Influences of acephate and mixtures with other commonly used pesticides on honey bee (Apis mellifera) survival and detoxification enzyme activities.

Acephate (organophosphate) is frequently used to control piercing/sucking insects in field crops in southern United States, which may pose a risk to honey bees. In this study, toxicity of acephate (formulation Bracket®97) was examined in honey bees through feeding treatments with sublethal (pollen residue level: 0.168 mg/L) and median-lethal (LC50: 6.97 mg/L) concentrations. Results indicated that adult bees treated with acephate at residue concentration did not show significant increase in mortality, but esterase activity was significantly suppressed. Similarly, bees treated with binary mixtures of acephate with six formulated pesticides (all at residue dose) consistently showed lower esterase activity and body weight. Clothianidin, λ-cyhalothrin, oxamyl, tetraconazole, and chlorpyrifos may interact with acephate significantly to reduce body weight in treated bees. The dose response data (LC50: 6.97 mg/L) revealed a relatively higher tolerance to acephate in Stoneville bee population (USA) than populations elsewhere, although in general the population is still very sensitive to the organophosphate. In addition to killing 50% of the treated bees acephate (6.97 mg/L) inhibited 79.9%, 20.4%, and 29.4% of esterase, Glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities, respectively, in survivors after feeding treatment for 48 h. However, P450 activity was elevated 20% in bees exposed to acephate for 48 h. Even though feeding on sublethal acephate did not kill honey bees directly, chronic toxicity to honey bee was noticeable in body weight loss and esterase suppression, and its potential risk of synergistic interactions with other formulated pesticides should not be ignored.

Different response of acetylcholinesterases in salt- and detergent-soluble fractions of honeybee haemolymph, head and thorax after exposure to diazinon.

Organophosphate pesticide diazinon is a specific inhibitor of acetylcholinesterase (AChE), which is a common neurotoxicity biomarker in environmental studies. In honeybees, AChE exists in two forms having different physiological roles, one existing as a soluble form and the other as membrane-bound. In most studies AChE activity has been analysed without paying considerable attention to different forms of AChE. In this study, we exposed honeybees Apis mellifera carnica for 10days to diazinon via oral exposure and analysed the total AChE activities in salt soluble (SS) and detergent soluble (DS) fractions. We assumed that SS fraction would preferentially contain the soluble AChE, but the DS fraction would contain only membrane AChE. On the contrary, our results showed that SS and DS fractions both contain soluble and membrane AChE and the latter has considerably higher activity. Despite this we obtained a differential response of AChE activity in SS and DS fractions when exposed to diazinon. The head/thorax AChE activity in DS fraction decreased, while the head/thorax AChE activity in SS fraction increased at sublethal concentrations. The AChE activity in honeybee hemolymph shown here for the first time is a salt soluble enzyme. Its activity remained unaltered after diazinon treatment. In conclusion, we provide evidence that varying results regarding AChE activity alterations upon stressor exposure are obtained when extracted through different procedures. In further environmental studies with honeybees this differential response of AChE activity should be given considerable attention because this affects the outcome of ecotoxicity study.

Altered incubation temperatures between embryonic Days 7 and 13 influence the weights and the mitochondrial respiratory and enzyme activities in breast and leg muscles of broiler embryos.

Altering incubation temperature during embryogenesis has an impact on chicken embryo growth, but the underlying molecular mechanisms are not understood; the present study was performed to address these changes. Broiler eggs were incubated at low (36.8°C), control (37.8°C), and high (38.8°C) temperatures between Embryonic Day (ED) 7 and 10 or ED 10 and 13, which cover critical periods of embryonic myogenesis. The embryos were then dissected immediately after treatment on ED 10 or 13 to assess body, liver, and heart weights as well as to analyze breast and leg muscle fibers for their mitochondrial respiratory activity (MRA). Breast muscle samples were additionally used to evaluate the activity of enzymes involved in energy metabolism and cell-cycle progression. ED-10 embryos incubated at 38.8°C showed elevated weights (body, liver, and heart), MRA, and activities of lactate dehydrogenase and cytochrome oxidase compared to the ED-10 embryos incubated at 36.8°C. Similarly, the ED-13 embryos incubated at 38.8°C showed elevated body weight, MRA, and activities of glycogen phosphorylase, phosphofructokinase, and cytochrome oxidase compared to their 36.8°C counterparts. Embryos incubated at the normal temperature (37.8°C), however, showed variable differences from those incubated at 38.8°C versus 36.8°C. Cell-cycle enzyme activities were not impacted by the different temperature treatments. Thus, an increase or decrease in the incubation temperature during embryonic broiler myogenesis results in altered embryo activity, muscle energy metabolism, and activity-dependent muscle growth.

Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes.

BACKGROUND: Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects. RESULTS: We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg(2+), the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). CONCLUSIONS: AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 µM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis.

Biochemical and toxicological properties of two acetylcholinesterases from the common bed bug, Cimex lectularius.

We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.

Multiple myeloma presenting initially with pleural effusion and a unique paraspinal tumor in the thorax.

We herein report an extremely rare case of a patient with IgD-lambda positive multiple myeloma presenting with myelomatous pleural effusion and ascites. A 58-year-old man visited our hospital with dyspnea as his initial symptom. His chest radiograph findings on admission revealed a left pleural effusion, and later, bilateral involvement. Computed tomography (CT) of the chest showed a paraspinal tumor with extension from the upper mediastinum to the abdomen. The cytological examination demonstrated myeloma cells in the pleural effusion and ascites, and histologically, in the pleura, an abdominal subcutaneous tumor and bone was observed. The pleural effusion was an exudate and slightly bloody. The ADA was 70 IU/L. Pleural effusion accompanying myeloma or primary pleural myeloma is very rare and, furthermore, the extremely rare findings of both myeloma cells in the ascites (although the ascites was mainly caused by liver cirrhosis) and a high ADA activity in the pleural fluid were also observed in this case.

Proline can be utilized as an energy substrate during flight of Aedes aegypti females.

In order to determine whether proline can be utilized as fuel during flight of Aedes aegypti, proline, alanine, and glutamine concentrations were monitored at 0, 30 and 60 min after flight using sugar-fed males and females, and blood meal-fed females. In sugar-fed and blood meal-fed females, flight lead to a significant decrease in proline and a significant increase in glutamine concentration in both hemolymph and thorax. Only during flight after a blood meal was a significant increase in the alanine concentration observed in hemolymph. After flight, the proline alanine and glutamine levels in the hemolymph and thorax from males did not change significantly. In addition, activities of enzymes related to amino acid metabolism were assayed in homogenates of cephalothorax and thorax from both sexes, and in fat body and midgut from females. In both sexes, the activities of all the enzymes studied were significantly higher in thorax than in cephalothorax. The levels of the enzymes involved in proline oxidation were higher in thorax than in fat body and midgut. These results suggest that proline can be used as an energy substrate for flight muscle of Ae. aegypti females. However, the elevation in glutamine levels observed in hemolymph and thorax after flight has not been reported in other insects that fuel flight using proline and may suggest an additional mechanism for shuttling ammonia between flight muscle and fat body is present in mosquitoes.

Glutathione S-transferases in housefly (Musca domestica): location of GST-1 and GST-2 families.

The two families of dipteran glutathione S-transferases (GST-1 and GST-2) were located separately by immunohistology on sections of adult houseflies. GST-1 was distributed in haemolymph cells, whereas GST-2 was found in the indirect flight muscles of the thorax and in the central nervous system. In the muscles, the distribution of GST-2 seemed to be uniform in cells, whereas in the brain and the thoracic ganglia GST-2 was found mainly in the cortical areas which are made up by cell bodies. Comparison of the GSTs' location between an insecticide susceptible strain of housefly and resistant ones indicated no variation due to resistance. Enzyme-linked immunosorbent assay tests were used to dose GST-2. In houseflies, there were 60 pmol of GST-2/fly, 80-90% being found in the thorax, about 10% in the head and the remainder in the abdomen. Furthermore, the roles of these GSTs are discussed in relation to their location and our knowledge on their catalytic activities or their transport ability in invertebrates and mammals.

