Haemolymph Volume Research Articles

Observations on the Primary Culture of Hemocytes of Penaeus

ABSTRACT A study of the primary culture of hemocytes of Penaeus was conducted to examine growth and maintenance of these cellular populations. Hemocytes rapidly attached to flask surfaces and could be maintained for approximately one month. A medium consisting of 2 × L-15, 20% Cellect Gold fetal calf serum, 20% AKN salt solution, and other supplements was suitable for long term hemocyte maintenance. Initial cell counts were found to be more representative of monolayer confluency than volume of hemolymph collected. Bacterial contamination of cell cultures was common, but could be controlled by the addition of antibiotics to the growth medium. Contaminants were identified as members of the genera Vibrio, Pseudomonas, Flavobacterium, Aeromonas, or Proteus. Attempts to subculture hemocyte monolayers using enzymatic and mechanical methods were unsuccessful. Additional investigations concluded that hemocyte cryoprotection by DMSO was poor when cells were frozen at –20 or -70°C, that monolayer integrity was optimal at a temperature range of 25–32°C, that hemocyte populations appear to contain heterologous surface residues, and that only 1-2% of fresh hemocytes are in an active proliferation cycle at the time of collection.

Differential haemotoxic effect of PCB congeners in the common shrimp, Crangon crangon

1. Specimens of the common shrimp, Crangon crangon, were exposed to sub-acute logarithmically separated concentrations (ranging from 0.05 μg to 500 μg 1 −1) of two structurally dissimilar PCB congeners (PCB 15 and PCB 77) for five days in vitro. 2. Mortality, recoverable haemolymph volume, haemocyte count, plasma protein and haemolymph osmolarity were determined for test animals together with levels of haemolymph cell prophenoloxidase, an indicator of immune potential. 3. Sub-acute concentrations of PCB 15, but not PCB 77, produced significant decreases in haemocyte count and phenoloxidase activity with a marked increase in recoverable haemolymph volume. 4. No effect was observed on haemolymph osmolarity or plasma protein levels with either PCB 77 or PCB 15.

Density-dependent phase polymorphism affects response to adipokinetic hormone in Locusta

1. 1. Haemolymph lipid levels were measured before and after injection of graded doses of corpus cardiacum (CC) extracts, and of synthetic adipokinetic hormone (AKH) I, to solitary and gregarious adult males of Locusta migratoria migratorioides. 2. 2. Gregarious locusts showed higher resting lipid levels and a consistently higher increase of haemolymph lipids, 90–100 min after injection. 3. 3. Haemolymph volume was lower in solitary than in gregarious locusts; therefore, the phase-dependent differences in haemolymph lipid levels and in adipokinetic response were not caused by differences in haemolymph volume. 4. 4. The authors conclude that solitary locusts have lower haemolymph lipid levels and a less intense response to AKH than gregarious locusts.

Effect of infection with Diplostomum spathaceum on the internal defense system of Lymnaea stagnalis

The effects of 3- and 6-month-old infections of the digenean Diplostomum spathaceum on the internal defense system of the pond snail Lymnaea stagnalis were investigated. Comparisons were made of several facets of the snail defense system in vitro. Total numbers of circulating hemocytes per unit volume of hemolymph were unaffected by infection but the relative proportions of round, intermediate, and spreading hemocytes were altered. The hemolymph of infected snails contained a higher proportion of immature round cell types and fewer spreading hemocytes. The phagocytic activity of circulating hemocytes was reduced by infection. The most dramatic effect of infection was a highly significant reduction of the agglutinating activity of snail hemolymph.

