Calcium signaling and regulation of ecdysteroidogenesis in crustacean Y-organs.

Crustacean Y-organs secrete ecdysteroid molting hormones. Ecdysteroids are released in increased amount during premolt, circulate in hemolymph, and stimulate the events in target cells that lead to molting. During much of the molting cycle, ecdysteroid production is suppressed by molt-inhibiting hormone (MIH), a peptide neurohormone produced in the eyestalks. The suppressive effect of MIH is mediated by a cyclic nucleotide second messenger. A decrease in circulating MIH is associated with an increase in the hemolymphatic ecdysteroid titer during pre-molt. Nevertheless, it has long been hypothesized that a positive regulatory signal or stimulus is also involved in promoting ecdysteroidogenensis during premolt. Data reviewed here are consistent with the hypothesis that an intracellular Ca2+ signal provides that stimulus. Pharmacological agents that increase intracellular Ca2+ in Y-organs promote ecdysteroidogenesis, while agents that lower intracellular Ca2+ or disrupt Ca2+ signaling suppress ecdysteroidogenesis. Further, an increase in the hemolymphatic ecdysteroid titer after eyestalk ablation or during natural premolt is associated with an increase in intracellular free Ca2+ in Y-organ cells. Several lines of evidence suggest elevated intracellular calcium is linked to enhanced ecdysteroidogenesis through activation of Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, thereby lowering intracellular cyclic nucleotide second messenger levels and promoting ecdysteroidogenesis. Results of transcriptomic studies show genes involved in Ca2+ signaling are well represented in Y-organs. Several recent studies have focused on Ca2+ transport proteins in Y-organs. Complementary DNAs encoding a plasma membrane Ca2+ ATPase (PMCA) and a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) have been cloned from crab Y-organs. The relative abundance of PMCA and SERCA transcripts in Y-organs is elevated during premolt, a time when Ca2+ levels in Y-organs are likewise elevated. The results are consistent with the notion that these transport proteins act to maintain the Ca2+ gradient across the cell membrane and re-set the cell for future Ca2+ signals.

Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

Modulation of the PGE2-Mediated Pathway in the Eclosion Blocking Effect of Flumethrin and Terpenoid Subfraction Isolated from Artemesia nilagirica in Rhipicephalus annulatus.

Prostaglandins are a group of important cell-signaling molecules involved in the regulation of ovarian maturation, oocyte development, egg laying and associated behaviors in invertebrates. However, the presence of prostaglandin E2 (PGE2), the key enzymes for PGE2 biosynthesis and its interference by drugs were not investigated previously in the ovary of ticks. The present study was undertaken to assess the modulation of the PGE2-mediated pathway in the eclosion blocking effect of flumethrin and terpenoid subfraction isolated from Artemisia nilagirica in Rhipicephalus annulatus ticks. The acaricidal activities and chemical profiling of the terpenoid subfraction were performed. The localization of the cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) enzymes and the quantification of PGE2 in the ovaries of the ticks treated with methanol (control), flumethrin and terpenoid subfraction were also undertaken. In addition, the vitellogenin concentration in hemolymph was also assayed. Both flumethrin and the terpenoid subfraction of A. nilagirica elicited a concentration-dependent inhibition of fecundity and blocking of hatching of the eggs. The COX1 could not be detected in the ovaries of treated and control ticks, while there was no significant difference observed in the concentration of vitellogenin (Vg) in them. The presence of PGES in the oocytes of control ticks was confirmed while the immunoreactivities against PGES were absent in the vitellogenic oocytes of ticks treated with flumethrin and terpenoid subfraction. The levels of PGE2 were below the detection limit in the ovaries of the flumethrin-treated ticks, while it was significantly lower in the ovaries of the terpenoid subfraction-treated ticks. Hence, the prostaglandin E synthase and PGE2 were identified as very important mediators for the signaling pathway for ovarian maturation and oviposition in ticks. In addition, the key enzyme for prostaglandin biosynthesis, PGES and the receptors for PGE2 can be exploited as potential drug targets for tick control. The detection of PGES by immunohistochemistry and quantification of PGE2 by LC-MSMS can be employed as valuable tools for screening newer compounds for their eclosion blocking acaricidal effects.

Silencing of a putative alanine aminotransferase (ALT) gene influences free amino acid composition in hemolymph and fecundity of the predatory bug, Cyrtorhinus lividipennis Reuter.

