Hemolymph Levels Research Articles

Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila.

ABSTRACTThe destruction of pancreatic β cells leads to reduced insulin secretion and eventually causes diabetes. Various types of cellular stress are thought to be involved in destruction and/or malfunction of these cells. We show that endoplasmic reticulum (ER) stress accumulation in insulin-producing cells (IPCs) generated diabetes-like phenotypes in Drosophila. To promote the accumulation of extra ER stress, we induced a dominant-negative form of a Drosophila ER chaperone protein (Hsc70-3DN) and demonstrate that it causes the unfolded-protein response (UPR) in various tissues. The numbers of IPCs decreased owing to apoptosis induction mediated by caspases. The apoptosis was driven by activation of Dronc, and subsequently by Drice and Dcp-1. Accordingly, the relative mRNA-expression levels of Drosophila insulin-like peptides significantly decreased. Consistent with these results, we demonstrate that glucose levels in larval haemolymph were significantly higher than those of controls. Accumulation of ER stress induced by continuous Hsc70-3DN expression in IPCs resulted in the production of undersized flies. Ectopic expression of Hsc70-3DN can induce more efficient ER stress responses and more severe phenotypes. We propose that ER stress is responsible for IPC loss and dysfunction, which results in diabetes-related pathogenesis in this Drosophila diabetes model. Moreover, inhibiting apoptosis partially prevents the ER stress-induced diabetes-like phenotypes.

Changes in vitellogenin expression caused by nematodal and fungal infections in insects.

This study examined the expression and role of vitellogenin (Vg) in the body of the firebug Pyrrhocoris apterus (Heteroptera, Insecta) during infection elicited by two entomopathogenic organisms, the nematode Steinernema carpocapsae and the fungus Isaria fumosorosea Infection by S. carpocapsae significantly upregulated Vg mRNA expression in the male body. The corresponding increase in Vg protein expression was also confirmed by electrophoretic and immunoblotting analyses. Remarkably, in females, the opposite tendency was noted. Nematodal infection significantly reduced both Vg mRNA and Vg protein expression levels in fat body and hemolymph, respectively. We speculate that infection of reproductive females reduces Vg expression to a level that is still sufficient for defense, but is insufficient for reproduction. This circumstance reduces energy expenditure and helps the individual to cope with the infection. Importantly, purified Vg significantly inhibited growth of Xenorhabdus spp., an entomotoxic bacteria isolated from S. carpocapsae. However, the effect of Vg against I. fumosorosea was not so obvious. The fungus significantly stimulated Vg gene expression in males; however, a similar increase was not recapitulated at the protein level. Nevertheless, in females, both mRNA and protein Vg levels were significantly reduced after the fungal infection. The obtained data demonstrate that Vg is probably an important defense protein, possibly with a specific activity. This considerably expands the known spectrum of Vg functions, as its primary role was thought to be limited to regulating egg development in the female body.

The cholesterol-hydroxyecdysone-vitellogenin pathway is involved in the longevity of trophocytes and oenocytes of queen honey bees (Apis mellifera)

Trophocytes and oenocytes in the abdomen of honey bees do not divide after eclosion; however, trophocytes and oenocytes of queen bees have a longer lifespan and maintain better cellular function than those of worker bees. To explore this phenomenon, we assayed the molecules involved in the cholesterol-hydroxyecdysone-vitellogenin (Vg) pathway in the trophocytes and oenocytes of young and old worker and queen bees. The results showed that Vg and cholesterol levels in hemolymph and cholesterol levels, 20-hydroxyecdysone (20E) levels, and the messenger RNA levels of cytochrome P450 314A1 20-hydroxylase (Cyp314A1), ecdysone receptor isoform A (EcR-A), ecdysone receptor isoform B1 (EcR-B1), ultraspiracle (USP), ecdysone-induced protein 74 (E74), ecdysone-induced protein 75 (E75), broad-complex (BR-C), Vg, and Vg receptor (VgR) in trophocytes and oenocytes were increased in queen bees compared with worker bees. These findings indicated that queen bees have higher expression of molecules in the cholesterol-hydroxyecdysone-Vg pathway than worker bees.

