The prophenoloxidase system and in vitro interaction of Trypanosoma rangeli with Rhodnius prolixus and Triatoma infestans haemolymph

The presence of the prophenoloxidase (proPO) system in the haemolymph of Rhodnius prolixus and Triatoma infestans and the role played by Trypanosoma rangeli in the in vitro activation of proPO were tested. Both R. prolixus and T. infestans whole blood preparations showed a very active ProPO system. The proPO cascade of the two insect species were differentially activated by microbial-derived extracts: laminarin was a better activator of T. infestans haemolymph than of R. prolixus blood, and lipopolysaccharides from Shigella flexneri or Pseudomonas aeroginosa caused significant proPO activation of T. infestans haemolymph but not of R. prolixus preparations. For the two insect species, neither T. rangeli from culture nor parasite lysates were able to trigger proPO activation. The presence of the parasite in R. prolixus haemolymph/laminarin assays, however, significantly reduced the level of proPO activation to that of spontaneous activating controls. The immobilization of T. rangeli in vitro in haemolymph preparations occurred in both insect species and was dependent on the proPO activation intensity. Our results suggest that the susceptibility of R. prolixus to T. rangeli haemocoel infection may be explained, at least in part, by the suppression of the insect immune defence system i.e., inhibition of proPO in the presence of this protozoan parasite.

Temperature dependent growth and inorganic ion composition of haemolymph of juvenile Sepia officinalis, 2006

B Body wet weight and mantle length of juvenile Sepia officinalis were monitored over a peroid of five weeks. The animals had hatched in our aquarium system in Bremerhaven, Germany at 16°C and were descendants of individuals collected in the Oosterschelde estuary, Netherlands. Animals were kept in natural sea water at 10 or 17°C and fed small live shrimp (Palaemonetes varians) ad libitum daily. At the end of the experiment some animals kept at 17°C were sacrificed using ethanol. Haemolymph was withdrawn from the head vein using syringe and needle. Haemolymph samples were stored at -20°C until Na+, Cl-, K+, Mg2+, Ca2+ and SO42- concentrations were determined using ion chromatography. Mean body weight more that tripled at 17°C during the investigation period, while growth was impared by exposue to 10°C. Haemolymph ion concentrations were similar to those in sea water, except for sulphate. The concentration of this ion in the haemolymph was more that ten times lower than in sea water.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and ambient magnesium concentration in early life history stages of Paralomis granulosa (Decapoda, Anomura, Lithodidae)

Marine brachyuran and anomuran crustaceans are completely absent from the extremely cold (-1.8 °C) Antarctic continental shelf, but caridean shrimps are abundant. This has at least partly been attributed to low capacities for magnesium excretion in brachyuran and anomuran lithodid crabs ([Mg2+]HL = 20-50 mmol/L) compared to caridean shrimp species ([Mg2+]HL = 5-12 mmol/L). Magnesium has an anaesthetizing effect and reduces cold tolerance and activity of adult brachyuran crabs. We investigated whether the capacity for magnesium regulation is a factor that influences temperature-dependent activity of early ontogenetic stages of the Sub-Antarctic lithodid crab Paralomis granulosa. Ion composition (Na+, Mg2+, Ca2+, Cl-, [SO4]2-) was measured in haemolymph withdrawn from larval stages, the first and second juvenile instars (crabs I and II) and adult males and females. Magnesium excretion improved during ontogeny, but haemolymph sulphate concentration was lowest in the zoeal stages. Neither haemolymph magnesium concentrations nor Ca2+:Mg2+ ratios paralleled activity levels of the life stages. Long-term (3 week) cold exposure of crab I to 1 °C caused a significant rise of haemolymph sulphate concentration and a decrease in magnesium and calcium concentrations compared to control temperature (9 °C). Spontaneous swimming activity of the zoeal stages was determined at 1, 4 and 9 °C in natural sea water (NSW, [Mg2+] = 51 mmol/L) and in sea water enriched with magnesium (NSW + Mg2+, [Mg2+] = 97 mmol/L). It declined significantly with temperature but only insignificantly with increased magnesium concentration. Spontaneous velocities were low, reflecting the demersal life style of the zoeae. Heart rate, scaphognathite beat rate and forced swimming activity (maxilliped beat rate, zoea I) or antennule beat rate (crab I) were investigated in response to acute temperature change (9, 6, 3, 1, -1 °C) in NSW or NSW + Mg2+. High magnesium concentration reduced heart rates in both stages. The temperature-frequency curve of the maxilliped beat (maximum: 9.6 beats/s at 6.6 °C in NSW) of zoea I was depressed and shifted towards warmer temperatures by 2 °C in NSW + Mg2+, but antennule beat rate of crab I was not affected. Magnesium may therefore influence cold tolerance of highly active larvae, but it remains questionable whether the slow-moving lithodid crabs with demersal larvae would benefit from an enhanced magnesium excretion in nature.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and magnesium in adult Paralomis granulosa (Decapoda, Anomura, Lithodidae)

