The anal portion as a salt-excreting organ in a seawater mosquito larva,A?des togoi Theobald

Summary Larvae of the marine mosquito,Aëdes togoi Theobald, tolerate environmental salinities ranging from fresh water to 300% sea water. When they were transferred from fresh water to sea water, sodium concentration in the haemolymph increased for the first 2 days and decreased to the seawater-adapted level within 4 days. When transferred from sea water to fresh water, the sodium concentration decreased markedly for the first 2 days and attained the freshwater adapted level after 4 days. When the larvae in sea water were ligated near the anus, they died within 3 days, showing an increased sodium level in the haemolymph. The larvae ligated at the neck lost considerable body weight and died within 4 days.When the anal portion, a terminal portion of the hindgut, was catheterized, the larvae maintained in sea water showed an increase in haemolymph sodium. The anal portion epithelium of the larvae adapted to 100 and 150% sea water demonstrated a strong positive reaction to the histochemical assay for chloride ions, whereas the reaction was negative or weakly positive in freshwater adapted larvae. In the larvae with the anal papillae ligated, a slight increase in haemolymph sodium occurred while in sea water. The anal papillae were weakly positive to chloride ions. Unlike salt-water mosquito larvae of the other species, in which the rectum is considered to be involved in hyperosmotic urine production and the anal papillae appear to be the extrarenal organ, the anal portion inA. togoi larvae seems to play an important role in excretion of excess ions when placed in hyperosmotic media.     

The osmotic response of salmon louse,Lepeophtheirus salmonis (Copepoda: Caligidae), during the transition from sea water to fresh water

Summary The osmotic changes in haemolymph and body tissues of the ectoparasitic salmon louse,Lepeophtheirus salmonis, have been studied upon transfer from sea water (SW) to dilute sea water (37% SW), and then to fresh water (FW). The parasite shows osmoconformity in SW but hyperosmotic regulation in 37% SW regardless of whether it is attached to the salmon host or free swimming in the water. The same conclusion is reached by haemolymph Cl^– measurements. In FW, the osmotic tolerance and response of attached and free swimming parasites differ: Attached animals maintain steady haemolymph osmolality and Cl^– concentration and survive for at least 1 week, while free swimming parasites quickly become diluted and start to die within 8 h.Acclimation to 37% SW is accompanied by changes in body tissue water content and in the content of ninhydrin positive substances and specific amino acids which suggest the presence of cell volume regulation. Glycine is the dominating free amino acid in the cephalothorax tissues but alanine, proline and taurine also occur in high amounts. Lysine is found to increase significantly during FW acclimation of attached parasites. A breakdown of cell volume regulation is suggested to limit the survival of attached salmon louse in fresh water.Abbreviations FW fresh water - NPS ninhydrin positive substances - SW sea water     

Some aspects of osmoregulation in a stenohaline freshwater catfish, Heteropneustes fossilis (Bloch), in different salinities

Transfer of the catfish, Heteropneustes fossilis , to 10% sea water (101 mosmol l⁻¹) or to 0·4% NaCl (140 mosmol l⁻¹) does not evoke any change in plasma osmolarity from the normal freshwater values. There is, however, a reduction in urine flow rate (UFR) and increase in urine osmolarity without any change in the rate of osmolar clearance. In isosmotic (25% sea water or 0·7% NaCl) and in hyperosmotic (30% sea water or 1·1% NaCl) media there is a significant increase in plasma osmolarity accompanied by marked reduction in glomerular filtration rate (GFR), UFR and free water clearance. The results suggest that the catfish cannot effectively osmoregulate in isosmotic or hyperosmotic media and that the inability of the renal tubules to increase reabsorption of water and to reduce free water clearance may account for the restricted range of salinity tolerance of this catfish. Also, in the hyperosmotic media, plasma levels of cortisol are lowered while in the proximal pars distalis the corticotrophs appear active, suggesting increased utilization and clearance of cortisol. Prolactin-secreting cells, however, are degranulated and chromophobic in catfish maintained in hyperosmotic environment.     

