Influence of Asafoetida Extract on the Virulence of the Entomopathogenic Nematode Steinernema carpocapsae and Its Symbiotic Bacterium Xenorhabdus nematophila in the Host Pyrrhocoris apterus.

Nematode-microbe symbiosis plays a key role in determining pathogenesis against pests. The modulation of symbiotic bacteria may affect the virulence of entomopathogenic nematodes (EPNs) and the biological management of pests. We tested the influence of asafoetida (ASF) extract on the virulence of Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila, in Pyrrhocoris apterus. A total of 100 mg of ASF killed 30% of EPNs in 48 h, while P. apterus remained unaffected. The EPNs pre-treated with 100 mg of ASF influenced P. apterus's mortality by 24-91.4% during a period of 24 to 72 h. The topical application of ASF acted as a deterrent to S. carpocapsae, lowering host invasion to 70% and delaying infectivity with 30% mortality for 168 h. Interestingly, Steinernema's symbiotic bacterium, Xenorhabdus, remained unaffected by ASF. An in vitro turbidity test containing 100 mg of ASF in a medium increased the growth rate of Xenorhabdus compared to a control. A disc diffusion assay confirmed the non-susceptibility of Xenorhabdus to ASF compared to a positive control, streptomycin. Pro-phenol oxidase (PPO) and phenol oxidase (PO) upregulation showed that ASF influences immunity, while EPN/ASF showed a combined immunomodulatory effect in P. apterus. We report that ASF modulated the virulence of S. carpocapsae but not that of its symbiotic bacterium, X. nematophila, against P. apterus.

Andrographolide: A Herbal-Chemosynthetic Approach for Enhancing Immunity, Combating Viral Infections, and Its Implication on Human Health.

Plants consistently synthesize and accumulate medically valuable secondary metabolites which can be isolated and clinically tested under in vitro conditions. An advancement with such important phytochemical production has been recognized and utilized as herbal drugs. Bioactive andrographolide (AGL; C20H30O5) isolated from Andrographis paniculate (AP) (Kalmegh) is a diterpenoid lactones having multifunctional medicinal properties including anti-manic, anti-inflammatory, liver, and lung protective. AGL is known for its immunostimulant activity against a variety of microbial infections thereby, regulating classical and alternative macrophage activation, Ag-specific antibody production during immune disorder therapy. In vitro studies with AGL found it to be effective against multiple tumors, neuronal disorders, diabetes, pneumonia, fibrosis, and other diverse therapeutic misadventures. Generally, virus-based diseases like ZIKA, influenza A virus subtype (H1NI), Ebola (EBOV), Dengue (DENV), and coronavirus (COVID-19) epidemics have greatly increased scientific interest and demands to develop more effective and economical immunomodulating drugs with minimal side effects. Trials and in vitro pharmacological studies with AGL and medicinally beneficial herbs might contribute to benefit the human population without using chemical-based synthetic drugs. In this review, we have discussed the possible role of AGL as a promising herbal-chemo remedy during human diseases, viral infections and as an immunity booster.

Adipokinetic Hormones Enhance the Efficacy of the Entomopathogenic Fungus Isaria fumosorosea in Model and Pest Insects.

Insect adipokinetic hormones (AKHs) are neuropeptides with a wide range of actions, including the control of insect energy metabolism. These hormones are also known to be involved in the insect defence system against toxins and pathogens. In this study, our aim was to demonstrate whether the application of external AKHs significantly enhances the efficacy of the entomopathogenic fungus Isaria fumosorosea in a model species (firebug Pyrrhocoris apterus) and pest species (Egyptian cotton leafworm Spodoptera littoralis and pea aphid Acyrthosiphon pisum). It was found that the co-application of Isaria with AKHs significantly enhanced insect mortality in comparison to the application of Isaria alone. The mode of action probably involves an increase in metabolism that is caused by AKHs (evidenced by the production of carbon dioxide), which accelerates the turnover of Isaria toxins produced into the infected insects. However, several species-specific differences probably exist. Intoxication by Isaria elicited the stimulation of Akh gene expression and synthesis of AKHs. Therefore, all interactions between Isaria and AKH actions as well as their impact on insect physiology from a theoretical and practical point of view need to be discussed further.

Collisional mechanism of ligand release by Bombyxmori JHBP, a member of the TULIP / Takeout family of lipid transporters.

