JNK Signaling in Drosophila Aging and Longevity.

The evolutionarily conserved c-Jun N-terminal kinase (JNK) signaling pathway is a critical genetic determinant in the control of longevity. In response to extrinsic and intrinsic stresses, JNK signaling is activated to protect cells from stress damage and promote survival. In Drosophila, global JNK upregulation can delay aging and extend lifespan, whereas tissue/organ-specific manipulation of JNK signaling impacts lifespan in a context-dependent manner. In this review, focusing on several tissues/organs that are highly associated with age-related diseases-including metabolic organs (intestine and fat body), neurons, and muscles-we summarize the distinct effects of tissue/organ-specific JNK signaling on aging and lifespan. We also highlight recent progress in elucidating the molecular mechanisms underlying the tissue-specific effects of JNK activity. Together, these studies highlight an important and comprehensive role for JNK signaling in the regulation of longevity in Drosophila.

Knockdown of CYP301B1 and CYP6AX1v2 increases the susceptibility of the brown planthopper to beta-asarone, a potential plant-derived insecticide.

The cytochrome P450 monooxygenases of insects play crucial roles in the metabolic detoxification of insecticides. Our previous finding showed that two cytochrome P450 genes, both CYP301B1 and CYP6AX1v2, in the BPH underwent overexpression due to ß-asarone. In this study, we investigated the molecular characteristics, expression patterns and functions of these two cytochrome P450 genes. The results showed that CYP301B1 had the highest expression level in the eggs, while CYP6AX1v2 was expressed in macropterous female adults. Moreover, the expression level of CYP301B1 in the head was higher than that in the integument, fat body and gut. The expression level of CYP6AX1v2 in the fat body and gut was higher than that in head and integument. Importantly, silencing CYP301B1 and CYP6AX1v2 separately could increase the sensitivity, resulting in significant higher mortality of BPH following treatment with ß-asarone. Our findings indicated that CYP301B1 and CYP6AX1v2 could contribute to the resistance of BPH to ß-asarone, and these two genes may be involved in the detoxification metabolism of ß-asarone in BPH.

Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function.

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.

Identification, genomic organization and expression pattern of glutathione transferase in Pardosa pseudoannulata.

The pond wolf spider, Pardosa pseudoannulata, is one of the dominant natural enemies in farmlands and plays important roles in controlling a range of insect pests. The spider is less sensitive to many insecticides than the target pests such as the brown planthopper, Nilaparvata lugens. The different sensitivity to a certain insecticide between species is mostly attributed to the differences in both molecular targets and detoxification enzymes. As one of the most important detoxification enzymes, glutathione transferases (GSTs) play a key role as phase II enzyme in the enzymic detoxification in organisms. Until now, there are few studies on spiders' GSTs, limiting the understanding of insecticide selectivity between insect pests and natural enemy spiders. In this study, based on the transcriptome and genome sequencing of P. pseudoannulata, thirteen full-length transcripts encoding GSTs were identified and analyzed. Interestingly, Delta family, which is thought to be specific to the Insecta, was identified in P. pseudoannulata. Further, vertebrate/mammalian-specific Mu family was also identified in P. pseudoannulata. The mRNA expression levels of cytosolic GSTs in different tissues were determined, and most GST genes were abundant in the gut and the fat body. To investigate GST candidates involving in insecticide detoxification, the mRNA levels of cytosolic GSTs were tested after spiders' exposure to either imidacloprid or deltamethrin. The results showed that PpGSTD3 and PpGSTT1 responded to at least one of these two insecticides. The present study helped understand the function of GSTs in P. pseudoannulata and enriched the genetic information of natural enemy spiders.

Assessing the toxicant effect of spontaneously volatilized 4-vinylcyclohexane exposure in nymphs of the lobster cockroach nauphoeta cinerea.

Vinylcyclohexene (VCH) is an environmental contaminant well known for its ovotoxicant effects in several organisms. However, the mechanisms underlying the toxicity of VCH as well as its harmful effects toward other organs are until unclear. In this work, we assess some endpoint signals of toxicity induced by volatilized VCH exposure using nymphs of the lobster cockroach Nauphoeta cinerea. Nymphs were exposed to VCH via inhalation for 70 days. The levels of volatilized VCH were quantified by headspace gas chromatography and the concentration varied between 3.41 and 7.03 nmol/µl. VCH inhalation caused a reduction of 35% in the survival rate of the exposed animals. Nymphs exposed to volatilized VCH for 35 and 70 days had a reduction in the body weight gain of 1.8- and 2.6-fold, respectively with a reduction in dissected head, fat body, and maturing reproductive organs. The exposure did not change water consumption, excepting on the 20th day (with a 3-fold change) and decreased the food intake significantly. Regarding biochemical markers, we found that the activity of GST from the dissected organs was increased by volatilized VCH after both 35 and 70 days of exposure. The fat body presented the most prominent GST activity especially after 35 days of exposure with 1.6-fold higher than the control group. Exposure also caused an increase in RS levels in the fat body of 1.35-fold and 1.47-fold after 35 and 70 days, respectively and did not affect the activity of the AChE from the head. Our findings support the harmful impact of volatilized VCH inhalation, highlighting the cockroach N.cinerea as a valuable insect model to investigate environmental toxicants.