In Asian rice systems, Cyrtorhinus lividipennis Reuter is an important predator that preys on rice planthopper eggs and young nymphs, as a primary food source. Alanine aminotransferase (ALT) acts in many physiological and biochemical processes in insects. We cloned the full-length complementary DNA of C. lividipennis ClALT. Expression analysis showed higher expression in the fat body and midgut compared to other tissues. It is expressed in all C. lividipennis developmental stages and at least four organs. Silencing of ClALT by RNA interference significantly decreased the ClALT enzyme activity and ClALT expression compared to dsGFP-treated controls at 2 days after emergence (DAE). Silencing of ClALT influenced free hemolymph amino acid compositions, resulting in a reduction of Aspartic acid (Asp) and Alanine (Ala) proportions, and increased Cysteine (Cys) and Valine (Val) proportions in females at 2 DAE. dsClALT treatments led to decreased soluble total protein concentrations in ovary and fat body, and to lower reduced vitellogenin (Vg) expression, body weight, and the numbers of laid eggs. The double-stranded RNA viruse treatments also led to prolonged preoviposition periods and hindered ovarian development. Western blot analysis indicated that silencing ClALT also led to reduced fat body Vg protein abundance at 2 DAE. These data support our hypothesis that ClALT influences amino acid metabolism and fecundity in C. lividipennis.

Biochemical characterization of the cyclooxygenase enzyme in penaeid shrimp.

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

Disruption of the cytochrome P450 CYP6BQ7 gene reduces tolerance to plant toxicants in the red flour beetle, Tribolium castaneum.

In insects, the cytochrome P450 CYP6B family plays key roles in the detoxification of toxic plant substances. However, the function of CYP6 family genes in degrading plant toxicants in Tribolium castaneum, an extremely destructive global storage pest, have yet to be elucidated. In this study, a T. castaneum CYP gene, TcCYP6BQ7, was characterized. TcCYP6BQ7 expression was significantly induced after exposure to essential oil of the plant Artemisia vulgaris (EOAV). Spatiotemporal expression profiling revealed that TcCYP6BQ7 expression was higher in larval and adult stages of T. castaneum than in other developmental stages, and that TcCYP6BQ7 was predominantly expressed in the brain and hemolymph from the late larval stage. TcCYP6BQ7 silencing by RNA interference increased larvae mortality in response to EOAV from 49.67% to 71.67%, suggesting that this gene is associated with plant toxicant detoxification. Combined results from this study indicate that the CYP6 family gene TcCYP6BQ7 likely plays a pivotal role in influencing the susceptibility of T. castaneum to plant toxicants. These findings may have implications for the development of novel therapeutics to control this agriculturally important pest.

Transcriptome analysis reveals the potential mechanism of dietary carotenoids improving antioxidative capability and immunity of juvenile Chinese mitten crabs Eriocheir sinensis.

Carotenoids are known to be involved in the regulation of the antioxidative capability, immune response and stress resistance in crustacean species; however, very limited information is available on their underlying molecular mechanisms. This study performed transcriptome sequencing of hemolymph and hepatopancreas of juvenile Chinese mitten crabs (Eriocheir sinensis) that fed with three diets, i.e. diet A containing 90 mg kg-1 dry weight of astaxanthin, diet B containing 200 mg kg-1 dry weight of ß-carotene and control diet without supplementation of dietary carotenoids. The results showed that there were 2955 and 497 differentially expressed genes (DEGs) in the hemolymph between the astaxanthin treatment and control groups, and between the ß-carotene treatment and control groups, respectively. Moreover, compared with the control group, 833 and 1886 DEGs were obtained in the hepatopancreas of the astaxanthin treatment and the ß-carotene treatment groups, respectively. The DEGs in the three groups were enriched in 255 specific KEGG metabolic pathways according to KEGG enrichment analysis. Through this study, a series of key genes involved in Nrf2 signalling, ROS production, intracellular antioxidant enzymes and chaperones were significantly affected by dietary carotenoids. Dietary carotenoids also significantly altered the expression levels of immune-related molecules associated with signal transduction, prophenoloxidase cascade, apoptosis, pattern recognition proteins/receptors and antimicrobial peptides. In conclusion, this transcriptomic study provides valuable information for understanding the molecular mechanism and potential pathway of dietary carotenoids improved the antioxidative capability and immunity of juvenile E. sinensis.