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction. In the hemolymph of Lepidoptera, these lipophilic sesquiterpenic hormones are transported from their site of synthesis to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). Lepidopteran JHBPs belong to a recently uncovered, yet very ancient family of proteins sharing a common lipid fold (TULIP domain) and involved in shuttling various lipid ligands. One important, but poorly understood aspect of JHs action, is the mechanism of hormone transfer to or through the plasma membranes of target cells. Since many membrane-active peptides and proteins, such as the pore-forming bacterial toxins, are activated by low pH or interaction with phospholipid membranes, we have examined the effect of these factors on JH binding by JHBPs. The affinity of Bombyx mori and Manduca sexta JHBPs for JH III was determined by the DCC assay, equilibrium dialysis, and isothermal titration calorimetry, and found to be greatly reduced at low pH, in agreement with previous observations. Loss of binding was accompanied by changes in fluorescence and near-UV CD spectra, indicating significant changes in protein structure in the environment of aromatic residues. The apparent dissociation rate constant (koff) of the JHBP-JH III complex was greater at acidic pH, suggesting that low pH favors ligand release by opening of the binding pocket. The affinity of recombinant B. mori JHBP (rBmJHBP) was also decreased in the presence of anionic phospholipid vesicles. Measurements of steady-state fluorescence anisotropy with the lipophilic probe TMA-DPH demonstrated that rBmJHBP specifically interacts with anionic membranes. These results suggest the existence of a collisional mechanism for ligand release that may be important for delivery of JHs to the target cells, and could be relevant to the function of related members of this emerging family of lipid-transport proteins.

Adipokinetic hormone promotes infection with entomopathogenic fungus Isaria fumosorosea in the cockroach Periplaneta americana.

The adipokinetic hormones (AKHs) are known to be involved in insect immunity, thus their role in the cockroach Periplaneta americana infected with the entomopathogenic fungus Isaria fumosorosea was examined in this study. The application of I. fumosorosea resulted in a significant increase in both Akh gene expression and AKH peptide levels. Further, co-application of I. fumosorosea with Peram-CAH-II significantly enhanced cockroach mortality compared with the application of I. fumosorosea alone. The mechanism of AKH action could involve metabolic stimulation, which was indicated by a significant increase in carbon dioxide production; this effect can increase the turnover and thus efficacy of toxins produced by I. fumosorosea in the cockroach's body. I. fumosorosea treatment resulted in a significant decrease in haemolymph nutrients (carbohydrates and lipids), but co-application with Peram-CAH-II restored control level of lipids or even further increased the level of carbohydrates. Such nutritional abundance could enhance the growth and development of I. fumosorosea. Further, both I. fumosorosea and Peram-CAH-II probably affected oxidative stress: I. fumosorosea alone curbed the activity of catalase in the cockroach's gut, but co-application with Peram-CAH-II stimulated it. Interestingly, the hormone alone had no effect on catalase activity. Taken together, the results of the present study demonstrate the interactions between the fungus and AKH activity; understanding this relationship could provide insight into AKH action and may have practical implications for insect pest control in the future.

Responses of sericotropin to toxic and pathogenic challenges: possible role in defense of the wax moth Galleria mellonella.

This study describes defense functions of the insect neuropeptide sericotropin, which is recognized as an agent that stimulates silk production in some lepidopteran larvae. Sericotropin, expressed in brain tissue of the wax moth Galleria mellonella in all developmental stages, is not expressed in silk glands, indicating its tissue specificity. Fluorescence microscopy confirmed the presence of sericotropin in the brain-subesophageal complex being predominantly and densely distributed under the plasmatic membrane and in axonal parts of neurons. Injection of venom from Habrobracon hebetor and topical application of the entomopathogenic nematode (EPN) Steinernema carpocapsae with symbiotic bacteria Xenorhabdus spp. into or onto G. mellonella larvae resulted in upregulation of the sericotropin gene and peptide, suggesting a role for sericotropin in defense and immunity. Accordingly, two synthetic fragments of sericotropin killed entomotoxic Xenorhabdus spp. bacteria in a disc diffusion antimicrobial test. Further, total metabolism, monitored by carbon dioxide production, significantly decreased after application of either venom or EPN, probably because of muscle impairment by the venom and serious cell damage caused by EPN, especially in the midgut. Both venom and EPN upregulated expression of genes encoding antimicrobial peptides gallerimycin and galiomicin in Galleria brain; however, they downregulated prophenoloxidase and phenoloxidase activity in hemolymph. These results suggest that sericotropin is a multifunctional peptide that plays an important role in G. mellonella defense and immunity.

Beneficial effect of adipokinetic hormone on neuromuscular paralysis in insect body elicited by braconid wasp venom.

The effect of Habrobracon hebetor venom and the role of the adipokinetic hormone (AKH) in poisoned adult females of the firebug Pyrrhocoris apterus were studied 24 and 48h after treatments. