Hemolymph Glucose Concentration Research Articles

Comparative transcriptome analysis provides a glance into the regulatory of insulin-like peptide 1 gene in the Pacific white shrimp Litopenaeus vannamei

Insulin-like peptides (ILPs) have been reported to exert significant and diverse regulatory effects through the insulin signaling pathway in invertebrates. However, there is a large research gap in the functional analysis of ILP genes in crustaceans. In this study, we combined RNA interference (RNAi) technology with transcriptome sequencing to investigate the regulatory effects of the ILP1 gene in the Pacific white shrimp Litopenaeus vannamei. After two weeks of continuous RNAi of LvILP1, the shrimp in the dsLvILP1 group exhibited reduced body length gain, weight gain, molting rate, and hemolymph glucose concentration compared to the control dsEGFP group, indicating that knockdown of LvILP1 slowed shrimp growth, molting, and metabolism. Comparative transcriptome analysis of three tissues revealed that knockdown of LvILP1 inhibited muscle growth and development by modulating the mTOR and MAPK signaling pathways, and prevented the renewal of epidermal tissue through the Hippo signaling pathway. Meanwhile, it also suppressed certain growth signals in the hepatopancreas, such as juvenile hormone (JH) synthesis and 20-hydroxyecdysteroid (20E) signal activation. Additionally, it promoted the conversion pathway of some sugars into amino acid metabolism instead of energy metabolism after glycolysis in the hepatopancreas. Interestingly, knockdown of LvILP1 also affected carbohydrate metabolism by regulating the intracellular and extracellular transport of glucose and trehalose, as well as regulating the expression levels of key metabolic genes in three shrimp tissues. This disruption led to a decrease in blood glucose levels and suppressed energy production, ultimately providing less energy for shrimp growth and molting. This study provides valuable insights into the underlying genetic mechanism through which ILP1 regulates glucose metabolism and growth in shrimp. It enhances our understanding of the structure and function of the insulin pathway in crustaceans and serves as an important reference for the identification of candidate genes in molecular genetic breeding of shrimp.

Effect of paprika extracts on growth performance, haemolymph chemistry, intestinal microbiota and antioxidant enzyme activities of white-leg shrimp (Litopenaeus vannamei).

This study aims to evaluate the effects of paprika extract on the survival rate, growth performance and stimulation of the innate immune system of Litopenaeus vannamei. In this experiment, 240 healthy shrimp (3.22 ± 0.12 g) were randomly divided into four groups. The shrimp were fed diets with different concentrations of paprika oil extracts (0%, 0.5%, 1% and 2%) for 8 weeks. The results showed that growth performance, urea, uric acid, creatinine, cholesterol levels, aspartate aminotransferase and alkaline phosphatase activities were not significantly affected by adding paprika extract to the shrimp diet (p > 0.05). Diets containing 1% and 0.5% paprika extract showed the highest levels of total protein and triglyceride, respectively (p < 0.05). There was a significant decrease in haemolymph glucose concentration in shrimp-fed diets containing 1% and 2% paprika extract (p < 0.05). Moreover, a diet containing 0.5% paprika extract resulted in the highest levels of total heamocyte count, hyaline cells and large-granular cells in shrimp (p < 0.05). Higher catalase and superoxide dismutase activities were also exhibited in the paprika groups (p < 0.05). Vibrio sp. bacteria were not significantly reduced by paprika extract in the intestines of L. vannamei (p > 0.05). A significant decrease in heterotrophic bacteria was observed with increasing extract concentrations (p < 0.05). The shrimp culture industry can utilize paprika extract as a cost-effective, efficient and environmentally friendly immune stimulant at a concentration of 0.5%.

