Hemolymph Concentrations Research Articles

A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2–6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6–12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.

Effect of 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on sperm quality and sperm quantity in male mud spiny lobster (Panulirus polyphagus).

The present study aimed to determine the effect of 17α-hydroxyprogesterone (17α-OHP) and 17α-hydroxypregnenolone (17α-OHPL) on sperm quality and sperm quantity in male mud spiny lobster (Panulirus polyphagus). The mean of sperm quality was increased in 17α-OHP and 17α-OHPL treated hormones. In 17α-OHP injected animals, the mean of sperm quantity of dose 0.01 μg g(-1) b.wt. was increased than 17α-OHPL. Meanwhile, 17α-OHP and 17α-OHPL concentrations were lower when injected with the hormones but 17α-OHP was higher at only day 15 (dose 0.01 and 0.1 μg g(-1) b.wt.). For 17α-OHPL, the hormone was a prohormone in the body of P. polyphagus and only required smaller to increase the sperm quantity. Besides, when the higher dose of 17α-OHPL (0.1 μg g(-1) b.wt.) was used in P. polyphagus, the development of P. polyphagus was inhibited and decreased the sperm quantity and 17α-OHPL concentration in hemolymph was lower. Injection of 17α-OHP in P. polyphagus has increased the sperm quality and quantity for both 17α-OHP dosage of 0.01 and 0.1 μg g(-1) b.wt. However, injection of 17α-OHPL in P. polyphagus has decreased the sperm quantity only, also for both dosage of 0.01 and 0.1 μg g(-1) b.wt. and lower on hormone concentration.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; comparison of the alkali-labile phosphate method (ALP) and a species-specific immunoassay (ELISA)

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17β-estradiol (57, 164 and 512ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus. The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed.

Excretion of NaCl and KCl loads in mosquitoes. 2. Effects of the small molecule Kir channel modulator VU573 and its inactive analog VU342.

The effect of two small molecules VU342 and VU573 on renal functions in the yellow fever mosquito Aedes aegypti was investigated in vitro and in vivo. In isolated Malpighian tubules, VU342 (10 μM) had no effect on the transepithelial secretion of Na(+), K(+), Cl(-), and water. In contrast, 10 μM VU573 first stimulated and then inhibited the transepithelial secretion of fluid when the tubules were bathed in Na(+)-rich or K(+)-rich Ringer solution. The early stimulation was blocked by bumetanide, suggesting the transient stimulation of Na-K-2Cl cotransport, and the late inhibition of fluid secretion was consistent with the known block of AeKir1, an Aedes inward rectifier K(+) channel, by VU573. VU342 and VU573 at a hemolymph concentration of about 11 μM had no effect on the diuresis triggered by hemolymph Na(+) or K(+) loads. VU342 at a hemolymph concentration of 420 μM had no effect on the diuresis elicited by hemolymph Na(+) or K(+) loads. In contrast, the same concentration of VU573 significantly diminished the Na(+) diuresis by inhibiting the urinary excretion of Na(+), Cl(-), and water. In K(+)-loaded mosquitoes, 420 μM VU573 significantly diminished the K(+) diuresis by inhibiting the urinary excretion of K(+), Na(+), Cl(-), and water. We conclude that 1) the effects of VU573 observed in isolated Malpighian tubules are overwhelmed in vivo by the diuresis triggered with the coinjection of Na(+) and K(+) loads, and 2) at a hemolymph concentration of 420 μM VU573 affects Kir channels systemically, including those that might be involved in the release of diuretic hormones.

Sex-specific divergence for body size and desiccation-related traits in Drosophila hydei from the western Himalayas

