Observations on egg production in the monogenean Entobdella soleae

Egg production in the monogenean Entobdella soleae increases as adult parasites grow. Medium-sized adults (anterior hamulus length 550–600 μm; total body length approx. 5 mm) produced about 30 eggs per day at 12°C. Parasites with anterior hamuli exceeding 650 μm in length (total body length about 6 mm) may produce more than 60 eggs per day. In smaller adults, eggs tend to spend longer in the ootype after assembly of the shell and the ootype remains empty for longer periods of time. The time taken to assemble a single egg is relatively constant (4–6 min) in adults of all sizes and there is evidence of a small but significant increase in egg size as parasites increase in size. Some eggs were retained in the uterus for as long as 168 min but others spent less than 5 s in the uterus, indicating that the uterus is no more than a passageway for these eggs to the outside world. The egg cell (or zygote) precedes the vitelline cells as they progress along the ovo-vitelline duct to the ootype.     

The chemical nature of host hatching factors in the monogenean skin parasites Entobdella soleae and Acanthocotyle lobianchi

Fully-developed eggs of the monogenean Entobdella soleae from the skin of the common sole (Solea solea) hatch when treated with dilute solutions of urea or ammonium chloride in sea water. There is some evidence that arginine may stimulate hatching but the eggs do not respond when treated with sea water solutions containing trimethylamine oxide or glutamine. Sole skin mucus contains sufficient urea to stimulate hatching but insufficient ammonia. Solutions of urea in sea water stimulate hatching in the monogenean parasite Acanthocotyle lobianchi found on ray skin. Sea water solutions containing ammonium chloride and trimethylamine oxide failed to hatch the eggs of A. lobianchi and the eggs were also insensitive to various amino acids made up at concentrations found in host mucus. Experiments with urease confirmed that urea in ray ventral skin mucus is the host hatching factor for A. lobianchi. Skin mucus from the common sole failed to stimulate hatching in A. lobianchi. The role as hatching factors of excretory products in host gill effluent, skin mucus and urine is discussed.     

Early development and microhabitat of the monogenean Horricauda rhinobatidis, with observations on the related Troglocephalus rhinobatidis, from Rhinobatos batillum from Queensland,Australia

The small monocotylid monogenean Horricauda rhinobatidis is abundant on the gills of its host Rhinobatos batillum, whereas the larger, related monogenean Troglocephalus rhinobatidis is comparatively uncommon. Young specimens of Horricauda live between the host's secondary gill lamellae. Post-oncomiracidia have 14 marginal booklets but as the larvae develop these are supplemented first by a pair of hamuli and then by muscular ventral loculi followed by six forwardly-directed, dorsal spines. By impaling secondary gill lamellae these spines may serve to prevent parasites from being dislodged by gill ventilating currents. Before reaching sexual maturity the parasites leave the secondary gill lamellae and establish themselves in the septal canals. It is uncommon to find more than one adult specimen of Horricauda in each septal canal. The significance of this in relation to sperm exchange is discussed. Like Troglocephalus, Horricauda has eight head sacs, and the suggestion is made that these sacs may play a part in feeding.     

The attachment of the monogenean parasite Callorhynchicola multitesticulatus to the gills of its holocephalan host Callorhynchus milii

Llewellyn J. and Simmons J. E. 1984. The attachment of the monogenean parasite Callorhynchicola multitesticulatus to the gills of its holocephalan host Callorhynchus milii. International Journal for Parasitology14: 191–196. C. multitesticulatus when juvenile (up to about 10 mm long) attaches itself by four pairs of clamps to the secondary gill lamellae of its host the elephant fish Callorhynchus milii. However, in adult parasites the haptor invades the host tissue and the stalk connecting the haptor with the body-proper becomes surrounded by a sleeve of fibrous host inflammatory reaction tissue. The stalk is provided with annuli with anteriorly-directed flanges which are pressed into the walls of the sleeve of host tissue when the diameter of the stalk increases as a result of the contraction of well-developed sub-tegumentary longitudinal muscles.     

Ultrastructure of the anterior alimentary tract of a monogenean, Diclidophora merlangi

The anterior alimentary tract of Diclidophora merlangi is composed of a complex series of morphologically distinct epithelia interconnected by septate desmosomes and penetrated by the openings of numerous unicellular glands. The mouth and buccal cavity are lined by an infolding of modified body tegument, distinguished by uniciliate sense receptors, buccal gland openings, and in the buccal region by a dense, spiny appearance. The prepharynx is covered by an irregularly folded epithelium and, for part of its length, by the luminal cytoplasm of the prepharyngeal gland cells. The epithelium is syncytial and pleiomorphic, and regional variation in structure is common. A separate epithelium invests the lips of the pharynx and its free surface is greatly amplified by numerous, dense lamellae of varying dimensions. The lip epithelium is continuous with cytoplasmic processes of cells located external to the pharynx. A further, distinct epithelium borders the pharynx lumen and is composed of discrete cytoplasmic units connected by short septate desmosomes. The oesophagus is lined by a modified caecal epithelium, lacking haematin cells, and, in places, is perforated by the openings of oesophageal gland cells; it is continuous with the syncytial connecting tissue of the gut caeca.     