Effects Density and Salinity of Artificial Brackish Water on the Growth and Physiological Perfomance of Whiteleg Shrimp (Litopenaeus vannamei Boone, 1931)

The increase in shrimp culture causes problems with the quality of seawater which is a source of water for whiteleg shrimp culture it self. The use of artificial brackish water as a culture medium is an effort to increase the availability of water sources for whiteleg shrimp culture. This study aims to (1) determine the optimum stocking density of whiteleg shrimp culture using artificial brackish water salinity based on carrying capacity, (2) determine mineral content (Mg, Ca, Na and K) in artificial brackish water, (3) study the effect of salinity on the growth and blood glucose concentration of whiteleg shrimp cultivated in artificial brackish water, (4) examine the effect of salinity on hemolymph osmolarity of whiteleg shrimp cultivated in artificial brackish water. This study used an experimental method consisting of two stages. Each stage was arranged using a completely randomized design (CRD). Obtained stocking densities of SD-50% (20 shrimps/50L), SD (40 shrimps/50L) and SD+50% (60 shrimps/50L). The best growth and performance were obtained in the treatment of 20 individuals, namely survival (SR) 87%, specific growth rate (SGR) 3.26%/day and hemolymph glucose concentration of 30.73 mg/dl. In the next stage of the research, the salinity treatments tested were seawater, 10 ppt, 15 ppt, 20 ppt and 25 ppt. The best shrimp performance was obtained in treatment 20 shrimps/50 L, namely survival rate 70.00%, specific growth rate (SGR) 1.55%/day, feed conversion ratio (FCR) 1.72, glucose haemolymp 73.44 mg/dl. The optimal osmotic work level (OWL) value is obtained at a salinity of 15-20 ppt.

STARVATION DURING DEVELOPMENT AFFECTS METABOLISM IN DROSOPHILA

Aim. To investigate how starvation during early stage of fly development affects carbohydrate metabolism in imago flies and their progeny of F1 generation. Methods. Wild-type Canton-S strain Drosophila melanogaster flies were used in all experiments. Flies of parental and offspring generations were used for the determination of glycogen and glucose content using the diagnostic kit Glucose-Mono-400-P according to the manufacturer's instructions. Results represent as the mean ± SEM of 3-4 replicates per group. According Student's t-test significant difference between groups was P&lt;0.05. Graphing and statistical analysis were performed by using GraphPad Prism. Results. Starvation during development significantly influenced the level of hemolymph and body glucose in imago flies of parental generation. Hemolymph glucose concentration was lower by 34% (P=0.008) and 32% (P=0.033) in experimental females and males, respectively, as compared to control groups. Starvation during development led to lower level of body glucose in adult parental flies of both sexes. Adult males F1, generated by parents that were starved during development, showed 3-fold lower glycogen content, as compared to control. Conclusions. Starvation at early stage of development led to lower hemolymph glucose and body glucose level in imago flies. Moreover, parental starvation decreased glycogen pool in F1 males.

Identification of Putative Molecules for Adiponectin and Adiponectin Receptor and Their Roles in Learning and Memory in Lymnaea stagnalis.

Adiponectin enhances insulin sensitivity, which improves cognition in mammals. How adiponectin affects the mechanism's underlying cognition, however, remains unknown. We hypothesized that experiments using the pond snail Lymnaea stagnalis, which has long been used in learning and memory studies and in which the function of insulin-like peptides affect learning and memory, could clarify the basic mechanisms by which adiponectin affects cognition. We first identified putative molecules of adiponectin and its receptor in Lymnaea. We then examined their distribution in the central nervous system and changes in their expression levels when hemolymph glucose concentrations were intentionally decreased by food deprivation. We also applied an operant conditioning protocol of escape behavior to Lymnaea and examined how the expression levels of adiponectin and its receptor changed after the conditioned behavior was established. The results demonstrate that adiponectin and adiponectin's receptor expression levels were increased in association with a reduced concentration of hemolymph glucose and that expression levels of both adiponectin and insulin-like peptide receptors were increased after the conditioning behavior was established. Thus, the involvement of the adiponectin-signaling cascade in learning and memory in Lymnaea was suggested to occur via changes in the glucose concentrations and the activation of insulin.

