Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis.

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura.

Insect gustatory receptors (GRs) aid in the precise identification of deterrent or stimulant compounds associated with food, mating, and egg-laying. Thus, they are promising targets for developing efficient insecticides. Here, 61 GRs in the chemosensory organs of Spodoptera litura larvae and adults were identified. Among them, SlitGR206 exhibited larval labium (LL)-specific expression characteristics. To explore the role of SlitGR206, a bacterial expression system was established to produce high-quality double-stranded RNA (dsRNA) and suppress SlitGR206 expression in LL. Subsequent behavioral assessments revealed that SlitGR206 silencing influenced larval feeding preferences and absorption. Moreover, it was found to reduce the ability of larvae to forage the five crucial host odorants. These findings demonstrate that SlitGR206 likely plays an indirect regulatory role in host recognition, consequently affecting foraging behavior. This provides a crucial foundation for the analysis of functional diversity among insect GRs and the precise development of nucleic acid pesticides in the future.

Hypoxia delays steroid-induced developmental maturation in Drosophila by suppressing EGF signaling.

Animals often grow and develop in unpredictable environments where factors like food availability, temperature, and oxygen levels can fluctuate dramatically. To ensure proper sexual maturation into adulthood, juvenile animals need to adapt their growth and developmental rates to these fluctuating environmental conditions. Failure to do so can result in impaired maturation and incorrect body size. Here we describe a mechanism by which Drosophila larvae adapt their development in low oxygen (hypoxia). During normal development, larvae grow and increase in mass until they reach critical weight (CW), after which point a neuroendocrine circuit triggers the production of the steroid hormone ecdysone from the prothoracic gland (PG), which promotes maturation to the pupal stage. However, when raised in hypoxia (5% oxygen), larvae slow their growth and delay their maturation to the pupal stage. We find that, although hypoxia delays the attainment of CW, the maturation delay occurs mainly because of hypoxia acting late in development to suppress ecdysone production. This suppression operates through a distinct mechanism from nutrient deprivation, occurs independently of HIF-1 alpha and does not involve dilp8 or modulation of Ptth, the main neuropeptide that initiates ecdysone production in the PG. Instead, we find that hypoxia lowers the expression of the EGF ligand, spitz, and that the delay in maturation occurs due to reduced EGFR/ERK signaling in the PG. Our study sheds light on how animals can adjust their development rate in response to changing oxygen levels in their environment. Given that hypoxia is a feature of both normal physiology and many diseases, our findings have important implications for understanding how low oxygen levels may impact animal development in both normal and pathological situations.

Characterization of myoinhibitory peptide signaling system and its implication in larval metamorphosis and spawning behavior in Pacific abalone.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.

Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Molecular-Weight-Dependent Degradation of Plastics: Deciphering Host-Microbiome Synergy Biodegradation of High-Purity Polypropylene Microplastics by Mealworms.

The biodegradation of polypropylene (PP), a highly persistent nonhydrolyzable polymer, by Tenebrio molitor has been confirmed using commercial PP microplastics (MPs) (Mn 26.59 and Mw 187.12 kDa). This confirmation was based on the reduction of the PP mass, change in molecular weight (MW), and a positive Δδ13C in the residual PP. A MW-dependent biodegradation mechanism was investigated using five high-purity PP MPs, classified into low (0.83 and 6.20 kDa), medium (50.40 and 108.0 kDa), and high (575.0 kDa) MW categories to access the impact of MW on the depolymerization pattern and associated gene expression of gut bacteria and the larval host. The larvae can depolymerize/biodegrade PP polymers with high MW although the consumption rate and weight losses increased, and survival rates declined with increasing PP MW. This pattern is similar to observations with polystyrene (PS) and polyethylene (PE), i.e., both Mn and Mw decreased after being fed low MW PP, while Mn and/or Mw increased after high MW PP was fed. The gut microbiota exhibited specific bacteria associations, such as Kluyvera sp. and Pediococcus sp. for high MW PP degradation, Acinetobacter sp. for medium MW PP, and Bacillus sp. alongside three other bacteria for low MW PP metabolism. In the host transcriptome, digestive enzymes and plastic degradation-related bacterial enzymes were up-regulated after feeding on PP depending on different MWs. The T. molitor host exhibited both defensive function and degradation capability during the biodegradation of plastics, with high MW PP showing a relatively negative impact on the larvae.

Fermented Protaetia brevitarsis Larvae Improves Neurotoxicity in Chronic Ethanol-Induced-Dementia Mice via Suppressing AKT and NF-κB Signaling Pathway.