A possible ciliary photoreceptor in a juvenile polyopisthocotylean monogenean, Sphyranura sp.

A pair of multiciliate, saccular structures containing multilayered lamellar bodies is reported in the head region of freshly hatched juveniles of the polyopisthocotylean monogenean Sphyranura sp. from Necturus maculosus. Similar structures have been recorded in only one other monogenean, in the oncomiracidium of Entobdella soleae, and the looser packing of the lamellar bodies in Sphyranura has permitted a more detailed ultrastructural study. A comparison is made with similar structures found in digenean miracidia.     

Ontogeny of the epicranial portion of the dorsal fin in Solea solea and Scophthalmus maximus (Teleostei, Pleuronectiformes)

A study of laboratory-reared larvae of Solea solea (Soleidae) and Scophthalmus maximus (Scophthalmidae) indicated that the epicranial portion of the dorsal fin results from the anterior displacement of proximal pterygiophores during ontogeny. The adult epicranial formula is attained early during ontogeny, and the anterior displacement is finalized after the passage of the migrating eye. In both species, the first two proximal pterygiophores fuse to form an erisma that is particularly long and well-developed in Solea. Moreover, in Solea, the neural spine of the second abdominal vertebra curves over the otic region, and the neural arch of the first vertebra remains incomplete. Received: August 3, 2000 / Revised: August 10, 2001 / Accepted: October 12, 2001     

Seasonal distribution and duration of the planktonic stage of Dover sole, Solea solea, larvae in the Bay of Biscay: an hypothesis

The possibility of deriving a prediction about the effect of seasonal variations in the duration of the planktonic larval phase on the dispersal of larval Dover sole was investigated. During six cruises, from February to May 1992, the distribution of sole larvae was studied along a 100-km transect, from the offshore spawning grounds to the coastal nurseries of the Bay of Biscay (France). Samples ( n = 189) were collected with a suprabenthic sampler, and vertical profiles of water temperature and salinity were recorded simultaneously. Counts of otolith increments of larval stage 4b (onset of metamorphosis) were used to estimate the duration of the planktonic life. Duration of the larval phase decreases by about 15 days (37%) with water temperature increase (between 8° in February and 11.2° C in May). Sole larvae occur from the coastal area to 100 km offshore. Within the same cruise, no difference in the duration of the planktonic life was observed between the larvae caught in the onshore and the offshore area. In spite of seasonal differences in abundance, the extent and the shape of the larval distributions during the period of study suggest that the seasonal variations in the duration of the planktonic life did not affect the larval distribution.     

Gland cells associated with the alimentary tract of a monogenean, Diclidophora merlangi

Three distinct groups of unicellular glands open into the buccal cavity, prepharynx, and oesophagus, respectively, of Diclidophora merlangi. Buccal glands produce a dense, acidophilic secretion of protein-rich droplets that are discharged from duct-like extensions of the glands by eccrine secretion. Prepharyngeal glands are extensive and contain basiphilic secretory droplets of varying density and characterized by the presence of one or two dense core-like inclusions. The droplets are PAS-positive and reactive for protein, and accumulate in the gland cell apices which form part of the lining to the prepharynx lumen. They are released in large numbers by apocrine secretion or, individually, by an eccrine secretory mechanism. Oesophageal glands are acidophilic and contain electronlucid, PAS-positive droplets that develop a crystalline appearance prior to release via either eccrine or apocrine secretion. Differences in ultrastructure and histochemistry indicate the glands are also functionally separate, and their probable role in feeding and extracellular digestion of blood in the worm is discussed.     

Trypanosoma vivax: Absence of host protein on the surface coat

The possible presence of host serum proteins on the surface of Trypanosoma vivax stock Zaria Y486 was studied. Intact washed bloodstream forms from mice were not lysed or neutralized by antisera against mouse serum proteins. Serum against T. vivax prepared in rabbits against an antigen which was a water-soluble trypanosome extract, failed to cross-react with mouse serum when tested by immunoelectrophoresis and immunodiffusion. The T. vivax antigen failed to cross-react with three different anti-mouse sera when tested by the same techniques.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of ¹²⁵I-surface-labeled parasites showed the presence of a cluster of proteins ranging in molecular weights between 57,000 and 45,000 daltons. None of these proteins was precipitated by anti-mouse serum protein sera. The serum against T. vivax precipitated a protein of 50,000 daltons molecular weight.     

The role of sense organs in the feeding behaviour of juvenile sole and plaice

An infra-red video recording system was used to observe and compare the feeding activity of juvenile sole and plaice. Experiments in light or dark and chemical ablation of the neuromasts with streptomycin sulphate allowed all or a limited set of senses to be used by the fishes. Both species were able to feed on dead prey when only chemical stimuli were available. Plaice showed a greater dependence on vision for feeding but sole relied principally on chemoreception and mechanoreception. When any one sense was removed plaice became less active whereas sole became more active.     