Amelioration of ammonia-induced intestinal oxidative stress by dietary Clostridium butyricum in giant freshwater prawn (Macrobrachium rosenbergii)

The use of Clostridium butyricum in crustacean aquaculture for anti-abiotic stress is yet unknown. Feeds were formulated containing 0, 125, 250, 500, and 1000 mg/kg Clostridium butyricum (2 × 107 CFU/g), respectively. The giant freshwater prawns (Macrobrachium rosenbergii) were fed for 8 weeks in triplicate. The results showed that C. butyricum-supplemented groups improved growth performance significantly with the optimum level at 610 mg/kg. Ammonia stress reduced hemolymph glucose, total protein, total cholesterol, and triglyceride concentrations while dietary C. butyricum significantly increased hemolymph glucose and total protein levels after the ammonia challenge. Ammonia stress increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels, and the treatments supplemented with C. butyricum had considerably enhanced levels of iNOS and NO after stress. Treatment with C. butyricum increased the level of superoxide dismutase (SOD), and decreased the level of malondialdehyde (MDA) and superoxide anion, with the 125 mg/kg treated groups having the extreme value. Furthermore, C. butyricum-treated groups reduced the expression of HSPs after ammonia stress while the ammonia stress induced the expression of HSP60, HSP70, and HSP90. Dietary C. butyricum elevated the expression of peroxiredoxin-5 and toll in response to ammonia stress. The results indicate that dietary supplementation with 125–500 mg/kg of C. butyricum (2 × 107 CFU/g) improved biochemical and antioxidant features as well as intestinal immunity of M. rosenbergii under ammonia challenge by activating the toll signal pathway.

A comparative study of natural variation in hemolymph glucose levels under different dietary sugar conditions in Drosophila melanogaster and D. simulans.

Physiological responses to environmental changes play important roles in adaptive evolution. In particular, homeostatic regulatory systems that maintain constant circulating glucose levels are crucial in animals. However, variation in circulating glucose levels and the genetic effects on phenotypic variation in natural populations remain to be clarified. Here, we investigated the hemolymph glucose levels in natural populations of Drosophila melanogaster and its sibling species, D. simulans, in Japan. We quantified hemolymph glucose concentrations in third instar larvae of 27 lines for each species, which were reared on either glucose-free or glucose-rich food. In both species, genetic variation was not a major component of phenotypic variation on either glucose-free or glucose-rich food. The hemolymph glucose concentrations were much higher in D. simulans than in D. melanogaster. Genetic variance was larger in D. simulans than in D. melanogaster. The observed differences between the two species may be associated with the much more recent colonization history of D. simulans populations in Japan and/or the tolerance to environmental stresses. Our findings suggest that natural selection acting on hemolymph glucose levels in D. melanogaster is different from that in D. simulans.

Optimal Niacin Requirement of Oriental River Prawn Macrobrachium nipponense as Determined by Growth, Energy Sensing, and Glycolipid Metabolism.

Niacin is indispensable for the growth and development of aquatic animals. However, the correlations between dietary niacin supplementations and the intermediary metabolism of crustaceans are still poorly elucidated. This study explored the effects of different dietary niacin levels on the growth, feed utilization, energy sensing, and glycolipid metabolism of oriental river prawn Macrobrachium nipponense. Prawns were fed with different experimental diets containing graded niacin levels (15.75, 37.62, 56.62, 97.78, 176.32, and 339.28 mg/kg, respectively) for 8 weeks. Weight gain, protein efficiency, feed intake, and hepatopancreas niacin contents all maximized in the 176.32 mg/kg group with significance noted with the control group (P <0.05), whereas the opposite was true for feed conversion ratio. Hepatopancreas niacin concentrations increased significantly (P < 0.05) as dietary niacin levels increased, and peaked at the 339.28 mg/kg group. Hemolymph glucose, total cholesterol, and triglyceride concentrations all maximized in the 37.62 mg/kg group, while total protein concentration reached the highest value in the 176.32 mg/kg group. The hepatopancreas mRNA expression of AMP-activated protein kinase α and sirtuin 1 peaked at the 97.78 and 56.62 mg/kg group, respectively, and then both decreased as dietary niacin levels increased furtherly (P < 0.05). Hepatopancreas transcriptions of the genes related to glucose transportation, glycolysis, glycogenesis, and lipogenesis all increased with increasing niacin levels up to 176.32 mg/kg, but decreased significantly (P < 0.05) as dietary niacin levels increased furtherly. However, the transcriptions of the genes related to gluconeogenesis and fatty acid β-oxidation all decreased significantly (P < 0.05) as dietary niacin levels increased. Collectively, the optimum dietary niacin demand of oriental river prawn is 168.01-169.08 mg/kg. In addition, appropriate doses of niacin promoted the energy-sensing capability and glycolipid metabolism of this species.

