Development of the neo-morphic air breathing organs in three genera of estuarine gobies

The rudiment of the neo-morphic organ for O2 uptake arises in the form of a gill mass formed by the gill material of the embryonic 5th gill arch. Ectodermal induction to the gill mass takes place in the post-embryonic stage of development to produce a respiratory epithelium of the neo-morphic air breathing organ. The respiratory epithelium of the opercular chamber and the buccal cavity is formed by the cells of the gill mass alone. The respiratory epithelium of the suprabranchial chamber is formed by the cells of the gill mass as well as the gill lamellae derived from the dorsal aspects of the functional gill arches (1 to 4). Extension of the suprabranchial chamber into the buccal region anteriorly is a device to increase the respiratory surface area available for O2 uptake by air. The epithelial position of the blood capillaries in the suprabranchial chamber of Periophthalmodon schlosseri signifies terrestrial nature of the fish.     

Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year

Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.     

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.     

Neurotransmitter receptors on gill muscle fibers and the gill peripheral nerve plexus in Aplysia.

Isolated pinnules of the gill of Aplysia contract when dopamine (DA) is perfused through the bath. The contraction is not blocked by high-Mg2+ seawater, and reflects excitatory receptors for DA on the smooth muscle cells of the gill. The pinnule often shows irregular, spontaneous contractions which are blocked by high-Mg2+ seawater and 30 mM CoCl2. These contractions reflect spontaneous activity of a peripheral nerve plexus. No other transmitter was found to be directly excitatory on the muscle fibers, although there are inhibitory receptors for serotonin (5-HT). Tactile stimulation of the pinnule evoked a two-component contractile reflex contraction due to activation of the peripheral nerve plexus. Acetylcholine, octopamine, and 5-HT but not several other transmitters depressed these responses, presumably due to inhibitory receptors on the neurons of the peripheral plexus.     

Cyclic Adenosine Monophosphate in the Nervous System of Aplysia californica : II. Effect of serotonin and dopamine

Serotonin and dopamine, both likely transmitter substances in Aplysia, stimulated formation of adenosine-3',5' monophosphate (cAMP) in ganglia, connectives, and identified nerve cell bodies. This widespread distribution suggests that receptors for the response are localized throughout the nervous system, as is adenyl cyclase. Both synthesis of cAMP-³H from precursor previously labeled in incubations with adenine-³H and total content of cAMP were stimulated up to 15-fold. The acetylcholine analogue carbachol, glutamate, norepinephrine, and histamine were inactive. Full stimulation occurred within 2–4 min of applying serotonin; the extent of the effect was half maximal at 6µ serotonin. Even in the continued presence of serotonin, the increased cAMP diminished with time. When serotonin was removed, tissue remained refractory for 15–20 min; sensitivity returned after 25 min. Serotonin stimulated cAMP after removal of extracellular Na, K, or Cl and in isotonic sucrose, with all extracellular ions removed. Elevating Mg, which blocked the stimulation of cAMP caused by synaptic activity, did not affect the response to serotonin. Thus the response appeared to be independent of transmitter release and of changes in synaptic potentials and current flow. The role of cAMP in neuronal functioning remains to be determined. Conditions which markedly increased cAMP in neurons, however, did not affect the rate of RNA synthesis, nor did they alter the distribution of phosphorylated adenine or uridine nucleotides.     

Stimulation-depletion of serotonin and noradrenaline from vesicles of sympathetic nerves in the pineal gland of the rat

Summary The pineal gland of the rat receives a rich nervous supply originating from the superior cervical ganglia. These fibers contain serotonin in addition to their neurotransmitter, noradrenaline. Cytochemical studies at the ultrastructural level have shown that both amines are present in the cores of the granular vesicles that are characteristic of these nerves. It is presently shown that the bilateral electrical stimulation of the preganglionic fibers innervating the ganglia markedly reduces the number of small sites reacting cytochemically for both noradrenaline and serotonin, these sites corresponding to the cores of small granular vesicles, while the larger reactive sites (cores of large vesicles) remain unaltered. The vesicles are retained in nerve terminals after stimulation, as observed in conventionally processed tissues, although with altered sizes and shapes. Apart from these cytochemical and structural changes, nerve stimulation also reduces the endogenous noradrenaline content of the pineal gland. Thus, both noradrenaline and serotonin are released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo. This suggests the possibility that several substances with presumed transmitter or modulatory functions might be simultaneously released by nerve impulses from a given nerve terminal.     

