Heterochrony and the development of the escape response: prehatching movements in the rainbow trout Oncorhynchus mykiss

Teleost fishes produce coordinated escape responses (C-starts) at hatching. This implies that essential swimming morphologies and motor behaviors develop during the incubation interval while the embryo is in the chorion. We examined prehatching motor behaviors in rainbow trout Oncorhycus mykiss (considered morphologically mature at hatching) and compared this species with zebrafish Danio rerio (considered morphologically immature) and assessed two hypotheses concerning the development of escape behavior. (1) Escape behavior is associated with the formation of key elements of the musculoskeletal and nervous systems; thus, the escape response appears early in ontogeny, when these elements form. (2) Escape behavior is not directly associated with the formation of underlying morphological elements; instead, it appears at hatching (i.e. when needed). We find that rainbow trout, like zebrafish, respond to touch early in the incubation interval, but do not demonstrate a complete C-start (including the second, propulsive stage) until shortly before hatching. At hatching, rainbow trout and zebrafish are similar in the degree of development of the chondocranium, paired fins and visceral arches (which comprise the larval jaw and gill support); however, rainbow trout have incipient rays in their unpaired fins (dorsal, anal and caudal), whereas zebrafish retain the embryonic fin fold. Although rainbow trout are more mature in axial swimming morphology at hatching, the essential neural and musculoskeletal systems that produce a coordinated escape response are functional at hatching in both species. This finding supports the evolutionary hypothesis that an effective escape response is critical for the survival of newly hatched teleost fishes.     

Immunocytochemistry of somatotrophs,gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries

Summary The chronological appearance of endocrine cells in the pituitary of sea-bream (Sparus auratus) larvae was studied using antisera against salmon prolactin, trout growth hormone, salmon gonadotropin and N-terminal human adrenocorticotropin. The larval pituitary (1–12 days after hatching) was oval in shape and was composed of a dense mass of cells with few neurohypophysial fibres. By 60 days after hatching it began to resemble the adult and was divisible into a distinct rostral pars distalis containing prolactin and adrenocorticotropin cells; a proximal pars distalis containing somatotrophs and gonadotrophs and a pars intermedia. Cells immunoreactive with antisera against growth hormone were observed immediately after hatching (2 days post-fertilization). Weakly staining prolactin cells were observed 2 days later in the region corresponding to the rostral pars distalis. Cells immunoreactive with anti-gonadotropin and anti-adrenocorticotropin sera were observed in the pituitary 6 and 8 days after hatching, respectively. All the cell-types studied were immunoreactive from the time they were first identified until the final samples 90 days after hatching.     

Allometric relations of total volumes of prolactin cells and corticotropic cells to body length in the annual cyprinodont Cynolebias whitei: effects of environmental salinity,stress and ageing

Summary An analysis of the allometric relations of the total volumes occupied by prolactin (PRL) and corticotropic (ACTH) cells (PRL volume and ACTH volume, respectively) to body length and a study of the immunocytochemical staining intensity of PRL and ACTH cells were used to determine the differences in activity of PRL and ACTH cells in freshwater-reared and in saltwater-reared Cynolebias whitei during the entire lifespan of this annual cyprinodont fish. An inflection in the allometric relation of PRL volume to body length was observed in fish of one-week old. The relatively large PRL volume in younger fish may be related to PRL cell activity before hatching. No inflections were observed in the allometric relations of PRL volume and ACTH volume to body length at the onset of maturation and the onset of ageing, indicating that the increased pituitary growth in maturing and ageing C. whitei is not the result of changes in PRL or ACTH cells. The slope of the allometric relation of PRL volume to body length in freshwater-reared fish was significantly steeper than the slope in saltwater-reared fish. The PRL volume in adult freshwater-reared fish was eight times larger than that in saltwater-reared fish of the same length. The intensity of immunocytochemical staining of saltwater PRL cells was significantly reduced. These volumetric and staining differences correspond to the low functional demand put upon PRL cells in saltwater-adapted fish. In contrast, the slope of the allometric relation of ACTH volume to body length and the intensity of immunocytochemical staining of ACTH cells were similar in freshwater-reared and in saltwater-reared fish. It is concluded that the functional demand put upon ACTH cells is similar in freshwater-reared and saltwater-reared C. whitei; the involvement of ACTH cells in the osmoregulation of the fish in both environments is similar.     