Telomerase activity is upregulated in the fat bodies of pre-diapause bumblebee queens (Bombus terrestris).

The attrition of telomeres, the ends of eukaryote chromosomes, and activity of telomerase, the enzyme that restores telomere length, play a role in the ageing process and act as indicators of biological age. A notable feature of advanced eusocial insects is the longevity of reproductive individuals (queens and kings) compared to those from non-reproductive castes (workers and soldiers) within a given species, with a proposed link towards upregulation of telomerase activity in the somatic tissues of reproductive individuals. Given this, eusocial insects provide excellent model systems for research into ageing. We tested telomerase activity and measured telomere length in Bombus terrestris, which is a primitively eusocial insect species with several distinct features compared to advanced social insects. In somatic tissues, telomerase activity was upregulated only in the fat bodies of pre-diapause queens, and this upregulation was linked to heightened DNA synthesis. Telomere length was shorter in old queens compared to that in younger queens or workers. We speculate that (1) the upregulation of telomerase activity, together with DNA synthesis, is the essential step for intensifying metabolic activity in the fat body to build up a sufficient energy reserve prior to diapause, and that (2) the lifespan differences between B. terrestris workers and queens are related to the long diapause period of the queen. A possible relationship between telomere length regulation and TOR, FOXO, and InR as cell signaling components, was tested.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

Comparison of Lactate Dehydrogenase Activity in Hive and Forager Honeybees May Indicate Delayed Onset Muscle Soreness - Preliminary Studies.

Active skeletal muscles produce lactate. H+ is generated during lactate neutralization in the Cori cycle, which leads to muscle acidosis and soreness (the so-called Delayed Onset Muscle Soreness, DOMS) in vertebrates. The aim of the study was to determine the activities/concentrations of compounds involved in the Cori cycle in worker and forager bees. Muscles, fat bodies, and hemolymph from 1- and 14-day-old workers and foragers were collected and assayed for the protein, lactate, glucose, NAD+, and NADH concentrations and lactate dehydrogenase (LDH) activity. Both lactate concentration and LDH activity in the hemolymph, muscles, and fat bodies increased with age. The concentrations of NAD+ and NADH in the tissues decreased with ageing/senescence, whereas protein concentrations increased until day 14 of bee's life and then decreased in foragers. The concentration of glucose decreased in the hemolymph and muscles and increased in the fat bodies. Elevated lactate concentrations in foragers may indicate transition from the aerobic to the anaerobic phase and development of metabolic acidosis that may eventually lead to muscle damage/soreness and shorter lifespan. When analyzing flight dynamics, load mass, and bee behavior, changes in the concentrations of Cori cycle compounds should be taken into account.

Global transcriptome profiling and functional analysis reveal that tissue-specific constitutive overexpression of cytochrome P450s confers tolerance to imidacloprid in palm weevils in date palm fields.

BACKGROUND: Cytochrome P450-dependent monooxygenases (P450s), constituting one of the largest and oldest gene superfamilies found in many organisms from bacteria to humans, play a vital role in the detoxification and inactivation of endogenous toxic compounds. The use of various insecticides has increased over the last two decades, and insects have developed resistance to most of these compounds through the detoxifying function of P450s. In this study, we focused on the red palm weevil (RPW), Rhynchophorus ferrugineus, the most devastating pest of palm trees worldwide, and demonstrated through functional analysis that upregulation of P450 gene expression has evolved as an adaptation to insecticide stress arising from exposure to the neonicotinoid-class systematic insecticide imidacloprid. RESULTS: Based on the RPW global transcriptome analysis, we identified 101 putative P450 genes, including 77 likely encoding protein coding genes with ubiquitous expression. A phylogenetic analysis revealed extensive functional and species-specific diversification of RPW P450s, indicating that multiple CYPs actively participated in the detoxification process. We identified highly conserved paralogs of insect P450s that likely play a role in the development of resistance to imidacloprid: Drosophila Cyp6g1 (CYP6345J1) and Bemisia tabaci CYP4C64 (CYP4LE1). We performed a toxicity bioassay and evaluated the induction of P450s, followed by the identification of overexpressed P450s, including CYP9Z82, CYP6fra5, CYP6NR1, CYP6345J1 and CYP4BD4, which confer cross-resistance to imidacloprid. In addition, under imidacloprid insecticide stress in a date palm field, we observed increased expression of various P450 genes, with CYP9Z82, CYP4BD4, CYP6NR1 and CYP6345J1 being the most upregulated detoxification genes in RPWs. Expression profiling and cluster analysis revealed P450 genes with multiple patterns of induction and differential expression. Furthermore, we used RNA interference to knock down the overexpressed P450s, after which a toxicity bioassay and quantitative expression analysis revealed likely candidates involved in metabolic resistance against imidacloprid in RPW. Ingestion of double-stranded RNA (dsRNA) successfully knocked down the expression of CYP9Z82, CYP6NR1 and CYP345J1 and demonstrated that silencing of CYP345J1 and CYP6NR1 significantly decreased the survival rate of adult RPWs treated with imidacloprid, indicating that overexpression of these two P450s may play an important role in developing tolerance to imidacloprid in a date palm field. CONCLUSION: Our study provides useful background information on imidacloprid-specific induction and overexpression of P450s, which may enable the development of diagnostic tools/markers for monitoring the spread of insecticide resistant RPWs. The observed trend of increasing tolerance to imidacloprid in the date palm field therefore indicated that strategies for resistance management are urgently needed.