Flexibility of Physiological Traits Underlying Inter-Individual Growth Differences in Intertidal and Subtidal Mussels Mytilusgalloprovincialis

Mussel seed (Mytilusgalloprovincialis) gathered from the intertidal and subtidal environments of a Galician embayment (NW, Spain) were maintained in the laboratory during five months to select fast (F) and slow (S) growing mussels. The physiological basis underlying inter-individual growth variations were compared for F and S mussels from both origins. Fast growing seemed to be a consequence of greater energy intake (20% higher clearance and ingestion rate) and higher food absorption rate coupled with low metabolic costs. The enhanced energy absorption (around 65% higher) resulted in 3 times higher Scope for Growth in F mussels (20.5±4.9 J h⁻¹) than S individuals (7.3±1.1 J h⁻¹). The higher clearance rate of F mussels appears to be linked with larger gill filtration surface compared to S mussels. Intertidal mussels showed higher food acquisition and absorption per mg of organic weight (i.e. mass-specific standardization) than subtidal mussels under the optimal feeding conditions of the laboratory. However, the enhanced feeding and digestive rates were not enough to compensate for the initial differences in tissue weight between mussels of similar shell length collected from the intertidal and subtidal environments. At the end of the experiment, subtidal individuals had higher gill efficiency, which probably lead to higher total feeding and absorption rates relative to intertidal individuals.     

Effects of municipal effluents on serotonin and dopamine levels in the freshwater mussel Elliptio complanata

Sex differentiation and gametogenesis represent critical steps in the reproductive process and are subject to hormonal control by serotonin, dopamine and steroids such as estradiol-17beta and testosterone. The purpose of this study sought to examine the endocrine-disrupting activity that a primary-treated municipal effluent might have on the metabolism of biogenic amine levels. First, serotonin receptors transfected in Chinese hamster ovary (CHO) cells were used to screen for the presence of serotonin receptor agonist or antagonist. Second, one group of Elliptio complanata mussels were exposed to single compounds likely to be found in municipal wastewaters and another group was exposed in situ to the municipal effluent plume for 90 days in experimental cages. Results showed that solid phase C-8 extracts of surface water downstream a municipal effluent could activate the transport of serotonin by receptors at a distance of at least 5 km from its outfall thereby indicating the presence of serotonin mimics in the effluent dispersion plume. Levels of serotonin and monoamine oxidase (MAO) activity in nerve ganglia of mussels exposed for 90 days to the municipal effluent were, respectively, reduced and increased at a distance 10-km downstream. Injections of estradiol-17beta and nonylphenol in mussels decreased the levels of serotonin and dopamine, but increased MAO activity in the gonad and nerve ganglia. Exposure to estrogenic chemicals present in municipal effluents may therefore alter the normal metabolism of serotonin and dopamine, both of which are involved in sexual differentiation in bivalves and fish. Chemicals acting through E2 receptor-mediated pathways and serotonin receptors are likely to cause the observed effects.     

The symbiotic water mite Unionicola formosa (Acari: Unionicolidae) ingests mucus and tissue of its molluscan host

Unionicola formosa is a symbiotic water mite that passes most of its life cycle in the mantle cavity of freshwater mussels. Although mites of this genus are often referred to as parasitic, little is known about their nutritional biology. A few species reportedly pierce the gill of a host mussel and ingest tissue or hemolymph. The present study was undertaken to identify possible sources of nutrition for U. formosa. To determine if mites ingested particulate matter in the mucous strand produced by a mussel during feeding, mussels with resident mites were exposed to a suspension of fluorescent microspheres. There was no evidence that U. formosa ingested the beads. Histochemical staining did, however, indicate a mucous material present in the midgut of the mites. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic assays revealed a high molecular weight component, consistent with a mucopolysaccharide, present both in the mussel gill and the mites. Results from western blots and an immunoaffinity binding assay with antibodies against mussel gill tissue and hemolymph also indicated that mites ingested host tissue. Whereas U. formosa probably does not ingest particulate material acquired by its host's suspension feeding, it is apparent that this mite utilizes host mucus, gill tissue, or hemolymph for at least part of its nutrition.     

First demonstration of the neuroepithelial cells and their chemical code in the accessory respiratory organ and the gill of the sharptooth catfish,Clarias gariepinus: A preliminary study

Available studies that have examined O₂ sensing in fish have indicated that oxygen-sensitive neuroepithelial cells (NECs) are O₂ sensors in the gills and initiate cardiorespiratory reflexes in aquatic vertebrates. This is the first study describing the occurrence of NECs in accessory respiratory organs in the air-breathing catfish Clarias gariepinus. Immunocytochemical stainings with specific neuronal markers such as nNOS, VAchT, 5-HT and TH have been shown to be very useful for location and distribution of these cells in the gill fans and suprabranchial chamber that take origin from the transformation of the gill tissue. But the response of these putative O₂ chemoreceptors, their role in the respiratory reflexes and their innervation await investigation.     

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log(10) improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in <5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.     