Acute and long-term manganese exposure and subsequent accumulation in relation to idiopathic blindness in the American lobster, Homarus americanus.

Manganese (Mn) is a hypoxic reactive metal commonly found in marine sediments. Under hypoxic conditions the metal becomes fully reduced to Mn2+ and is biologically available to the benthic community for uptake. Mn is also a potent neurotoxin and it may play a role in the etiology of idiopathic blindness that has been observed in American lobsters. An acute exposure study was designed to expose American lobster, Homarus americanus, to 0, 20, 80, 150, and 300 mg L-1 (ppm) for 96 hs to explore disparities in Mn accumulation among several tissues: optic nerve, brain, hepatopancreas, muscle, hemolymph, gill, and exoskeleton. These concentrations were based on realistic pore-water concentrations (20 mg L-1), high sediment concentrations (80 mg L-1), and unrealistically high concentrations to determine lethality (150 and 300 mg L-1). A positive correlation between Mn accumulation and exposure concentration was observed in all tissues examined. In the internal tissues, manganese concentrations showed a high affinity towards brain, optic nerve, and hemolymph. In the exoskeleton and gills, Mn concentrations were also high, possibly because of internal uptake as well as external adsorption. Concentrations of Mn in tissues from the acute exposure study followed the accumulation pattern: hemolymph > gill > exoskeleton > optic nerve > brain > hepatopancreas = muscle. A long-term exposure study lasting seven weeks was designed to investigate the potential link between high Mn exposure and idiopathic blindness, a condition that affects an estimated 50 % of the adult American lobster population off Southern New England (SNE), USA. A comparison of these exposure studies showed evidence of time-dependent Mn accumulation in brain, muscle, exoskeleton, and gill tissue. Although the relationship between Mn exposure and blindness was not apparent, there was a modest trend in the development of blindness (Chi-square, p = 0.102) in animals exposed to a high concentration (150 mg L-1) of the metal. With no mortalities occurring in the acute study and only one mortality in the long-term study, it is highly unlikely that Mn is acutely toxic to American lobsters at environmentally relevant concentrations. Its potential role in idiopathic blindness remains to be determined.

Facile synthesis of haemocyanin-capped zinc oxide nanoparticles: Effect on growth performance, digestive-enzyme activity, and immune responses of Penaeus semisulcatus.

ZnO nanoparticles (NPs) synthesized using haemocyanin (Hc-ZnONPs) purified from haemolymph of Penaeus semisulcatus were characterized using various techniques. HR-TEM and SEM microscopy indicated Hc-ZnONPs had a typical size of 20-50 nm and were spherical. The objective of current investigation was to assess the effects of dietary supplementation of Hc-ZnONPs on the development and activity of digestive and metabolic enzymes, as well as the antioxidant levels in P. semisulcatus. Trial basal diets were supplemented with Hc-ZnONPs at rates of 0, 10, 20, 40, 60, and 80 mg kg-1 (dry feed weight) and were fed to P. semisulcatus for 30 d. For 60 mg kg-1 Hc-ZnONPs-supplemented feed, significantly (P < 0.05) enhanced endurance, development, and activity of the digestive enzyme were observed. The enzymatic antioxidants and metabolic enzymes activities in the muscle exhibited no significant changes when 10-60 mg kg-1 Hc-ZnONPs-supplemented feed was fed to P. semisulcatus. Conversely, feeding the P. semisulcatus with 80 mg kg-1 Hc-ZnONPs produced a harmful outcome, with significant increase in the enzymatic antioxidants and metabolic enzymes. Consequently, 80 mg kg-1 Hc-ZnONPs was identified as lethal to P. semisulcatus. Hence, it is proposed that the diet of P. semisulcatus can be supplemented with up to 60 mg kg-1 Hc-ZnONPs for improving the endurance, development and immunity.

Effect of sub-chronic dietary L-selenomethionine exposure on reproductive performance of Red Swamp Crayfish, (Procambarus clarkii).