Dietary Corn Starch Levels Regulated Insulin-Mediated Glycemic Responses and Glucose Homeostasis in Swimming Crab (Portunus trituberculatus).

Carbohydrate is the cheapest source of energy among the three major nutrient groups, an appropriate amount of carbohydrates can reduce feed cost and improve growth performance, but carnivorous aquatic animals cannot effectively utilize carbohydrates. The objectives of the present study are aimed at exploring the effects of dietary corn starch levels on glucose loading capacity, insulin-mediated glycemic responses, and glucose homeostasis for Portunus trituberculatus. After two weeks of feeding trial, swimming crabs were starved and sampled at 0, 1, 2, 3, 4, 5, 6, 12, and 24 hours, respectively. The results indicated that crabs fed diet with 0% corn starch exhibited lower glucose concentration in hemolymph than those fed with the other diets, and glucose concentration in hemolymph remained low with the extension of sampling time. The glucose concentration in hemolymph of crabs fed with 6% and 12% corn starch diets reached the peak after 2 hours of feeding; however, the glucose concentration in hemolymph of crabs fed with 24% corn starch attained the highest value after 3 hours of feeding, and the hyperglycemia lasted for 3 hours and decreased rapidly after 6 hours of feeding. Enzyme activities in hemolymph related to glucose metabolism such as pyruvate kinase (PK), glucokinase (GK), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly influenced by dietary corn starch levels and sampling time. Glycogen content in hepatopancreas of crabs fed with 6% and 12% corn starch first increased and then decreased; however, the glycogen content in hepatopancreas of crabs fed with 24% corn starch significantly increased with the prolongation of feeding time. In the 24% corn starch diet, insulin-like peptide (ILP) in hemolymph reached a peak after 1 hour of feeding and then significantly decreased, whereas crustacean hyperglycemia hormone (CHH) was not significantly influenced by dietary corn starch levels and sampling time. ATP content in hepatopancreas peaked at 1 h after feeding and then decreased significantly in different corn starch feeding groups, while the opposite trend was observed in NADH. The activities of mitochondrial respiratory chain complexes I, II, III, and V of crabs fed with different corn starch diets significantly increased first and then decreased. In addition, relative expressions of genes related to glycolysis, gluconeogenesis, glucose transport, glycogen synthesis, insulin signaling pathway, and energy metabolism were significantly affected by dietary corn starch levels and sampling time. In conclusion, the results of the present study reveal glucose metabolic responses were regulated by different corn starch levels at different time points and play an important role in clearing glucose through increased activity of insulin, glycolysis, and glycogenesis, along with gluconeogenesis suppression.

Analytical validation of a portable human Accu-Chek glucometer in honeybee hemolymph.

Glucose and trehalose are the main energy sources used by honeybees (Apis mellifera) for daily activities. However, there is no validated point-of-care method to reliably measure both sugars. We performed an analytical validation of a portable human glucometer (Accu-Chek; Roche) for glucose measurement in honeybee hemolymph compared to a reference method (GluCH, UniCel DxC 600; Beckman Coulter). We used 30 pooled hemolymph samples collected from the antennae of anesthetized honeybees and diluted 1:4 in 0.9% saline. We evaluated dilution linearity, spike recovery, and inter- and intra-assay imprecision. Glucose concentration was measured over time (2 h, 4 h, 8 h, 12 h, 1 d, 2 d, 3 d, 7 d, 21 d, 28 d) at various storage temperature (25°C, 4°C, −20°C, −80°C). The trehalose concentration was measured indirectly by trehalase hydrolyzation. Glucose concentrations measured by both instruments had a strong correlation (0.985, p < 0.0001) and a bias of −7.33 mmol/L (±1.96SD: 13.70 to −28.36), with linear agreement at <20 mmol/L (physiologic value: 100 mmol/L). The accuracy of the glucometer decreased at >20 mmol/L. Recovery of 115–130% of diluted spikes indicated good specificity. Inter- and intra-assay imprecision were 2.50% and 2.21%, respectively. Glucose concentrations fluctuated in stored samples dependent on time and temperature; however, glucose concentrations were constant with storage at −80°C for ≥28 d. The Accu-Chek glucometer is an adequate instrument to measure honeybee glucose concentration in hemolymph diluted with 0.9% NaCl, with good accuracy and precision at <20 mmol/L. Hemolymph storage at −80°C is suitable for long-term conservation of glucose.