Neuroendocrine and metabolic responses of Pacific whiteleg shrimp Penaeus vannamei exposed to hypoxia stress

Neuroendocrine mechanisms involved in crustacean stress response are different and less understood than those of vertebrates. Although indirect evidence suggests the participation of catecholamines (CA), few studies have analyzed their levels during stress response in crustaceans. This study examined CA levels in hemolymph and tissue of whiteleg shrimp Penaeus vannamei besides other biochemical indicators in response to hypoxia (0.8-1.0 mg L -1 ). Shrimp were stressed by hypoxia exposure, and their responses were analyzed at several intervals (10-480 min). Hemolymph glucose levels decreased gradually due to a higher demand not compensated by the gluconeogenesis process from hepatopancreas or muscle after 10 min. Lactate levels in hemolymph showed a 5-fold increase after 10 min, and a correlation with CA levels in hemolymph was observed suggesting its possible role in signaling catecholaminergic activation from eyestalk in a period from 10 to 120 min. Protein levels in muscle and hepatopancreas increased gradually throughout the trial indicating the possible use of another energetic substrate as arginine phosphate. A decrease in triacylglyceride levels denotes its preferential utilization as an immediate energy source in the scape response during the first minutes of hypoxia exposure. Heart adrenaline and noradrenaline levels increased rapidly at the beginning of the trial showing a correlation with the use of triacylglycerides and carbohydrates in hepatopancreas and muscle (r = 0.89 and r = 0.93, P ˂ 0.05, respectively). This manuscript reports evidence of CA participation in response to stress due to hypoxia and discusses possible adaptation mechanisms. Normal 0 21 false false false ES-MX JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-fareast-language:EN-US;}

Hemolymph Glucose Levels as a Measure of Crayfish Stress: A Methodology Using a Human Glucometer

The objective of this study was to develop a methodology for measuring hemolymph glucose levels in crayfish of the genus Orconectes using a human glucometer. A secondary objective was to confirm through the use of this methodology that subjecting crayfish to acute stress in the form of a short‐term exposure to a basic solution or to chronic stress in the form of prolonged exposure to a nitrate‐containing solution, would increase hemolymph glucose levels. To measure the effects of acute exposure to a basic solution on crayfish hemolymph glucose levels, crayfish were placed in a beaker containing a basic solution (Ringer's buffer, pH=8) or a control solution (Ringer's buffer, pH=7) for a period of 10 seconds, after which time they were removed from the beakers and hemolymph was collected with a syringe. A ReliOn Prime glucometer (Wal‐Mart Stores, Inc.) was used to measured glucose levels (mg/dl) directly from the hemolymph. In order for the glucose levels to be within the sensitivity of the glucometer, hemolymph was mixed with high glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco) in a 3:1 ratio. Glucose levels were determined by subtracting the levels of glucose measured in the DMEM solution from the glucose levels measured in the DMEM+hemolymph solution and correcting for the dilution factor. Crayfish acutely stressed through immersion in a basic solution had significantly higher hemolymph glucose levels compared to control crayfish (45.8 ± 5.9 vs 12.4 ± 3.8 mg/dl, two‐tailed t‐test; p<0.0001). To measure the effects of chronic exposure to a solution containing nitrate on crayfish hemolymph glucose levels, crayfish were housed in control tanks (no nitrate) or tanks containing 14 mg/L nitrate for one week, after which time hemolymph was drawn and glucose levels measured using the methodology described above. Crayfish chronically stressed through exposure to a nitrate‐containing solution had significantly higher hemolymph glucose levels compared to control crayfish (45 ± 14.3 vs 28.4 ± 8.8 mg/dl, two‐tailed t‐test; p=0.045). These results show that implementing this methodology human glucometers can be used to determine crayfish hemolymph glucose levels, making them an easy, inexpensive, and reliable alternative for this type of measurement. We were also able to show that hemolymph glucose levels are increased in both chronically and acutely stressed crayfish compared to control crayfish. These experiments can be used in physiology classrooms to demonstrate and discuss the role of the autonomic nervous system in an animal's response to stressful environmental conditions.Support or Funding InformationThis research was funded by the Denison University Department of Biology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