Sex-specific-differences are a widespread source of genetic variation in various Drosophila species. In the present study, we have examined desiccation survival in males and females of Drosophila hydei from colder and drier montane conditions of the western Himalayas (altitudinal populations; 600–2202m). In contrast with most other studies in drosophilids, D. hydei males exhibited comparatively higher desiccation resistance despite smaller body size compared to females. Accordingly, we tested the physiological basis of such adaptations in both sexes of D. hydei. Body size traits (wing length, wet weight and dry weight) were ~1.2 fold higher in females than males. However, desiccation resistance was 10 to 13h higher in males than females. These differences matched enhanced storage of trehalose content (~1.2 fold), higher hemolymph content (~1.2 fold) and enhanced cuticular lipid mass (~1.5 fold) in males than females. Water loss before succumbing to death (dehydration tolerance) was much higher in males (~81%) than females (~64%). A greater loss of hemolymph water until death under desiccation stress was associated with higher desiccation resistance in males. Further, there were lacks of differences in the rate of water loss, rate of trehalose utilization and rate of hemolymph depletion between the sexes in D. hydei. Therefore, sex-specific differences in desiccation resistance of D. hydei were independent of body size as well as the exhaustion of metabolite reserves and rather were caused by the higher dehydration tolerance as well as higher acquisition of hemolymph and trehalose contents.

Sex-specific differences in the physiological basis of water conservation in the fruit flyDrosophila hydeifrom the western Himalayas

In the cosmopolitan fruit fly Drosophila hydei Sturtevant, 1921 (Diptera: Drosophilidae), the relative abundance of males is significantly higher than females, but the physiological basis of such sex-specific differences are largely unknown. For wild populations of D. hydei, we found seasonal changes (summer versus autumn) in desiccation-related traits, but the desiccation tolerance of males was higher than that of females in all seasons. For desiccation-related traits, we tested whether thermal developmental acclimation at three temperatures (17, 21, and 28 °C) matched seasonal changes observed under wild conditions. Male flies showed significantly higher trait values for desiccation resistance, cuticular lipid mass, hemolymph content, carbohydrate content, and dehydration tolerance compared with females when reared at lower or higher temperatures despite the lack of significant sex-specific differences in the total body-water content of flies reared at a particular growth temperature. We observed plastic changes in the amount of cuticular lipids consistent with corresponding differences in the rate of water loss. Treatment of cuticular surface with organic solvent (hexane) supported the role of cuticular lipids in affecting transcuticular water loss. We found significant thermal plastic effects for desiccation-related traits of D. hydei, but the sexual dimorphism was in the opposite direction, i.e., males were more desiccation resistant than females in D. hydei, whereas the reverse is true for many other Drosophila species. Our results suggest that sex-specific differences in the level of desiccation resistance in D. hydei are good predictors of relative abundance levels of male and female flies under wild conditions.

Laboratory Evaluation of the Biochemical Parameters in the Haemolymph of the Lepidopteran Larvae after Stinging by the Potter Wasp, Eumenes Conica (Insecta: Hymenoptera)

Potter wasps belong to the subfamily Eumeninae of the family Vespidae. Potter wasp is a common name given for a group of caterpillar hunting wasp which builds the pot-shaped mud nests. Initially the wasp constructs the nest and then starts hunting for its prey, the caterpillars. The prey is stung and paralyzed by the wasp then brought to the nest. It is perhaps a very highly specific behavior of these wasps. The female wasp lays her egg on the prey. The egg is firmly attached to the prey and the larva hatching out sucks the haemocoel which oozes out from the prey. In the present study, the biochemical changes in the haemolypmh of Helicoverpa armigera (Lepidoptera: Noctuidae), before and after stinging by the potter wasp were observed. There is considerable change in the nutritional physiology of parasitized prey. The successful development of the parasitoid depends on the concentration of the host haemolymph. Hosts do not survive and thus parasitoids play an important role in regulating the population of the hosts

Function of an anti-lipopolysaccharide factor (ALF) isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection

In this study, a 780-bp full-length cDNA encoding Macrobrachium rosenbergii anti-lipopolysaccharide factor (MrALF) from hemocytes was cloned and identified. The ALF isoform exhibited immune activities, and its concentration in hemolymph was determined. An in vivo expression study showed that the ALF mRNA level of hemocytes could be induced by lipopolysaccharides (LPSs) in an exposure time-dependent manner. Purified recombinant MrALF (rMrALF) expressed in the yeast Pichia pastoris SMD1168 eukaryotic protein expression system demonstrated antibacterial activity against the Gram-negative prawn pathogen Aeromonas hydrophila (minimum inhibitory concentration (MIC)=0.806μM, minimum bactericidal concentration (MBC)=1.606μM) but not the Gram-positive pathogen Lactococcus garvieae exposed to 25.696μM of rALF. However, rMrALF can bind to Gram-negative and -positive bacteria. An in vivo expression study demonstrated that the ALF concentrations in prawn hemocytes and plasma were 0.176μM and 0.168μM, respectively; following LPS treatment for 6h, the corresponding concentrations were 0.133μM in hemocytes and 0.272μM in plasma. Furthermore, the percentage of hemocytes phagocytosing bacteria cells was higher in hemoyctes previously treated with MrALF than those treated with sterile medium. These results suggest that in the innate immune response of M. rosenbergii, the MrALF from hemocytes may play an opsonin during a bacterial invasion.

