Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development

The presence of immunocompetent cells was studied in the larval and adult stages of the spotted wolffish, Anarhichas minor. In situ hybridization with a probe complementary to the secretory Igmu-chain was used to localize immunoglobulin producing cells or plasma cells in organs from adult fish and the appearance of these cells in lymphoid tissues during juvenile development. Plasma cells were located in pronephros, spleen, gut, gills and skin of adult wolffish. In juveniles, the first plasma cells were detected in the kidney 1 week post-hatching and the appearance in other lymphoid organs was in the order spleen, gut and thymus. No plasma cells were detected in skin and gills during the sampling period of juveniles (<10 cm). Our study confirmed that plasma cells are present in both the systemic and mucosal compartments of adult fish but during ontogeny there is an earlier appearance of plasma cells in the gut compared to gill and skin compartments.     

Ontogenesis of oxygen chemoreception in aquatic vertebrates

In aquatic vertebrates, peripheral O(2) chemoreceptors initiate compensatory physiological and behavioural responses to hypoxia, beginning at very early stages of development, to maintain sufficient gas exchange across the skin or gills. This review highlights the morphological and physiological studies, particularly those of zebrafish, that have contributed to the current understanding of the development of O(2) chemoreception and the response to hypoxic challenges in embryonic and larval stages of fish and amphibians. The gills appear to be the primary site of O(2) chemoreception in developing aquatic vertebrates and initiate ventilatory changes, and adult-like O(2)-sensitive neuroepithelial cells (NECs) are found in the gills in larval stages of zebrafish and Xenopus laevis. However, evidence from zebrafish studies indicates that extrabranchial O(2) chemoreceptors appear before gill NECs and regulate responses to hypoxia that develop earlier. The developmental and evolutionary significance of the internal migration of O(2)-chemoreceptive sites with changes in respiratory organs is also discussed.     

Location and morphology of chloride cells during the post-embryonic development of the european sea bass,Dicentrarchus labrax

Location and morphology of chloride cells were studied in the sea bass ( Dicentrarchus labrax) from hatching to the juvenile stage to determine the development of the adult osmoregulatory function as seen in adult fish. During the studied developmental sequence changes were observed in the location, number, size and structure of these cells, that were studied by microscopy (light, scanning electron, transmission electron and confocal) and immunocytochemistry. Chloride cells were found on the tegument and on the gills. They were present on the tegument already at hatching, before the development of the gills. Their density as well as their association in multicellular complexes decreased during the postembryonic development. In old larvae and in juveniles, cutaneous chloride cells were associated with the fins, the developing scales and the lateral line. Gills developed gradually during the prelarval stage and the gill arches were present at mouth opening. At that time chloride cells were already numerous on the gill arches. In older larvae, during the progressive development of the gill filaments, chloride cells were numerous on these structures and formed multicellular complexes. Several stages in the differentiation of these cells were studied, including the development of the tubulovesicular system at the end of the prelarval stage, as well as the stratification appearance of the cytoplasm that was concomitant with the considerable development of the tubular system and its association with the endoplasmic reticulum during the larval period. The involvement of different epithelia in the osmoregulatory process during the postembryonic development of this species, as well as the role of chloride cells during successive developmental stages, is discussed.     

Implications for Osmorespiratory Compromise by Anatomical Remodeling in the Gills of Arapaima gigas

The gill structure of the Amazonian fish Arapaima gigas, an obligatory air breather, was investigated during its transition from water breathing to the obligatory air breathing modes of respiration. The gill structure of A. gigas larvae is similar to that of most teleost fish; however, the morphology of the gills changes as the fish grow. The main morphological changes in the gill structure of a growing fish include the following: (1) intense cell proliferation in the filaments and lamellae, resulting in increasing epithelial thickness and decreasing interlamellar distance; (2) pillar cell system atrophy, which reduces the blood circulation through the lamellae; (3) the generation of long cytoplasmic processes from the epithelial cells into the intercellular space, resulting in continuous and sinuous paracellular channels between the epithelial cells of the filament and lamella that may be involved in gas, ion, and nutrient transport to epithelial cells; and (4) intense mitochondria‐rich cell (MRC) proliferation in the lamellar epithelium. All of these morphological changes in the gills contribute to a low increase of the respiratory surface area for gas exchange and an increase in the water–blood diffusion distance increasing their dependence on air‐breathing as fish developed. The increased proliferation of MRCs may contribute to increased ion uptake, which favors the regulation of ion content and pH equilibrium. Anat Rec, 296:1664–1675, 2013. © 2013 Wiley Periodicals, Inc.     