JH biosynthesis and hemolymph titers in adult male Aedes aegypti mosquitoes

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low in late pupae, significantly increased during the first 24 h after adult eclosion, and then remained relatively constant during the first six days after adult eclosion. Feeding high sugar diets resulted in an increase of JH synthesis and titers, and starvation significantly decreased JH synthesis, but this effect could be reversed by changing the males back to a high sugar diet. JH synthesis rates were similar in virgin and mated males, but hemolymph JH levels were different in well-nourished virgin and mated males. Starvation resulted in a significant reduction in insemination rates; with well-nourished males inseminating 2 times more females than water-fed. Giving a 20% sugar meal for 24 h to those mosquitoes that were previously starved for 6 days, caused a significant rise in insemination rates, restoring them to levels similar to those recorded for 20% fed males. These results suggest that nutrition plays a role on male fecundity, and this effect might be mediated by JH.

Effects of adipokinetic hormone and its related peptide on maintaining hemolymph carbohydrate and lipid levels in the two-spotted cricket, Gryllus bimaculatus.

Adipokinetic hormone (AKH) regulates energy homeostasis in insects by mobilizing lipid and carbohydrate from the fat body. Here, using RNA sequencing data, we identified cDNAs encoding AKH (GbAKH) and its highly homologous hormone AKH/corazonin-related peptide (GbACP) in the corpora cardiaca of the two-spotted cricket, Gryllus bimaculatus. RT-PCR revealed that GbAKH and GbACP are predominantly expressed in the corpora cardiaca and corpora allata, respectively. Phylogenetic analysis confirmed that the identified GbAKH and GbACP belong to the clades containing other AKHs and ACPs, respectively. Injection of synthetic GbAKH and GbACP elevated hemolymph carbohydrate and lipid levels and reduced food intake significantly. In contrast, knockdown of GbAKH and GbACP by RNA interference increased the food intake, although hemolymph lipid level was not altered. Collectively, this study provides evidence that ACP regulates hemolymph carbohydrate and lipid levels in cricket, possibly collaborative contribution with AKH to the maintenance of energy homeostasis.

Ca2+ levels in Daphnia hemolymph may explain occurrences of daphniid species along recent Ca gradients in Canadian soft-water lakes.

Calcium levels are declining in eastern North American and western European lakes. This widespread issue is affecting the composition of crustacean zooplankton communities, as the presence and abundance of several calcium-rich daphniid species are declining, while two other daphniids, D. catawba and D. ambigua, that apparently tolerate low calcium environments, are prospering. The physiological basis for low calcium tolerance of these daphniids is unknown. In this study the presence of one Ca-rich (D. pulicaria) and one Ca-poor (D. ambigua) daphniid species in Canadian Shield lakes is assessed in relation to lake water Ca levels. The occurrence of D. ambigua was independent of Ca levels in Ontario lakes, whereas D. pulicaria was more likely to occur in lakes with relatively more Ca. In the laboratory, D. ambigua maintained lower levels of hemolymph Ca2+ across a range of low Ca levels (0.7 to 7 mg l−1) compared with D. pulicaria. The hemolymph pH remained steady across this Ca gradient in D. ambigua while it was significantly more acidic in D. pulicaria in the two lowest Ca treatments. While Ca2+ uptake was observed adjacent to the surface of D. ambigua individuals, Ca2+ loss was observed for D. pulicaria assayed under moderately high Ca levels. Based on these observations we propose that D. ambigua is able to survive in low Ca lakes by maintaining low free ionic Ca2+ levels in the hemolymph which minimizes the Ca gradient across the body wall in low Ca water thus limiting overall Ca loss and facilitating Ca2+ uptake.