Effect of dietary potassium on growth, nitrogen metabolism, osmoregulation and immunity of pacific white shrimp (Litopenaeus vannamei) reared in low salinity seawater

An 8 weeks feeding experiment was conducted to determine the effect of dietary potassium on the growth and physiological acclimation of Pacific white shrimp (Litopenaeus vannamei) reared in diluted seawater (salinity 4). Six semi-purified practical diets containing 0.59, 0.96, 1.26, 1.48, 1.74, and 2.17 g potassium K+ per 100 g diet were formulated, respectively. The survival and feed conversion rate did not show significant difference among groups of shrimps given these diets (P>0.05). The shrimps fed the diets containing 0.96–1.48 g K+ per 100 g diet gained the highest weight, specific growth rate, and protein efficiency ratio. Their ammonium-N excretion rate as well as hemolymph concentration of Na+ and Cl− were significantly lower than those of the control (P<0.05), but a reverse trend was observed for their gill Na+/K+-ATPase. Moreover, the shrimps fed with 1.48 g K+ per 100 g diet were the highest in hemolymph urea level, and the phenoloxidase and lysozyme activities were significantly higher than those of the control (P<0.05). The growth and physiological response of the test shrimps suggested that diet containing 1.48 g K+ per 100 g diet improved the growth of L. vannamei in low-salinity seawater, and enhanced the physiological acclimation of the organism.

Influence of the porewater geochemistry on Fe and Mn assimilation in Laternula elliptica at King George Island (Antarctica)

A high input of lithogenic sediment from glaciers was assumed to be responsible for high Fe and Mn contents in the Antarctic soft shell clam Laternula elliptica at King George Island. Indeed, withdrawal experiments indicated a strong influence of environmental Fe concentrations on Fe contents in bivalve hemolymph, but no significant differences in hemolymph and tissue concentrations were found among two sites of high and lower input of lithogenic debris. Comparing Fe and Mn concentrations of porewater, bottom water, and hemolymph from sampling sites, Mn appears to be assimilated as dissolved species, whereas Fe apparently precipitates as ferrihydrite within the oxic sediment or bottom water layer prior to assimilation by the bivalve. Hence, we attribute the high variability of Fe and Mn accumulation in tissues of L. elliptica around Antarctica to differences in the geochemical environment of the sediment and the resulting Fe and Mn flux across the benthic boundary.

Divergence of water balance mechanisms and acclimation potential in body color morphs ofDrosophila ananassae

Drosophila ananassae is a desiccation sensitive species, but the physiological basis of its abundance in the drier subtropical areas is largely unknown. We tested the hypothesis whether body color morphs of D. ananassae differ in the mechanistic basis of water conservation as well as desiccation acclimation potential, consistent with their distribution under dry or wet habitats. We observed reduced rate of water loss consistent with the greater desiccation potential of dark morph as compared with light morph, despite lack of quantitative differences in cuticular lipid mass between them. Dark morph evidenced greater wet and dry mass (∼1.17-fold) as well as higher hemolymph content (∼1.70-fold) and (∼17%) dehydration tolerance to sustain longer survival under desiccation stress (LT50 17.5 hr) as compared with light morph (LT50 4.3 hr). We found significant differences in the storage of energy metabolites in the body color morphs of D. ananassae, that is, carbohydrate content was significantly higher (∼0.18 mg/mg dry mass) in the dark morph as compared to light morph, but greater (∼0.05 mg/mg dry mass) body lipid content was evident in the light morph. Under desiccation stress, dark and light morphs utilized mainly carbohydrates but also lipids to a lesser extent. However, the rate of utilization of energy metabolites did not vary between dark and light morphs. Further, the dark morph consumed higher energy content derived from carbohydrates under desiccation stress as compared with the light morph. Finally, we found contrasting patterns of acclimation to desiccation stress in the two body color morphs, that is, increase in desiccation survival (4.7 hr), as well as in dehydration tolerance (∼6%) due to acclimation of the dark morph but no such effects were observed in the light morph. Thus, divergence in water balance mechanisms as well as acclimation potential reflects evolved physiological adaptations of the dark morph under drier but of the light morph to wet climatic conditions.