Chronology of the appearance of β, A,and α mitochondria‐rich cells in the gill epithelium during ontogenesis of the brown trout (Salmo trutta)

Three types of mitochondria‐rich (MR) cells, the α, β, and accessory cells, are observed in the gill epithelium of juvenile and adult freshwater teleosts. In addition to numerous mitochondria, their cytoplasm contains a network of membranous tubules, the tubular system, connected to the laterobasal plasma membrane. Because they are believed to play a role in ionic regulation, it is of interest to examine the order of appearance and the ultrastructural characteristics of such cells during the embryogenesis and larval life of the brown trout. Gills of embryos and fry maintained in freshwater were thus removed at different stages and prepared for transmission and scanning electron microscopic examination. One week before hatching, cells resembling the β cells of juvenile and adult teleosts appeared first among the epithelial cells located at the base of the filaments in the gills of the brown trout larva. In addition to their tubular system, they contained numerous and large apical structures seemingly originating from the Golgi apparatus. At approximately hatching time, small pear‐shaped cells were seen to be closely apposed to the lateral side of the β cells; they were usually devoid of apical structures and were considered to be accessory cells. After yolk sac resorption, additional cells, the α cells, were present along the lamellae. In contrast to the β cells, they only exhibited poorly developed apical structures. The possible role of these three types of MR cells in osmoregulation during fish development is discussed. Anat Rec 259:301–311, 2000. © 2000 Wiley‐Liss, Inc.     

The epithelium of the tongue of Ambystoma mexicanum: Ultrastructural and histochemical aspects

The distribution pattern of taste buds and goblet cells and histochemical and ultrastructural aspects of the tongue epithelium of Ambystoma mexicanum are here described. This study is also concerned with the developmental stages and origins of the epithelial cells. Pavement cells and goblet cells of the stratum superficiale are replaced by basal cells of the stratum germinativum in larvae and neotenous adults. The pavement cells of the larvae are characterized by a marginal layer of mucin grana. Decompaction of the mucins occurs immediately before extrusion in the adult. The larval goblet cell type (type I), which is also present in the adult, contains unfused grana of irregular shape. At the tip of the tongue, a further type (type II) of goblet cells is found. In the type II cells the intracellular secretory grana fuse to a single homogeneous mass. Leydig cells of the tongue epithelium are discerned by light microscopy first in the semi-adult, apparently correlated with partial metamorphosis. In the course of ontogenesis and induced metamorphosis the secretion changes to neutral glycoconjugates. The mucins of the pavement cells change first followed by those of the goblet cells. The glands of the secondary tongue show a dorso-ventral pattern of varying secretory qualities. Taste buds are found at the anterior margin of the tongue and along the base of the gill clasps in the early larva. They are already distributed all over the tongue at the end of the early larval phase.     

Late development and fertility of adult worms derived from gamma-irradiated first-stage larvae of Angiostrongylus cantonensis

Effects of gamma ray irradiation on the first-stage larvae of Angiostrongylus cantonensis were studied. Compared to the non-treated controls, infection of rats with third-stage larvae which developed from irradiated first-stage larvae resulted in reduced recovery rates of adults. There was also a change in the male-female adult worm ratio and a reduction in larval output per female in relation to increasing irradiation dosage. Morphological changes in the adults were noted.     

The life cycle of <Emphasis Type="Italic">Stephanoprora aylacostoma</Emphasis> n.sp. (Digenea: Echinostomatidae), parasite of the threatened snail <Emphasis Type="Italic">Aylacostoma chloroticum</Emphasis> (Prosobranchia,Thiaridae), in Argentina