A low capacity for regulation of extracellular Mg2+ has been proposed to exclude reptant marine decapod crustaceans from temperatures below 0°C and thus to exclude them from the high Antarctic. To test this hypothesis and to elaborate the underlying mechanisms in the most cold-tolerant reptant decapod family of the sub-Antarctic, the Lithodidae, thermal tolerance was determined in the crab Paralomis granulosa (Decapoda, Anomura, Lithodidae) using an acute stepwise temperature protocol (-1°, 1°, 4°, 7°, 10°, and 13°C). Arterial and venous oxygen partial pressures (Po2) in hemolymph, heartbeat and ventilation beat frequencies, and hemolymph cation composition were measured at rest and after a forced activity (righting) trial. Scopes for heartbeat and ventilation beat frequencies and intermittent heartbeat and scaphognathite beat rates at rest were evaluated. Hemolymph [Mg2+] was experimentally reduced from 30 mmol/L to a level naturally observed in Antarctic caridean shrimps (12 mmol/L) to investigate whether the animals remain more active and tolerant to cold (-1°, 1°, and 4°C). In natural seawater, righting speed was significantly slower at -1° and 13°C, compared with acclimation temperature (4°C). Arterial and venous hemolymph Po2 increased in response to cooling even though heartbeat and ventilation beat frequencies as well as scopes decreased. At rest, ionic composition of the hemolymph was not affected by temperature. Activity induced a significant increase in hemolymph [K+] at -1° and 1°C. Reduction of hemolymph [Mg2+] did not result in an increase in activity, an increase in heartbeat and ventilation beat frequencies, or a shift in thermal tolerance to lower temperatures. In conclusion, oxygen delivery in this cold-water crustacean was not acutely limiting cold tolerance, and animals may have been constrained more by their functional capacity and motility. In contrast to earlier findings in temperate and subpolar brachyuran crabs, these constraints remained insensitive to changing Mg2+ levels.

Acid-base balance and changes in haemolymph properties of the South African rock lobsters, Jasus lalandii, a palinurid decapod, during chronic hypercapnia

Few studies exist reporting on long-term exposure of crustaceans to hypercapnia. We exposed juvenile South African rock lobsters, Jasus lalandii, to hypercapnic conditions of pH 7.3 for 28 weeks and subsequently analysed changes in the extracellular fluid (haemolymph). Results revealed, for the first time, adjustments in the haemolymph of a palinurid crustacean during chronic hypercapnic exposure: 1) acid-base balance was adjusted and sustained by increased bicarbonate and 2) quantity and oxygen binding properties of haemocyanin changed. Compared with lobsters kept under normocapnic conditions (pH 8.0), during prolonged hypercapnia, juvenile lobsters increased bicarbonate buffering of haemolymph. This is necessary to provide optimum pH conditions for oxygen binding of haemocyanin and functioning of respiration in the presence of a strong Bohr Effect. Furthermore, modification of the intrinsic structure of the haemocyanin molecule, and not the presence of molecular modulators, seems to improve oxygen affinity under conditions of elevated pCO2.

