Increase In Hemolymph Osmolality Research Articles

Metabolites, ions, and the mechanisms behind seasonal cold hardening of Pyrochroa coccinea (Pyrochroidae) larvae

The larvae of the black headed cardinal beetle Pyrochroa coccinea, overwinters under the bark of dead logs in northern European dioecious forests, and are thus exposed to temperatures below the melting point of their bodily fluids. Here we explore the mechanisms behind their seasonal cold hardening by characterising field samples collected monthly throughout the year. Both the lower lethal temperature and supercooling point dropped as much as 10℃ in the second half of November, reaching values around –15℃ by the beginning of December. This change was accompanied by a 320 mosmol/kg increase in hemolymph osmolality, which is a doubling compared to the summer levels. We used NMR metabolomics to identify and measure the absolute concentrations of the responsible cryoprotective C-H containing metabolites in the hemolymph. The largest increase was found to be in either glucose or trehalose, with an average total increase of 120 mM. Proline, alanine, and choline concentrations were found to increase by around 10 mM each. Contrarily, phosphocholine and phosphoethanolamine were halved, resulting in a total decrease of around 50 mM. These measurements were complemented with ion exchange chromatography measurements. This allowed us to account for all the osmotic pressure in the summer hemolymph, and the measured concentration changes explained as much as 40 % of the observed osmolality increase upon cold hardening. Preliminary results indicate that the remainder may be explained by non-colligative protein contributions.

Metabolic rate and osmoregulation in desert spring amphipods

Desert springs contain many endemic taxa and are of conservation concern due to anthropogenic activities that are expected to increase environmental salinity. Understanding the nature of osmoregulation is necessary to predict how non-vagile aquatic organisms will respond to changes. In the Chihuahuan Desert, the amphipod genus Gammarus Fabricius, 1775 is composed of two lineages. These lineages have species that currently inhabit springs ranging from 0.4 to 7.8 parts per thousand (ppt). All Gammarus in this region are of conservation concern because each is endemic to a single spring system. We exposed individuals of Gammarus colei Walters, Cannizzaro, and Berg in Walters, Cannizzaro, Trujillo and Berg, 2020 and Gammarus seideli Cannizzaro, Walters and Berg, 2018 species occupying low-salinity springs, to the range of salinities found in the Chihuahuan Desert. We measured metabolic rates as [Formula: see text] to examine the energetic cost of osmoregulation. We also measured the hemolymph osmolality of G. colei and compared that with the isosmotic line to determine the degree of osmoregulation. Neither species increased its metabolic rate across increasing salinities. However, G. colei showed an increase in hemolymph osmolality. Despite the divergence (∼66 million years ago) between the two lineages, it appears their physiological tolerances have converged, suggesting that both lineages may be able to persist when exposed to moderate salinity changes.

Hemolymph osmolality in mollusk Lymnaea stagnalis during acute experimental hyperglycemia

It has been established that the development of hyperglycemia, accompanied by a multiple increase in the hemolymph glucose content (up to 4.0 – 4.5 mmol/L), doesn’t lead to any significant changes in Lymnaea’ s hemolymph osmolality. Incubation of mollusks in 100 mmol/L aqueous solution of polyethylene glycol 200 for 2 h causes a 1.4-fold increase in hemolymph osmolality (up to 210 mosmol/kg) compared to the control level.

The chloride channel Bestrophin‐1 acts in the Malpighian tubule and hindgut of Drosophila melanogaster to maintain salt and water balance

