Bahram Sayyaf Dezfuli

Inflammatory response to Dentitruncus truttae (Acanthocephala) in the intestine of brown trout

Brown trout, Salmo trutta L., were infected with the acanthocephalan Dentitruncus truttae with the most affected areas being the anterior (near the pyloric caeca) and middle intestine. The parasite attached with a proboscis which usually penetrated the mucosa, lamina propria, stratum compactum, stratum granulosum and, sometimes, the muscularis layer. Around the parasites body was an area of inflammatory tissue. At the point of attachment the lamina propria was thickened and the stratum compactum, stratum granulosum and muscularis layer were disrupted by proboscis penetration. Rodlet cells were more numerous in infected fish (P<0.01), and were found in the epithelial layer away from the worm. Infected intestines had larger numbers of mast cells (P<0.01), often in close proximity to, and inside, the blood capillaries and associated with fibroblasts of the muscularis layer and the stratum granulosum. Their migration toward the site of infection was suggested. Intense degranulation of mast cells was encountered in all intestinal layers especially near the parasites body. Immunohistochemical tests were conducted on sections of intestinal tissue of uninfected and infected fish revealing the presence of met-enkephalin and serotonin (5-HT) in immuno-related cells of the intestine wall. Infected trout had larger numbers of elements positive to met-enkephalin and serotonin antisera. These data provided evidence for the role of the immune system of brown trout in the modulation of the inflammatory response to D. truttae. Results are discussed with respect to host immune response to an intestinal helminth.

Costs of intraspecific and interspecific host sharing in acanthocephalan cystacanths

Larval helminths often share individual intermediate hosts with other larval worms of the same or different species. In the case of immature acanthocephalans capable of altering the phenotype of their intermediate hosts, the benefits or costs of host sharing can be evaluated in terms of increased or decreased probability of transmission to a suitable definitive host. Competitive interactions among the immature stages of acanthocephalans within the intermediate host could create additional costs of host sharing, however. The effects of intraspecific and interspecific interactions were measured in 3 sympatric species of acanthocephalans exploiting a population of the amphipod Echinogammarus stammeri in the River Brenta, Italy. The strength of interactions was assessed from differences in the size achieved by infective cystacanths in the intermediate host. The size of Pomphorhynchus laevis cystacanths was not correlated with host size, whereas the size of Acanthocephallus clavula and Polymorphus minutus cystacanths increased with host size. Reductions in cystacanth size caused by intraspecific competition were only detected in P. laevis, but may also occur in both A. clavula and P. minutus. When co-occurring in the same amphipod with cystacanths of A. clavula, cystacanths of P. laevis attained a smaller size than when they occurred on their own. This effect was not reciprocal, with the size of A. clavula cystacanths not being affected. This supports earlier suggestions that it is adaptive for A. clavula to associate with P. laevis in amphipod intermediate hosts, with both species going to the same fish definitive hosts. In contrast, cystacanths of P. laevis achieved their largest size when they co-occurred in an amphipod with a cystacanth of P. minutus, which has a different definitive host (i.e. birds). These findings suggest that the net benefits of sharing an intermediate host can only be estimated by taking into account both the effects on transmission success and the consequences for cystacanth development.

Immunocytochemical localization of piscidin in mast cells of infected seabass gill.

Annual losses of approximately 5-10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worms attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.

Intra- and interspecific density-dependent effects on growth in helminth parasites of the cormorant, Phalacrocorax carbo sinensis

The action of intra- and interspecific competition, mediated by density-dependent effects on growth, was investigated among the 3 helminth species found in the alimentary tract of 104 cormorants, Phalacrocorax carbo sinensis. Intraspecific density-dependent effects on worm sizes were observed in the abundant nematode Contracaecum rudolphii, as shown by a negative correlation between mean worm size and intensity of infection. Higher intensities of infection by C. rudolphii were also associated with more variable worm sizes in the nematode Syncuaria squamata, suggesting a one-sided and density-dependent interspecific effect. There was also clear evidence of some form of negative interaction between the nematode S. squamata and the acanthocephalan Southwellina hispida from two fronts. First, there was a strong negative correlation between the intensities of infection of the 2 species across hosts. Second, sizes of worms of 1 species became more variable as the number of worms of the other species per host increased, and vice versa. This interspecific density-dependent effect on growth was thus apparently symmetrical. We also found evidence that worm size is a predictor of egg output in the 3 helminth species, indicating that intra- and interspecific density-dependent effects on growth can affect population dynamics in these worms. These results illustrate the complex nature of density dependence in helminth growth, and how its effects can act both within and among species.

Parasite-altered behavior in a crustacean intermediate host: field and laboratory studies.

The effects of the acanthocephalan parasite Pomphorhynchus laevis on the behavior of its crustacean intermediate host, the amphipod Echinogammarus stammeri, were studied. A drift study revealed that infected amphipods were disproportionately represented in drift samples taken throughout a 24-hr period; infection with more than 1 parasite enhanced this effect. Infection also interacted with the daily timing of drift, with parasitized amphipods beginning to drift earlier in the evening. Two distinct behaviors quantified in laboratory settings may play a role in this increased drifting behavior: parasitized amphipods showed (1) an increased preference for an illuminated environment and (2) increased activity in comparison to nonparasitized conspecifics. These results are consistent with previous studies on the effects of P. laevis on another amphipod host, Gammarus pulex, and provide new data on the activity level of P. laevis-infected amphipods.