Growth variability of juvenile soles Solea solea and Solea senegalensis, and comparison with RNA : DNA ratios in the Tagus estuary, Portugal

Growth variability and condition of juvenile soles Solea solea and Solea senegalensis , were assessed through RNA : DNA estimates and compared to absolute growth rates. Higher mean cohort RNA : DNA ratios were observed for cohort I at the beginning of estuarine occurrence for both species (4·42 and 4·87, for S. solea and S. senegalensis respectively). Despite different estuarine colonization habits, no significant differences were observed between RNA : DNA monthly variation for both sole species within the same year ( P  > 0·05 for 2003 and 2004). Juvenile S. senegalensis showed significant differences between RNA : DNA ratios obtained for the two nursery areas ( P  < 0·001). The decrease of seasonal growth rates with fish age was similar to seasonal variation of mean RNA : DNA values. Thus the RNA : DNA pattern of juvenile S. solea and S. senegalensis reflected growth and estuarine colonization patterns.     

A new Perkinsus species (Apicomplexa,Perkinsea) from the abalone Haliotis ruber

A new protozoan of the genus Perkinsus is described from the muscle and hemolymph of the blacklip abalone, Haliotis ruber, from South Australia. It occurs in the muscle of the adductor and mantle, and free and in brownish masses in the hemolymph. Cells cultured in thioglycollate medium produced a prezoosporangium which stained blue-black with iodine. The parasite differs from Perkinsus marinus, the only other member of the class, in having a much larger trophozoite, an eosinophilic vacuoplast when present, a short discharge tube, and appears to be uninfective to oysters.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

Genomic regions of pufferfishes responsible for host specificity of a monogenean parasite, Heterobothrium okamotoi

The genetic mechanisms underlying host specificity of parasitic infections are largely unknown. After hatching, the larvae of the monogenean parasite, Heterobothrium okamotoi, attach to the gill filaments of hosts and the post-larval worms develop there by consuming nutrients from the host. The susceptibility to H. okamotoi infection differs markedly among fish species. While this parasite can grow on tiger pufferfish (also called fugu), Takifugu rubripes, it appears to be rejected by a close congener, grass pufferfish, Takifugu niphobles, after initial attachment to the gills. To determine the genetic architecture of the pufferfish responsible for this host specificity, we performed genome-wide quantitative trait loci analysis. We raised second generation (F₂) hybrids of the two pufferfish species and experimentally infected them with the monogenean in vivo. To assess possible differences in host mechanisms between early and later periods of infection, we sampled fish three h and 21 days after exposure. Genome scanning of fish from the 3 h infection trial revealed suggestive quantitative trait loci on linkage groups 2 and 14, which affected the number of parasites on the gill. However, analysis of fish 21 days p.i. detected a significant quantitative trait locus on linkage group 9 and three other suggestive quantitative trait loci on linkage groups 7, 18 and 22. These results indicated the polygenic nature of the host mechanisms involved in the infection/rejection of H. okamotoi. Moreover the analyses suggested that host factors may play a more important role during the growth period of the parasite than during initial host recognition at the time of attachment. Within the 95% confidence interval of the linkage group 9 quantitative trait locus in the fugu genome, there were 214 annotated protein-coding genes, including immunity-related genes such as Irak4, Muc2 and Muc5ac.     

Influence of DNA isolation from historical otoliths on nuclear-mitochondrial marker amplification and age determination in an overexploited fish, the common sole (Solea solea L.)

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole (Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material.     

Effect of the parasitoid, Campoletis sonorensis, on the growth of its host, Heliothis virescens

Growth of Heliothis virescens was stopped following parasitism by the ichneumonid parasitoid, Campoletis sonorensis. The source of the active factor was found to be the lateral oviducts of the female parasitoid. The active factor appears to be a protein.     

Comments on the mechanism of attachment in species of the monogenean genus Gyrodactylus

Abstract. In species of the monogenean helminth Gyrodactylus , the opisthaptor is the main organ of attachment to the host. The opisthaptor comprises two large centrally positioned hooks or hamuli and sixteen peripherally distributed marginal hooks. This paper describes the functional morphology and the mechanism and sequence of attachment in this species. Information on the attachment process was gathered from observations of live gyrodactylids, from transmission electron microscopy, from scanning electron microscopy of skeletal elements, and by histochemical and X-ray elemental analysis of hook chemical composition. The marginal hooks provide the principal force of attachment whilst the hamuli are not actively employed in the process of attachment. Instead, the hamuli provide a system preventing accidental dislodgement and assist the action of the marginal hooks. Attachment is achieved by the alternating action of two systems of muscles attached respectively to the hamuli and to the marginal hooks. Relaxation or contraction of the muscles connected to the hamuli manoeuvres the hamuli over the extremities of the accessory ventral bar and allows them to pivot around their longitudinal axis, effectively raising or lowering the opisthaptoral dome. Under reduced opisthaptoral tension, the independent gaffing activity of the marginal hooks ensures a secure attachment to the host's epidermis. Repositioning of the hamuli then raises the opisthaptoral dome to tension the peripheral marginal hooks. The sequence of attachment is complete when all the muscles associated with the hooks are in a state of relaxation but are held securely and under tension by the surrounding, stretched, opisthaptoral dome.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.