Absence of developmental incompatibility in hybrids between rainbow trout and two subspecies of cutthroat trout

We examined the developmental rate of hybrids between rainbow trout (Salmo gairdneri) and two subspecies of cutthroat trout: westslope cutthroat trout (Salmo clarki lewisi) and Yellowstone cutthroat trout (Salmo clarki bouvieri). These taxa show considerable genetic divergence at 42 structural loci encoding enzymes; the mean Nei's d between the rainbow trout and the two species of cutthroat trout is 0.22. We used four measures of developmental rate: time of hatching and yolk resorption, rate of increase in activity of four enzymes, and time of initial detection of seven isozyme loci. The two cutthroat trout subspecies reached hatching and yolk resorption earlier than rainbow trout. Cutthroat trout had higher relative enzyme activities than rainbow trout from deposition of eye pigment to hatching. There was no difference in the rate of increase in enzyme activity or time of initial expression of these loci between these species. Hybrids showed developmental rates intermediate or similar to that of the parental species using all measures. Our results indicate an absence of regulatory and developmental incompatibility between these taxa.This research was supported by NSF Grants ISP-8011449 and BSR-8300039. M.M.F. was supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada.     

Histological observations on the pituitary gland and the glomerular body of the larvae of the marine teleost,Hexagrammos otakii

Summary In the larvae of the marine teleost, Hexagrammos otakii, the prolactin, ACTH, STH and pars-intermedia cells as well as the neurohypophysis and glomerular body showed no histological change at the time of hatching. These cells or organs do not seem to be specifically involved in the mechanisms of adjustment to the hypertonic osmotic stress at this time. The size of the nuclei of the prolactin cells increased in fresh-water (F. W.) and that of the ACTH cells decreased in 2/3, 1/2 and 1/3 sea-water (S.W.). The amount of chrome-alum haematoxylin-positive material in the neurohypophysis of fish immersed in 1/2 S.W., 1/3 S.W. and F.W. was less than that of the control fish. There was a difference in the glomerular ratio between the fish kept in F. W. and control fish. And the red blood cells in the glomerulus of the larvae kept in 1/2 and 1/3 S.W. apparently increased in number in comparison with the control fish.The author wishes to express his gratitude to Professor Hideshi Kobayashi for his valuable guidance during the course of this study. The author is indebted to Dr. Masaaki Inoue, Kanagawa Prefectural Fisheries Experimental Station, for supplying the eggs of Hexagrammos.     

Gene expression and intracellular localization of somatolactin in the pituitary of rainbow trout

The gene expression and intracellular localization of somatolactin (SL), a putative pituitary hormone structurally related to both growth hormone and prolactin, were investigated in the pituitary of rainbow trout, Oncorhynchus mykiss. Using an in situ hybridization technique, we demonstrated the gene expression of the SL molecule in cells bordering the neurohypophysial tissue in the pars intermedia. These cells were identified immunocytochemically as SL-cells on the adjacent section. Electron-microscopic immunocytochemistry by means of the protein A-gold technique, also revealed that the SL-immunoreactivity was located mostly on the secretory granules in SL-cells. Our findings clearly indicate that SL is biosynthesized and stored in the granules in these cells.     

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.     

The influence of social rank on adenohypophysial cell activity in Salmo irideus

Summary In nine cell types of the adenohypophysis in untreated adult rainbow trout, histologically different activity phases, seasonal changes in activity, and the relation between certain cell types and the interrenal gland, thyroid or gonads were investigated by light and, occasionally, by electron microscopy. Special attention was given to the effect of social rank on the synthetic activity in adenohypophysial cells of trout kept in small groups in which a social hierarchy with one (light) dominant and several (dark) submissives is established.Cell types in the rostral pars distalis were azocarminophil (I) or amphiphil (II). Proximal pars distalis cell types were slightly basophil (IV), orangeophil (V), strongly basophil (VI) or chromophobe (VII). In the pars intermedia, cell types were amphiphil (VIII) or very slightly basophil (IX). Type III was a non-secretory supporting (?) cell.Histologically different activity phases abounded in type IV cells, which mainly occurred in the proximal pars distalis but were also found dispersed in the rostral pars distalis, the pars intermedia and the neurohypophysis.Influences of social rank were pronounced in type IV cells. Phases with a high synthetic activity were exclusively found in submissive animals, phases with a low synthetic activity occurred in dominants. As a positive relation existed between type IV cell activity and the social rank dependent activity of the interrenal gland, it was suggested that type IV cells produce ACTH.In (dominant) male trout treated with DOCG or ACTH, colloid-containing type IV cell phases, reflecting accumulation of the secretory product, were found. This supported the earlier suggestion that ACTH in the trout is produced in the basophil type IV cells and not, as reported in the literature, in cells comparable to type II.The author is greatly indebted to Miss M.C. Wentzel, Miss I. Stulen, Mr. J. Veening and Dr. J.G. van Rhijn for their help with histological techniques, interrenal cell measurements and statistical aspects     

Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study

The ontogenetic development of IgM-containing cells is described as demonstrated by immunoperoxidase staining with a mouse anti-trout IgM monoclonal antibody and the differentiation of enzyme-histochemical markers in the non-lymphoid cells forming the stroma of the thymus, spleen and kidney of the rainbow trout. The first lymphoid cells staining with the monoclonal antibody occurred at day 4-5 after hatching in the renal lympho-haemopoietic tissue. By 1 month after hatching IgM-positive cells also appeared in the spleen and thymus. Enzyme-histochemical demonstration of the alkaline and acid phosphatase and non-specific σ-naphthyl acetate esterase enzymatic activities in the non-lymphoid cells indicated that a certain degree of maturation of the cellular stroma of the developing lymphoid organs of trout was reached before or at the time when IgM-expressing cells could be observed. The relationships of the stromal components of the various lymphoid organs to the development of IgM-positive cells, and the possible role of the renal lympho-haemopoietic tissue as a primary lymphoid organ for B-cell differentiation in the trout are discussed.     

Immunocytochemical devidence for hypophysial projections of the cells of the nucleus lateralis tuberis pars lateralis in the rainbow trout (Salmo gairdneri)

In the rainbow trout the pars lateralis is the most prominent part of the nucleus lateralis tuberis (NLT). To demonstrate a morphological relationship between this lateral part of the NLT and the pituitary, immunocytochemistry was applied as a staining method. Experiments were carried out on glutaraldehyde-picric acid-acetic acid-fixed brain sections of mature male and female rainbow trout using the peroxidase-anti-peroxidase immune technique with an antiserum against 27-S-methylglucagon as the first antibody. Most of the cells in the NLT/pars lateralis reacted with the antiserum. Axons from these cells enter the pituitary, extending exclusively in the numerous neurohypophysial digitations in the pars intermedia. No immunoreactive neurohypophysial protrusions were found in those parts of the adenohypophysis where the gonadotropic cells are located, indicating that the lateral part of the NLT is not directly involved in the control of gonadotropin secretion. In addition to cells of the NLT/pars lateralis only prolactin cells in the rostral pars distalis of the adenohypophysis reacted with the antiserum used.     

The occurrence and distribution of peptide- or 5-HT-containing nerves in the swimbladder of four different species of teleosts (Gadus morhua,Ctenolabrus rupestris,Anguilla anguilla,Salmo gairdneri)

Summary The innervation of the swimbladder in four different teleost species has been studied by the use of immunohistochemical methods. The teleosts examined belong to two different groups regarding their swimbladder morphology: physoclists (the cod, Gadus morhua and the goldsinny wrasse, Ctenolabrus rupestris) and physostomes (the eel, Anguilla anguilla and the rainbow trout, Salmo gairdneri). Vasoactive intestinal polypeptide-like immunoreactivity was demonstrated in nerves of the swimbladder walls of all four species, and in the gas glands of the cod and the goldsinny wrasse. Substance P-like immunoreactivity was shown in swimbladders of the cod, eel and rainbow trout but not the goldsinny wrasse. Immunoreactivity to met-enkephalin antiserum was revealed in the swimbladder walls of the eel and the goldsinny wrasse, while neurotensin-like immunoreactivity was present in the goldsinny wrasse and rainbow trout swimbladders. Neurotensin-like immunoreactivity was also seen in the gas gland of the goldsinny wrasse. 5-Hydroxytryptamine immunoreactivity was found in endocrine cells in the pneumatic duct of the eel and in the swimbladder walls of the goldsinny wrasse and the rainbow trout. In conclusion, all teleosts examined showed a very close resemblance in the peptidergic/tryptaminergic innervation of the swimbladder to that of the gut, inasmuch as the immunoreactivity present in the swimbladders always occurred in the gut of the same species.     

Effect of the prolactin-inhibiting substances bromocripton and lergotrile on hydromineral regulation in rainbow troutSalmo gairdneri