Regulation of Pulmonary Blood Flow and of Blood Pressure in a Mangrove Crab (Goniopsis Cruentata)

ABSTRACT The air-breathing mangrove crab Goniopsis cruentata ventilates the branchial chambers with its scaphognathites (SG). Ventilation is predominantly in the forward direction, but is punctuated by bouts of reversed pumping. Reversals are more frequent when crabs are in air than in water, and yet more frequent during respiratory stress (hypoxia or exercise). Reversed SG pumping is tightly coupled with bursts of impulses to the dorsal-ventral muscles (DVM) which span the anterolateral thorax. Phasic contractions of the DVMs increase the hemolymph pressure in the dorsal sinuses. These pressure pulses help drive hemolymph through the lungs. The coupled SG reversed ventilation and DVM-assisted increases in lung perfusion appear to be an adaptation to increase gas exchange at the lungs. When crabs are made hyper-or hypotensive by changes in hemolymph volume, the EMG activity of the DVMs dramatically decreases or increases, respectively. The resultant expansion or constriction of the dorsal sinuses is an effective baroreceptor reflex producing short-term adjustments in hemolymph pressure.

The Dorsoventral Muscles of Carcinus maenas: Evidence for Hydrostatic Pressure Control in a Crab

The dorsoventral muscles of crabs extend between the carapace and the roof of the branchial chamber. In Carcinus they insert dorsally onto a row of conspicuous white spots, permitting precise location of electromyogram (EMG) electrodes. Contraction raises the branchial roof and compresses extensions of the hemocoel within the branchiostegites. Tonic EMG activity at ~2-14 Hz was observed in resting, forward-ventilating crabs. Stepwise elevation of internal pressure, by injection of saline or by placing a weight on the carapace, reduced EMG frequency (often to zero for a few seconds) before establishing a new, lower tonic EMG frequency. Following infusion of 2 mL saline into the pericardium (mean crab weight 124 g), EMG frequency fell to 15% of the initial value (mean over a period of 12 s), increasing to 37% of initial rate after ~ 1 min. Reduction of hemolymph volume and pressure increased the EMG frequency (stable rate 153% of baseline value after removal of 2 mL, 175% in first 12 s). A significant incre...

The effect of aluminum in soft water at low pH on water balance and hemolymph ionic and acid-base regulation in the dragonflyLibellula julia Uhler

The physiological responses of last instarLibellula julia nymphs exposed for 96 (or 192) h to low pH (4.0 and 2.3) and elevated Al concentrations (0.3, 3, and 30 mg l−1) at low pH were investigated. To some extent, both low pH alone and Al at low pH were found to affect water balance (wet weight and hemolymph volume), ionic regulation (hemolymph osmolality and concentrations of Na+, Cl−, and K+), and acid-base balance (hemolymph pH). The extent and significance of these alterations are discussed.

Control of ‘Pulmonary’ Pressure and Coordination with Gill Ventilation in the Shore Crab Carcinus Maenas

ABSTRACT In the shore crab, Carcinus maenas (L.), forward ventilation creates negative pulses of hydrostatic pressure while reversed ventilation causes dramatic positive pressure fluctuations in the branchial chamber. These pressures are transmitted via the gills to the haemolymph of the open circulatory system. The branchiostegal sinus, which is a compliant chamber, may function as a reservoir for displaced haemolymph and may operate as an accessory pump driven by the action of the dorsoventral (DV) muscles. A band of dorsoventral muscles controls the volume of the branchiostegal sinuses. The muscular activity is coordinated with ventilatory activity and may assist in regulating pressure fluctuations caused by ventilatory pressure pulses. During a ventilatory reversal, the haemolymph displaced from the gills is added to the volume of haemolymph in the open circulatory system and this haemolymph may be accommodated in the branchiostegal sinus by relaxation of the DV muscles. Artificially regulating the pressure either in the branchial chamber or in the branchiostegal sinus reflexively alters DV muscle activity, which suggests the occurrence of baroreceptors in this crab. The branchiostegal nerve that innervates the DV muscles contains five neurones identified by cobalt backfills. Three of them are median and two are contralateral. The dendritic field of each neurone is confined to its respective hemiganglia. The electrical activity of one of the motoneurones in the branchiostegal nerve corresponds to the activity of the DV muscles. In vitro observations of the activity of branchiostegal motoneurones in relation to ventilatory motoneurone activity indicate that both are centrally coupled and support the hypothesis that the branchiostegal motoneurones are influenced by the ventilatory central pattern generator.