This study was investigated to examine the neuroprotective effect of fermented Protaetia brevitarsis larvae (FPB) in ethanol-induced-dementia mice. Consumption of FPB by mice resulted in improved memory dysfunction in the Y-maze, passive avoidance, and Morris water maze tests. FPB significantly decreased oxidative stress by regulating levels of malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) in brain tissues. In addition, FPB restored cerebral mitochondrial dysfunction by modulating levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP. In addition, FPB enhanced the cholinergic system via the regulation of acetylcholine (ACh) content, acetylcholinesterase (AChE) activity, and expressions of AChE and choline acetyltransferase (ChAT) in brain tissues. FPB ameliorated neuronal apoptosis through modulation of the protein kinase B (AKT)/B-cell lymphoma (BCL)-2 signaling pathway. Also, FPB improved inflammation response by down-regulating the toll-like receptor (TLR)-4/nuclear factor (NF)-κB pathway. Additionally, FPB ameliorated synaptic plasticity via the increase of the expressions of synaptophysin (SYP), postsynaptic density protein (PSD)-95, and growth-associated protein (GAP)-43. Treatment with FPB also reinforced the blood-brain barrier by increasing tight junctions including zonula occludens (ZO)-1, occludin, and claudin-1. In conclusion, these results show that FPB can improve cognitive impairment via AKT/NF-κB pathways in ethanol-induced-dementia mice.

Skin Anti-Aging Potential through Whitening and Wrinkle Improvement Using Fermented Oil Derived from Hermetia illucens Larvae.

As the aging population increases, so has interest among emerging seniors in anti-aging ingredients that enhance functionality by incorporating fermentation with natural materials. In this study, fermentation conditions for enhancing the functionality of Hermetia illucens larvae oil (HIO) were established, and its anti-aging potential was evaluated. First, the lipase activity and amount of lipid degradation products of the fermentation strains were measured in order to select Lactobacillus gasseri and Lactiplantibacillus plantarum as the strains with high fermentation ability. A fermentation period of 28 d and a fermentation method that uses only the strain culture medium were established by evaluating the fermentation degree after fermenting HIO with the selected strains. The whitening functionality test results of fermented HIO (FHIO) showed an increase of approximately 20% in extracellular tyrosinase inhibition activity compared with HIO. Additionally, within melanocytes, there was a 12% increase in tyrosinase inhibition activity and a 26% enhancement in melanin production inhibition ability. For wrinkle-improving functionality, it was observed that, for fibroblasts, there was a 10% increase in collagen production, a 9% increase in collagenase inhibition ability, and an 8% increase in elastase inhibition ability. Therefore, FHIO was confirmed to be an effective cosmetic raw material, with high functionality for anti-aging within the senior generation. This is achieved through increased whitening and wrinkle-improving functionality.

Identification of insect cuticular protein genes LCP17 and SgAbd5 from Helicoverpa armigera and evaluation their roles in fenvalerate resistance.

Insect cuticular protein (ICP) plays an important role in insect growth and development. However, research on the role of ICP in insecticide resistance is very limited. In this study, insect cuticular protein genes LCP17 and SgAbd5 were cloned and characterized in Helicoverpa armigera based on previous transcriptome data. The functions of LCP17 and SgAbd5 genes in fenvalerate resistance were assessed by RNA interference (RNAi), and their response to fenvalerate was further detected. The results showed that LCP17 and SgAbd5 were overexpressed in the fenvalerate-resistant strain comparing with a susceptible strain. The open reading frames of LCP17 and SgAbd5 genes were 423 bp and 369 bp, encoding 141 and 123 amino acids, respectively. LCP17 and SgAbd5 genes were highly expressed in the larval stage, but less expressed in the adult and pupal stages. The expression level of LCP17 and SgAbd5 genes increased significantly after fenvalerate treatment at 24 h. When the cotton bollworms larvae were exposed to fenvalerate at LD50 level, RNAi-mediated silencing of LCP17 and SgAbd5 genes increased the mortality from 50.68% to 68.67% and 63.89%, respectively; the mortality increased to even higher level, which was 73.61%, when these two genes were co-silenced. Moreover, silencing of these two genes caused the cuticle lamellar structure to become loose, which led to increased penetration of fenvalerate into the larvae. The results suggested that LCP17 and SgAbd5 may be involved in the resistance of cotton bollworm to fenvalerate, and LCP17 and SgAbd5 could serve as potential targets for H. armigera control.

Stage-specific exposure of Caenorhabditis elegans to cadmium identifies unique transcriptomic response cascades and an uncharacterized cadmium responsive transcript.