L-3,4-Dihydroxyphenylalanine (l-DOPA) induces neuroendocrinological, physiological, and immunological regulation in white shrimp, Litopenaeus vannamei

L-3,4-Dihydroxyphenylalanine (l-DOPA) is a precursor for dopamine (DA) synthesis. Assessments were conducted to analyze the effects of l-DOPA on mediating regulation of neuroendocrinological, immunological, and physiological parameters in the shrimp, Litopenaeus vannamei when they were individually injected with 0.01 N HCl or l-DOPA at 0.5 or 1.0 μmol shrimp−1 for 60, 120, and 240 min. For catecholamine synthesis evaluation, tyrosine hydroxylase (TH) and DA beta hydroxylase (DBH) activities, l-DOPA, DA, and norepinephrine (NE) levels in hemolymph were determined. The total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency in response to the pathogen, Vibrio alginolyticus were assessed for immune responses, and plasma glucose and lactate levels were for physiological response. Results showed that the TH activity, THC, hyaline cells (HCs), and semigranular cells (SGCs) at 120 min, DA levels at 60–240 min, PO activity in hemocytes per 50 μL of hemolymph at 60–120 min, and PO activity per granulocyte (granular cells (GCs) + SGCs) at 60 min significantly increased, but TH activity, l-DOPA levels, GCs, SGCs, and respiratory bursts in hemocytes per 10 μL of hemolymph at 60 min, respiratory bursts per hemocyte and SOD activity at 120 min, phagocytic activity at 60–240 min, and the clearance efficiency at 60–120 min significantly decreased in shrimp injected with l-DOPA at 1.0 μmol shrimp−1. In another experiment, 60 min after shrimp had received l-DOPA at 0.5 or 1.0 μmol shrimp−1, they were challenged with an injection of V. alginolyticus at 2 × 105 colony-forming units (cfu) shrimp−1. The injection of l-DOPA at 1.0 μmol shrimp−1 also significantly increased the cumulative mortality of shrimp by 16.7%, compared to the HCl-challenged control after 120 h. These results suggest that l-DOPA administration at 1.0 μmol shrimp−1 can mediate the transient regulation of neuroendocrinological, immunological, and physiologic responses resulting in immunosuppression, which in turn promoted the susceptibility of L. vannamei to V. alginolyticus.

Neonicotinoid pesticides and nutritional stress synergistically reduce survival in honey bees.

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD50) could alter bee survival, food consumption and haemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (-50%). Nutritional and pesticide stressors reduced also food consumption (-48%) and haemolymph levels of glucose (-60%) and trehalose (-27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection.

Transgenic corn decreased total and key storage and lipid transport protein levels in honey bee hemolymph while seed treatment with imidacloprid reduced lipophorin levels

Pollen substitutes are currently widely used in apiculture because of a lack of natural forage and an increasing need for bees for pollination services. A major concern in the confection of bee diets is whether specific ingredients, such as GMO grain, or contaminants such as pesticides that are applied to crops, negatively impact bee health. We measured hemolymph protein levels and the lifespan of honey bees fed on diets containing non-hybrid (IAC-Airan), hybrid (AG 7088) or transgenic (AG 8088YG) maize, the seeds of which had been treated with fipronil, imidacloprid or left untreated. Corn from these plantings was harvested, dried and finely ground, and then used for the preparation of corn-based diets. Groups of 100 newly emerged bees were placed in cages, where the diets were offered ad libitum. Honey bee lifespan was similar for all the corn diets, though shorter than for bees fed beebread and longer than for bees fed only honey. Total protein in the hemolymph of bees fed with transgenic maize was 15% lower than in bees fed on non-hybrid maize; lipophorin and vitellogenin, were more reduced, by over 30%. Irrespective of corn variety, imidacloprid resulted in over 25% lower levels of lipophorin compared with the control. Lower storage and transport protein levels would compromise the ability of the bees to rear brood and develop an adequate immune response to pathogen challenge. Consequently, transgenic maize, as well as any maize produced from seed treated with imidacloprid should not be included as ingredients in honey bee diets.