EFFECT OF STARVATION ON LIPOPHORIN DENSITY IN FIFTH INSTAR LARVAL Manduca sexta

Lipophorin (Lp) is a major insect lipoprotein and is responsible for lipid transport between organs. In this study, the effect of starvation on Lp properties was analyzed in larval Manduca sexta during the fifth instar. Lp hemolymph concentrations in larvae at days 1 and 2 were around 2-3 mg/ml and at day 3 it increased to 8 mg/ml. When larvae were starved for 24 h, they did not grow, but their body mass and hemolymph volume did not decrease significantly. Differences in Lp densities were observed. In fed larvae, from days 1 to 4, two major Lp populations were found with densities of 1.124 ± 0.002 (high density Lp-larval1 , HDLp-L1 ) and 1.141 ± 0.002 g/ml (HDLp-L2 ). When larvae were starved for 24 h, only one Lp population was present, with density 1.114 ± 0.001 g/ml (HDLp-Ls ). When larvae were abdominally ligated at day 1 or 2 of fifth instar, only HDLp-Ls was found after 24 h, indicating that the formation of this HDLp population was not dependent on any factor released by head. On the other hand, larvae that were ligated at day 3 showed the same Lp populations as the fed ones. In 24-h starved larvae, lipid load in Lp was higher as compared to the fed controls. In 24-h ligated larvae Lp lipid content increased when ligation was performed on day 1 or 2, but not on day 3. So, different responses to starvation can be observed depending on the developmental phase of the same larval instar.

Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca: Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography

The activity of lactate dehydrogenase and the concentrations of glucose in the hemolymph and of glycogen in the digestive gland and cephalopedal mass of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis were evaluated. Additionally, high performance liquid chromatography (HPLC) was used to determine the hemolymph concentrations of some carboxylic acids (oxalic, piruvic, lactic and succinic). After one, two and three weeks of infection, the snails were dissected to collect the hemolymph and separate the tissues. A significant reduction of the levels of glucose in the hemolymph was observed as of the first week of infection in relation to the control group. The lactate dehydrogenase activity of the infected group was significantly higher than the average of the control group. This increase was accompanied by a reduction of the levels of piruvic acid and an increase in the levels of lactic acid in the hemolymph of the parasited snails, confirming the acceleration of the anaerobic metabolism, necessary for the host to obtain energy and maintain its redox balance. In parallel, there was a decrease in the glycogen content of the storage tissues, with that reduction being significantly greater in the cephalopedal mass than the digestive gland, demonstrating that in this interaction system, the mobilization of glycogen was not sufficient to maintain and reestablish the normal glycemia of the infected snails.

Sex-specific divergence for adaptations to dehydration stress in Drosophila kikkawai

Several studies on diverse Drosophila species have reported higher desiccation resistance of females, but the physiological basis of such sex-specific differences has received less attention. We tested whether sex-specific differences in cuticular traits (melanic females and non-melanic males) of Drosophila kikkawai correspond with divergence in their water balance mechanisms. Our results are interesting in several respects. First, positive clinal variation in desiccation resistance was correlated with cuticular melanisation in females but with changes in cuticular lipid mass in males, despite a lack of differences between the sexes for the rate of water loss. Second, a comparative analysis of water budget showed that females of the northern population stored more body water as well as hemolymph content and exhibited greater dehydration tolerance than flies from the southern tropics. In contrast, we found no geographical variation in the males for water content and dehydration tolerance. Third, an ~10-fold increase in the rate of water loss after organic solvent treatment of male D. kikkawai suggested a role of cuticular lipids in cuticular transpiration, but had no effect in the females. Fourth, geographical differences in the storage of carbohydrate content (metabolic fuel) were observed in females but not in males. Interestingly, in females, the rate of utilization of carbohydrates did not vary geographically, but males from drier localities showed a 50% reduction compared with wetter localities. Thus, body melanisation, increased body water, hemolymph, carbohydrate content and greater dehydration tolerance confer greater desiccation resistance in females, but a reduced rate of water loss is the only possible mechanism to cope with drought stress in males. Finally, acclimated females showed a significant increase in drought resistance associated with higher trehalose content as well as dehydration tolerance, while males showed no acclimation response. Thus, sex-specific differences in desiccation resistance of D. kikkawai are associated with divergence in some water balance strategies, despite a lack of differences in the rate of water loss between the two sexes.