The intestinal parasite Pomphorhynchus laevis (Acanthocephala) interferes with the uptake and accumulation of lead (210Pb) in its fish host chub (Leuciscus cephalus)

Uninfected chub as well as fish experimentally infected with the acanthocephalan parasite Pomphorhynchus laevis were exposed to (210)Pb(2+) for up to 38 days and the uptake and distribution of lead within different fish organs and the parasites was determined at various time points. Highest metal concentrations were detected in the acanthocephalans, followed by intestine, bile, liver, gill and muscle of the fish host. Infected chub had significantly lower (210)Pb levels in the gills on day 17 (P< or =0.01), in the bile on day 24 (P< or =0.05) and in the gills as well as in the intestine on day 38 compared with uninfected fish. A subsequent polynomial regression revealed that lead levels for the infected fish ranged below the levels determined for uninfected fish during most of the exposure period. This is the first proof that P. laevis reduces lead levels in the bile thereby diminishing or even impeding the hepatic intestinal cycling of lead, which may reduce the amount of metals available for the fish organs. This is especially important for ecotoxicological research. For example, organisms used as accumulation indicators may erroneously indicate low levels of pollution if they are infected with parasites which alter their pollutant uptake mechanisms. Additionally, the results gave further experimental evidence for acanthocephalans as accumulation indicators for metals.

SPERMIOGENESIS AND SPERM ULTRASTRUCTURE OF CYATHOCEPHALUS TRUNCATUS (PALLAS, 1781) KESSLER, 1868 (CESTODA: SPATHEBOTHRIIDEA)

Spermiogenesis and sperm ultrastructure of adult Cyathocephalus truncatus, a member of presumably basal group of “true” cestodes (Eucestoda), have been examined for the first time by using transmission electron microscopy. The process of sperm formation corresponds in basic pattern to that of the Pseudophyllidea. In addition, the 2 pairs of electron-dense attachment zones are present in median cytoplasmic process of C. truncatus. However, mature spermatozoa of C. truncatus differ significantly from those of the pseudophyllideans, especially in the morphology of the proximal and distal spermatozoon extremities. The proximal extremity of the mature spermatozoon lacks a crested body, which is present in more derived cestodes and some pseudophyllideans. The distal end of the mature spermatozoa exhibits different morphology in the gametes from testes and those from receptaculum seminis. New for the Eucestoda is a finding that a lateral cytoplasmic extension creates the distal end of the spermatozoa from testes, resembling sperm of some Monogenea and Digenea. In contrast, the distal extremity of the spermatozoa from receptaculum seminis contains only a nucleus. Despite the above-mentioned peculiarities, the ultrastructural data on sperm/spermiogenesis suggest close relationships of the Spathebothriidea and Pseudophyllidea.

Vitellogenesis in two spathebothriidean cestodes

Vitellogenesis in two spathebothriidean tapeworms, dixenous adult Cyathocephalus truncatus and monoxenous progenetic Diplocotyle olrikii, has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Each vitelline follicle consists of vitellocytes at various stages of development and one irregularly shaped interstitial cell. Projections of the interstitial cell enclose the vitellocytes and extend as a cytoplasmic sheath on the follicular periphery. An outer thin fibrous layer (= extracellular lamina) covers the cytoplasmic sheath in C. truncatus, but lacks in D. olrikii. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules that gradually fuse and give rise to the large shell globule clusters. Morphology of shell globule clusters differs slightly in both species. In addition, single “lamellar” granules are present in the cytoplasm of vitellocytes of C. truncatus, but not in D. olrikii. Both electron lucent and electron dense lipid droplets are present in the vitellocytes of C. truncatus, whereas only electron dense lipids occur in D. olrikii. A single lipid droplet turns up occasionally in the nuclei of some of the vitellocytes of C. truncatus. The ultrastructural features of vitellogenesis in spathebothriideans resemble those reported previously in “lower” cestodes, especially in pseudophyllideans.

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.

Intestinal immune response of Silurus glanis and Barbus barbus naturally infected with Pomphorhynchus laevis (Acanthocephala).

Immunopathological and ultrastructural studies were conducted on the intestine of barbel Barbus barbus and sheatfish Silurus glanis that were naturally infected with the acanthocephalan Pomphorhynchus laevis. Enteric helminths often cause inflammation of the digestive tract, inducing the recruitment of different types of immune cells at the site of infection. The results of our study clearly demonstrated that mast cells (MC) were the dominant immune cells which occur at the site of inflammation in both hosts. MC were associated with fibroblasts and were found in close proximity to, and inside, the capillaries of the intestine, thus, migration of mast cells via the bloodstream was suggested. Significant degranulation of MC was present. Immunohistochemical staining revealed met‐enkephalin and serotonin (5‐HT) in intestinal MC of both uninfected and infected barbel and the absence of the antimicrobial peptides piscidin 3 and piscidin 4 in both species. Data are discussed with respect to host immune response to an intestinal helminth and compared with other host‐parasite systems.