Age/stage sensitivity is considered a significant factor in toxicity assessments. Previous studies investigated cadmium (Cd) toxicosis in Caenorhabditis elegans, and a plethora of metal-responsive genes/proteins have been identified and characterized in fine detail; however, most of these studies neglected age sensitivity and stage-specific response to toxicants at the molecular level. This present study compared the transcriptome response between C. elegans L3 vs L4 larvae exposed to 20 µM Cd to explore the transcriptional hallmarks of stage sensitivity. The results showed that the transcriptome of the L3 stage, despite being exposed to Cd for a shorter period, was more affected than the L4 stage, as demonstrated by differences in transcriptional changes and magnitude of induction. Additionally, T08G5.1, a hitherto uncharacterized gene located upstream of metallothionein (mtl-2), was transcriptionally hyperresponsive to Cd exposure. Deletion of one or both metallothioneins (mtl-1 and/or mtl-2) increased T08G5.1 expression, suggesting that its expression is linked to the loss of metallothionein. The generation of an extrachromosomal transgene (PT08G5.1:: GFP) revealed that T08G5.1 is constitutively expressed in the head neurons and induced in gut cells upon Cd exposure, not unlike mtl-1 and mtl-2. The low abundance of cysteine residues in T08G5.1 suggests, however, that it may not be involved directly in Cd sequestration to limit its toxicity like metallothionein, but might be associated with a parallel pathway, possibly an oxidative stress response.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis.

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.

Investigating the toxic mechanism of iron oxide nanoparticles-induced oxidative stress in tadpole (Duttaphrynus melanostictus): A combined biochemical and molecular study.

Metal oxide nanomaterials have toxicity towards aquatic organisms, especially microbes and invertebrates, but little is known about their impact on amphibians. We conducted a study on Duttaphrynus melanostictus (D. melanostictus) tadpoles to explore the chronic toxicity effects of iron oxide nanoparticles (IONPs) and the underlying mechanisms of IONPs-induced oxidative stress. IONPs exposure led to increased iron accumulation in the blood, liver, and kidneys of tadpoles, significantly affecting blood parameters and morphology. Higher IONPs concentrations (10 and 50 mg L-1) triggered reactive oxygen species generation, resulting in lipid peroxidation, oxidative stress, and pronounced toxicity in tadpoles. The activity levels of antioxidant enzymes/proteins (SOD, CAT, albumin, and lysozyme) decreased after IONPs exposure, and immunological measures in the blood serum were significantly reduced compared to the control group. Molecular docking analysis revealed that IONPs primarily attached to the surface of SOD/CAT/albumin/lysozyme through hydrogen bonding and hydrophobic forces. Overall, this study emphasizes the ability of IONPs to induce oxidative damage by decreasing immunological profiles such as ACH50 (34.58 ± 2.74 U mL-1), lysozyme (6.94 ± 0.82 U mL-1), total Ig (5.00 ± 0.35 g dL-1), total protein (1.20 ± 0.17 g dL-1), albumin (0.52 ± 0.01 g dL-1) and globulin (0.96 ± 0.01 g dL-1) and sheds light on their potential toxic effects on tadpoles.

A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.

Differences in ingestion and biodegradation of the melamine formaldehyde plastic by yellow mealworms Tenebrio molitor and superworms Zophobas atratus, and the prediction of functional gut microbes.

Plastics biodegradation by insect larvae is considered as a new strategy for plastic wastes treatment. To uncover the biodegradation of a more complex chemical polymer of melamine formaldehyde (MF) by insect larvae, two worm species of yellow mealworm Tenebrio molitor and superworm Zophobas atratus were fed on MF foam as sole diet for 45 days with sole bran diet as control. Although the MF foam consumption by yellow mealworms of 0.38 mg/d/g-larvae was almost 40% higher than that by superworms of 0.28 mg/d/g-larvae, a similar decrease of survival rates in both species were obtained at about 58%, indicating the adverse effects on their growth. Depolymerization and biodegradation of MF foam occurred in both larval guts, but was more extensive in yellow mealworms. MF foam sole diet influenced gut bacterial and fungal microbiomes of both larvae species, which were assessed by Illumina MiSeq on day 45. Compared to the bran-fed group, both gut bacterial and fungal communities significantly changed in MF-fed groups, but differed in the two larvae species. The results demonstrated a strong association between the distinctive gut microbiome and MF foam degradation, such as unclassified Enterobacteriaceae, Hyphopichia and Issatchenkia. However, sole MF foam diet negatively influenced worms, like lower survival rates and gut abnormalities. In summary, MF foam could be degraded by both yellow mealworms and superworms, albeit with adverse effects. Gut microbes were strongly associated to MF foam degradation, especially the gut fungi.

The glutathione biosynthesis is involved in metamorphosis, antioxidant function, and insecticide resistance in Tribolium castaneum.