The effect of nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes on hemolymph proteins, sugars, and lipids in Spodoptera exigua larvae

We determined the physiological effects of joint and separate nucleopolyhedrovirus (NPV) infection and parasitism by the endoparasitoid Microplitis pallidipes Szepligeti on biochemical events in the noctuid Spodoptera exigua (Hubner). The results indicated that in parasitized larvae, compared to healthy larvae, total protein concentration in host hemolymph began to decline and total sugar concentration significantly increased by the first day, while lipid content in the host body significantly increased by the second day after parasitization. Meanwhile, in jointly infected and parasitized hosts, compared to parasitized larvae, total protein concentration was consistently higher, total sugar concentration was consistently lower, and lipid content became higher by the second day after treatment. In virus-infected larvae, compared to healthy larvae, total protein concentration sharply declined during the first two days but increased by the third, while total sugar concentration increased on the second and third days after virus infection but decreased at other observation times, and lipid content began to increase by the second day after virus infection. Finally, in larvae that were both parasitized and virus-infected, compared to just virus-infected larvae, total protein concentration increased during the first two days but decreased by the third, total sugar concentration increased only on the first and fourth days, and lipid content decreased significantly on the first day but began to increase by the second day after treatment. These findings led us to conclude that parasitization inhibited protein mobilization but stimulated sugar mobilization in host hemolymph, and promoted lipid mobilization in the host body, while Spodoptera exigua NPV infection stimulated protein mobilization induced by parasitization but inhibited sugar mobilization induced by parasitization.

Rapid effects of humidity acclimation on stress resistance in Drosophila melanogaster

We tested the hypothesis whether developmental acclimation at ecologically relevant humidity regimes (40% and 75% RH) affects desiccation resistance of pre-adults (3rd instar larvae) and adults of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Additionally, we untangled whether drought (40% RH) acclimation affects cold-tolerance in the adults of D. melanogaster. We observed that low humidity (40% RH) acclimated individuals survived significantly longer (1.6-fold) under lethal levels of desiccation stress (0–5% RH) than their counter-replicates acclimated at 75% RH. In contrast to a faster duration of development of 1st and 2nd instar larvae, 3rd instar larvae showed a delayed development at 40% RH as compared to their counterparts grown at 75% RH. Rearing to low humidity conferred an increase in bulk water, hemolymph content and dehydration tolerance, consistent with increase in desiccation resistance for replicates grown at 40% as compared to their counterparts at 75% RH. Further, we found a trade-off between the levels of carbohydrates and body lipid reserves at 40% and 75% RH. Higher levels of carbohydrates sustained longer survival under desiccation stress for individuals developed at 40% RH than their congeners at 75% RH. However, the rate of carbohydrate utilization did not differ between the individuals reared at these contrasting humidity regimes. Interestingly, our results of accelerated failure time (AFT) models showed substantial decreased death rates at a series of low temperatures (0, −2, or −4°C) for replicates acclimated at 40% RH as compared to their counter-parts at 75% RH. Therefore, our findings indicate that development to low humidity conditions constrained on multiple physiological mechanisms of water-balance, and conferred cross-tolerance towards desiccation and cold stress in D. melanogaster. Finally, we suggest that the ability of generalist Drosophila species to tolerate fluctuations in humidity might aid in their existence and abundance under expected changes in moisture level in course of global climate change.