Changes in antifungal defence systems during the intermoult period in the Colorado potato beetle.

Susceptibility to the fungus Metarhizium robertsii and changes in host defences were evaluated in different stages of the intermoult period (4-6 h, 34-36 h and 84-86 h post moult in IV larval instars) of the Colorado potato beetle. A significant thickening of the cuticle during larval growth was accompanied by decreases in cuticle melanization, phenoloxidase activity and epicuticular hydrocarbon contents (C28-C32). At the same time, a decrease in the conidial adhesion rate and an increase in resistance to the fungus were observed. In addition, we recorded significant elevation of the encapsulation rate and total haemocyte counts in the haemolymph during the specified period. The activity of detoxification enzymes decreased in the haemolymph but increased in the fat body during larval growth. No significant differences in the fatty acid content in the epicuticle were observed. The role of developmental disorders in susceptibility to entomopathogenic fungi is also discussed.

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

AMP-activated protein kinase protects against anoxia in Drosophila melanogaster.

During anoxia, proper energy maintenance is essential in order to maintain neural operation. Starvation activates AMP-activated protein kinase (AMPK), an evolutionarily conserved indicator of cellular energy status, in a cascade which modulates ATP production and consumption. We investigated the role of energetic status on anoxia tolerance in Drosophila and discovered that starvation or AMPK activation increases the speed of locomotor recovery from an anoxic coma. Using temporal and spatial genetic targeting we found that AMPK in the fat body contributes to starvation-induced fast locomotor recovery, whereas, under fed conditions, disrupting AMPK in oenocytes prolongs recovery. By evaluating spreading depolarization in the fly brain during anoxia we show that AMPK activation reduces the severity of ionic disruption and prolongs recovery of electrical activity. Further genetic targeting indicates that glial, but not neuronal, AMPK affects locomotor recovery. Together, these findings support a model in which AMPK is neuroprotective in Drosophila.

Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana.

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.

NONSULFATED SULFAKININ CHANGES METABOLIC PARAMETERS OF INSECT FAT BODY MITOCHONDRIA.

We investigated the effect of neuropeptide, the nonsulfated sulfakinin (SK) Zopat-SK-1 (pETSDDYGHLRFa) on the mitochondrial oxidative metabolism in the Zophobas atratus larval fat body. Mitochondria were isolated from beetle fat bodies 2 and 24 h after hormone injection. The administration of 20 pmol of Zopat-SK-1 to feeding larvae led to decreased mitochondrial oxidative activities in larval fat body. Diminished activities of citrate synthase and the cytochrome pathway, that is, nonphosphorylating and phosphorylating respiration during succinate oxidation, were observed. However, the effect of Zopat-SK-1 was more pronounced in fat body of insects after 24 h since hormone application. In hormone-treated larval fat bodies, mitochondrial respiration was decreased at the level of respiratory chain and the TCA cycle as well as at the level of mitochondrial biogenesis, as indicated by decreased activities of mitochondrial marker enzymes in fat body homogenates. The inhibition of succinate oxidation may indicate the role of Zopat-SK-1 in the regulation of mitochondrial complex II activity. Moreover, decreased respiratory chain activity was accompanied by the reduced activity of mitochondrial energy-dissipating pathway, uncoupling protein 4. The observed decrease in mitochondrial oxidative metabolism may reflect the Zopat-SK-1-induced reduction in the metabolic rate of larval fat body linked to actual energetic demands of animal.

Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif.

Drosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-ß import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.

Adipose triglyceride lipase (Atgl) mediates the antibiotic jinggangmycin-stimulated reproduction in the brown planthopper, Nilaparvata lugens Stål.

The antibiotic jinggangmycin (JGM) is an agrochemical product widely used in China for controlling rice sheath blight, Rhizoctonia solani. Unexpectedly, it stimulates reproduction in the brown planthopper (BPH), Nilaparvata lugens (Stål). However, the underlying molecular mechanisms of the stimulation are unclear. The present investigation demonstrates that adipose triglyceride lipase (Atgl) is one of the enzymes involved in the JGM-stimulated reproduction in BPH. Silence of Atgl in JGM-treated (JGM + dsAtgl) females eliminated JGM-stimulated fecundity of BPH females. In addition, Atgl knockdown significantly reduced the protein and glycerin contents in the ovaries and fat bodies of JGM + dsAtgl females required for reproduction. We conclude that Atgl is one of the key enzymes responsible for JGM-stimulated reproduction in BPH.

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura.

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate-binding motifs were predicted in the protein. Modeling of the 3-D structure showed that the protein consisted of eight α-helixes linked with loops, with no ß-sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.

The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease.

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.