ObjectiveThe Malpighian tubule and hindgut are iono‐ and osmoregulatory epithelia in insects. The tubule secretes KCl‐rich fluid, and ions and water are subsequently reabsorbed in the hindgut. We characterized the role of the chloride channel, Bestrophin‐1 (Best1), in the tubule and hindgut of Drosophila melanogaster.MethodsWe studied flies carrying a germline mutation in Best1, Best1c04106, as well as Best1c04106 flies with genomic rescue, and flies with tissue‐specific knockdown (quantified by qRT‐PCR) or rescue of Best1. Malpighian tubule ion flux was measured ex vivo, and immunostaining was performed using antibodies to HA‐tagged Bestrophin‐1. Flies were assayed for lethality on normal vs. 0.3 M‐supplemented NaCl (high‐salt) diet and after water restriction. Excretion was quantified by counting excretory drops. Hemolymph and excretory drop osmolality were measured using a vapor pressure osmometer. Water weight was determined by measuring flies’ weight before and after baking at 60 °C.ResultsBest1c04106 mutant Malpighian tubules had decreased fluid secretion and transepithelial ion flux that was rescued by expression of wild‐type Best1 in the K+‐secreting principal cell. Principal cell Best1 knockdown decreased transcript levels by 80% and decreased fluid secretion and ion flux. We previously showed that NKCC is required for principal cell K+ secretion. Here, we immunolocalized both Best1 and NKCC to the principal cell basolateral membrane. There was no further decrease in ion transport in Best1 knockdown tubules when NKCC was mutated, suggesting that Best1 allows Cl− entering through the NKCC to recycle across the basolateral membrane. Best1c04106 mutant flies also had increased lethality on high‐salt diet. Hemolymph osmolality increased in control flies fed high‐salt diet, but Best1c04106 mutants had decreased osmolality on both normal and high salt diet, and increased number and osmolality of excretory drops. As a consequence, Best1c04106 mutant flies were dehydrated and died when water restricted. The Best1c04106 mutant phenotypes were rescued by genomic rescue (which recapitulates normal Best1 expression throughout the body), or targeted rescue in the Malpighian tubule and hindgut.ConclusionBest1 is required for normal ion transport in the Drosophila Malpighian tubule, and in the tubule and hindgut for iono‐ and osmoregulation, where ion reabsorption through Best1 in high‐salt conditions allows an increase in hemolymph osmolality to prevent osmotic water loss and dehydration.Support or Funding InformationNIH DK110358

Evidence that dopamine is involved in neuroendocrine regulation, gill intracellular signaling pathways and ion regulation in Litopenaeus vannamei.

The transport of ions and ammonia in gills may be regulated by neuroendocrine factors. In order to explore the mechanism of dopamine (DA) regulation, we investigated hemolymph neuroendocrine hormones, gill intracellular signaling pathways, ion and ammonia transporters, hemolymph osmolality and ammonia concentration in Litopenaeus vannamei after injection of 10-7 and 10-6mol DA per shrimp. The data showed a significant increase in crustacean hyperglycemic hormone (CHH) concentration at 1-12 h and a transient significant decrease in corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol concentration under DA stimulation. The up-regulation of guanylyl cyclase (GC) mRNA, cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) concentration, together with the down-regulation of DA receptor D4 mRNA and up-regulation of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), diacylglycerol (DAG) and protein kinase C (PKC) concentration suggested the activation of complicated intracellular signaling pathways. The expression of cAMP response element-binding protein (CREB), FXYD2 and 14-3-3 protein mRNA was significantly increased by PKA regulation. The increase in Na+/K+-ATPase (NKA) activity and the stabilization of V-type H+-ATPase (V-rATPase) activity were accompanied by an up-regulation of K+ channel, Na+/K+/2Cl- cotransporter (NKCC), Rh protein and vesicle associated membrane protein (VAMP) mRNA, resulting in an increase in hemolymph osmolality and a decrease in hemolymph ammonia concentration. These results suggest that DA stimulates the secretion of CHH and inhibits the release of cortisol, which activates intracellular signaling factors to facilitate ion and ammonia transport across the gills, and may not affect intracellular acidification.

The springtail Megaphorura arctica survives extremely high osmolality of body fluids during drought.

The springtail Megaphorura arctica Tullberg 1876 is widespread in the arctic and subarctic regions where it can be abundant along beaches. This species survives winters using cryoprotective dehydration as a cold tolerance strategy during which it becomes drastically dehydrated. Several studies have investigated the physiological responses associated with water loss of M. arctica under exposure to freezing temperatures, but little is known of the dynamics of body water and hemolymph osmolality when subjected to gradually increasing drought stress at temperatures above the freezing point. Therefore, an experiment was conducted in which M. arctica was subjected to relative humidities (RH) decreasing from fully saturated conditions to ca. 89%RH over a period of 30 days. During the experiment water content of springtails decreased from about 3 to ca. 0.5mgmg-1 dry weight. Alongside with water loss, trehalose concentrations increased from nearly nothing to 0.12mg mg-1 dry weight, which contributed to an increase in hemolymph osmolality from ca. 250mOsm to at least 7Osm. All springtails survived water loss down to 0.7mgmg-1 dry weight and hemolymph osmolality of ca. 4Osm, and about 60% of the springtails survived with only 0.5mg watermg-1 dry weight and osmolality of ca. 7Osm.At this level of dehydration, Differential Scanning Calorimetry analysis showed that most, but not all, osmotically active water was lost. It is discussed that the extensive dehydration must be associated with high concentrations of salts potentially causing denaturation and precipitation of cellular proteins. M. arctica is remarkably tolerant of dehydration, but because it does not endure loss of the osmotically inactive water it cannot be categorized as a truly anhydrobiotic species.

Differential effects of water loss and temperature increase on the physiology of fiddler crabs from distinct habitats

Temperature is one of the main environmental constraints to organism distribution, affecting physiology and survival. Organisms that inhabit the intertidal zone are exposed to temperature variation and, with climate change, they should face different conditions which include higher temperatures, leading to higher rates of water loss through evaporation and then fitness reduction or mortality. Here we tested the effects of desiccation and increased temperature in two fiddler crabs species that occupy distinct habitats in regard to vegetation cover and position on the intertidal zone and thus may respond differently to these stressors. Leptuca thayeri, which is restricted to the mid-tide zone and vegetated areas, had higher desiccation and mortality rates than Minuca rapax, a generalist species, when exposed to desiccation for 120 min. Also, compared to M. rapax, L. thayeri had a more permeable carapace. Temperature elevation of 10 °C and 20 °C for 72 h caused no mortality in either species. However, there were changes in hemolymph osmolality and muscle hydration in both species. Leptuca thayeri osmolality was low in the intermediate temperature, suggesting that at this temperature this species has a better salt secretion capability. Minuca rapax, however, had an increase in hemolymph osmolality at the highest temperatures with no LDH increase, which indicates that osmotic control in this species is more sensitive to temperature increase. Our results show that L. thayeri suffers more from desiccation, due to a more permeable carapace. However, because of this higher permeability L. thayeri is capable of lowering its temperature more than M. rapax. As temperature elevation produces great physiological changes in M. rapax, a reduced ability to keep a low temperature can be an issue for this species if temperature increases. However, higher water loss to keep body temperature low may decrease L. thayeri survivability in the same scenario.

Immunocytochemical localization of V-H(+) -ATPase, Na(+) /K(+) -ATPase, and carbonic anhydrase in gill lamellae of adult freshwater euryhaline shrimp Macrobrachium acanthurus (Decapoda, Palaemonidae).

Physiological (organismal), biochemical, and molecular biological contributions to the knowledge of the osmoregulatory plasticity of palaemonid freshwater shrimps has provided a fairly complete model of transporter localization in their branchial epithelium. Direct immunological demonstration of the main enzymes in the gill epithelia of adult palaemonids is, however, still incipient. The diadromous freshwater shrimp Macrobrachium acanthurus was exposed to increased salinity (25‰ for 24 hr), and its responses at the systemic level were evaluated through the assays of hemolymph osmolality and muscle hydration, and at cellular and subcellular levels through the activity and localization of the V-H(+) -ATPase, the Na(+) /K(+) -ATPase, and the carbonic anhydrase. Results showed an increase in hemolymph osmolality (629 ± 5.3 mOsm/kg H2 O) and a decrease in muscle hydration (73.8 ± 0.5%), comparing values after 24 hr in 25‰ with control shrimps in freshwater (respectively 409.5 ± 15.8 mOsm/kg H2 O and 77.5 ± 0.4%). V-H(+) -ATPase was localized in pillar cells, whereas Na(+) /K(+) -ATPase in the septal cells. The main novelty of this study was that carbonic anhydrase was localized in the whole branchial tissue, in pillar and septal cells. Exposure to high salinity for 24 hr led to no detectable changes in their localization or in vitro activity. Immunolocalization data corroborated the literature and current models of palaemonid gill ion transport. The absence of changes reinforces the need for the constant expression of these enzymes to account for the euryhalinity of these shrimps.