Domoic acid toxicokinetics in Dungeness crabs: New insights into mechanisms that regulate bioaccumulation

Domoic acid (DA) is an excitatory neurotoxic amino acid produced by several marine algal species and is the causative agent of amnesic shellfish poisoning. Profound differences in the toxicokinetics of DA have been identified in a wide variety of shellfish. We characterized the toxicokinetics of DA in Dungeness crabs (Metacarcinus magister) after oral and intravascular dosing (IV) using a variety of doses ranging from 0.1 to 20mg/kg. After a 1mg/kg oral dose, DA disappeared from the foregut within 2h and largely accumulated in the hepatopancreas, with hemolymph and other tissues having 100–1000 times lower concentrations. After IV dosing, hemolymph concentrations of DA were unexpectedly high and toxicokinetic analysis indicated the steady-state volume of distribution (Vss) was 123–197ml/kg, which is well below the hemolymph volume of 350ml/kg for crabs. This indicated only limited extravascular distribution of DA was occurring after IV injection, which is surprising considering the capacity of the hepatopancreas to sequester DA after oral dosing. Additional studies measured the partitioning of DA in hepatopancreas cellular and subcellular fractions. The subcellular distribution of DA was primarily associated with the S8 fraction and could be filtered through a 30,000MW cut-off filter, indicating DA was not appreciably bound to macromolecules. Interestingly, very little (<0.4%) of the total hepatopancreas DA tissue content was associated with the cellular fraction isolated after dissociation and separation from tissue fragments. The in vivo and in vitro results led us to hypothesize that DA uptake and distribution is regulated by crustacean orthologs of ATP-binding cassette (ABC) type transporters. We tested this hypothesis by co-exposing crabs to DA and known inhibitors of ABC transporters (verapamil, cyclosporine A and MK-571) and through in vitro studies using isolated hepatopancreas tissue and mixed cell suspensions prepared from hepatopancreas tissue. The in vivo results were inconclusive in that the toxicokinetics of DA was not consistently altered by co-administration of the inhibitors. Two exceptions were MK-571, which significantly increased the total body clearance of DA and co-administration of verapamil, which significantly increased the hepatopancreas tissue content of DA 24h after IV injection. Isolated pieces of hepatopancreas tissue were able to readily absorb DA from incubation media, but mixed cell suspensions did not. The absorption of DA or lack thereof was largely unaffected by co-incubation with verapamil although cell suspensions appeared to accumulate small quantities of DA in the presence of verapamil. Collectively, the results of this study suggest DA accumulates in the extracellular spaces of the hepatopancreas, such as the tubular lumen. Under natural circumstances, crabs feeding on contaminated shellfish would be expected to readily absorb DA, which is then stored and slowly eliminated in urine. If the DA exposure level exceeds the storage capacity of the tissue (as occurred with the 20mg/kg dose), breakthrough occurs resulting in much higher systemic exposure and potential for DA toxicity.

In vivo effect of Neuropeptide F on ecdysteroidogenesis in adult female desert locusts (Schistocerca gregaria)

Neuropeptides are important regulatory factors that mediate key life processes, both in vertebrates and invertebrates. Many insect neuropeptides display pleiotropic activities, which means that they can influence multiple aspects of insect physiology. In the fruit fly, Drosophila melanogaster, Neuropeptide F (NPF) mediates diverse physiological processes, such as learning, stress responses, feeding and male courtship behavior. In locusts, only a truncated form of the predicted “full-length” NPF, the nonapeptide “trNPF”, has been isolated. This nonapeptide previously proved to be biologically active, since it was shown to influence food intake and weight increase, as well as oocyte growth in adult female desert locusts (Schistocerca gregaria [Forskål]). In the present study, we have further analyzed the effect of trNPF on female reproductive physiology in S. gregaria. We confirmed that daily trNPF injections in adult females elicit an increase of oocyte size. In addition, an RNAi-mediated knockdown of the Schgr-NPF precursor transcript in adult female locusts resulted in the opposite effect, i.e. significantly smaller oocytes. Moreover, we discovered that daily injections of trNPF in adult female S. gregaria, caused higher ecdysteroid titers in the ovaries and accelerated the appearance of ecdysteroid peaks in the hemolymph of these animals. The RNAi-based knockdown of the Schgr-NPF precursor transcript clearly resulted in reduction of both hemolymph and ovarian ecdysteroid concentrations, confirming the stimulatory effects of trNPF injections on adult female ecdysteroid levels. The observed results are discussed in relation to previous reports on NPF activities in locusts and other insects.

