Honeybee symbiont Bombella apis could restore larval-to-pupal transition disrupted by antibiotic treatment.

Numerous studies have demonstrated the vital roles of gut microbes in the health, immunity, nutrient metabolism, and behavior of adult worker honeybees. However, a few studies have been conducted on gut microbiota associated with the larval stage of honeybees. In the present study, we explored the role of a gut bacterium in larval development and larval-pupal transition in the Asian honeybee, Apis cerana. First, our examination of gut microbial profiling showed that Bombella apis, a larvae-associated bacterium, was the most dominant bacterium colonized in the fifth instar larvae. Second, we demonstrated that tetracycline, an antibiotic used to treat a honeybee bacterial brood disease, could cause the complete depletion of gut bacteria. This antibiotic-induced gut microbiome depletion in turn, significantly impacted the survivorship, pupation rate and emergence rate of the treated larvae. Furthermore, our analysis of gene expression pattens revealed noteworthy changes in key genes. The expression of genes responsible for encoding storage proteins vitellogenin (vg) and major royal jelly protein 1 (mrjp1) was significantly down-regulated in the tetracycline-treated larvae. Concurrently, the expression of krüppel homolog 1(kr-h1), a pivotal gene in endocrine signaling, increased, whilethe expression of broad-complex (br-c) gene that plays a key role in the ecdysone regulation decreased. These alterations indicated a disruption in the coordination of juvenile hormone and ecdysteroid synthesis. Finally, we cultivated B. apis isolated from the fifth instar worker larval of A. cerana and fed tetracycline-treated larvae with a diet replenished by B. apis. This intervention resulted in a significant improvement in the pupation rate, emergence rate, and overall survival rate of the treated larvae. Our findings demonstrate the positive impact of B. apis on honeybee larvae development, providing new evidence of the functional capacities of gut microbes in honeybee growth and development.

Expression and functional analysis of an odorant binding protein PopeOBP16 from Phthorimaea operculella (Zeller).

Odorant binding proteins (OBPs) are essential proteins in the peripheral olfactory system, responsible for odorant recognition and transport to olfactory receptors. Phthorimaea operculella (potato tuber moth) is an important oligophagous pest on Solanaceae crops in many countries and regions. PopeOBP16 is one of the OBPs in potato tuber moth. This study examined the expression profiles of PopeOBP16. The results of qPCR indicated that PopeOBP16 was highly expressed in the antennae of adults, especially in males, suggesting that it may be involved in odor recognition in adults. The electroantennogram (EAG) was used to screen candidate compounds with the antennae of P. operculella. The relative affinities of PopeOBP16 to 27 host volatiles and two sex pheromone components with the highest relative EAG responses were examined with competitive fluorescence-based binding assays. PopeOBP16 had the strongest binding affinity with the plant volatiles: nerol, 2-phenylethanol, linalool, 1,8-cineole, benzaldehyde, ß-pinene, d-limonene, terpinolene, α-terpinene, and the sex pheromone component trans-4, cis-7, cis-10-tridecatrien-1-ol acetate. The results provide a foundation for further research into the functioning of the olfactory system and the potential development of green chemistry for control of the potato tuber moth.

Genome-wide identification and characterization of the chemosensory relative protein genes in Rhus gall aphid Schlechtendalia chinensis.

BACKGROUND: The Rhus gall aphid Schlechtendalia chinensis specially uses the only species Rhus chinensis and certain moss species (Mniaceae) as its primary host plant and secondary host plants, respectively. Rhus galls are formed on the primary host by the sucking of aphids, and used in traditional medicine as well as other various areas due to their high tannin contents. Chemoreception is critical for insect behaviors such as host searching, location and identification of mates and reproductive behavior. The process of chemoreception is mediated by a series of protein gene families, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs). However, there have been no reports on the analysis of molecular components related to the chemoreception system of S. chinensis at the genome level. RESULTS: We examined the genes of eight OBPs, nine CSPs, 24 ORs, 16 GRs, 22 IRs, and five SNMPs in the S. chinensis genome using homological searches, and these chemosensory genes appeared mostly on chromosome 1. Phylogenetic and gene number analysis revealed that the gene families, e.g., ORs, GRs, CSPs and SNMPs in S. chinensis, have experienced major contractions by comparing to Myzus persicae, while the two gene families OBPs and IRs had slight expansion. The current results might be related to the broader host range of M. persicae versus the specialization of S. chinensis on only a host plant. There were 28 gene pairs between genomes of S. chinensis and Acyrthosiphon pisum in the chemoreceptor gene families by collinear comparison. Ka/Ks ratios (< 1) indicated that the genes of S. chinensis were mainly affected by purification selection during evolution. We also found the lower number and expression level of chemoreception genes in S. chinensis than in other 11 aphid species, such as ORs, GRs and IRs, which play an important role in host search. CONCLUSION: Our study firstly identified the genes of the different chemosensory protein gene families in the S. chinensis genome, and analyzed their general features and expression profile, demonstrating the importance of chemoreception in the aphid and providing new information for further functional research.