A new species, Stephanoprora aylacostoma is described and its life cycle was resolved experimentally. The prosobranch snail Aylacostoma chloroticum Hylton Scott, collected in the Yacyretá dam, Province of Misiones, Argentina, was found naturally infected with large-tailed cercariae possessing a prepharyngeal body and corpuscles in the excretory system but lacking collar spines. Metacercariae, which encysted on the gills of experimentally infected fishes Cnesterodon decemmaculatus (Jenyns) and Poecilia reticulata (Peters) (Poecilidae), developed collar spines after 10 days. Tetragonopterid fishes Moenckhausia dichroura (Kner), Astyanax erythropterus (Holmberg) and Hyphesobrycon serpae (Durbin in Eigenmann) were found infected naturally. Sexually mature adults were recovered from domestic chicks at day 7 post-exposure. Eggs shed in chick faeces developed to miracidia within 13–15 days; sporocysts were found on the gills of snails. The new species differs from other species of the genus in its larger eggs, in the smaller, slender body and smaller collar spines of the adult and in the morphological and biological features of the larval stages.     

Immunization with Trichinella spiralis Korean Isolate Larval Excretory–Secretory Antigen Induces Protection and Lymphocyte Subset Changes in Rats

Protection and immune responses were studied in rats immunized with Trichinella spiralis muscle stage larval excretory–secretory antigen (ES Ag) without adjuvant. Protection was assessed by the degree of adult worm burden and the yield of muscle (diaphragmatic) larvae after challenge infection with live larvae. Lymphocyte subsets were identified by flow cytometry in the spleen and peripheral blood. Cytokine production and specific IgG, IgG1 and IgG2a antibody responses were measured. Immunization with ES Ag produced highly significant protection against adult stages (98.4%) and muscle larvae (82.9%). Th2 type cytokines (IL‐10, IL‐4) were predominant. Anti‐muscle stage larval ES Ag antibody was significantly elevated in the order IgG2a > IgG1 > IgG on the 2^nd day after final immunization and on the 7^th day after challenge infection. Expression of CD4⁺ and the CD4⁺/CD8⁺ ratio from spleen and blood were significantly increased compared to the control. These results demonstrate that immunization with T. spiralis antigen can elicit robust immune response, resulting in complete protection against infective larvae, and this protection can be achieved without the use of any adjuvant.     

Development of Embryonic Gill Vasculature in the Yellow Stingray,Urobatis Jamaicensis

Corrosion casting was utilized to examine the development of gill vasculature in embryonic yellow stingrays, Urobatis jamaicensis (formerly Urolophus jamaicensis). The most marked changes in vascular configuration of the gills occur in the earliest castable stages of gestation. These changes included development of afferent external gill filament vessels and progression from paired dorsal aortae to a single fused dorsal aorta. Internal gill vasculature was found to nearly match that of an adult by the time the external gill filaments had fully regressed and yolk sac had been exhausted (>47 mm disc width). Examination of embryo casts also revealed characteristics of the branchial vasculature not previously reported in adult specimens. These include the presence of pre‐lamellar sphincters, intertrematic branches, afferent distributing arteries, which supply blood to many afferent filament arteries resulting in greater interconnection of the filaments, and observation that the afferent branchial artery in the first hemibranch supplies blood directly to afferent filament arteries on the dorsal half of this arch. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Toxocara canis in experimentally infected silver and arctic foxes

In two experiments, thirty-six farm foxes of two species were inoculated with various doses of infective Toxocara canis eggs or tissue larvae isolated from mice. In experiment I, six adult arctic foxes (Alopex lagopus; 11-month old) were each inoculated with 20,000 eggs and sacrificed 100, 220, or 300 days post infection (dpi), while ten silver fox cubs (Vulpes vulpes; 6–9-week old) were infected with varying doses of eggs (30–3000) and necropsied 120 dpi. In experiment II, two groups of five cubs and two groups of five adult silver foxes received both a primary inoculation and either one or two challenge inoculations: primary inoculation (day 0) with 400 embryonated eggs were administered to five cubs and five adults and another five cubs and five adults received 400 larvae. At 50 dpi, the first challenge inoculation (400 eggs) was inoculated in all animals. At 100 dpi, three animals from each group were necropsied. The remaining two animals in each group were received a second challenge inoculation of 400 tissue larvae on 100 dpi and were subsequently necropsied at 150 dpi. In both experiments, the highest numbers of larvae per gram (lpg) of tissue was found in the kidneys (100–300 dpi). In adult foxes receiving a high dose (20,000 eggs), increasing larval burdens were found in the kidneys over the course of the experiment (up to 300 dpi). The larval migration from the lungs to other tissues appeared to be dose-dependent with the highest larval burdens found in adult foxes. The faecal egg excretion, larval burden and intestinal worm burdens decreased from the first to the second challenge infection.     