Haemolymph inorganic ion composition of crustaceans from the Southern Ocean

Brachyuran and anomuran decapod crabs do not occur in the extremely cold waters of the Antarctic continental shelf whereas caridean and other shrimp-like decapods, amphipods and isopods are highly abundant. Differing capacities for extracellular ion regulation, especially concerning magnesium, have been hypothesised to determine cold tolerance and by that the biogeography of Antarctic crustaceans. Magnesium is known to have a paralysing effect, which is even more distinct in the cold. As only few or no data exist on haemolymph ionic composition of Sub-Antarctic and Antarctic crustaceans, haemolymph samples of 12 species from these regions were analysed for the concentrations of major inorganic ions (Na+, K+, Ca2+, Mg2+, Cl-, SO4 2-) by ion chromatography. Cation relationships guaranteed neuromuscular excitability in all species. Sulphate and potassium correlated positively with magnesium concentration. The Antarctic caridean decapod as well as the amphipods maintained low (6-20% of ambient sea water magnesium concentration), Sub-Antarctic brachyuran and anomuran crabs as well as the Antarctic isopods high (54-96% of ambient sea water magnesium concentration) haemolymph magnesium levels. In conclusion, magnesium regulation may explain the biogeography of decapods, but not that of the peracarids.

(Table 1) Ion concentrations in the haemolymph of Apherusa glacialis after incubation at different medium salinities

Amphipods living at the underside of Arctic sea ice are exposed to varying salinities due to freezing and melting, and have to cope with the resulting osmotic stress. Extracellular osmotic and ionic regulation at different salinities, thermal hysteresis, and supercooling points (SCPs) were studied in the under-ice amphipod Apherusa glacialis. The species is euryhaline, capable to regulate hyperosmotically at salinities S(R) < 30 g/kg, and osmoconforms at salinities S(R) >= 30 g/kg. Hyperosmotic regulation is an adaptation to thrive in low-salinity meltwater below the ice. Conforming to the ambient salinity during freezing reduces the risk of internal ice formation. Thermal hysteresis was not observed in the haemolymph of A. glacialis. The SCP of the species was -7.8 ± 1.9°C. Several ions were specifically downregulated ([Mg2+], [SO4]2-), or upregulated ([K+], [Ca2+]) in comparison to the medium. Strong downregulation of [Mg2+], is probably necessary to avoid an anaesthetic effect at low temperatures.

The binding of closantel to ovine serum albumin, and homogenate fractions of Haemonchus contortus

Closantel binds to the serum proteins of the host and affects blood sucking parasites when they ingest the brood of treated hosts. Closantel binds specifically to ovine serum albumin (K-a of 9.3 x 10(6)M(-1)) at site I, the warfarin/phenylbutazone binding site of albumin Closantel also binds to invertebrate haemocyanin and haemolymph. The strongest binding of closantel in homogenates of H. contortus is found in fractions containing soluble proteins. This binding is of low affinity and, because the site itself is not fully denaturable, it may not be proteinaceous. There is no detectable difference in binding affinity between homogenate fractions from closantel susceptible and resistant isolates of adult or larval worms suggesting that closantel resistance is not due to changes in the closantel receptor or carrier. (C) 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Solution structure of BSTI: A new trypsin inhibitor from skin secretions of Bombina bombina

The three-dimensional solution structure of BSTI, a trypsin inhibitor from the European frog Bombina bombina, has been solved using H-1 NMR spectroscopy. The 60 amino acid protein contains five disulfide bonds, which were unambiguously determined to be Cvs (4-38), Cys (13-34), Cys (17-30), Cys (21-60), and Cys (40-54) by experimental restraints and subsequent structure calculations. The main elements of secondary structure are four beta -strands, arranged as two small antiparallel beta -sheets, The overall fold of BSTI is disk shaped and is characterized by the lack of a hydrophobic core. The presumed active site is located on a loop comprising residues 21-34, which is a relatively disordered region similar to that seen in many other protease inhibitors. However, the overall fold is different to other known protease inhibitors with the exception of a small family of inhibitors isolated from nematodes of the family Ascaris and recently also from the haemolymph of Apis mellifera. BSTI may thus be classified as a new member of this recently discovered family of protease inhibitors.

Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells ill a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Dinoflagellate symbiosis: strategies and adaptations for the aquisition and fixation of inorganic carbon

Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O-2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome. this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3- and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.