Functional disparity of four pheromone-binding proteins from the plum fruit moth Grapholita funebrana Treitscheke in detection of sex pheromone components.

Grapholita funebrana, also known as the plum fruit moth, is an oligophagous pest species that causes enormous economic losses of the fruits of Rosaceae. An eco-friendly method for the control of G. funebrana besides chemical control has not yet been developed. The sex pheromone communication system plays an important role in moth courtship and mating, in which pheromone-binding proteins (PBPs) are critical. In this research, we identified four PBPs, namely, GfunPBP1.1, GfunPBP1.2, GfunPBP2, and GfunPBP3, from the antennae of G. funebrana. The results of real-time quantitative PCR (RT-qPCR) showed that all four GfunPBPs were overwhelmingly expressed in the antennae and that GfunPBP1.2 and GfunPBP2 showed male-biased expression patterns, whereas GfunPBP1.1 and GfunPBP3 were equally expressed between sexes. The results of ligand-binding assays illustrated that although all four recombinant GfunPBPs (rGfunPBPs) had binding activity with the tested sex pheromone compounds, their preferred ligands were significantly different. rGfunPBP2 had the strongest binding affinity to Z8-12:Ac and Z8-12:OH; rGfunPBP1.1 preferred to bind Z8-14:Ac, Z10-14:Ac, and 12:OH more than to the other three GfunPBPs; and rGfunPBP1.2 exhibited stronger binding affinity to E8-12:Ac than to the other rGfunPBPs. Molecular docking results demonstrated that hydrophobic forces, especially van der Waals forces and hydrogen bonds, were the most important forces that maintained GfunPBP-pheromone ligand complexes. This study will improve our understanding of the sex pheromone recognition mechanisms of G. funebrana and promote the development of novel strategies for controlling G. funebrana.

CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.

Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.

Tyrosine hydroxylase involved in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

BACKGROUND: Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. RESULTS: The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. CONCLUSIONS: The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.

RNAi suppression of the nuclear receptor FTZ-F1 impaired ecdysis, pupation, and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Fushi-tarazu factor 1 (FTZF1) is an ecdysone-inducible transcription factor that plays a vital role during the metamorphosis in insects. In this study, we functionally characterized HvFTZ-F1 in H. vigintioctopunctata, a dreadful solanaceous crop pest, by using a dietary RNA interference technique. The HvFTZ-F1 expression levels were elevated in the 1st and 2nd-instars before molting and declined immediately after ecdysis. The HvFTZ-F1 silencing led to high mortality in the 1st instars, while the expression of the osmosis-regulative gene, HvAQPAn.G, was significantly increased in the 1st instars. HvFTZ-F1 silencing downregulated the Halloween and 20E-related genes, decreased the ecdysteroids titer, suppressed the expression of pigmentation-related genes, and reduced the catecholamines titer. In the 4th instars, HvFTZ-F1 silencing caused 100% mortality by arresting the development at the prepupal stage and preventing new abdominal cuticle formation. In the female adults, HvFTZ-F1 silencing caused an evident decrease in fecundity, prolonged the pre-oviposition period, reduced the number of eggs and hatching rate, severely atrophied the ovaries. Moreover, the 20E-related genes and the dopamine synthesis genes were suppressed in the dsHvFTZ-F1-treated females. Overall, our results revealed that HvFTZ-F1 regulates ecdysis, pupation, and reproduction in H. vigintioctopunctata, thereby could be a promising molecular target for the development of RNAi-based biopesticides to control H. vigintioctopunctata.