The regulation of haemolymph calcium concentration of the crab Carcinus maenas (L.).

After acclimation, Carcinus can maintain calcium balance in dilute (35-100%) but not in low calcium sea water. 71% of total haemolymph calcium (9-54 +/- 0-42 mM) was in ionic form as compared with 90-9%(9-9mM) in sea water. On acclimation to dilute sea water the calcium activity of the haemolymph was greater than that of the medium, the difference being maintained by active calcium uptake. Carcinus is highly permeable to Ca2+, influx from sea water being 0-513 +/- 0-07 mumoles g-1 h-1 and the time constant for calcium influx 4-3 +/- 0-48 h. Calcium space represented ca. 25% wet body weight independent of body size or salinity of acclimation medium.     

Toxicological impact of butralin,glyphosate-isopropylammonium and pendimethalin herbicides on physiological parameters of Biomphalaria alexandrina snails

ABSTRACT

Biomphalaria alexandrina snails have been used as bioindicators for freshwater qaulity and the effects of some herbicides such as butralin, glyphosate-isopropylammonium and pendimethalin). In the present study the effect of these three herbicides on snail biochemistry was examined. The results indicated that the herbicides increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the haemolymph of B. alexandrina snails and significantly decreased total protein and albumin content. Light microscopical examinations of haemocytes monolayers of B. alexandrina snails showed three different cell types (small cells, granulocytes and hyalinocytes). All three herbicides caused abnormalities in cell shapes. Flow cytometric analysis of haemocytes from B. alexandrina demonstrated that circulating haemocyte populations could be divided into two main subtypes differing in their granularity (granulocytes or hyalinocytes) and size (large and small cells). In addition, the flow cytometric analysis showed that the total number of dead haemocytes in the haemolymph was significantly increased in treated groups compared to the control group. Phagocytosis in groups treated with the herbicides was highly significantly increased compared to the control indicating a very strong response of the treated snails. The results of the alkaline comet assay of DNA damage demonstrated that these herbicides have a genotoxic effect.     

Habitat and phylogeny influence salinity discrimination in crocodilians: implications for osmoregulatory physiology and historical biogeography

Crocodylids are better adapted than alligatorids, through a suite of morphological specializations, for life in hyperosmotic environments. The presence of such specializations even in freshwater crocodylids has been interpreted as evidence for a marine phase in crocodylid evolution, consistent with the trans-osceanic migration hypothesis of crocodilian biogeography. The ability to discriminate fresh water from hyperosmotic sea water, and to avoid drinking the latter, is known to be an important osmoregulatory mechanism for estuarine crocodylids. This study was undertaken to determine whether the ability to discriminate between hyper- and hypo-osmotic salinities is determined by habitat, as it is in other normally freshwater reptiles, or whether, like morphological adaptations associated with estuarine life, it has a phylogenetic basis. Two species of freshwater alligatorid were found to drink fresh water and hyperosmotic sea water indiscriminately, while an estuarine population of a normally freshwater alligatorid species drank only fresh water. This indicated that salinity discrimination is determined at least in part by habitat. However, all three crocodylid species tested drank fresh water but not hyperosmotic sea water, suggesting that, in crocodilians, the ability to distinguish between fresh water and sea water is influenced by phylogeny as well as by habitat. The implications of this result are discussed in the context of two alternate hypotheses for the historical biogeography of the Crocodilia.     