Differential effects of predator cues versus activation of fight-or-flight behaviour on reproduction in the cricket Gryllus texensis

How prey animals determine predation risk remains uncertain. We propose that one signal of high predation risk is repeated activation of fight-or-flight behaviour. We activated escape runs in the cricket Gryllus texensis by blowing air on the cerci. Escape runs were induced for 5 min, three times per day, three times per week for 4 weeks. Repeated fight-or-flight behaviour led to a loss in mass and decreased life span, suggesting a decline in somatic maintenance. However, there was an increase in egg laying, which we interpret as terminal reproductive investment. Stress responses remained robust. Octopamine (OA), a stress neurohormone in insects, increased in concentration in the haemolymph after running, and the magnitude of the increase was the same even after repeated activation (i.e. there was no habituation of the response). There was also no increase in basal OA haemolymph levels. In a second experiment, crickets were exposed to a mantid (predator, Tenodera sinensis), a walking stick (nonpredator, Carausius morosus), or an empty container. None of the crickets exhibited fight-or-flight behaviour. However, mantid-exposed crickets decreased egg laying. There was no decrease in life span or mass. There was no change in basal levels of OA, or in the magnitude of the OA increase after running. These results are consistent with the hypothesis that repeated fight-or-flight behaviour induces reproductive responses that would be adaptive for a shortened life span. These responses differ from those produced by predator cues alone. Even short-lived animals, such as crickets, appear to alter reproduction depending on the relative predation risk and their residual reproductive potential.

Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes.

Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which—together with confocal microscopy and flow cytometry—allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

Pre-treatment with melatonin decreases abamectin induced toxicity in a nocturnal insect Spodoptera litura (Lepidoptera: Noctuidae)

AimOxidative stress is an important component of the mechanism of pesticide toxicity. The aim of the present study was to investigate the time-dependent melatonin effects against abamectin-induced oxidative stress in a S.litura model. Larvae were divided into 5 different groups; (1) control group,(2) Melatonin group (4.3×10−5M/100ml diet), (3) Abamectin group 1.5ml/L, (4) Pre-melatonin treated group (PM) (4.3×10−5M/100ml diet) before abamectin exposure 1.5ml/L, (5) Post-melatonin treated group (TM) after abamectin exposure. Melatonin was supplemented via artificial diet in PM and TM animals during 24h. Main methodsMidgut, fatbody, and hemolymph, were collected for the analysis of oxidative stress markers (Total ROS, GSH, nitrite, TBARS, LPO), antioxidant enzyme levels (SOD, GST, CAT, POX, APOX) in fifth instar larvae. Midgut damage was examined by using morphological analysis. Key findingsOur results observed that ABA group showed significant changes (p<0.001) in the ROS and carbonyl content in midgut. The increase of antioxidant enzyme levels (SOD, CAT, POX, and APOX) in midgut was led by the continuous free radical scavenger cascade of melatonin. Significant (p<0.01) increases in CAT and APOX levels were seen in the fatbody of PM and TM treated insects. SignificanceIn conclusion, the results of the study revealed that abamectin toxicity generates oxidative stress in the insect, while pre-melatonin treatment reduces this damage due to its antioxidant properties, especially POX levels in midgut, fatbody, and hemolymph. Therefore, indoleamine can play a vital role curtailing the abamectin toxicity in time dependent manner in S.litura.

Changes in light and dark periods affect the arylalkylamine N-acetyl transferase, melatonin activities and redox status in the head and hemolymph of nocturnal insect Spodoptera litura

The present study was conducted to describe the impact of circadian rhythm on melatonin levels and redox statusunder three photoperiods (12L:12D, 0L:24D, and 24L:0D) in head and hemolymph of Spodoptera litura. Melatonin is an powerful antioxidant and controls the reproduction of organisms. In this study, melatonin levels, Arylalkylamine N-acetyltransferase(AA-NAT), and antioxidant enzyme activities were analyzed. Results showed melatonin, AA-NAT levels in hemolymph were significantly (p < 0.05) higher during the dark period than during LL regime. HPLC chromatogram of the insect head and hemolymph showed 5 peaks while hemolymph showed 6 peaks in LD, and LLregimes. The day–night changes of melatonin increased the antioxidant enzymes (GST, CAT, POX) persisted in the insect hemolymph, but were suppressed by constant light. The present study leads us to speculate that synthesis and release of melatonin in the S.litura head occur as circadian rhythm and light has an inhibitory effect on melatonin synthesis.