The limits of drought-induced rapid cold-hardening: Extremely brief, mild desiccation triggers enhanced freeze-tolerance in Eurosta solidaginis larvae

Rapid cold-hardening (RCH) is a highly conserved response in insects that induces physiological changes within minutes to hours of exposure to low temperature and provides protection from chilling injury. Recently, a similar response, termed drought-induced RCH, was described following as little as 6h of desiccation, producing a loss of less than 10% of fresh mass. In this study, we investigated the limits and mechanisms of this response in larvae of the goldenrod gall fly Eurosta solidaginis (Diptera, Tephritidae). The cold-hardiness of larvae increased markedly after as few as 2h of desiccation and a loss of less than 1% fresh mass, as organismal survival increased from 8% to 41% following exposure to −18°C. Tissue-level effects of desiccation were observed within 1h, as 87% of midgut cells from desiccated larvae remained viable following freezing compared to 57% of controls. We also demonstrated that drought-induced RCH occurs independently of neuroendocrine input, as midgut tissue desiccated ex vivo displayed improved freeze-tolerance relative to control tissue (78–11% survival, respectively). Finally, though there was an increase in hemolymph osmolality beyond the expected effects of the osmo-concentration of solutes during dehydration, we determined that this increase was not due to the synthesis of glycerol, glucose, sorbitol, or trehalose. Our results indicate that E. solidaginis larvae are extremely sensitive to desiccation, which is a triggering mechanism for one or more physiological pathways that confer enhanced freeze-tolerance.

Physiological responses of two acorn barnacles, Tetraclita japonica and Megabalanus volcano, to summer heat stress on a tropical shore

Species living on rocky shores are subject to periodic tidal emersions and can experience intense thermal stress during daytime low tides. Tetraclita japonica and Megabalanus volcano are common intertidal barnacles in Hong Kong, with T. japonica occupying the high to mid-intertidal zone where its lower limits overlap with the upper limits of M. volcano, which is found in the lower intertidal to subtidal zone. In this study, daily profiles of body temperature and hemolymph osmolality on the shore, and responses to thermal challenge under laboratory conditions were compared between the two species to investigate their behavioral and physiological responses to heat and desiccation stress. On-shore measurements of body temperature revealed contrasting thermal profiles, with T. japonica from the upper shore experiencing greater thermal stress than T. japonica and M. volcano from the lower shore. Aerial exposure of the barnacles was associated with an increase in hemolymph osmolality. M. volcano, however, showed a greater increase in hemolymph osmolality than T. japonica at similar shore levels. When the two species were exposed to rock temperatures of 40°C for 5h, more individuals (94%) of T. japonica entered a coma state, and at an earlier time than M. volcano (36%). All T. japonica individuals resumed activity when re-immersed in water, whereas in contrast, all M. volcano that went into a coma state did not recover and died within 24h. Quiescence and reversible coma during the early stages of acute heat stress, as observed in T. japonica, could therefore be adaptive responses to cope with thermal stress in the upper rocky shore.

Cyclomorphosis in Tardigrada: adaptation to environmental constraints

Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice formation proceeds rapidly at a crystallization temperature of around -20 degrees C, revealing extensive supercooling in both stages, while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage specimens (350-500 microm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was approximately 950 mOsm kg(-1). Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.

