Osmoregulatory disturbances induced by the parasitic barnacle Loxothylacus texanus (Rhizocephala) in the crab Callinectes rathbunae (Portunidae)

The osmoregulatory response of the blue crab Callinectes rathbunae parasitized with the rhizocephalan barnacle Loxothylacus texanus, and subjected to sudden salinity changes, was experimentally measured in the laboratory. Parasitized and control crabs were exposed to salinity changes every 3 h and their hemolymph osmolality measured. Two experiments, one with increasing salinity conditions (5‰, 12‰, 19‰, 25‰) and a second one with decreasing salinities (35‰, 25‰, 15‰, 5‰) were conducted. The results show that L. texanus significantly alters the hemolymph osmolality of C. rathbunae maintaining it at lower than normal levels. In the increasing salinity trial, the hypoosmotic hemolymph condition of parasitized crabs was present at all salinities tested, whereas in the decreasing salinity trial a significant effect was found only at salinities of 5‰ and 15‰. Since C. rathbunae is constantly subjected to abrupt salinity changes in the tropical estuaries where it occurs, moving into high salinity areas may be the only way to cope with the impact of L. texanus.     

Effect of ambient extracellular glutamate on <Emphasis Type="Italic">Drosophila</Emphasis> glutamate receptor trafficking and function

Measurements suggest that the hemolymph glutamate concentrations in Drosophila are relatively high. This raises the possibility that extracellular glutamate could be an important regulator of glutamatergic transmission in vivo. Using voltage clamp electrophysiology, we found that synaptic currents in D. melanogaster larval neuromuscular junctions are reduced by extracellular glutamate (EC50: ~0.4 mM), such that only 10–30% of receptors were functionally available in 1 mM extracellular glutamate. The kinetics of synaptic currents were also slowed in a dose-dependent fashion (EC50: ~1 mM), consistent with the idea that extracellular glutamate preferentially removes the fastest-desensitizing receptors from the functional pool. Prolonged exposure (several hours) to extracellular glutamate also triggers loss of glutamate receptor immunoreactivity from neuromuscular junctions. To determine whether this receptor loss requires that glutamate bind directly to the lost receptors, we examined glutamate-dependent loss of receptor immunoreactivity in larvae with glutamate receptor ligand binding mutations. Our results suggest that glutamate-dependent receptor loss requires binding of glutamate directly to the lost receptors. To determine whether lost receptor protein is degraded or merely redistributed, we used immunoblots. Results suggest that glutamate receptor protein is redistributed, but not degraded, after prolonged exposure to high extracellular glutamate. K. Chen and H. Augustin contributed equally to this work.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

肌注和药饵给药下诺氟沙星在南美白对虾血淋巴中药代动力学

本文分析了盐度1‰和15‰下诺氟沙星肌肉注射给药(剂量10mg/kg)和盐度15‰下药饵口服给药(剂量15mg/kg和30mg/kg)后南美白对虾血淋巴中药代动力学。在盐度1‰和15‰下,南美白对虾肌注给药后血药浓度的变化趋势基本相似,血药浓度与时间关系曲线适合用二室模型来描述,其药动学方程分别为C0=35.422×e-9.778t 4.363×e-0.165t和C0=35.144×e-13.335t 7.888×e-0.608t,但两盐度下部分药动学参数差别较大。药饵口服给药时,给药剂量与血药浓度未呈明显的正相关,且血药浓度—时间关系曲线均表现出双峰现象。以30mg/kg剂量药饵口服给药后,药峰出现的时间分别为给药后4h和12h,峰浓度分别为2.86μg/mL和2.04μg/mL;以15mg/kg剂量给药时也得到相似的双峰现象,但第二峰浓度出现的时间为8h。     

泥蚶(Tegillarca granosa Linnaeus)血淋巴液凝集素的分离纯化及其性质研究

泥蚶是一种重要的海产经济贝类,其血淋巴液经硫酸铵二步分级沉淀后,再经Sephadex G- 100凝胶过滤和Sepharose 4B亲和层析纯化制得泥蚶血淋巴液凝集素。经测定,该凝集素分子量约为123Kda,为两个亚基的蛋白质,其相对分子量分别为15 KDa和16 KDa,分子中含5．02％的糖。在氨基酸组成中,天门冬氨酸(Asp)含量最高,其次是谷氨酸(Glu)和组氨酸(His),不含蛋氨酸(Met)。泥蚶血淋巴液凝集素对多种天然或经酶修饰的人或动物红细胞具有不同的凝集作用,其中对兔红细胞的凝集活性最大。半乳糖和乳糖对其凝集活性具有抑制作用。凝集活性依赖于Ca2 ,在pH7．0较稳定,热稳定性不高,在30℃-70℃时凝集效价由原来的25下降为21,当温度超过80℃以后,凝血活性完全丧失。     

Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta

The pathway for the synthesis of diacylglycerol in larval Manduca sexta midgut was studied. Fifth instar larvae were fed with [9,10–³H]–oleic acid–labeled triolein and the incorporation of the label into lipid intermediates was analyzed as a function of time. The results showed that the triacylglycerol was hydrolyzed to fatty acids and glycerol in the midgut lumen. In midgut tissue, the labeled fatty acids were rapidly incorporated into phosphatidic acid, diacylglycerol and triacylglycerol, but no significant labeling of monoacylglycerol was observed. Dual-labeling experiments were performed in order to characterize the kinetics of diacylglycerol biosynthesis in the midgut, its incorporation into hemolymph lipophorin and its clearance from hemolymph. The results were best described by a model in which the rate-limiting step in diacylglycerol biosynthesis was the uptake of fatty acid from the lumen of the midgut. Once in the cell the fatty acid was rapidly incorporated in phosphatidic acid and diacylglycerol. Diacylglycerol was converted to triacylglycerol or exported into hemolymph. The interconversion of diacylglycerol and triacylglycerol was fairly rapid, suggesting that triacylglycerol serves as a reservoir from which diacylglycerol can be produced. This mechanism permits the cell to maintain a low steady-state concentration of diacylglycerol and yet efficiently absorb fatty acids from the lumen of the midgut.     

Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.     

In vitro chemotaxis of Brugia pahangi infective larvae to the sera and hemolymph of mammals and lower animals

The jird (Mongolian gerbil) is a highly susceptible experimental host for the lymphatic filarial nematode, Brugia pahangi. The chemotactic activity of serum from this host for B. pahangi infective larvae was compared in vitro to that of sera or hemolymph of a wide variety of other organisms including mammals, reptiles, fishes and invertebrates. The range of the Chemotactic Index (CI) was from 96.0 for the jird to 56.2 for a snail. An average of CI of saline control was 4.5. Significant chemotactic activity was present in many organisms, especially mammals, but was not closely related to either the phylogenetic position of the organism and to its known susceptibility as definitive host for B. pahangi. Migratory response was diminished in a consistent way by serial dilution of sera of humans, jirds and fetal bovine serum. Pre-incubation of larvae in fetal bovine serum inhibited migration, especially towards the sera of humans. Inhibition could be reversed by rinsing larvae in saline, longer rinse periods resulting in greater recovery of CI. These results are the first to suggest the activity of the specific amphid chemoreceptors in the chemotaxis of the infective larvae of B. pahangi.     

Morphological and quantitative aspects of nodule formation in hemolymph of the blowfly Chrysomya megacephala (Fabricius, 1794)

Insects manifest effective immune responses that include both cellular and humoral components. Morphological and quantitative aspects of cellular and humoral cooperation during nodule formation in Chrysomya megacephala hemolymph against Saccharomyces cerevisae yeast cells were demonstrated for the first time. The analyses were performed in non-injected larvae (NIL), saline-injected larvae (SIL) and yeast-injected larvae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24, 36, or 48-h post-injection. Morphological aspects of YIL nodulation were investigated using transmission electron microscopy (TEM). Quantitative analyses consisted of total (THC) and differential hemocyte counts (DHC) in all the groups and total yeast count (TYC) in YIL, which were performed in an improved Neubauer chamber. Nodule formation was initiated at approximately 2-h post-injection. Twelve hours after the injection, TEM revealed the presence of an amorphous membrane, at the same time that circulating hemocyte number decreased significantly contrasting the increase of yeast number. Our results showed the ability of C. megacephala hemolymph to perform humoral encapsulation when hemocyte population is insufficient to eliminate the microorganisms, warranting consideration in future investigations on the relative roles played by cellular and humoral elements of innate immunity of this calliphorid.