WATER-REGULATORY BEHAVIOUR IN TERRESTRIAL GASTROPODS

1. Terrestrial snails and slugs are exceedingly susceptible to dehydration due to evaporative water loss from the integument and lung surface, and the deposition of a dilute mucous trail. Active slugs can lose 30–40% of their initial body weight (IBW) within 2 h. 2. Both field and laboratory studies have revealed that habitat selection by snails and slugs is well correlated with the availability of water. In addition, numerous species display homing behaviour, returning directly to their moist secluded daytime resting sites at dawn. 3. Several aspects of locomotor activity are affected by body hydration and environmental conditions such as relative humidity. Moist conditions result in termination of aestivation in snails and a generally higher level of activity in both snails and slugs. In contrast, severe dehydration initiates aestivation in snails and an increase in the intensity and duration of circadian locomotor activity in slugs. 4. Huddling behaviour is a specialized example of the general preference of slugs for moist habitats. When groups of slugs are exposed to dry environmental conditions, they form closely packed aggregations. This response results in a decrease in the rate of dehydration of the individual slugs. 5. When slugs have been dehydrated to about 90% IBW, rhythmic closures of the pneumostome are initiated. As dehydration progresses there is a reduction in the open diameter of the pneumostome. These responses reduce the total exposure of the lung surface and thereby evaporative water loss. In slugs dehydrated to about 80 % IBW, these responses can result in a 7 % reduction in water loss. 6. When slugs have been dehydrated to about 65% IBW (67·6 ± 4·3% IBW) they move on to a moist surface, assume a characteristic flattened posture and remain quiescent while water is absorbed through the surface of the foot. Once they are rehydrated (to 93·5 ± 12·4% IBW) they move off the moist surface. Thus there exists a specific dehydration threshold for the initiation of contact-rehydration and a rehydration set-point at which the response is terminated. 7. Both initiation and termination of contact-rehydration are controlled by variations in haemolymph osmotic pressure. The behaviour can be experimentally initiated by injection of hyperosmotic mannitol solution and terminated by injection of dilute saline. 8. Contact-rehydration involves bulk flow of water through an epithelial paracellular pathway in the integument of the foot. The rate of absorption of [¹⁴C]inulin during contact-rehydration is similar to that of water. The efficacy of water movement through the pathway is modulated by body hydration, the pathway being opened only in dehydrated slugs. 9. By means of the dual-limit control of contact-rehydration slugs can behaviourally regulate their body hydration and haemolymph osmolality within the tolerable hydration range described by the upper and lower limits.     

Coagulation of sea urchin egg cytoplasm by high salt concentrations

Unfertilized sea urchin eggs exposed to hyperosmotic salt solutions in excess of 1.75 M undergo a form of intracellular coagulation known as black cytolysis, similar to that seen in eggs injured by freezing. The process can be simulated by the microinjection of hypertonic salt into the cell suspended in isotonic solution in the absence of volume reduction. Black cytolysis during hyperosmotic stress can be attributed to the entry of concentrated extracellular solution through a membrane made permeable by excessive osmotic stress.     

Hyperosmotic readjustment of the crab,Hemigrapsus edwardsi

Summary Daily changes in the composition of the haemolymph and muscle tissue ofHemigrapsus edwardsi, following transfer to hyperosmotic saline, were investigated. The haemolymph approached a new equilibrium, with respect to ionic constituents, within 48 h. The muscle fibre water content, expressed in kg/kg dry weight, fell initially and then rose. This was associated with a rise in the amount (per kg tissue dry weight) of intracellular ninhydrin-positive substances (mainly amino acids) which was substantially complete within four days. It was concluded that this crab is capable of relatively rapid intracellular readjustment to hyperosmotic stress.     

Hydromineral regulation in the saline water corixid Trichocorixa reticulata (Hemiptera: Corixidae)

The aquatic corixid Trichocorixa reticulata (Guerin-Meneville) inhabits coastal marshes, brackish water ponds and salt ponds of high salinity, suggesting the presence of well developed mechanisms for hydromineral regulation.Groups of corixids acclimated in salinities ranging from fresh water to just above 300% sea water (100‰) were analyzed for total body water content, haemolymph ionic and osmotic levels, and haemolymph free amino acids.Results indicate an excellent ability to maintain haemolymph Na⁺, Cl^?, Mg²⁺ and K⁺ hyperosmotic to the medium at low salinities and hyposmotic at high salinities. Calcium appears to conform closely to changes in external medium, becoming hyposmotic at very high salinities (80‰).Total haemolymph osmotic pressure was well regulated, the freezing point depression varying from 0.75°C in distilled water to 1.15°C in salinities of 100‰. Total body water was maintained at approx. 75% of the total animal wet weight at all salinities tested.Free amino acids were maintained between 40–60 mM in all tests and did not appear to change with salinity.     