BACKGROUND: Reduced glutathione (GSH) synthesis is vital for redox homeostasis, cell-cycle regulation and apoptosis, and immune function. The glutamate-cysteine ligase catalytic subunit (Gclc) is the first and rate-limiting enzyme in GSH synthesis, suggesting the potential use of Gclc as a pesticide target. However, the functional characterization of Gclc, especially its contribution in metamorphosis, antioxidant status and insecticide resistance, is unclear in Tribolium castaneum. RESULTS: In this study, we identified and cloned Gclc from T. castaneum (TcGclc) and found that its expression began to increase significantly from the late larvae (LL) stage (3.491 ± 0.490-fold). Furthermore, RNA interference-mediated knockdown of TcGclc resulted in three types of aberration (100% total aberration rate) caused by the downregulation of genes related to the 20-hydroxyecdysone (20E) pathway. This deficiency was partially rescued by exogenous 20E treatment (53.1% ± 3.2%), but not by antioxidant. Moreover, in the TcGclc knockdown group, GSH content was decreased to 62.3%, and total antioxidant capacity, glutathione peroxidase and total superoxide dismutase activities were reduced by 14.6%, 83.6%, and 82.3%, respectively. In addition, treatment with different insecticides upregulated expression of TcGclc significantly compared with a control group during the late larval stage (P < 0.01). CONCLUSION: Our results indicate that TcGclc has an extensive role in metamorphosis, antioxidant function and insecticide resistance in T. castaneum, thereby expanding our understanding of GSH functions and providing a scientific basis for pest control. © 2024 Society of Chemical Industry.

Black soldier fly larvae meal as a potential modulator of immune, inflammatory, and antioxidant status in gilthead seabream juveniles.

This study aimed to evaluate the effects of fish meal (FM) replacement with defatted Hermetia illucens larvae meal (HM) on the hematological profile, immune parameters, intestinal inflammatory status, and antioxidant response in gilthead seabream juveniles. Four diets were formulated, replacing FM with HM at 0%, 22%, 60%, and 100% levels, corresponding to an inclusion level of 15 (diet HM15), 30 (diet HM30), and 45% (diet HM45), respectively. Over 67 days, fish were fed these diets until apparent visual satiation. Results showed no significant differences in immune parameters or hematological profiles, except for a decrease in hemoglobin and hematocrit levels. In the liver, glucose-6-phosphate dehydrogenase and glutathione peroxidase decreased linearly with HM content, especially at 100% replacement. Glutathione reductase activity was also reduced with HM inclusion, being lower in fish fed diet HM30 compared to the control. Fish fed diet HM15 showed lower hepatic superoxide dismutase activity, while catalase activity and lipid peroxidation remained unaffected. In the intestine, antioxidant enzyme activity was not influenced by HM, but lipid peroxidation linearly decreased with HM inclusion, being lower in the HM30 diet compared to the control. The inclusion of HM reduced the expression of intestinal pro-inflammatory genes (interleukin-1ß and cyclooxygenase-2) while the expression of transforming growth factor ß was higher in fish fed diet HM30 compared to the control and HM45 diets. In conclusion, up to 45% dietary inclusion of HM showed no adverse effects, improving liver antioxidant status, reducing intestinal oxidative stress, and regulating inflammatory gene expression.

Aminoacylase efficiently hydrolyses fatty acid amino acid conjugates of Helicoverpa armigera potentially to increase the pool of glutamine.

One of the most prevalent bioactive molecules present in the oral secretion (OS) of lepidopteran insects is fatty acid amino acid conjugates (FACs). Insect dietary components have influence on the synthesis and retaining the pool of FACs in the OS. We noted differential and diet-specific accumulation of FACs in the OS of Helicoverpa armigera by using Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry. Interestingly, we identified FACs hydrolyzing enzyme aminoacylase (HaACY) in the OS of H. armigera through proteomic analysis. Next, we have cloned, expressed, and purified active recombinant HaACY in the bacterial system. Recombinant HaACY hydrolyzes all the six identified FACs in the OS of H. armigera larvae fed on host and non-host plants and releases respective fatty acid and glutamine. In these six FACs, fatty acid moieties vary while amino acid glutamine was common. Glutamine obtained upon hydrolysis of FACs by HaACY might serve as an amino acid pool for insect growth and development. To understand the substrate choices of HaACY, we chemically synthesized, purified, and characterized all the six FACs. Interestingly, rHaACY also shows hydrolysis of synthetic FACs into respective fatty acid and glutamine. Our results underline the importance of diet on accumulation of FACs and role of aminoacylase(s) in regulating the level of FACs and glutamine.

V-ATPase E mediates Cry2Ab binding and toxicity in Helicoverpa armigera.

Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.