The effect of selenium (Se) on the reproductive system has been investigated in both humans and vertebrates, but few studies of female fertility and reproduction in invertebrate have been reported. This study is aimed to investigate the effect of SeMet on growth performance and reproductive system after crayfish were fed with graded levels of dietary SeMet (0, 1.49, 3.29, 10.02, 30.27 or 59.8 µg Se/g dry weight) for 60 days. Crayfish treated with the high levels of SeMet (10.02, 30.27 and 59.76 µg Se/g) exhibited decreasing FW and CL in both male and female. Interestingly, Se accumulation was higher in ovary than in other tissues, suggesting that ovary may serve as a target organ for Se accumulation. We found that dietary Se concentration of 10.02 µg Se/g significantly improved the spawning rate, promoted the synchronized spawning, and up-regulated the expressions of mRNA of cdc2 and vitellogenin, with significantly increased E2 and VTG concentrations in hemolymph of female crayfish. However, a marked decrease of the E2 contents and spawning rate was observed in the groups treated with 30.27 and 59.76 µg Se/g diets. In conclusion, the results of this study indicated that the Se had maximum accumulation in ovary, affecting the reproductive capacity by intervening the expression of cdc2 and vitellogenin in the reproductive system. The LOAEL to induce FW was observed in crayfish fed with 10.02 µg Se/g diet, and its value can cause toxicity within the range of natural concentration, so the addition of Se in the feed should be within 10.02 µg Se/g.

Dosage and frequency effects of the microalgae Dunaliella sp. on the diet of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

Carotenoid sources in shrimp diets have shown to be effective for improving survival, growth, reproductive capacity, stress resistance, and also for diminishing disease. Dunaliella sp. is known to have high levels of ß-carotenes, which works as pro-vitamin A, enhancing the immune response in shrimp. However, the administration of Dunaliella sp. in shrimp diet needs to be evaluated to determine the appropriate dose and frequency of administration needed to optimize performance in cultured white shrimp. Diets with three different concentrations of Dunaliella sp. flour (1.5, 2 and 3%) were tested, and each one was administered at three different time frequencies: daily, and at 3- and 7-days intervals. Shrimp fed for 20 days were then infected with Vibrio parahaemolyticus (1 × 106 CFU/mL). Hemolymph parameters including protein, glucose, lactate, cholesterol and triglycerides were analyzed to evaluate shrimp stress status. Additionally, L. vannamei innate non-specific immune response was examined by evaluating the activity of prophenoloxidase (proPO), phenoloxidase (PO) and superoxide dismutase (SOD) in hemolymph; shrimp survival was also recorded. Survival after infection with V. parahaemolyticus was higher for shrimp fed with diets consisting of 2% Dunaliella sp. administered every 3 and 7 days. Shrimp fed a diet consisting of 2% or 3% Dunaliella sp. administered every third day showed positive physiological and immune responses to infection. A decrease in lipid oxidation in plasma triglycerides was observed at 48 h post inoculation in shrimp fed at all diets regimes due to Dunaliella sp. antioxidant action. Experimental results suggest the importance of Dunaliella sp. dosage and feeding frequency in L. vannamei diet to improve the survival and immune response.

Adipokinetic hormone signaling determines dietary fatty acid preference through maintenance of hemolymph fatty acid composition in the cricket Gryllus bimaculatus.

Adipokinetic hormone (AKH), an analog of mammalian glucagon, functions in supplying the required energy by releasing lipids and carbohydrates from the fat body into the hemolymph. Our previous study showed that AKH receptor (AKHR) knockdown in the two-spotted cricket Gryllus bimaculatus decreased hemolymph lipid levels and increased its feeding frequency. To reveal underlying mechanisms by which AKH signaling modulates lipid homeostasis, we analyzed the fatty acid composition as the lipid structure in the crickets. AKH administration significantly increased the proportion of unsaturated fatty acids (USFAs) to total fatty acids with decrease of the saturated fatty acids (SFAs) in hemolymph, while these proportions were inversely changed in RNA interference-mediated AKHR-knockdowned (AKHRRNAi) crickets. Interestingly, knockdown of hormone-sensitive lipase (Hsl) by RNAi (HslRNAi) affected the proportion of USFAs and SFAs in a similar manner to that observed in AKHRRNAi crickets. AKH administration in HslRNAi crickets did not change hemolymph fatty acid composition, indicating that AKH signaling critically altered fatty acid composition in the hemolymph through Hsl. In addition, a choice assay revealed that AKHRRNAi significantly increases the preference of USFAs. These data indicate that hemolymph lipid level and composition were modulated by AKH signaling with a complementary feeding behavior toward USFAs.