Summary Plasma Na⁺, K⁺ and osmotic pressure were measured in rainbow trout (Salmo gairdneri) following the administration of the prolactin-inhibiting substances Lergotrile and Bromocripton. Both drugs elicited a significant fall in plasma Na⁺ concentrations although a significant response to Bromocripton was apparent only in trout acclimated to distilled water.The changes in plasma Na⁺ levels and in some cases of plasma osmotic pressure following administration of the prolactin-inhibiting substances were consistent with the hypothesis that prolactin acts to maintain plasma Na⁺ levels in this salmonid species in the same manner as in other teleosts. However, since the changes in plasma ions were small (albeit significant) is is proposed that prolactin may play a less important role in osmotic and/or ionic regulation in this species than it does in other teleostean species. Conversely, it may be that the drugs effects a less complete blockage of prolactin secretion than they appear to do in mammals.Ovine prolactin, administered with the drugs, effected a partial retention of plasma Na⁺ in Lergotrile-injected fish but did not significantly modify the effect of Bromocripton. These findings are discussed in light of the proposed action of the drugs, namely that of inhibiting the release of endogenous prolactin.Both Bromocripton and Lergotrile caused a significant fall in hematocrit values. Since plasma osmotic pressure values and plasma K⁺ concentrations were not markedly affected by the drugs (except for a significant (P<0.01) reduction in plasma osmotic pressure in the Bromocriptons-injected groups maintained in distilled water) it was thought that these changes were due to a reduction in the number of blood cells in the peripheral circulation rather than to an influx of water in response to the inhibition of prolactin.     

Influence of hydrologic attributes on brown trout recruitment in low-latitude range margins

Factors controlling brown trout Salmo trutta recruitment in Mediterranean areas are largely unknown, despite the relevance this may have for fisheries management. The effect of hydrological variability on survival of young brown trout was studied during seven consecutive years in five resident populations from the southern range of the species distribution. Recruit density at the end of summer varied markedly among year-classes and rivers during the study period. Previous work showed that egg density the previous fall did not account for more than 50% of the observed variation in recruitment density. Thus, we expected that climatic patterns, as determinants of discharge and water temperature, would play a role in the control of young trout abundance. We tested this by analyzing the effects of flow variation and predictability on young trout survival during the spawning to emergence and the summer drought periods. Both hatching and emergence times and length of hatching and emergence periods were similar between years within each river but varied considerably among populations, due to differences in water temperature. Interannual variation in flow attributes during spawning to emergence and summer drought affected juvenile survival in all populations, once the effect of endogenous factors was removed. Survival rate was significantly related to the timing, magnitude and duration of extreme water conditions, and to the rate of change in discharge during hatching and emergence times in most rivers. The magnitude and duration of low flows during summer drought appeared to be a critical factor for survival of young trout. Our findings suggest that density-independent factors, i.e., hydrological variability, play a central role in the population dynamics of brown trout in populations from low-latitude range margins. Reported effects of hydrologic attributes on trout survival are likely to be increasingly important if, as predicted, climate change leads to greater extremes and variability of flow regimes.     

Modulation of fish phagocytic cells by N-terminal peptides of proopiomelanocortin (NPP)

N-terminal peptide of proopiomelanocortin (NPP, or pro-gamma-MSH) has shown to exhibit biological activity such as stimulation of adrenal mitogenesis and prolactin release-inhibiting factor activity. Structurally, studies reveal a significant difference between fish NPP from that of tetrapods, as NPPs from carp and salmonid lack gamma-MSH. Thus, fish NPP may exhibit functions different from that of mammals. The activation of phagocytic cells by NPP was analysed using rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio. Rainbow trout and carp macrophages incubated with chum salmon NPP significantly enhanced the production of superoxide anion in comparison with control macrophages (without hormones). Both rainbow trout and carp macrophages had shown increased phagocytosis when stimulated administered with NPP. The above results were complemented by in vivo studies where NPP was administered to rainbow trout and carp. NPP significantly increased superoxide anion production as well as phagocytosis in macrophages. These results show that NPP in lower vertebrates activates the function of the phagocytic cells.     

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum     

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.     

Cytochemical demonstration of acid phosphatase activity in the pineal organ of the rainbow trout,Salmo gairdneri

Summary Activity of acid phosphatase (ACP) was investigated cytochemically in the pineal organ of the rainbow trout, Salmo gairdneri. Intense reaction product for ACP activity was observed (1) in lysosomes varying in size and shape and (2) in endoplasmic reticulum associated with the Golgi complex of (i) the pineal photoreceptor and supporting cells, (ii) vascular endothelial cells, and (iii) macrophages inhabiting pineal lumen, parenchymal epithelium and perivascular spaces. This localization of ACP is discussed with particular reference to the capacity for lysosomal digestion in a pineal organ combining photoreceptive and secretory functions, and lacking a blood-brain barrier, as holds true for the pineal of the rainbow trout. Taking advantage of its capacity for endocytotic uptake and lysosomal digestion, the pineal organ of the rainbow trout may serve as a barrier between the blood circulation and the cerebrospinal-fluid compartment. Furthermore, the macrophages may be considered as an essential component in pineal function of fish.Fellow of the Alexander von Humboldt Foundation.Fellow of the Alexander von Humboldt Foundation.