Water-Orientation Behavior in the Terrestrial GastropodLimax maximus:The Effects of Dehydration and Arginine Vasotocin

When specimens of the terrestrial slug Limax maximus are dehydrated to 70% of their initial body weight (IBW), they exhibit water-orientation behavior that is characterized by both an increase in locomotor activity and a preference for the moist arm of a Y maze. Water orientation was elicited in fully hydrated slugs by injection of 10⁻⁶ M arginine vasotocin (AVT, calculated final hemolymph concentration). Injection of 10⁻⁷ M AVT elicited water-orientation behavior in slugs dehydrated to only 80% IBW, demonstrating that the effects of AVT and dehydration are additive. Injection of slugs with the octapeptides angiotensin II (AII) and arginine vasopressin (AVP), at concentrations ranging from 10⁻⁶ to 10⁻⁸ M, failed to elicit water orientation. Water orientation does not seem to be mediated solely by the alterations in hemolymph volume or osmolality that occur during dehydration. Rather, it appears that a dehydration-induced increase in hemolymph ionic concentration is involved in initiating water orientation.

Influence of physiological condition on cadmium transport from haemolymph to hepatopancreas inCarcinus maenas

Accumulation and binding of cadmium in the tissues of the shore crabCarcinus maenas (L.) were investigated in a series of laboratory experiments. The relation between the physiological condition of individual crabs and their ability to transport cadmium from haemolymph to hepatopancreas was specifically addressed. Cadmium was removed from the haemolymph with a half-life of approximately 10 h, and half of the cadmium removed from the haemolymph was taken up in the hepatopancreas. The efficiency with which individual crabs transported cadmium from the haemolymph to the hepatopancreas was strongly related to physiological parameters, such as concentrations of calcium and magnesium in the hepatopancreas and haemolymph volume and haemolymph protein concentration. The transport of cadmium from the haemolymph to the hepatopancreas was saturated in crabs exposed to more than 2 to 4 mg Cd l−1 in the sea water. In the hepatopancreas of unexposed crabs, cadmium was bound mainly in the insoluble tissue fraction (40%) and in the protein fraction with a molecular weight of approximately 6000 D (50%). Exposure to 0.25 to 1.5 mg Cd l−1 for 2 w led to dose-dependent increases of the amounts of cadmium bound in the high-molecular weight protein fraction and in the insoluble tissue fraction. Modes of internal cadmium transport and accumulation in tissues and variability in physiological parameters are discussed.

Haemolymph pressures in barnacles: a comparative study

Haemolymph pressures have been measured for several Australasian thoracican barnacles. The list included six lepadomorphs and four balanomorphs, with particular interest centred on Catomerus polymerus , a primitive chthamaloid in which certain lepadomorph features are vestigially retained. The results were incorporated with those from the literature to present a more comprehensive data set for thoracicans. In general, lepadomorphs have higher haemolymph pressures than balanomorphs and their maximum pressures are sustainable. Lepadomorphs with high absolute pressures (range, 38-350 cm H 2 O ) were all intertidal species from exposed shores, highly armoured in both capitulum and stalk, with few haemolymph spaces in the stalk. These animals exhibited limited stalk elongation/shortening and bending. The other lepadomorphs were less well armoured, their peduncular cuticle was not reinforced and there were many haemolymph spaces interspersed amongst the peduncular tissues. Much lower absolute pressures were generated (range, 6-116 cm H 2 O) and these animals exhibited considerable elongation/contraction or bending (or both) of the stalk. C . polymerus has reasonably high haemolymph pressures, which overlap with those measured in some of the lepadomorphs. This chthamaloid has a low haemolymph volume, most of the available space enclosed by the wall plates and basis being occupied by tissue. C . polymerus generated very high maximum pressures, which were sustainable. This balanomorph, retaining evidence of scalpelloid ancestry, is adapted to survive in the high intertidal pounded by surf. Tesseropora rosea , another surf barnacle of a more advanced balanomorph form, also has high maximum pressure, although lower than that of C. polymerus . It too has low haemolymph volume. The remaining balanomorphs examined contain significant haemolymph volume, exhibited low absolute pressures (range, 3-19 cm H 2 O) and, except for Balanus hameri , non-sustainable maximum pressures. The implications of an expansive haemolymph system and low absolute pressures in balanomorph evolution are discussed.