Probiotic (protexin) modulates glucose level in sucrose-induced hyperglycaemia in Harwich strain Drosophila melanogaster

BackgroundProbiotics are beneficial microorganisms that alter microbial flora to promote human and animal health by ameliorating the physiological and psychological hitches and distress. The aim of the study was to investigate the protective effect of probiotic (protexin) in sucrose induced hyperglycaemia in Harwich strain Drosophila melanogaster.MethodsFor lethal concentration, different concentrations of probiotic were fed on fresh breaded D. melanogaster for two weeks in two phases. The concentrations varied from 10 to 1000 mg. For increment in haemolymph glucose level, Insta-larva of 540 D melanogaster was divided into six groups of thirty larva (n = 30) each in triplicate. Baseline glucose level was determined by administering group normal diet, while group 2–6 were fed with a normal diet containing 30% sucrose. For body weight, Eppendorf microtubes were weighed and anaesthetised flies were transferred into the tubes which were, re-weigh to get the actual weight of the flies before and after sucrose intake. Treatment supplement was introduced after flies emerged to be three days old. Group I: (Normal Control) received a normal cornmeal diet 30 g. Group II: (positive control) received normal diet + 30% sucrose only. Group III; received 10 mg of metformin/30 g diet. Group IV received 250 mg of probiotic/30 g of normal diet. Group V received 500 mg of probiotic/30 g normal diet. Group VI received 1000 mg of probiotic/30 g normal diet. All treatments lasted for 7 days. At the end of the treatment period, flies were immobilized and anaesthetised in ice and homogenised vigorously in 0.1 M phosphate buffer, pH 7.4. Eppendorf microtubes were weighed and anesthetized flies were transferred into them, and re-weighed with appropriate micro-litre of phosphate buffered saline (PBS) added and the flies were squashed to get the supernatant. The resulting homogenates were centrifuged at 10,000×g, 4 °C for 10 min. The supernatant was separated from the pellets into labelled tubes and used for the various biochemical assays. Data were expressed as mean ± standard error of mean (SEM) and subjected one-way analysis of variance (ANOVA), followed by Tukey post-hoc test for multiple comparisons between groups. Statistical package Graph Pad Prism version 8.1 was used for statistical analysis and values of p ˂ 0.05 were considered significant. Lethal concentration, logic probit test software was used.ResultsThe dosage of probiotic ranging from 10 to 1000 mg were not toxic to the fruit-fly. The LC50 of protexin after seven days was found to be greater than 1000 mg. Significantly (p < 0.05) decreased in hemolymph glucose concentration was recorded for the base-line (phase one) in the normal control group, compared to diabetic-induced groups. There was a significant (p < 0.05) decline in body weight and blood glucose level in all the treatment groups, when compared to the diabetic untreated group.ConclusionsAdministration of sucrose diet increased the blood glucose level and body weight in diabetic D. melanogaster flies, mitigated by administration of probiotics (protexins).

The Role of Insulin-like Peptide in Maintaining Hemolymph Glucose Homeostasis in the Pacific White Shrimp Litopenaeus vannamei.