RNA-Sequencing of Drosophila melanogaster Head Tissue on High-Sugar and High-Fat Diets.

Obesity has been shown to increase risk for cardiovascular disease and type-2 diabetes. In addition, it has been implicated in aggravation of neurological conditions such as Alzheimer's. In the model organism Drosophila melanogaster, a physiological state mimicking diet-induced obesity can be induced by subjecting fruit flies to a solid medium disproportionately higher in sugar than protein, or that has been supplemented with a rich source of saturated fat. These flies can exhibit increased circulating glucose levels, increased triglyceride content, insulin-like peptide resistance, and behavior indicative of neurological decline. We subjected flies to variants of the high-sugar diet, high-fat diet, or normal (control) diet, followed by a total RNA extraction from fly heads of each diet group for the purpose of Poly-A selected RNA-Sequencing. Our objective was to identify the effects of obesogenic diets on transcriptome patterns, how they differed between obesogenic diets, and identify genes that may relate to pathogenesis accompanying an obesity-like state. Gene ontology analysis indicated an overrepresentation of affected genes associated with immunity, metabolism, and hemocyanin in the high-fat diet group, and CHK, cell cycle activity, and DNA binding and transcription in the high-sugar diet group. Our results also indicate differences in the effects of the high-fat diet and high-sugar diet on expression profiles in head tissue of flies, despite the reportedly similar phenotypic impacts of the diets. The impacted genes, and how they may relate to pathogenesis in the Drosophila obesity-like state, warrant further experimental investigation.

A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus.

Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420bp, including an ORF of 2169bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and 'EEVD' motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca.

Dietary alpha-ketoglutarate increases cold tolerance in Drosophila melanogaster and enhances protein pool and antioxidant defense in sex-specific manner.

Alpha-ketoglutarate (AKG) is an important intermediate in Krebs cycle which bridges the metabolism of amino acids and carbohydrates. Its effects as a dietary supplement on cold tolerance were studied in Drosophila melanogaster Canton S. Two-day-old adult flies fed at larval and adult stages with AKG at moderate concentrations (5-10mM) recovered faster from chill coma (0°C for 15min or 3h) than control ones. The beneficial effect of AKG on chill coma recovery was not found at its higher concentrations, which suggests hormetic like action of this keto acid. Time of 50% observed mortality after 2h recovery from continuous cold exposure (-1°C for 3-31h) (LTi50) was higher for flies reared on 10mM AKG compared with control ones, showing that the diet with AKG enhanced insect cold tolerance. In parallel with enhancement of cold tolerance, dietary AKG improved fly locomotor activity. Metabolic effects of AKG differed partly in males and females. In males fed on AKG, there were no differences in total protein and free amino acid levels, but the total antioxidant capacity, catalase activity and low molecular mass thiol content were higher than in control animals. In females, dietary AKG promoted higher total antioxidant capacity and higher levels of proteins, total amino acids, proline and low molecular mass thiols. The levels of lipid peroxides were lower in both fly sexes reared on AKG as compared with control ones. We conclude that both enhancement of antioxidant system capacity and synthesis of amino acids can be important for AKG-promoted cold tolerance in D. melanogaster. The involvement of AKG in metabolic pathways of Drosophila males and females is discussed.

Impact of fluorescent silicon nanoparticles on circulating hemolymph and hematopoiesis in an invertebrate model organism.

Silicon nanoparticles (SiNPs) have attractive potential applications in biological and medical fields, and yet their impact on animals is still controversial, and there have been no reports of their effects on hematopoiesis. In this study, the effects of SiNPs on hemocytes and hematopoiesis were investigated by administering SiNPs via a vascular injection into an invertebrate model, the silkworm. Our results show that the ability of SiNPs to enter different types of circulating hemocytes and their impact on those hemocytes differed significantly. Rapid accumulation of SiNPs was observed in granulocytes, oenocytoids, and spherulocytes, which have immune functions in the circulating hemolymph, whereas SiNPs did not easily enter prohemocytes, which can differentiate into granulocytes, oenocytoids, and spherulocytes and replenish them. The SiNPs that entered the hemocytes initiated autophagy and apoptosis via the lysosomal/mitochondrial pathway. High-dose SiNPs weakly stimulated lysosomal activity in hematopoietic organs, but did not lead to a significant increase in reactive oxygen species or severe autophagy or apoptosis in the organ tissues. We suggest that the damage caused by high-dose SiNPs to hematopoiesis is self-healing, because few SiNPs entered the hematopoietic stem cells in the circulating hemolymph, so the damage to the hematopoietic tissues was limited.