A NEW INTEGRATED STRATEGY FOR CONTROLLING THE DESERT LOCUST SCHISTOCERCA GREGARIA (FORSKAL)

Nowadays, there is an upsurge in the locust and grasshopper populations in many parts of the world. Any way despite of the increase use of agrochemicals, that reduced the attack by insects. These chemicals are still represents a high risk to field workers and consumers. Accordingly, the use of biological control in these days as a particle solution is too desirable as the only alternative solution to the problem of chemical pesticides usage for long time. Our study revealed that the mortality percentage of the first nymphal instar of the desert locust was 100% after 7, 10 And 19 days when treated with mixture of conc. 5 x 1010 added to 100ppm μl of 10% consult (inhibitor compound), and Metarhizium anisopliae fungal spores at concentrations 5 ×1010, 5 ×109 Spores/ml., respectively. On the other hand, mortality percentage reached 98 and 80% after 6 days by treatment with 100ppm and 0.1ppm of the inhibitor compound respectively. Also, the mortality percentage of the second nymphal instar attained 100% after 20 days by treatment with Conc. 5 x 109 Spores/ml. of fungal spores. Amazingly, the mortality percentage was 100% after 17 days for the third nymphal instar, this by mixing the fungal spores at higher Conc. 5 x 1010 Spores/ml. With the insecticide at Conc. of 100ppm interestingly, the third and fourth nymphal instar, were less sensitive toward lower concentration of insecticide than that of fungal spores usage. Any way, the biochemical pattern revealed that the carbohydrate concentration in haemolymph of infected adult locust was higher than that in the adult non infected control. On the other hand lipid contents and total cholesterol, of the growth inhibitor treated adult insect was sharply increased after 3 days of treatment. Moreover, results showed that, the treatment with fungal spores reduced both the lipid and total cholesterol contents in the insect nymph, while total protein in the nymph of adult locust insect was increased with treatment by both fungal spores and growth inhibitor as a mixture. Also results revealed that the mortality percentage was 100% in both cases of adult and immature insect after 35 days and, 72% after 24 days. Moreover the treatment with mixture of both fungal spores and growth inhibitor reduced the hatchability percentage up to 47.2% in case of treated immature insects compared with 95.5 to the control. However, results also showed that the percentage was 16.9% in case of adult when compared with control. Also sterility percentage was 93.12% in case of immature insect to control, and 97.82% for mature ones compared with the control.

Effect of Pollution History on Immunological Responses and Organ Histology in the Marine Mussel Mytilus edulis Exposed to Cadmium

The effect of previous toxicant exposure (i.e., exposure history) on an organism's response to re-exposure to the toxicant is of considerable interest. The marine mussel Mytilus edulis was collected from reference and polluted sites in southwest England, and groups of mussels from each site were exposed to 20 μg/L CdCl2 for 0, 1, 4, and 8 days and compared with unexposed controls. End points evaluated were tissue metal and electrolyte concentrations, haemolymph chemistry, haemocyte characteristics [counts, neutral red uptake (NRU), and phagocytosis], histology, and expression of metallothionein gene (mt10) expression in digestive glands. Field-collected animals differed by collection site for some end points at time zero, at which time tissue Fe and Pb concentrations were greater and NRU and condition index lower in mussels from the polluted site. Subsequent exposure to cadmium (Cd) in the laboratory caused Cd accumulation mainly in digestive gland, but there were no site-specific effects on tissue trace-metal concentrations. NRU, phagocytosis, and haemolymph Na(+) and K(+) concentrations differed among sites and Cd treatment, but there were no clear trends. Exposure to Cd resulted in lower Ca(2+) concentrations in gill, digestive gland, and haemolymph in animals from the polluted site compared with controls (Kruskal-Wallis, p ≤ 0.05). Lesions, including necrosis, inflammation, and neoplasia, were observed in animals from the polluted site, but the frequency of these lesions appeared to decrease unexpectedly after Cd exposure. Expression of mt10 increased 3-fold in Cd-exposed animals from the polluted site compared with all other groups (Kruskal-Wallis, p = 0.01). We conclude that Cd exposure affected some immune responses in M. edulis, but pre-exposure history influenced toxicological outcomes of Cd exposure in the laboratory.