DEPLETION OF VESICLES FROM FROG NEUROMUSCULAR JUNCTIONS BY PROLONGED TETANIC STIMULATION

Curarized cutaneous pectoris nerve muscle preparations from frogs were subjected to prolonged indirect stimulation at 2/sec while recording from end plate regions. At the ends of the periods of stimulation, the curare was removed and the preparations were fixed for electron microscopy or treated with black widow spider venom to determine the degree to which their stores of transmitter had been depleted. After 6–8 hr of stimulation the nerve terminals were almost completely depleted of their stores of transmitter and of their population of vesicles. Most of the transmitter release occurred during the first 4 hr of stimulation, and after this time most (about 80%) of the fibers were depleted of about 80% of their transmitter. The organization of the nerve terminals in 4-hr preparations appeared normal and the terminals still contained many vesicles. When peroxidase was present in the bathing medium, terminals from stimulated preparations showed many vesicles that contained peroxidase, whereas the rested control preparations showed few such vesicles The fact that after 4 hr the total number of vesicles is not markedly changed while a large fraction (up to 45%) contained peroxidase suggests that in our experiments vesicles were continuously fusing with and reforming from the axolemma.     

Expression of recombinant goldfish glutamic acid decarboxylase 65 and evidence for differential pH and PLP responsiveness compared to the human enzyme

Variability of taurine (2-aminoethane sulfonic acid) was studied as a function of size in the mussel Mytilus galloprovincialis and tissue specificity. Isometric and/or allometric relationships were established with regard to total soft mass of the mussels between 20 and 60 mm shell length. Relative amounts of taurine dropped significantly with increasing mass of whole soft tissues with an allometric coefficient value of -0.15. The inverse relationship between taurine and increasing size of mussels was confirmed for gill epithelium and labial palp (allometric coefficient values of -0.16 and -0.10, respectively), tissues that, in turn, represented isometric functions with regard to total soft mass. Although relative amounts of taurine were significantly different in digestive gland, mantle and foot, relationships with increasing size of mussels remained unchanged in these tissues. Gill area of mussels was related to soft mass with an allometric coefficient of 0.70 by 2D Image Analysis, but increased to 0.85 when introducing a third dimension, i.e. gill thickness. Results are discussed according to gill structure analysis and taurine functionality.     

The effect of environmental salinity on the level of heat shock proteins in gill epithelium of Mytilus edilis L. mussel

The composition and the level of heat shock proteins in the gill epithelium cells of mussels Mytilus edulis L. from the White Sea under different levels of environmental salinity were studied by the method of immunoblotting. In mussels maintained under normal salinity (26%), constitutive Hsp70 and protein of about 40 kDa were revealed. After long-term (11?C14 days) acclimation to 14 and 35?? of the level Hsp70 in gill epithelium cells increased. Hsp70 induction was also observed in cells of isolated gills after salinity shock at 14% for 3 and 24 h.     

ABCB- and ABCC-type transporters confer multixenobiotic resistance and form an environment-tissue barrier in bivalve gills

Aquatic organisms and, in particular, filter feeders, such as mussels, are continuously exposed to toxicants dissolved in the water and, presumably, require adaptations to avoid the detrimental effects from such chemicals. Previous work indicates that activity of ATP-binding cassette (ABC) transporters protects mussels against toxicants, but the nature of these transporters and the structural basis of protection are not known. Here we meld studies on transporter function, gene expression, and localization of transporter protein in mussel gill tissue and show activity and expression of two xenobiotic transporter types in the gills, where they provide an effective structural barrier against chemicals. Activity of ABCB/MDR/P-glycoprotein and ABCC/MRP-type transporters was indicated by sensitivity of efflux of the test substrate calcein-AM to the ABCB inhibitor PSC-833 and the ABCC inhibitor MK-571. This activity profile is supported by our cloning of the complete sequence of two ABC transporter types from RNA in mussel tissue with a high degree of identity to transporters from the ABCB and ABCC subfamilies. Overall identity of the amino acid sequences with corresponding homologs from other organisms was 38-50% (ABCB) and 27-44% (ABCC). C219 antibody staining specific for ABCB revealed that this transporter was restricted to cells in the gill filaments with direct exposure to water flow. Taken together, our data demonstrate that ABC transporters form an active, physiological barrier at the tissue-environment interface in mussel gills, providing protection against environmental xenotoxicants.     

Pathology of acute poisoning with the insecticide Sevin in the bent-nosed clam, Macoma nasuta

Toxicity tests of 96-hr duration of the insecticide Sevin were done with adult bent-nosed clams, Macoma nasuta. Sevin concentrations of 15, 20, 25, and 30 mg/liter were used in duplicate tests. The criterion of “death” was the inability of clams to retract siphons or to close valves. About half of the animals so affected were removed from the test solutions and returned to clean sea water to observe if recovery occurred, and others were preserved for histological examination.No “dead” clams recovered within 96 hr after return to clean water. The histopathology consisted primarily of necrosis of epithelial tissue of the gill, mantle, siphon, and suprabranchial gland, and the severity of damage was directly related to the test concentrations. Vacuolization, rupture, and pycnosis of cells occurred. The gills were the most severely affected organs. Epithelial cells of the gill filaments bearing the frontal, laterofrontal, and lateral cilia were sloughed as early as 24 hr after the beginning of exposure to Sevin. About 50% of the exposed clams had lost one or both siphons and also the epithelia on still attached segments within 96 hr of exposure. There were no deaths of control clams, and their tissues were normal.     