Isolation and characterization of the corticotropin-releasing factor-related diuretic hormone receptor in Rhodnius prolixus.

Rhodnius prolixus, the vector of human Chagas disease, is a hemipteran insect that undergoes rapid post-feeding diuresis following ingestion of a blood meal that can be up to 10 times its initial body weight. Corticotropin-releasing factor-related diuretic hormone (Rhopr-CRF/DH) and serotonin are neurohormones that are synergistic in increasing rates of fluid secretion by Malpighian tubules during this rapid post-feeding diuresis. A Rhopr-CRF/DH receptor transcript has now been isolated and characterized from fifth instar R. prolixus. The receptor is a family B1 (secretin) G protein-coupled receptor (GPCR) and was deorphaned in a heterologous cellular system using Chinese hamster ovary (CHO) cells stably expressing a promiscuous G-protein (Gα16). This assay was also used to demonstrate the presence of Rhopr-CRF/DH in the haemolymph of R. prolixus in response to blood-gorging. Two additional cell lines were used in this heterologous assay to verify that the cyclic adenosine monophosphate (cAMP) pathway and not the inositol triphosphate (IP3) pathway was stimulated upon activation of the receptor. Lastly, quantitative PCR demonstrated strong receptor expression in digestive tissues, upper Malpighian tubules and reproductive tissues. Identification of the Rhopr-CRF/DH receptor now provides tools for a more detailed understanding into the precise coordination of diuresis and other physiological processes in R. prolixus.

Plant phenolics are detoxified by prophenoloxidase in the insect gut.

Plant phenolics are a group of important secondary metabolites that are toxic to many animals and insects if ingested at high concentrations. Because most insects consume plant phenolics daily, they have likely evolved the capacity to detoxify these compounds. Here, we used Drosophila melanogaster, Bombyx mori and Helicoverpa armigera as models to study the metabolism of plant phenolics by prophenoloxidases. We found that insect foreguts release prophenoloxidases into the lumen, and that the survival of prophenoloxidase-deletion mutants was impaired when fed several plant phenolics and tea extracts. Using l-DOPA as a model substrate, biochemical assays in large Lepidopteran insects demonstrated that low levels of l-DOPA are rapidly metabolized into intermediates by phenoloxidases. Feeding with excess l-DOPA showed that the metabolic intermediate 5,6-dihydroxyindole reached the hindgut either by passing directly through the midgut, or by transport through the hemolymph. In the hindgut, 5,6-dihydroxyindole was further oxidized by prophenoloxidases. Intermediates exerted no toxicity in the hemocoel or midgut. These results show that plant phenolics are not toxic to insects unless prophenoloxidase genes are lost or the levels of phenolics exceed the catalytic activity of the gut prophenoloxidases.

Effects of Oral Exposure to Fungicides on Honey Bee Nutrition and Virus Levels.

Sublethal exposure to fungicides can affect honey bees (Apis mellifera L.) in ways that resemble malnutrition. These include reduced brood rearing, queen loss, and increased pathogen levels. We examined the effects of oral exposure to the fungicides boscalid and pyraclostrobin on factors affecting colony nutrition and immune function including pollen consumption, protein digestion, hemolymph protein titers, and changes in virus levels. Because the fungicides are respiratory inhibitors, we also measured ATP concentrations in flight muscle. The effects were evaluated in 3- and 7-d-old worker bees at high fungicide concentrations in cage studies, and at field-relevant concentrations in colony studies. Though fungicide levels differed greatly between the cage and colony studies, similar effects were observed. Hemolymph protein concentrations were comparable between bees feeding on pollen with and without added fungicides. However, in both cage and colony studies, bees consumed less pollen containing fungicides and digested less of the protein. Bees fed fungicide-treated pollen also had lower ATP concentrations and higher virus titers. The combination of effects we detected could produce symptoms that are similar to those from poor nutrition and weaken colonies making them more vulnerable to loss from additional stressors such as parasites and pathogens.

Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.