Insulin-like peptide (ILP) has been identified in various crustaceans, but whether it has a similar function in regulating hemolymph glucose as vertebrate insulin is unclear. We analyzed the components of hemolymph sugar in the Pacific white shrimp, Litopenaeus vannamei, and investigated the changes of hemolymph glucose concentration and the expressions of ILP and glucose metabolism genes under different treatments. We found glucose was a major component of hemolymph sugar in shrimp. Starvation caused hemolymph glucose to rise first and then decline, and the raised hemolymph glucose after exogenous glucose injection returned to basal levels within a short time, indicating that shrimp have a regulatory mechanism to maintain hemolymph glucose homeostasis. In addition, injections of bovine insulin and recombinant LvILP protein both resulted in a fast decline in hemolymph glucose. Notably, RNA interference of LvILP did not significantly affect hemolymph glucose levels, but it inhibited exogenous glucose clearance. Based on the detection of glucose metabolism genes, we found LvILP might maintain hemolymph glucose stability by regulating the expression of these genes. These results suggest that ILP has a conserved function in shrimp similar to insulin in vertebrates and plays an important role in maintaining hemolymph glucose homeostasis.

Dietary thiamine requirement and its effects on glycolipid metabolism in oriental river prawn (Macrobrachium nipponense)

A 10-week feeding trial was conducted to investigate the optimal dietary thiamine requirement of oriental river prawn Macrobrachium nipponense with the effects on glycolipid metabolism evaluated. A total of 960 prawns with an average weight of 0.18 g were selected and randomly fed six semi-purified diets containing graded levels of thiamine (14.10, 35.20, 43.30, 61.40, 95.60 and 165.00 mg/kg, respectively) in an indoor water circulation system. Each diet was tested in 4 replicates. Final weight, weight gain, specific growth rate, protein efficiency rate, hemolymph thiamine and triglycerides concentrations all increased significantly (P < 0.05) with increasing dietary thiamine levels up to 61.40 mg/kg, and then decreased. A similar result was also observed in hemolymph transketolase activities except for the fact that the maximum value was observed in the 95.70 mg/kg group (P < 0.05). However, the opposite was true for hemolymph glucose and pyruvate concentrations with no statisticaldifference observed (P > 0.05). As dietary thiamine levels increased, the expressions of hexokinase and acetyl-CoA carboxylase both increased significantly (P < 0.05), and peaked at the 95.70 mg/kg group. Similar results were also observed in the expressions of glucose transporter 2, pyruvate kinase, glycogen synthase and fatty acid synthase except that the maximum values were all observed in the 61.40 mg/kg group. Furthermore, the expressions of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, carnitine palmitoyltransferase I and hormone-sensitive triglayceride lipase all decreased significantly (P < 0.05) with increasing dietary thiamine levels up to 61.40 mg/kg, and then plateaued. Based on the broken-line regression analysis of growth performance and hemolymph thiamine content, the optimal thiamine requirement of M.nipponense was estimated to be 66.80 and 67.57 mg/kg, respectively.

Effects of handling during experimental procedures on stress indices in the green shore crab, Carcinus maenas (L)

ABSTRACT Stress due to handling is often an unavoidable feature of experimental investigations. In some cases, appropriate settling times are not considered, and as such, physiological responses caused by handling may become additive with those of experimental treatments. This study investigated the effect of different handling procedures on the acute physiological responses of green shore crab (Carcinus maenas). Handling, such as would occur during transport around a research facility or transfer during experimental procedure, was designated as light (10 min emersion) or severe (10 min emersion with shaking). Oxygen consumption (MO2) and haemolymph glucose and haemolymph L-lactate concentrations were elevated post-handling, the magnitude of the change related to the severity of handling stress. Glucose and L-lactate concentrations peaked within 1 h and returned to basal levels within 6 h, but MO2 remained elevated for 10 h, reflecting the additional energy required to oxidize L-lactate and replenish energy reserves. Differences between light and severe handling treatments showed that vibration (shaking) was a major contributor to the stress response, rather than the experimental emersion. This was confirmed in a second experiment where crabs were handled without emersion, and MO2 remained elevated for 14 h. In this experiment, the most pronounced increase in MO2 and metabolic parameters occurred in crabs that were physically touched and moved rapidly from the holding to experimental tanks. Here the touch, as well as vibration and visual stimuli, provoked a fight-flight response in the crabs. Stress responses were also evident in crabs gently transferred by containers. The fact that transferring crabs with no physical touching and minimal visual and vibrational stimuli still evoked a stress response, albeit less pronounced, supports a recommendation that crustaceans should be left to settle in the apparatus for at least 12 h after handling before experimental procedures are initiated.