Comparative glucose tolerance studies in the freshwater snail Biomphalaria glabrata: influence of starvation and infection with the trematode Schistosoma mansoni

The dynamic reactions of B. glabrata on intravasal loads of 20-1000 micrograms glucose.g live fresh wt-1 were studied in standard fed snails (SFS). Starved snails (SS) and snails infected with S. mansoni (IS) were given 100-500 micrograms glucose.g fresh live wt-1. Mean hemolymph glucose level was 12.1 mg.100 ml-1 in SFS. It was not significantly lower in SS (10.7 mg.100 ml-1), but significantly reduced in IS (8.5 mg.100 ml-1). Since hemolymph volumes were significantly increased in SS, the amount of circulating glucose (pool) did not change (75 micrograms.g body weight-1) compared to SFS (62 micrograms.g-1). It was, however, reduced to 41 micrograms.g-1 in IS. In SFS the circulating glucose pool had to be doubled to induce significantly elimination of the injected glucose. Tripled pools were eliminated with half-times of 45 min, whereas lower and higher glucose loads were eliminated significantly slower (half-times: 80-105 min). Glucose tolerance of SS was reduced: half-times were doubled, and metabolization of injected glucose was reduced. Since tissue fresh weights were lowered by 40%, absolute incorporation of 14C from labeled glucose was lowered, but specific incorporation (per mg) was higher than in SFS and IS. Glucose tolerance of IS was increased: metabolic clearance rates rose by 70% and half-times were shortened by 30%, though absolute and specific rates of 14C incorporation were lowered. However, IS lost 25% of the label to the water, whereas SFS lost 12% and SS only lost 8%. Using the antimetabolite 2-deoxyglucose, 80% of the losses proved to be glucose in IS, and 50% in SFS. The present results suggest the existence of a glucostatic regulation in B. glabrata with lower sensitivity and capacity than in mammals. As to glucose tolerance, the often reported parallelism in metabolic shifts induced by starvation and parasitic infection was not confirmed.

Heart Rate and Hemolymph Pressure Responses to Hemolymph Volume Changes in the Land Crab Cardisoma guanhumi: Evidence for "Baroreflex" Regulation

Hemolymph volume in restrained land crabs (Cardisoma guanhumi) was altered in 5% increments by rapid infusion of filtered seawater into, or rapid withdrawal of hemolymph from, an infrabranchial sinus. Associated changes in intracardiac hemolymph pressure and in heart rate were measured. Elevation of hemolymph volume resulted in a brief (<20 s) rise in hemolymph pressure followed by a return toward control levels. Heart rate changed in a reciprocal fashion to hemolymph pressure, briefly decreasing during and immediately following infusion and then rising back toward control levels. The net effect after infusion or withdrawal was a readjustment in both hemolymph pressure and heart rate to stable new levels. A significant inverse correlation between heart rate and hemolymph pressure was evident, with heart rate decreasing approximately 2 beats · min⁻¹ · mmHg⁻¹ rise in hemolymph pressure. Collectively, these data indicate that changes in hemolymph pressure result in reflex adjustments in heart rate in a manner analogous to baroreceptor reflexes in vertebrates.