Insect acetyl-CoA carboxylase: enzyme activity during adult development and after feeding in the tsetse fly, Glossina morsitans.

Acetyl-CoA carboxylase (EC 6.1.4.2) activity in the adult tsetse fly (Glossina morsitans) increased 2-3 days after pupation to reach a plateau of between 0.4 and 0.6 mumol/min/mg after 7 days, and between 0.6 and 0.8 mumol/min/mg after 6 days in the abdomens of male and female flies, respectively. The enzyme showed a 50-70% increase in specific activity within 20 hr after a blood meal in previously starved flies. Lipogenesis and acetyl-CoA carboxylase activity were detected in the thorax, the abdominal cuticle and, in greatest quantity, in the fat body.

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.

The cyclic AMP phosphodiesterase encoded by the Drosophila dunce gene is concentrated in the mushroom body neuropil.

Drosophila dunce (dnc) flies are defective in learning and memory as a result of lesions in the gene that codes for a cAMP-specific phosphodiesterase (PDE). Antibodies to the dnc PDE showed that the most intensely stained regions in the adult brain were the mushroom body neuropil--areas previously implicated in learning and memory. In situ hybridization demonstrated that dnc RNA was enriched in the mushroom body perikarya. The mushroom bodies of third instar larval brains were also stained intensely by the antibody, suggesting that the dnc PDE plays an important role in these neurons throughout their development. The role of the dnc PDE in mushroom body physiology is discussed, and a circuit model describing a possible role of the mushroom bodies in mediating olfactory learning and memory is presented.

Developmental changes in phosphodiesterase activity and hormonal response in the prothoracic glands of Manduca sexta.

Prothoracicotropic hormone (PTTH) stimulates ecdysteroid secretion by the prothoracic glands of Manduca sexta in a cAMP-dependent manner. However, larval and pupal glands differ markedly in the degree to which PTTH stimulates cAMP accumulation, suggesting a stage-specific difference in phosphodiesterase activity. The present study was designed to determine if and when such a difference arose during development, and its effect on PTTH-stimulated ecdysteroid secretion. The results reveal that soluble phosphodiesterase activity in the prothoracic glands changes significantly during the course of the fifth (last) larval instar, with a marked increase in activity occurring at the onset of prepupal development. Phosphodiesterase activity, particularly in the soluble cell fraction, is inversely correlated with PTTH-stimulated cAMP accumulation. Hormone-stimulated ecdysteroid secretion does not require cAMP accumulation, but does appear to require detectable cAMP synthesis as measured in the presence of phosphodiesterase inhibitors. The amount of ecdysteroid secreted, however, is not proportional to the amount of cAMP synthesized but rather is more closely correlated with developmental changes in glandular protein content.

A calmodulin-sensitive adenylate cyclase in the prothoracic glands of the tobacco hornworm, Manduca sexta.

The Ca2+/calmodulin (CaM) dependence of adenylate cyclase activity in Manduca sexta prothoracic glands was investigated. Membrane fractions from two developmental stages were used, day 3 of the last larval instar and day 0 of the pupal stage, both of which respond to the neuropeptide prothoracicotropic hormone (PTTH) with increased cAMP production dependent on extracellular Ca2+. The data revealed that both larval and pupal prothoracic gland membrane fractions have a Ca2+/CaM-dependent adenylate cyclase which is inhibited by CaM antagonists and EGTA. The larval adenylate cyclase shows a multiphasic response to Ca2+/CaM, with a 2-fold stimulation between 0.02 and 0.01 microM, a further increase in adenylate cyclase activity at concentrations greater than 2 microM and a potentiation of NaF-stimulated activity at doses greater than 0.1 microM Ca2+/CaM. Pupal prothoracic gland membrane fractions exhibit only the second phase of stimulation. Stimulation by the GTP analogs GTP-gamma-S and Gpp(NH)p is dependent on CaM in larval, but not in pupal membrane fractions, suggesting a role for CaM in Gs protein-mediated regulation of adenylate cyclase. However, adenylate cyclase activity in glands from both stages is dependent on CaM, supporting our initial premise that Ca2+ is required for cAMP synthesis in the prothoracic glands.