PLA2 mediates the innate immune response in Asian corn borer, Ostrinia furnacalis.

The eicosanoid signaling pathway mediates insect immune reactions to a wide range of stimuli. This pathway begins with the biosynthesis of arachidonic acid (AA) from the hydrolysis of phospholipids catalyzed by phospholipase A2 (PLA2 ). We report here that the PLA2 inhibitor, dexamethasone (DEX), impaired the innate immune response including nodulation, encapsulation, and melanization in Ostrinia furnacalis larvae, while AA partially reversed these effects of DEX. We cloned a full-length complementary DNA encoding a PLA2 , designated as OfsPLA2 , from O. furnacalis. The open reading frame of OfsPLA2 encodes a 195-amino acid residue protein with a 22-residue signal peptide. Sequence alignment analyses indicated that O. furnacalis PLA2 might be a Group III secretory PLA2 . The highest transcript levels of OfsPLA2 were detected in the fat body, and its transcript levels increased dramatically after infection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. Recombinant OfsPLA2 significantly induced prophenoloxidase (PPO) activation in larval hemolymph in the presence of Ca2+ and encapsulation of agarose beads. Injection of recombinant OfsPLA2 into larvae resulted in increased transcript levels of attacin, defencin, and moricin-3 genes. Our results demonstrate the involvement of the eicosanoid signaling pathway in the innate immune response of O. furnacalis larvae and provide new information about the roles of O. furnacalis secretory PLA2 in activating PPO and antimicrobial peptide production.

Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus.

BACKGROUND: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. METHODS AND FINDINGS: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. CONCLUSIONS AND SIGNIFICANCE: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.

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.

Identification of candidate chemosensory receptors in the antennal transcriptome of Tropidothorax elegans.

Chemosensory receptors in the dendritic membrane of olfactory cells are critical for the molecular recognition and discrimination of odorants. Tropidothorax elegans is a major pest of agricultural, ornamental, and medicinal plants. However, very little is known about olfactory genes in T. elegans. The purpose of this study was to obtain chemosensory receptor genes by sequencing the antennal transcriptome of T. elegans using Illumina sequencing technology. We identified 153 candidate chemosensory receptors, including 121 olfactory receptors (including one olfactory receptor co-receptor), 10 ionotropic receptors (including one IR8a and one IR25a), and 22 gustatory receptors (GRs). TeleOR76, 104 and 112 displayed more highly expression level than TeleOrco. Other TeleGR genes were expressed at very low levels except TeleGR1 and 20. TeleIR76b was the most highly expressed among TeleIR genes. Our results provide valuable biological information for studies of the olfactory communication system of T. elegans.

Tachykinin signaling inhibits task-specific behavioral responsiveness in honeybee workers.

Behavioral specialization is key to the success of social insects and leads to division of labor among colony members. Response thresholds to task-specific stimuli are thought to proximally regulate behavioral specialization, but their neurobiological regulation is complex and not well understood. Here, we show that response thresholds to task-relevant stimuli correspond to the specialization of three behavioral phenotypes of honeybee workers in the well-studied and important Apis mellifera and Apis cerana. Quantitative neuropeptidome comparisons suggest two tachykinin-related peptides (TRP2 and TRP3) as candidates for the modification of these response thresholds. Based on our characterization of their receptor binding and downstream signaling, we confirm a functional role of tachykinin signaling in regulating specific responsiveness of honeybee workers: TRP2 injection and RNAi-mediated downregulation cause consistent, opposite effects on responsiveness to task-specific stimuli of each behaviorally specialized phenotype but not to stimuli that are unrelated to their tasks. Thus, our study demonstrates that TRP signaling regulates the degree of task-specific responsiveness of specialized honeybee workers and may control the context specificity of behavior in animals more generally.

In Silico Studies of Lamiaceae Diterpenes with Bioinsecticide Potential against Aphis gossypii and Drosophila melanogaster.