Quantitation of Antisporozoite Immunoglobulins in the Hemolymph of Anopheles Stephensi after Bloodfeeding

Passage of rat antibodies induced by Plasmodium falciparum circumsporozoite protein (anti-CS IgG) from the bloodmeal into the hemocoel of uninfected Anopheles stephensi mosquitoes was quantified using enzyme-linked immunosorbent assay (ELISA) techniques. Anti-CS IgG were present in hemolymph immediately upon cessation of mosquito feeding. Titers peaked at 3 hr post-ingestion then declined steadily, becoming negligible at 18 hr. Substantial titers were present in the bloodmeal at 24 hr post-ingestion. By 48 hr, anti-CS IgG in both mosquito hemolymph and bloodmeal were virtually absent. Estimated quantities of anti-CS IgG in the hemolymph at 3 hr post-ingestion were 905-958 ng/ml, representing approximately 0.5% of that present in the host serum. Rat IgG subclasses 1, 2a, and 2b passed into hemolymph more readily than did IgM and possibly IgG2c. Hemolymph volume of unengorged mosquitoes (0.53 microliters) increased after a bloodmeal (0.73 microliters at 3 hr post-ingestion), suggesting that anti-CS IgG may move into the hemocoel in an aqueous solution.

Haemolymph protein composition and copper levels in decapod crustaceans

Variations in haemolymph protein composition and concentration, in copper content and copper distribution in the tissue of decapod crustaceans are reviewed. Haemocyanin is the major haemolymph constituent (> 60%); the remaining proteins (in order of concentration) include coagulogen, apohaemocyanin, hormones and antisomes. Moulting, nutritional state, infection, hypoxia and salinity fluctuations are the major factors affecting the relative proportions and total quantities of the haemolymph proteins. With regard to haemocyanin, the changes in concentration during the moult cycle are principally associated with changes in haemolymph volume, rather than with changes in total haemocyanin content due to synthesis or catabolism. The role of the midgut gland in regulating haemolymph copper and haemocyanin concentration has been re-evaluated. More than 50% of the whole body copper load is stored in the haemolymph. In contrast, less than 3% of the copper load resides in the midgut gland. The latter has little potential for regulating haemolymph copper levels, at least in the short term (hours to a few days), though it may be involved in regulating haemocyanin levels over longer periods (weeks to months). The total copper content of the haemolymph remains within a narrow range, except during starvation when levels may decrease. Consequently, variations in the copper content of soft tissues, which constitute only 20% of decapod dry weight, do not significanlty alter whole body copper concentrations. Evidence that copper released following haemocyanin catabolism becomes bound to metallothionein for later use in the resynthesis of haemocyanin is reviewed and found to be inconclusive. The amount of copper that can be stored in this way is trivial compared with the amount of copper required to permit significant changes in haemolymph haemocyanin concentration. Average tissue copper requirements, calculated during the present study, are approx. 4 times higher than previous theoretical estimates.

Hemocyanin levels in the giant keyhole limpet, Megathura crenulata, from the coast of California

1. 1. The concentration of hemocyanin in the hemolymph of the giant keyhole limpet Megalhura crenulata, has been determined. 2. 2. In 56 specimens of M. crenulata the hemocyanin concentration ranged from 1.53 to 10.81 g/1 with a mean of 5.45 g/1 and a standard deviation of 2.71 g/1. There is no correlation between the hemocyanin concentration and the weight of the animal. The hemolymph volume and the colour of the mantle are not related to the hemocyanin level either. 3. 3. The hemocyanin concentration shows a seasonal variation, the means and the standard deviations for February, April, July, and September being 6.48 ± 2.58, 4.44 ± 1.72, 2.62 ± 1.05, and 7.65 ± 1.54 g/1, respectively. 4. 4. The lability of the hemocyanin concentration in Megathura (7-fold variation) is less pronounced than it is in California Haliotis, but comparable to that of the Australian abalones. 5. 5. It is difficult to reconcile the wide fluctuations observed in the concentration of hemocyanin with an essential respiratory task for this protein.