Volume regulation during dehydration of desert beetles

In arid areas in East Africa, dietary water is available only during the rainy seasons. Since the rainy seasons are separated by dry seasons, which may last for many months and in extreme cases for more than a year, the beetles may lose more than 80% of their body water. The water loss takes place mainly at the expense of the extracellular fluid, i.e. as the haemolymph volume drops to zero, the cell volume is only moderately reduced. The protection of cell volume at the expense of the haemolymph requires that solutes are removed from the haemolymph. The solutes are either excreted from the body or sequestered within the body in an osmotically inactive state. In predatory beetles of the family Carabidae, where Na is the dominating extracellular solute, Na is excreted, but it can easily be replaced from the diet. In most herbivorous beetles, such as the Tenebrionidae, which feed on a low Na diet, and which have low extracellular Na levels, Na is usually, but not always, deposited within the body. Free amino acids are moved from haemolymph to cells, but some seem to be made osmotically inactive by polymerization to peptides. As beetles become rehydrated, the peptides are rapidly depolymerized and the amino acids released to the haemolymph. Another factor, which may be important in the stabilisation of cell volume, is the colloid osmotic contribution of intracellular proteins, which may have a steep increase in their osmotic activity with increasing concentration.     

THE EFFECT OF SALT CONCENTRATION OF THE MEDIUM ON THE RATE OF OSMOSIS OF WATER THROUGH THE MEMBRANE OF LIVING CELLS

1. Using the unfertilized egg of the sea urchin, Arbacia, as osmometer, it was found that the rate with which water enters or leaves the cell depends on the osmotic pressure of the medium: the velocity constant of the diffusion process is higher when the cell is in concentrated sea water, and lower when the sea water medium is diluted with distilled water. Differences of more than tenfold in the value of the velocity constant were obtained in this way. When velocity constants are plotted against concentration of medium, a sigmoid curve is obtained. 2. These results are believed to indicate that cells are more permeable to water when the osmotic pressure of the medium is high than when it is low. This relation would be accounted for if water should diffuse through pores in a partially hydrated gel, constituting the cell membrane. In a medium of high osmotic pressure, the gel is conceived to give up water, to shrink, and therefore to allow widening of its pores with more ready diffusion of water through them. Conversely, in solutions of lower osmotic pressure, the gel would take up water and its pores become narrow.     

Transport of water from concentrated to dilute solutions in cells of Nitella

The transport of water from concentrated to dilute solutions which occurs in the kidney and in a variety of living cells presents a problem of fundamental importance. If the cell acts as an osmometer we may expect to bring about such transport by creating an inwardly directed osmotic drive which is higher in one part of the cell than in other regions of the same cell. The osmotic drive is defined as the difference between internal and external osmotic pressure. Experiments with Nitella show that this expectation is justified. If water is placed at one end of the cell (A) and 0.4 M sucrose with an osmotic pressure of 11.2 atmospheres at the other end (B) water enters at A, passes along inside the cell, and escapes at B leaving behind at B the solutes which cannot pass out through the protoplasm. Hence the internal osmotic pressure becomes much higher at B than at A. When 0.4 M sucrose at B is replaced by 0.3 M sucrose with an osmotic pressure of 8.1 atmospheres we find that water enters at B, passes along inside the cell, and escapes at A so that water is transported from a concentrated to a dilute solution although the difference in osmotic pressure of the 2 solutions is more than 8 atmospheres. The solution at B thus becomes more concentrated. It is evident that if metabolism produces a higher osmotic pressure and consequently a higher inwardly directed osmotic drive in one region of the cell as compared with other parts of the same cell water may be transferred from a concentrated to a dilute solution so that the former solution becomes still more concentrated.     

Gill Na(+), K(+)-ATPase in a series of hyper-regulating gammarid amphipods: enzyme characterisation and the effects of salinity acclimation

The enzyme Na(+), K(+)-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na(+), K(+)-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na(+), K(+)-ATPase activity in G. pulex were 100mM Na(+), 15mM K(+), 15mM Mg(2+) and 5mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na(+), K(+)-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na(+), K(+)-ATPase activity occurred. This implicates the involvement of gill Na(+), K(+)-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.     