Effect of E-field at frequency 50 Hz on protein, glucose, and triglycerides concentration in honeybee hemolymph

The development of technology contributes to the increase in the exposure of living organisms to the E-field. While searching for food in the environment, the honeybee is exposed to the influence of this factor. The aim of our research was to determine the E-field effect with a frequency of 50 Hz at various intensities and exposure times on the concentration of protein, glucose, and triglycerides in the honeybee hemolymph. The analysis was carried out on hemolymph taken from at the age of 2 days old worker honeybees. The research showed changes in the tested parameters. Change related to exposure time and field intensity was noticed. The lowest protein concentration in the hemolymph of worker honeybee was observed in all control groups (0.13 mg/mL). The highest in the experimental groups were 23.0 kV/m and 34.5 kV/m with 12 h exposure time (1.04 mg/mL and 1.25 mg/mL). In all experimental groups, statistically significant lower values of glucose concentration in the hemolymph of worker honeybee were observed than in the control groups (23.43–23.55 mmol/l). The highest concentration of triglycerides was observed in the control groups (1.55 mmol/l). Lower but not statistically significant values were observed in the 5.0 kV/m 1 h, 11.5 kV/m 1 and 3 h, and 23.0 kV/m 3 h groups, while the differences were statistically significant in the other groups. The results of the research confirm our earlier observations that the E-field can induce similar reactions in the body to the pathogen. Changes in these important indicators of the body’s nutritional status such as protein, glucose, and triglyceride levels can have long-term effects.

Thermal stress responses of two sympatric crayfishes in Louisiana, Procambarus clarkii Girard, 1852 and Procambarus zonangulus Hobbs &amp; Hobbs, 1990 (Decapoda: Astacoidea: Cambaridae)

Abstract Water temperature is an important abiotic component in farmed and wild crayfish habitats as it influences individual and population characteristics including growth, metabolic activity, and overall population vigor. Thermal limits, however, have not been established for the two commercially important crayfishes in Louisiana, the red swamp crayfish Procambarus clarkii (Girard, 1852) and the southern white river crayfish P. zonangulus (Hobbs &amp; Hobbs, 1990). We aimed to determine the thermal stress responses for the two species. Thermal stress was examined by the critical thermal maxima (CTmax) for crayfish acclimated to 24, 27, or 30 °C for two weeks prior to experimentation. Water temperature in all acclimation trials was increased 1 °C h–1 and the end-point temperature for an individual was determined by the failure of righting response within 30 seconds. CTmax values (±SD) for P. clarkii acclimated at 24, 27, and 30 °C were 39.5 ± 0.5, 39.1 ± 0.3, and 39.4 ± 0.7 °C respectively, and 39.8 ± 0.8, 38.8 ± 0.8, and 39.0 ± 0.6 °C for P. zonangulus. CTmax did not differ significantly between species in any of the acclimation treatments. Thermal stress was also examined by measuring P. clarkii and P. zonangulus hemolymph glucose concentration every two hours for crayfish acclimated at 26 °C for two weeks and water temperature increased 1 °C h–1. Hemolymph glucose concentration began to increase at 36 °C in P. zonangulus and 38 °C in P. clarkii. Large increases in mean hemolymph glucose concentration (±SE) were observed in both P. clarkii (67.19 mg–dl ± 24.55) and P. zonangulus (74.11 mg–dl ± 18.04) at 40 °C.

Effects of dietary carbohydrate levels on growth, body composition, and gene expression of key enzymes involved in hepatopancreas metabolism in mud crab Scylla paramamosain