Regulation of hemolymph volume by uptake of sand capillary water in desiccated fiddler crabs, UCA pugilator and UCA pugnax

1. 1. Desiccated Uca pugilator and Uca pugnax can rehydrate on damp sand. 2. 2. The crabs use setal tufts between the second and third pairs of walking legs and branchial chamber suction of up to -43 mmHg to extract sand capillary water, which is absorbed by the gills and/or drunk. 3. 3. Desiccated crabs of both species have a limited ability to generate “free water” from sand capillary water which they obtain from the substratum by suction; thus, although they can regulate hemolymph volume, they cannot regulate hemolymph osmotic pressure by using sand capillary water. 4. 4. The most likely use of this mechanism by the crabs in nature is for obtaining water from the substratum for flotation deposit feeding.

Changes in the hemolymph of the stable fly, Stomoxys calcitrans, after a blood meal

AbstractThe stable fly hemolymph was analyzed after a blood meal. The hemolymph volume increased to approximately three times the pre‐feeding level 3–6 h after a blood meal and gradually returned to normal 18 h after the blood meal. The osmotic pressure decreased approximately 10% following a blood meal and gradually returned to normal with a pattern that was a mirror‐image of that of the hemolymph volume. Concentrations of cations and anions are not directly affected by the ingested blood, indicating a possible selective excretory mechanism.

Heart and circulatory functions in a spider (Eurypelma californicum): the effects of hydraulic force generation

In the tarantulaEurypelma californicum, the relationships between heart activity, circulation and the generation of hydraulic pressure for locomotion were studied. Several new techniques were employed. Mean resting heart rate was 21 beats min−1 rising to 90 beats min−1 after burst activity. Decay time to resting rates was related to the increase of heart rate. Post-recovery resting rates were usually elevated in comparison with rates after very long resting periods. A relative measure of heart amplitude was obtained. Four distinct patterns could be distinguished: (i) regular beats; (ii) short-term fluctuations of amplitude within a few heart beats; (iii) a slow rhythmic change of heart/pericardium filling, and (iv) non-periodic, stronger amplitude changes during periods of activity. During locomotion, heart rate rises with maximum rates often reached only minutes after the onset of activity. The rising phase is often characterized by irregularities and a reduction of heart amplitude. Prosomal hemolymph pressure in resting, restrained animals was 41±19 Torr, rising to ca. 90, and 217±48 Torr during walking and fast sprints, respectively. Values in unrestrained spiders were similar. Opisthosomal hemolymph pressures were ca. 20 Torr in resting animals, rising to 40–60 Torr during locomotion. Opisthosomal volume changes were measured. A small volume of hemolymph moved from the prosoma to the opisthosoma at the onset of locomotion, but following activity this volume quickly returned to the prosoma. The simultaneous measurement of carapace depression, opisthosomal volume changes and hemolymph pressures, and heart activity revealed the relationship between circulation and hydraulic force generation. The direction of hemolymph flow was also studied. In non-active animals, the heart occasionally changes its main pumping direction. During locomotion, hemolymph flow from the heart to the prosoma is often reduced or stopped. With a slight delay, hemolymph flow to the opisthosoma is increased. The critical pressure at which prosomal perfusion from the heart is halted is 50–70 Torr. It is concluded that anterior and posterior circulations are separate: hemolymph returning from the prosoma passes only through the anterior lungs, while hemolymph returning from the opisthosoma passes through the posterior lungs.

The influence of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: in vivo and in vitro diuresis and production of diuretic hormone.

The homoxenous flagellate Blastocrithidia triatomae disturbs excretion in Triatoma infestans. During the first 4 and 24 h after feeding, infected 5th instars excreted approximately a 2.5-fold smaller volume of urine. Consequently, about double the normal volume of haemolymph could be obtained from these infected bugs 24 h after feeding. Surprisingly, the in vitro secretion rates of Malpighian tubules from uninfected and infected bugs were nearly identical. In addition, the storage and release of diuretic hormone in infected bugs was sufficient to induce normal secretion rates by isolated Malpighian tubules. Therefore, the previously observed ultrastructural alterations in the upper Malpighian tubules of infected bugs do not result in a disturbed in vitro secretion, although in vivo excretion is reduced.