In vitro induction of lymphocyte responsiveness by aStrongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected withStrongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults ofS. vulgaris. In addition,S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by theS. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary forS. vulgaris mitogen-induced transformation of spleen cells. TheS. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells andS. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae ofS. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that theS. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the -globulinaemia, of which IgG(T) is a major component, inS. vulgaris infected horses.     

The experimental receptivity ofHelicella (Helicella) itala andCepaea nemoralis (Mollusca,Helicidae) to larvae ofMuellerius sp. andNeostrongylus linearis (Nematoda,Protostrongylidae) from chamois (Rupicapra rupicapra)

Two batches ofHelicella (H.) itala (adult specimens) and two ofCepaea nemoralis (adult and young specimens) were experimentally infected with larvae I (L-I) ofMuellerius sp. andNeostrongylus linearis obtained from the lungs and faeces ofRupicapra rupicapra. In assess larval development, the number and percentage of the total number of larvae (L-I+L-II+L-III) per mollusc were studied, together with the number and percentage of L-III per snail and the days on which the different larval stages were reached. The development ofMuellerius sp. andN. linearis was greater in larvae from faeces. For both species of molluscs, the values for the percentages of the total number of larvae and L-III were higher inN. linearis than inMuellerius sp., but there were no notable differences in the days on which the various larval stages were reached. Both nematodes achieved a greater degree of development in young specimens ofC. nemoralis than in adults. Whether the larvae came from facces or the lungs,H. (H.) itala was a better intermediate host thanC. nemoralis forMuellerius sp. andN. linearis.     

Intra-tissue localization of an antibacterial l-amino acid oxidase in the rockfish Sebastes schlegeli

The rockfish Sebastes schlegeli skin mucus contains a potent antibacterial protein, SSAP (S. schlegeli antibacterial protein), a novel l-amino acid oxidase with strict substrate specificity that acts against water-borne Gram-negative bacteria. We previously demonstrated that SSAP distributes in the skin and gills. Here we investigated the intra-tissue localization of SSAP in the tissues by in situ hybridization. Skin and gill sections were hybridized with digoxigenin-conjugated SSAP-specific RNA probe. SSAP mRNA-positive cells located near the basal membrane of skin epidermis and the gill epithelium. Furthermore, skin section was analyzed by immunohistochemistry and reacted with anti-SSAP antiserum as a primary antibody. The mucus layer and mucous cells in the skin were immunopositive. Skin and gill extracts produced hydrogen peroxide, responsible for antibacterial activity, in the presence of l-lysine. These results suggested that SSAP functions locally as a humoral defense factor in S. schlegeli skin and gills and prevents pathogenic bacterial invasion.     

Transmammary transmission ofStrongyloides ratti

The rate of transmammary transmission ofStrongyloides ratti was examined in albino rats in terms of the route of subcutaneous (s.c.) migration from the infection site (the skin) to the cranium. Inoculation sites nearer the cranium resulted in less frequent transmammary infection. The maximum number of adult worms was recovered from the sucklings when the mother was inoculated in her hindquarter and sucklings were allowed to feed for 30–36 h after inoculation (AI). Few worms were recovered from sucklings when they were allowed to nurse during periods of<24 h AI or>42 h AI. In lactating mothers, larval infection of the mammary glands was commonly observed, and these larvae showed an increased esophagus length. In nonlactating mothers, most larvae completed their migration to the cranium within 36 h AI.     

Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys,Petromyzon marinus L.

Immunocytochemistry with protein A-gold and routine electron microscopy were used to identify cell types within the endocrine pancreas of larvae, juvenile adults, and upstream-migrant adults of the sea lamprey, Petromyzon marinus. The larval pancreatic islets are composed only of insulin-immunoreactive B-cells, which are uniform in their fine structure. The cranial and caudal pancreatic tissue in both adult periods contains three cell types: B-cells, somatostatin-immunoreactive D-cells, and a third cell type of unknown content. No glucagon-immunoreactive cells are present in lampreys, but B-and D-cells exist in equal numbers in the pancreatic tissue of adults. The B-cells of adults have a fine structure similar to those in larvae. D-cells have secretory granules that are distinctly different from those both in B-cells and in the third cell type. Although B- and D-cells in lamprey pancreatic tissues have a basic morphological similarity to these cells in other vertebrates, their granules are generally of smaller dimensions. The inclusion of granules within large pleomorphic bodies in many D-cells indicates that granule turnover is common. Immunocytochemistry will be a useful tool for showing the relationship between the cells in the degenerating bile ducts and those of the developing adult pancreas.     