Physiological effects of saline waters on zander

Rapid transfer of zander Stizostedion lucioperca to hypoosmotic brackish water (mean osmolality 230 mOsmol kg–1 , c. 8 psu) significantly increased plasma chloride concentrations after 24 h compared to those transferred to fresh water, although plasma osmolality was not significantly affected. After 6 days, plasma osmolality was slightly elevated but stable plasma glucose and cortisol concentrations and blood haematocrit and haemoglobin suggest a lack of hormonal stress responses and resultant secondary effects. Rapid transfer of zander to a more saline environment, hyperosmotic to plasma (mean osmolality 462 mOsmol kg^‐1, c. 16 psu) induced a greater increase in plasma osmolality and chloride concentrations within 24 h, with a further rise after 6 days exposure, but all fish maintained a state of hypo‐osmoregulation both 24 h and 6 days after transfer. The initial osmotic disturbance (at 24 h) was accompanied by increased plasma glucose, blood haematocrit and haemoglobin and a decreased mean cell haemoglobin concentration (MCHC), suggesting an adrenergic stress response, but these parameters fully recovered within 6 days of exposure to this hyperosmotic environment with MCHC rising to exceed the level in freshwater fish. Zander did not survive rapid transfer to more hyperosmotic conditions (750 or 1001 mOsmol kg^‐1, 26‐35 psu), but they did survive exposure to simulated‘tidal cycles’ of rising and declining salinity, peaking after 6 h at c. 29 or 33 psu. Although osmotic disturbance was apparent after 6 h exposure and other physiological parameters suggested both adrenergic and corticosteroid components of a stress response, rapid recovery was apparent after return to fresh water. The results indicate that the zander, a non‐indigenous species in the U.K., has a high level of osmotic tolerance and a degree of hypo‐osmoregulation in saline environments not found in most stenohaline freshwater teleosts. This osmoregulatory ability could enable invasion of new U.K. river systems by using inshore marine environments of low salinity as saltwater bridges.     

The salinity tolerance of the invasive golden apple snail (Pomacea canaliculata)

We exposed snails of an invasive species of golden apple snail (Pomacea canaliculata) to five artificial sea water treatments at salinity levels of 0, 5, 10, 15 or 20 parts per thousand (ppt) to assess their salinity tolerance. We observed the behaviour, heart rate, total haemocyte counts, haemolymph ionic concentration and Na⁺/K⁺-ATPase activity in the mantle at 0, 12, 24, 48, 72 and 96 h post salinity exposures. The heart rate declined with increasing salinity, while Na⁺/K⁺-ATPase activity in the mantle presented a reverse trend, possibly to maintain normal osmolality. A trend of rising total haemocyte count was observed from 0 ppt and 5 ppt to 10 ppt salinities, while a sudden increase in the count was observed at 15 ppt and 20 ppt salinity groups. Furthermore, haemolymph Cl^?, Na⁺ and K⁺ concentrations increased directly with elevated salinity. An additional trial was performed to assess the growth performance of the snails under exposure to low salinities. During a 1 month trial, snails grew better at 5 ppt salinity treatment. Taken together, our results demonstrate that P. canaliculata can tolerate salt stress to some extent. The finding also obviously implies a possible invasive risk to estuaries.     

Growth enhancement of the macrophyte Ceratophyllum demersum in the presence of the snail Planorbis planorbis: the effect of grazing and chemical conditioning

• 1 The effects of epiphyton grazing and nutrient recycling by the freshwater snail Planorbis planorbis on the growth of the macrophyte Ceratophyllum demersum were examined in laboratory experiments.
• 2 Ceratophyllum plants grown in the presence of snails, or in water chemically conditioned by snails, were significantly longer, had more healthy nodes of leaves, and more growing tips than Ceratophyllum plants grown in isolation. There were no significant differences between the growth of plants that were grazed by snails, and those plants which received water chemically conditioned by snails.
• 3 Adult P. planorbis released 3.74 × 10^?4μg phosphate, 23.9 × 10^?4μg nitrate, 4.38 × 10^?4μg ammonia, and 0.79 × 10^?4μg of urea mg^?1 wet weight of snail h^?1. Measurements of these chemicals in the experimental systems suggested that ammonia was an important nutrient in stimulating the growth of Ceratophyllum.
• 4 Removal of epiphyton by grazing snails significantly prolonged the life of individual Ceratophyllum leaves compared to ungrazed leaves.