Surface area of the respiratory organs of the Climbing perch, Anabas testudineus (Pisces: Anabantidae)

Measurements have been made of the surface area of the gills and accessory respiratory organs of Anabas in the weight range 1–120 g, and the data analysed with respect to body weight using logarithmic transformations. The slope of the regression line for total gill area (0–615) is less than that found in most fish, the number of secondary lamellae/mm decreased more rapidly with body weight than for most water-breathing species (h = -0.152). The gill area of Anabas is relatively small but when the area of the accessory organs is added, the total respiratory area is of the same order as inactive water-breathing fish. The regression coefficient for combined areas of labyrinthine organs and lining of the suprabranchial chambers (0.713) exceeds that for the gills and together with other evidence (including estimates of diffusing capacity from morphological measurements), indicates an increasing importance of air-breathing of larger specimens. The average surface area of the accessory organs available for 1 ml of air within the suprabranchial chambers was found to be 2226 mm².     

Embryonic development characteristics and host mussel utilization of flat bitterling <Emphasis Type="Italic">Acheilognathus rhombeus</Emphasis> (Cyprinidae) during winter in Korea

This study aimed to investigate the embryonic development characteristics and host mussel utilization of Acheilognathus rhombeus to determine its adaptations or host utilization for improving survival during winter in the wild; the study was conducted from August 2014 to May 2015. Spawning of A. rhombeus occurred between mid-September and early November, peaking between late September and early October of 2014 when the water temperature was 19–21°C. The embryos of A. rhombeus were first found in early October of 2014 and remained inside the host mussel for 7 months. Development retardation and diapause in embryonic stages (stages C and D) occurred before eye pigmentation and continued until late March of 2015. Subsequently, the embryos resumed development (stage E) when the water temperature increased to about 10 °C. The positions during the development of A. rhombeus embryos in the mussels were mainly present in the farthest suprabranchial cavity and not in the water tube. The embryos were frequently found in the two inner demibranches of the four gills of the mussels regardless of their sex. We assume that embryonic diapause and use of suprabranchial cavity and inner demibranches of the gills of the mussels by A. rhombeus might be the optimal adaptation to maintain high embryonic survival in low water temperature during winter and to avoid interspecific competition.     

Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss)

Serotonin (5-hydroxytryptamine, 5-HT) occurs in a wide range of tissues throughout the body of the rainbow trout. Results reported here indicate that the main peripheral sources of serotonin are the intestinal tract and the gill epithelium (levels above 1500 ng/g). The high intestinal serotonin concentration is mostly due to serotoninergic nerve fibres, which are present at high density in the intestinal wall. Only about 2% of serotonin is associated with mucosal enterochromaffin cells. In the remaining tissues studied serotonin concentration was below 160 ng/g: the highest concentrations were seen in the anterior and posterior kidneys, followed by the liver, heart, and spleen. 5-Hydroxyindolacetic acid (5-HIAA) levels, except in plasma, were generally lower than serotonin levels, and were below our detection limits in heart, spleen and posterior kidney. Acute d-fenfluramine treatment (5 or 15 mg/kg i.p.) significantly increased 5-HIAA/5-HT ratio in the anterior intestine, pyloric caeca and plasma. Serotonin released from intestinal serotoninergic fibres in response to d-fenfluramine treatment is metabolized locally, and only a small part reaches the blood, from where it can be taken up and metabolized by other peripheral tissues, such as the liver and gill epithelium. The non-metabolized serotonin pool in the blood appears to be located extracellularly, not intracellularly as in mammals. In view of these findings, we present an overview of peripheral serotonin dynamics in rainbow trout.     

Gross structure of the respiratory organs and dimensions of the gill in the mud–skipper, Periophthalmodon schlosseri (Bleeker)

In Periopkrlialnwdon scldosseri the respiratory organs consist of the gills, the suprabranchial and opercular chambers. The gills are more suited for aerial than aquatic respiration as is shown by the presence of the vascular papillae, blood sinusesand dilated blood vessels in their lamellae. The gill lamellae possess a surface coat of sulphated mucopolysaccharides that prevents water loss during exposure to the air. The filaments of the outer hemibranchs in the first gill arch are reduced to nearly one quarter of those of its posterior hemibranch. The gill area in relation to body weight shows a high slope value ( b =0·93).