Identification and Characterization of BmVta1, a Bombyx mori (Lepidoptera: Bombycidae) Homologue for Vta1 That is Up-Regulated in Development.

Vps20-associated 1 (Vta1) positively regulates Vacuolar protein sorting 4 (Vps4) to disassemble endosomal sorting complex required for transport III (ESCRT-III) for repeated uses in multivesicular body (MVB) pathway, virus budding and other processes. Currently, these proteins have mainly been studied in yeast and mammalian cells, while identities of them in insects remain largely unknown. We previously identified BmVps4, a Vps4 homologue from Bombyx mori. Here, we report the identification of a homologue for Vta1, designated as BmVta1. The BmVta1 cDNA contains an open reading frame of 933 bp and encodes a protein of 311 amino acid residues. We cloned BmVta1, expressed it in Escherichia coli, and prepared mouse polyclonal antibodies. Like BmVps4, BmVta1 is well conserved as shown by sequence analysis. Both proteins are localized in cytoplasm as revealed by subcellular location analysis. Interestingly, as revealed by semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR), transcriptions of BmVta1 and BmVps4 are highly up-regulated during silkworm metamorphosis and embryogenesis but down-regulated during larva stages, and are of higher levels in head, silk gland and testis than in Malpighian tube, fat body and ganglion, indicating important and similar roles of them in silkworm development and in silkworm tissues and organs. However, compared to BmVps4, the transcription of BmVta1 changes less drastically during development and is of much higher levels in midgut, ovary and hemolymph, suggesting the existence of distinct requirements of them in silkworm development and in certain tissues and organs.

Acute Exposure to Worst-Case Concentrations of Amitraz Does Not Affect Honey Bee Learning, Short-Term Memory, or Hemolymph Octopamine Levels.

Amitraz, an acaricide used to treat Varroa destructor Anderson & Trueman, is one of the most commonly detected pesticides in honey bee (Apis mellifera L.) hives. Acaricides sometimes negatively impact honey bee cognition, but potential effects of amitraz on honey bee learning have been rarely studied. We topically exposed foragers to 95th percentile field-relevant levels of amitraz and, 24 h later, tested the ability of bees to associate a sucrose reward with a conditioned odor (learning response) using the proboscis extension response (PER). We then tested the ability of the bees to retain this memory 1 h and 2 h post-conditioning. Because amitraz is thought to affect octopamine metabolism in honey bees, and because octopamine is directly related to honey bee learning and memory, we also examined effects of exposure to amitraz on octopamine levels in honey bee hemolymph. We found that acute exposure to 95th percentile doses of amitraz had no impact on honey bee learning or short-term memory as measured by PER. Concentrations of octopamine in hemolymph from our low amitraz treatment were 1.4-fold higher than control levels, but other treatments had no effect. Our results from worst-case acute exposure experiments with worker bees in the laboratory suggest that typical field-relevant (within hive) exposures to amitraz probably have little effect on honey bee learning and memory.

Dopamine beta-hydroxylase participate in the immunoendocrine responses of hypothermal stressed white shrimp, Litopenaeus vannamei