     

Ultrastructure of the anal papillae of a salt-water mosquito larva, Aedes campestris

Anal papillae from fourth instar larvae of the salt-water mosquito Aedes campestris maintained in normal (hyperosmotic) lake water are made up of a single epithelial layer. The only type of cell present in this layer shows several ultrastructural features characteristic of transporting tissues. The apical plasma membrane (facing the external medium) is infolded into a series of lamellae which bear a particulate coat on the cytoplasmic surface. The basal plasma membrane (facing the haemolymph) is also highly infolded to form a system of interconnecting channels throughout the cell. These channels may be considerably dilated. Dense mitochondria are abundant in anal papillae cells and tracheoles frequently penetrate deeply into the cells. No qualitative or statistically significant quantitative differences are observed in the ultrastructure of anal papillae taken from A. campestris larvae maintained in diluted (hyposmotic) lake water. It is suggested that in both hyperosmotic and hyposmotic external media the anal papillae are actively engaged in ion transport and that adaptive changes in transport rates which have been demonstrated physiologically may require only minor structural modifications such as permeability changes or activation of enzyme systems already present in the cell membranes.     

The role of nemertean cerebral organs in salinity stress tolerance re‐examined in Paranemertes peregrina coe (hoplonemertea: Monostilifera)

The role of the cerebral organs of Paranemertes in salinity stress tolerance was investigated by measuring the effect of cerebral organ removal on volume regulation, and by observing the effects of hypo‐osmotic media on the cytology of the cerebral organs. Removal of the cerebral organs decreases the capacity for volume regulation, however, a similar change is seen in sham‐operated worms, indicating that the decreased capacity for regulation is due to the operation itself and not to interference with a physiological role of the cerebral organs. Cytological changes caused by in vivo exposure to dilute sea water are similar to those seen in worms fixed in hypo‐osmotic fixative and therefore do not likely represent a coordinated response of the organs to salinity stress. Also, no exchange of material between the cerebral organs and the vascular system, as reported in some other species (Ling, 1970; Ferraris, 1979), was observed. We conclude that in Paranemertes, the cerebral organs are not involved in osmoregulation.     

Adaptive patterns of osmotic and ionic regulation, and the invasion of fresh water by the palaemonid shrimps

To evaluate trends in the osmoregulatory behavior of neotropical, palaemonid shrimps, we investigated osmotic and ionic regulatory patterns in five species of Palaemon or Macrobrachium. The species' life histories depend on saline water to differing degrees, their habitats ranging from the marine/intertidal (P. northropi), through estuaries (P. pandaliformis) to coastal, freshwater streams (M. olfersii, M. potiuna) and inland, continental river systems (M. brasiliense). Hemolymph osmolality, chloride, sodium and magnesium concentrations were measured in shrimps exposed to experimental media ranging from fresh water (<0.5 per thousand ) to concentrated seawater (42 per thousand ) for up to 10 days. The marine and estuarine Palaemon species exhibit well-developed hyper/hypo-osmotic, sodium and chloride regulatory capabilities in mid-range salinities, tending to hyperconform in low salinities. The freshwater Macrobrachium species show variable hyperosmotic, sodium and chloride regulatory capacities, tending to hypoconform or unable to survive at higher salinities. All species hyper-regulate magnesium in fresh water, but hyporegulate strongly in saline media. Palaemonids from the saline habitats show the strongest osmoregulatory capabilities, and fresh water may have been gradually invaded by ancestral species with similar regulatory capacity. However, this regulatory plasticity has been lost to varying degrees in extant freshwater species.