This study estimated the effects of different dietary carbohydrate levels on growth performance, feed utilization, body composition, and the gene expression of key enzymes involved in hepatopancreas lipogenesis and glucose metabolism in mud crab Scylla paramamosain. Crabs with an initial mean weight of 1.84 ± 0.06 g were used in an 8-week experiment to test five diets (C6, C12, C18, C24, C30) formulated to contain increasing levels of carbohydrate, measured as 8.55%, 13.36%, 17.76%, 25.64% and 32.50%, respectively. The results showed that the levels 25.64% and 32.50% improved growth performance, with the best gains at 25.64%. The highest dietary carbohydrate level led to higher hemolymph glucose concentrations and more fat deposition in the body. Furthermore, real-time quantitative PCR analysis revealed that the mRNA expression of key enzymes in glycolytic and lipogenic metabolism (i.e. glucokinase, fructose-2,6-diphosphatase, and fatty acid synthetase) were significantly affected by the dietary carbohydrate level. The mRNA expression of glucokinase was lowest in the group fed the carbohydrate-rich C30 diet, and the mRNA of fructose-2 and 6-diphosphatase were remarkably expressed in the C24 group (P < .05). The mRNA expression of fatty acid synthase at the dietary carbohydrate levels 32.50% and 25.64% were respectively 10-fold and 5-fold greater than with 8.55% dietary carbohydrate. The overall data suggest that S. paramamosain can be fed 25.64%–32.50% carbohydrate to achieve satisfactory growth performance. A two-slope broken-line regression analysis of weight gain showed that 24.08% dietary carbohydrate would be optimal for this species reared under similar conditions.

Effects of hypoxia on immune responses and carbohydrate metabolism in the Chinese mitten crab, Eriocheir sinensis

In aquaculture, hypoxia is an important stressor that can inhibit the healthy development of Chinese mitten crab, Eriocheir sinensis. The aim of this work was to research on how hypoxia (1 mg/L) followed by reoxygenation affects the immune response and carbohydrate metabolism in the Chinese mitten crab. The activities of immune-related enzymes including lysozyme (LZM), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and phenoloxidase (PO) and concentration changes in haemolymph glucose, trehalose and hepatic glycogen were evaluated after 8 hr of hypoxia followed by 12 hr of reoxygenation. After 8 hr of hypoxia, the activities of LZM, PO, SOD and hepatic glycogen concentration significantly decreased to the minimum when compared to the control group, but the activities of ACP and AKP, and haemolymph glucose concentration significantly increased. After 12 hr of reoxygenation, the activities of LZM, ACP, AKP and SOD recovered to the normal level except SOD in plasma, while the PO, SOD in plasma and hepatic glycogen concentration were still significantly lower than the control level. No significant change was observed in trehalose concentration during hypoxia and reoxygenation periods. These results indicated that although the activities of ACP and AKP were likely increased to facilitate the hydrolysis of foreign matter, the overall strength of the immune response was decreased as LZM, PO and SOD activities were suppressed after exposure to hypoxic conditions. The energy stores of exposed crabs were reduced due to the expenditure of energy during exposure to hypoxic stress. In general, chronic hypoxia would impair the immunocompetence and increase the energy expenditure of Chinese mitten crab, further increased the risk of pathogen infection and feed coefficient, so the negative effects of hypoxia should not be ignored.

Insights into carbohydrate metabolism from an insulin-like peptide in Macrobrachium rosenbergii

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.

Induction of LTM following an Insulin Injection.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). One-day food-deprived snails (day 1 snails) show the best CTA learning and memory, whereas more severely food-deprived snails (5 d) do not express good memory. However, previous studies showed that CTA-LTM was indeed formed in 5-d food-deprived snails (day 5 snails), but its recall was prevented by the effects of food deprivation. CTA-LTM recall in day 5 snails was expressed following 7 d of feeding and then 1 d of food deprivation (day 13 snails). In the present study, we thus hypothesized that memory recall occurs because day 13 snails are in an optimal internal state. One day of food deprivation before the memory test in day 13 snails increased the mRNA level of molluscan insulin-related peptide (MIP) in the CNS. Thus, we further hypothesized that an injection of insulin into day 5 snails following seven additional days with access to food (day 12 snails) activates CTA neurons and mimics the food deprivation state before the memory test in day 13 snails. Day 12 snails injected with insulin could recall the memory. In addition, the simultaneous injection of an anti-insulin receptor antibody and insulin into day 12 snails did not allow memory recall. Insulin injection also decreased the hemolymph glucose concentration. Together, the results suggest that an optimal internal state (i.e., a spike in insulin release and specific glucose levels) are necessary for LTM recall following CTA training in snails.