feces.

Baylisascaris procyonis is a large ascarid nematode found in the small intestine of raccoons (Procyon lotor). Infection with larvae of B. procyonis can produce visceral, ocular, and neural larval migrans in humans. Infected raccoons can shed millions of eggs a day in their feces. However, it is unknown whether eggs are consistently shed or whether eggs occur at irregular intervals by the population of female nematodes within a host. We trapped, infected, and collected daily fecal samples from 11 raccoons maintained in captivity. Eggs from B. procyonis were obtained from anterior, central, and posterior sections of raccoon feces, isolated by flotation, and quantified under 100× magnification. Naturally infected raccoons were collected and used as a comparison with the experimentally infected group. All raccoons in the experimental group (n=11) became infected with B. procyonis after consuming one infected mouse. Additionally, differential egg deposition rates were observed among individual raccoons from the experimental and naturally infected groups. Mean number of eggs per gram of feces (means±SE) was 16,563±4,321, which was less than previously reported for the species. However, no differences (F(2,30)=0.84, P=0.45) were noted in mean number of eggs per gram of feces among fecal sections. Wildlife biologists, veterinarians, health officials, and researchers of B. procyonis should collect daily fecal samples for a minimum of 3 days before identifying a raccoon as negative for B. procyonis infection. However, it does not matter where within the fecal matter the sample is obtained.

Proteolytic activity in Hysterothylacium aduncum (Nematoda: Anisakidae), a fish gastrointestinal parasite of worldwide distribution.

Proteases have a significant role in the life cycle of parasites and the pathogen-host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolytic activity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolytic activity was detected in all the developmental stages studied and at all pH values within the range employed (2.0-7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0 h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0 h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolytic activity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.

Population biology of Contracaecum rudolphii sensu lato (Nematoda) in the great cormorant (Phalacrocorax carbo) from northeastern Poland.

Parameters related to the occurrence, aggregation, and population structure of the nematode Contracaecum rudolphii Hartwich, 1964 , in the great cormorant ( Phalacrocorax carbo ) from northeastern Poland were analyzed. A total of 491 birds of different ages (adults, immatures, and nestlings) was examined; the cormorants studied were taken from both fresh- and brackish water habitats. Contracaecum rudolphii were found in stomachs of 454 birds (92.5%); the 46,244 nematodes included third- (L3) and fourth-stage larvae (L4), and sub-adult and adult females and males. The distribution of nematodes in the host population were highly aggregated. The occurrence of C. rudolphii was significantly dependent on the host's age and habitat, as well as on season; the proportion of development stages differed significantly depending on birds' age and season (the latter only in adult birds). The infrapopulations of C. rudolphii in the adult cormorants showed distinct density-dependent correlations: that is, as the infrapopulation size increased, the number of adult females C. rudolphii decreased, and the proportion of larvae and sub-adult females increased. A higher proportion of larvae and sub-adult females characterized the component population structure of the nematodes in the cormorant nestlings, compared with adult birds, probably because of immune system deficiency in the immature birds, coupled with the development of the nematode population. Seasonal changes in the C. rudolphii population, observed in the adult cormorants, were not related to seasonality of the L3 occurrence in food; instead, the changes are believed to have resulted from independent processes of elimination of the oldest nematodes and their replacement by larval stages that subsequently matured.