Triggering of cryoprotectant synthesis in the woolly bear caterpillar (Pyrrharctia isabella Lepidoptera: Arctiidae)

Isabella tiger moths (Pyrrharctia isabella) overwinter as caterpillars (i.e., woolly bears) that can survive freezing at moderate subzero temperatures. We observed an increase in hemolymph osmolality for field-collected woolly bears during October (325 +/- 47 to 445 +/- 27 mOsmol/liter) and tested the influence of temperature and moisture levels on cryoprotectant production. Laboratory acclimation was done at 5 degrees C in moist conditions and at 25 degrees C acclimation in both dry and moist conditions. Body water contents were diminished by dehydration at 25 degrees C for 4 days (57 +/- 4%). Caterpillars collected in early October did not alter their hemolymph osmolality during cold acclimation, but caterpillars increased by 45% (to 647 +/- 90 mOsmol/liter) after 4 days at 5 degrees C following their collection in late October. Hemolymph composition was markedly changed in caterpillars experiencing dehydration at 25 degrees C (1042 +/- 200 mOsmol/liter; 507 +/- 225 mmol glycerol/liter), whereas caterpillars showed no change in their hemolymph composition when kept moist at 25 degrees C. Our experiments reveal that both dehydration and cold acclimation rapidly induce cryoprotectant synthesis in P. isabella caterpillars. J. Exp. Zool. 286:367-371, 2000.

The effects of temperature and moisture on survival capacity, cuticular permeability, hemolymph osmoregulation and metabolism in the scorpion, Centruroides hentzi (banks) (scorpiones, buthidae)

1. 1. The temperature and water relations of Centruroides hentzi females were investigated. At 12 and 72% relative humidity (RH), the lower and upper Lt 50 were -4.5 and 43.7°C, and -4.7 and 45.1°C, respectively. When exposed to high temperature stress, survivorship was significantly greater under mesic conditions. 2. 2. Cuticular water loss was higher under xeric conditions (12% RH), ranging from 0.061 mg/cm 2/hr at 30°C to 0.211 at 41°C. 3. 3. Exposure to dry air (0–5% RH) resulted in a significant increase in hemolymph osmolality: from 441 to 688 mOsm over a 5 day period. 4. 4. Mean oxygen consumption rates increased from 161.7 mm 3/g/hr at 34°C to 541.6 at 44°C. ATPase activity was significantly higher in animals acclimated and tested at 35°C.

Modification of cardiac activity in response to dehydration in the terrestrial slug, Limax maximus

The level of body hydration in the terrestrial slug Limax maximus modifies several aspects of behavior such as pneumostome activity, feeding responsiveness, huddling, and contact-rehydration. The relationship between water balance and pneumostome activity and respiratory function suggested that cardiac activity might also be affected. To pursue this possibility, intact slugs and isolated heart-central nervous system (CNS) preparations were used to investigate cardiac responses to the increase in hemolymph osmolality which occurs during dehydration. In intact animals, heart rate increased in response to progressive air-dehydration and to increases in hemolymph osmolality resulting from injections of hyperosmotic solutions of mannitol or NaCl. In isolated preparations, the heart or CNS were separately exposed to hyperosmotic saline. Exposure of the heart alone to hyperosmotic saline resulted in decreased heart rate while exposure of only the CNS resulted in an increase in heart rate. These observations suggest that the increase in heart rate that is observed in intact air-dehydrated slugs is primarily mediated by the CNS.

Surface mucus production in gastropods is dependent on environmental salinity and humidity

1. 1. Skin surface barrier mucus might play an important role in the transport of water and salt by mollusean skin. It is currently believed that surface mucus exerts its control by means of altering the ion gradients at the skin surface. 2. 2. The purpose of this study was to quantitate changes in mucus metabolism that might occur when gastropods are forced to alter salt and water transport in response to transfer to an osmotically stressful environment. 3. 3. We found that the amount of barrier mucus can be quantitated using the alcian blue method and that the amount of secreted soluble mucus is proportional to the amount of surface-associated barrier mucus, suggesting that quantitation of soluble mucus can be used as a tool to study changes in barrier mucus metabolism. 4. 4. The amount of barrier mucus increased as the environmental salinity was augmented and did so before an increase in hemolymph osmolality occurred. 5. 5. Transfer of gastropods from water to water-saturated air resulted in an increase in the quantitity of barrier mucus. 6. 6. Mild dehydration resulted in a significant decrease in the amount of surface-associated barrier mucus. 7. 7. These results support the hypothesis that skin surface mucus plays an important osmoregulatory role in molluscs.