Dopamine beta-hydroxylase (DBH) plays a critical role in catecholamine (CA) synthesis of neuroendocrine regulatory network, and is suggested to be involved in the immunoendocrine responses of invertebrate against bacterial challenge. DBH has been identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after hypothermal stress, pharmaceutical inhibition and gene silencing in the present study. Cloned DBH L. vannamei (LvDBH), belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by a DOMON domain, a Cu2_monooxygen domain and three glycosylation sites, and its expression was abundant in thoracic ganglia and haemocytes determined by quantitative real-time PCR. The effects of hypothermal stress showed that LvDBH expression in thoracic ganglia, haemocytes and hepatopancreas as well as the DBH contents in haemocytes and dopamine (DA) and norepinephrine (NE) levels in haemolymph are obviously up-regulated. L. vannamei receiving disulfiram for 30–120 min revealed the inhibition of DBH and NE contents in haemocytes and haemolymph respectively, but high level of DA in haemolymph was noticed. Besides, a significant decrease of LvDBH expression in thoracic ganglia, haemocytes and hepatopancreas were also observed. Subsequently, LvDBH expression was successfully silenced in thoracic ganglia, haemocytes and hepatopancreas of shrimp that received LvDBH-dsRNA for 3 days, and meanwhile, a decrease of DBH contents in haemocytes accompanied by decreased levels of NE and DA in haemolymph were also observed. These results indicate that LvDBH possesses the functional domains responsible for CAs synthesis, and therefore, inhibiting DBH contents in haemocytes by disulfiram and by LvDBH-dsRNA resulted in the impaired synthesis of NE from DA in haemolymph. These also suggest that the increased release of DA and NE in haemolymph for potential modulation of physiological or immunological responses is the consequence of the upregulated LvDBH expression and DBH contents in L. vannamei exposed to hypothermal stress.

Biochemical and histopathological changes of Biomphalaria alexandrina snails exposed to bisphenol A

ABSTRACTThe effects of sub-lethal concentrations of bisphenol A on some biochemical parameters in Biomphalaria alexandrina snail's hemolymph and tissues as well as on histological changes of the hermaphrodite gland were evaluated. Adult snails were exposed to sub-lethal concentrations for four weeks and samples of snails were investigated after two and four weeks. Total protein and albumin levels in hemolymph, as well as lipid peroxidation and glutathione contents of snails’ tissues, were measured. After two weeks of exposure, total protein content and albumin levels decreased in all groups. Elevation of lipid peroxidation levels was correlated with decreasing glutathione content in the treated snails. Histological examination of the hermaphrodite gland revealed deformations in eggs and affected sperm production.

Effects of salinity on short-term waterborne zinc uptake, accumulation and sub-lethal toxicity in the green shore crab (Carcinus maenas)

Waterborne zinc (Zn) is known to cause toxicity to freshwater animals primarily by disrupting calcium (Ca) homeostasis during acute exposure, but its effects in marine and estuarine animals are not well characterized. The present study investigated the effects of salinity on short-term Zn accumulation and sub-lethal toxicity in the euryhaline green shore crab, Carcinus maenas. The kinetic and pharmacological properties of short-term branchial Zn uptake were also examined. Green crabs (n=10) were exposed to control (no added Zn) and 50μM (3.25mgL−1) of waterborne Zn (∼25% of 96h LC50 in 100 seawater) for 96h at 3 different salinity regimes (100%, 60% and 20% seawater). Exposure to waterborne Zn increased tissue-specific Zn accumulation across different salinities. However, the maximum accumulation occurred in 20% seawater and no difference was recorded between 60% and 100% seawater. Gills appeared to be the primary site of Zn accumulation, since the accumulation was significantly higher in the gills relative to the hepatopancreas, haemolymph and muscle. Waterborne Zn exposure induced a slight increase in haemolymph osmolality and chloride levels irrespective of salinity. In contrast, Zn exposure elicited marked increases in both haemolymph and gill Ca levels, and these changes were more pronounced in 20% seawater relative to that in 60% or 100% seawater. An in vitro gill perfusion technique was used to examine the characteristics of short-term (1–4h) branchial Zn uptake over an exposure concentration range of 3–12μM (200–800μgL−1). The rate of short-term branchial Zn uptake did not change significantly after 2h, and no difference was recorded in the rate of uptake between the anterior (respiratory) and posterior (ion transporting) gills. The in vitro branchial Zn uptake occurred in a concentration-dependent manner across different salinities. However, the rate of uptake was consistently higher in 20% seawater relative to 60% or 100% seawater – similar to the trend observed with tissue Zn accumulation during in vivo exposure. The short-term branchial Zn uptake was found to be inhibited by lanthanum (a blocker of voltage-independent Ca channels), suggesting that branchial Zn uptake occurs via the Ca transporting pathways, at least in part. Overall, our findings indicate that acute exposure to waterborne Zn leads to the disruption of Zn and Ca homeostasis in green crab, and these effects are exacerbated at the lower salinity.