Sexual dimorphism and electrophoretic variation in the form I cyclic nucleotide phosphodiesterase from Drosophila melanogaster.

We have investigated the form I cyclic nucleotide phosphodiesterase (PDE) from Drosophila melanogaster and shown that whereas heads and male thoraces and abdomens contain high levels of Ca2+-stimulated enzyme, female thoraces and abdomens contain little Ca2+-stimulated activity. The electrophoretic patterns of form I PDE from these 3 sources have also been studied and reveal that heads, and male thoraces and abdomens, produce two bands of form I PDE both of which are stimulated by Ca2+. Extracts of female thoraces and abdomens, on the other hand, show only a single, faster running band of PDE activity which is only marginally stimulated by Ca2+, if at all. Surveying wild-type strains of Drosophila has revealed that one strain, Swedish, shows altered electrophoretic mobility of the PDE band from female thoraces and abdomens. The alteration is such that the Swedish PDE band runs more anodally than the Oregon-R and Canton-S PDE activities. Mixing experiments, using co-homogenization of heads with female thoraces and abdomens, yield a single faster running band on electrophoresis. This band contains only Ca2+-insensitive PDE. Attempts to reconstruct this loss of Ca2+-sensitive PDE without electrophoresis have failed. The Swedish electrophoretic variation of the PDE from female thoraces and abdomens has been found to be recessive with respect to the Canton-S phenotype, but the variation is observed to re-emerge and segregate with the third chromosome in the F2 generation. The results indicate that electrophoretic variation in the form I PDE is, by itself, insufficient to allow the location of the structural gene for this enzyme.

Multiple defects in the activity of adenylate cyclase from the Drosophila memory mutant rutabaga.

Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from approximately 10(-5) M to approximately 10(-3) M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole-body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.

The localization of honey bee thorax trehalase.

Differential and sucrose gradient centrifugation of honey bee thoraces, disrupted by gentle methods and using mannitol-triethanolamine-EDTA buffer at pH 6.5, showed that in the honey bee thorax 92-94.8% of the trehalase was mitochondrial. Since only 92-95% of the cytochrome c oxidase, a known mitochondrial enzyme, was found in the mitochondrial fraction by these methods, it was concluded that honey bee trehalase is totally mitochondrial. Significant amounts of 'microsomal' or 'soluble' trehalase were formed only by harsh methods of thorax disruption and similar 'microsomal' or 'soluble' trehalases were also formed by harsh treatment of purified whole mitochondria. They thus seem to be artifacts of the isolation procedure. Studies (using marker enzymes) with purified intact mitochondria which were dispersed by various chemical, enzymatic, and physical methods showed that the trehalase in the mitochondria was membrane bound and that it was bound to either the outside of the inner membrane or to one of the sides of the outer membrane.

Seasonal changes in the histoenzymatic distribution of acid and alkaline phosphatases activity in the B cells of the thoracic ganglion of fresh-water crab, Potamon magnum magnum (Pretzman).

The present study deals with acid and alkaline phosphatases activity determination in B cells of the thoracie ganglion of Potamon magnum magnum. Intense alkaline and acid phosphatases activity is seen in the neurosecretory granules as well as in the neural sheath. Acid phosphatase activity unlike alkaline phosphatase distribution, is seen in form of positive stained spherules showing different pattern of distribution. Alkaline phosphatase activity is linked with the metabolic process. The significance of these results is discussed.

Penetration and comparative metabolism of leptophos in susceptible and resistant houseflies.

The metabolism and rate of penetration of leptophos (O-methyl O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate) was determined in a susceptible strain and a strain of housefies which was 50-fold resistant to leptophos. Penetration of leptophos into resistant flies was substantially slower than into susceptible flies but large differences in metabolism, both quantitatively and qualitatively, were not observed. No difference was observed in the sensitivity of flyhead and thorax acetylcholinesterase to leptophos-oxon in vitro, and tolerance to leptophos by the resistant strain is explained in terms of decreased rates or penetration and minor differences in metabolism.