BACKGROUND: The growing demand for agricultural products has led to the misuse/overuse of insecticides; resulting in the use of higher concentrations and the need for ever more toxic products. Ecologically, bioinsecticides are considered better and safer than synthetic insecticides; they must be toxic to the target organism, yet with low or no toxicity to non-target organisms. Many plant extracts have seen their high insecticide potential confirmed under laboratory conditions, and in the search for plant compounds with bioinsecticidal activity, the Lamiaceae family has yielded satisfactory results. OBJECTIVE: The aim of our study was to develop computer-assisted predictions for compounds with known insecticidal activity against Aphis gossypii and Drosophila melanogaster. RESULTS AND CONCLUSION: Structure analysis revealed ent-kaurane, kaurene, and clerodane diterpenes as the most active, showing excellent results. We also found that the interactions formed by these compounds were more stable, or presented similar stability to the commercialized insecticides tested. Overall, we concluded that the compounds bistenuifolin L (1836) and bistenuifolin K (1931), were potentially active against A. gossypii enzymes; and salvisplendin C (1086) and salvixalapadiene (1195), are potentially active against D. melanogaster. We observed and highlight that the diterpenes bistenuifolin L (1836), bistenuifolin K (1931), salvisplendin C (1086), and salvixalapadiene (1195), present a high probability of activity and low toxicity against the species studied.

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine.

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.

Molecular cloning, expression, and characterization of BmSOD3 in silkworm (Bombyx mori).

Superoxide dismutases (SODs) play an essential role in eliminating excess reactive oxygen species and maintaining the redox balance of the immune system. To study the function of BmSOD3 in silkworm, 543-bp full-length complementary DNA-encoding BmSOD3 was cloned from silkworm. The BmSOD3 amino acids were compared to their homologs, and several highly conserved regions were analyzed. We also carried out phylogenetic analyses of the SOD gene. Our results showed that the BmSOD3 gene belonged with the ecCu/Zn SOD gene. The BmSOD3 gene was transformed into the pET28a vector for functional expression in Escherichia coli. The sodium salt-polyacrylamide gel electrophoresis results showed that the molecular weight of recombinant BmSOD3 was about 22 kDa. The recombinant protein BmSOD3 was purified to detect its properties. After purification analyses, the enzyme activity showed Cu/Zn SOD activity, and the specific activity of the purified enzyme was 0.51 U/mg. The BmSOD3 transcripts showed tissue-specific expression in the midgut and malpighian tubule. The immune microarray data for BmSOD3 showed an expression signal that had a strong response to the induction of four pathogens (Bacillus bombyseptieus, Beauveria bassiana, E. coli, and nuclear polyhedrosis virus), particularly after infection for 24 h, which indicates that the BmSOD3 gene plays a key role in response to bacterial, fungal, and viral invasion. The fusion protein also showed antibacterial activity against E. coli in vitro. Thus, the fusion protein BmSOD3 exhibits antibacterial activity and may be used in production to combat diseases caused by bacteria in silkworm.

Selenium accumulation in protein fractions of Tenebrio molitor larvae and the antioxidant and immunoregulatory activity of protein hydrolysates.

Although numerous types of organisms have been used to enrich selenium, a low-cost and efficient organism is yet to be identified. This study aimed to develop a new means of selenium enrichment using Tenebrio molitor larvae. Our results indicated that the total selenium content in larvae was increased 83-fold to 54.21 ± 1.25 µg/g, and of this content, organic selenium accounted for over 97% after feeding the larvae with 20 µg/g of sodium selenite. Selenium was distributed unequally in the protein fraction with following order: alkali-soluble protein-bound selenium (36.32%) > salt-soluble protein-bound selenium (19.41%) > water-soluble protein-bound selenium (17.03%) > alcohol-soluble protein-bound selenium (3.21%). Additionally, 81% of the selenium within the soluble proteins was distributed in subunits possessing molecular weights of <40 kDa. After hydrolysis by alcalase, the protein hydrolysate of selenium-enriched larvae possessing 75% selenium recovery exhibited stronger antioxidant and immunoregulatory activities than those of regular larvae.

Caffeic acid and caffeine attenuate toxicity associated with malonic or methylmalonic acid exposure in Drosophila melanogaster.