Micronucleus test in fish cells: a bioassay for in situ monitoring of genotoxic pollution in the marine environment

To evaluate the use of native fish species for assessing genotoxic pollution in the marine environment, micronucleus (MN) analysis was performed in peripheral blood erythrocytes and gill cells of the grey mullet (Mugil cephalus) from three sampling stations off the southeastern Mediterranean coast of Turkey. The frequencies of blebbed, notched, and lobed nuclei and binucleated cells also were evaluated in peripheral erythrocytes. The sampling sites were chosen on the basis of pollution levels; Karaduvar harbor, contaminated by different types of industrial effluents, and Mersin harbor, mainly contaminated by aromatic hydrocarbons, were selected as polluted areas. Erdemli harbor, a relatively unpolluted site, was used as the control area. Sampling was carried out at four different seasons. The frequencies of both micronuclei and other nuclear abnormalities (NAs) in mullets captured from polluted areas were significantly higher than those in mullets from the reference area. In general, gill cells had considerably higher MN frequencies than did erythrocytes, and genotoxic responses were higher in summer than in winter. The results of this study indicate that the MN test in fish is a suitable biomarker for in situ monitoring of genotoxic pollution in the marine environment. As demonstrated in this study, NAs other than micronuclei are also useful indices of chemical exposure and toxic responses. Therefore, measuring both micronuclei and NAs may increase the sensitivity of the test system.     

Lens regenerates by means of similar processes and timeline in adults and larvae of the newt Cynops pyrrhogaster

Background: It is widely accepted that juvenile animals can regenerate faster than adults. For example, in the case of lens regeneration of the newt Cynops pyrrhogaster, larvae and adults require approximately 30 and 80 days for completion of lens regeneration, respectively. However, when we carefully observed lens regeneration in C. pyrrhogaster at the cellular level using molecular markers in the present study, we found that lens regeneration during the larval stage proceeded at similar speed and by means of similar steps to those in adults. Results: We could not find any drastic difference between regeneration at these two stages, except that the size of the eyes was very different. Conclusions: Our observations suggested that larvae could regenerate a lens of the original size within a shorter time than adults because the larval lens was smaller than the adult lens, but the speed of regeneration was not faster in larvae. In addition, by repeatedly observing the regeneration in one individual transgenic newt that expressed fluorescence specifically in lens fiber cells in vivo and comparing the regeneration process at the embryonic, larval, and postmetamorphosis stages, we confirmed that the regeneration speed was the same at each of these stages in the same individual. Developmental Dynamics 241:1575–1583, 2012. © 2012 Wiley Periodicals, Inc.     

Effects of solvents and surfactant agents on the female and larvae of cattle tick <Emphasis Type="Italic">Boophilus microplus</Emphasis>

Many natural compounds have low water solubility and need to be dissolved in organic solvents, or surfactant agents must be used before addition into experimental systems. Therefore, it is necessary to determine their toxicity. Experiments were performed aiming to select solvents to be used in the bioassays, searching new acaricide agents from plants. Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the sensibility of B. microplus female and larvae to different solvents (acetone, methanol, ethanol and 1% dimethyl sulfoxide) and surfactant agents (1% Tween 80 and 5% Triton X-100) using the larval immersion test (LIT) and adult immersion test (AIT). In the AIT, the effect of the treatments on engorged females was assessed by measuring egg production and hatching rate. Acetone was toxic to the adults promoting mortality of 100%. Methanol and ethanol caused 45.3 and 14.2% of mortality, respectively. The other tested substances were not toxic to the engorged females of B. microplus. In the LIT, it was observed that the larvae were more resistant; after 48 h, about 100% of the larvae were alive in all the treatments except with acetone that caused a mortality of 10%.