Physiological adaptations to low temperature in freezing-tolerant Phyllodecta laticollis beetles

1. 1. Adult beetles of the species Phyllodecta laticollis, overwintering at exposed sites beneath loose bark of dry trees, were found to tolerate freezing to about −42°C in winter. 2. 2. Phyllodecta laticollis beetles are also freezing-tolerant in summer having a low temperature tolerance limit of about −9°C. 3. 3. The hemolymph of Phyllodecta laticollis contained nucleating agents the whole year through. The supercooling points of the beetles varied from about −5°C in summer to about −7°C in winter. 4. 4. The increase in the freezing tolerance of the beetles from summer to winter is correlated with an accumulation of glycerol to high concentrations; the body fluid of the beetles reaching osmolality values of about 2500 mOsm. The osmotic contribution of glycerol seems to account for almost the whole increase in hemolymph osmolality. In winter, the high cold-hardiness of the beetles seems to be due to the colligative properties of glycerol. 5. 5. From autumn/winter to spring, however, there is a shift in the cold-hardiness. Beetles having the same hemolymph osmolalities and glycerol concentrations tolerate about 10°C lower temperatures in spring than in autumn. This increased freezing tolerance in spring seems to be based on other mechanisms than colligative ones. 6. 6. The increased cold-hardiness in spring might be of great importance to Phyllodecta laticollis, in that the glycerol concentration might decrease during the day due to sun exposure at a time when the beetles still are likely to be exposed to low temperatures at night or sudden periods of cold weather. 7. 7. The total amino acid concentration of the beetles varied from about 165 mMolal in summer to about 300 mMolal in winter. In spring and autumn the amino acid concentration was the same, so that the shift in cold-hardiness of Phyllodecta laticollis could not be explained by differences in total amino acid concentrations.

Hemolymph oxygen transport, acid‐base status, and hydromineral regulation during dehydration in three terrestrial crabs, Cardisoma, Birgus, and Coenobita

AbstractHemolymph oxygen transport, acid‐base status, and hydromineral regulation have been measured at 28–30°C in Cardisoma carnifex, Birgus latro, and Coenobita brevimanus in their normal hydrated state, following a period of severe dehydration, and after a subsequent 24‐hour period of rehydration.Cardisoma and Birgus tolerated a maximum loss of body water of 20%, which developed in 3–4 days without water, and resulted in an 25–30% increase in hemolymph osmolality. Ion concentrations also increased considerably, with the largest percentage increases in ion concentration occurring for Mg++, Ca++, and K+.A metabolic acidosis and a resultant fall in HCO developed in Cardisoma and Birgus, but PCO2 showed no large changes, suggesting little respiratory compensation. Oxygenation of postbranchial hemolymph was not affected during dehydration, since P and hemolymph O2 capacity increased, the latter because of a hemoconcentration. Within 24 hours of rehydration, a restoration of the original levels of hemolymph ions and osmolality occurred, and a nearly complete return to normal acid‐base status developed.Coenobita was distinctive, since dehydration to the maximum tolerated 28% loss of body water appeared to be accompanied by a closer regulation of hemolymph ion levels. Only protein increased, which probably accounted for some of the rise in hemolymph osmolality. However, hemolymph O2 capacity fell, and that, in conjunction with a significant reduction in P, suggested a more severe disruption of O2 transport than in either Cardisoma or Birgus. The metabolic acidosis and the fall in HCO during dehydration were also more profound, and Coenobita showed the least recovery of these three species after 24 hours of rehydration.