The deficiency in the activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) leads to a condition called methylmalonic academia, which is characterised by the accumulation of methylmalonic (MMA), malonic (MA) or other organic acids. Importantly, we have recently found that supplementation with Ilex paraguariensis aqueous extract offered protection against toxicity associated with MMA or MA exposure to Drosophila melanogaster. Of note, caffeic acid (CA) and caffeine (CAF) were the major phytochemicals found in our Ilex paraguariensis crude extract. Therefore, here, we have exploited CA and/or CAF to test the hypothesis that supplementation with the isolated compounds (either alone or combined) could exert a protective effect against MMA or MA-induced toxicity in flies. Therefore, flies were exposed to MA (5 mM) or MMA (5 mM) and concomitantly treated with CA (1.39 µg/mL), CAF (1.27 µg/mL) or CA + CAF for 10 days for survival, and for 4 days for behavioural and biochemical assays. CA, CAF and CA + CAF treatments completely abolished the mortality associated with either MMA or MA exposure. Moreover, CA and CAF, either alone or combined, completely abolished behavioural changes, and completely protect against changes in thiobarbituric acid reactive substances (TBARS) levels, catalase (CAT) activity and MTT reduction ability, associated with MA or MMA exposure. In turn, CAF restored SOD activity in the head of flies exposed to MA or MMA. However, CA and CAF (either alone or combined) significantly decreased acetylcholinesterase (AChE) activity per se, while CAF alone protected from changes in AChE activity (in head tissue) associated with MA or MMA. Finally, CA and/or CAF were able to protect from a decrease in glucose and triglyceride levels associated with both MA and MMA exposures in haemolymph. Together, our data confirm the hypothesis that supplementation with CA and/or CAF offers protection against detrimental changes associated with MMA or MA exposure in flies, being responsible, at least in part, for the protective effect of I. paraguariensis crude extract which was reported previously.

Protective effect of Catharanthus roseus plant extracts against endosulfan and its isomers induced impacts on non-targeted insect model, Drosophila melanogaster and live brain cell imaging.

Endosulfan has been recognized as a highly controversial pesticide due to its acute toxicity, potential bioaccumulation, persistency, and long-range atmospheric transport. Several plant extracts act as antioxidant agents against wide-range of pesticide toxicity hazards through the free radicals scavenging properties. Plants' secondary metabolites are considered as efficient protective agents against various cellular toxic injuries. Understanding these properties of botanicals, several researchers currently focused on the detoxification and ameliorative potency of plant extracts against highly toxic chemicals. In our studies, we focused on the endosulfan total and its isomers (alpha and beta) induced changes on Drosophila melanogaster and their ameliorative effects by co-administrated with methanolic and aqueous extracts of Catharanthus roseus whole plant. We selected the 1/5th EC50 concentration of alpha-endosulfan, beta-endosulfan, and endosulfan (total) and co-administrated with 1/50th EC50 concentration of aqueous and methanolic extracts and evaluated their ameliorative effects, in terms of verifying the life stage activities, protein profiling and also by using live brain cells imaging. We finally concluded that, the methanolic and aqueous extracts inhibit the toxic impacts caused by endosulfan and its isomers and also increasing the survival rate of the test organism.

Anti-ageing effects of red ginseng on female Drosophila melanogaster.

Red ginseng (RG) was recently reported to extend the lifespan of Drosophila melanogaster. However, the mechanism underlying this effect has not yet been elucidated. The present study aimed to elucidate the molecular mechanisms of the RG-mediated prolongation of the lifespan of female D melanogaster. In this study, protein changes in 36-day-old female D melanogaster were identified using isobaric tag for relative and absolute quantitation (iTRAQ), and levels of differentially expressed proteins were verified by quantitative real-time PCR and Western blotting. Our studies have shown that RG concentrations of 12.5, 15 and 17.5 mg/mL significantly prolonged the lifespan. Eleven proteins were up-regulated and 46 were down-regulated between the RG and control groups; and Pebp1 expression was significantly down-regulated. In addition, AKT and p-AKT were down-regulated, and ERK, p-ERK and Raf1 were up-regulated by RG. Therefore, RG significantly prolonged the lifespan of female D melanogaster by reducing the expression of Pebp1, up-regulating ERK and inhibiting the AKT pathway. RG may be a potential drug for anti-ageing treatment.

Zucchini consensus motifs determine the mechanism of pre-piRNA production.

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.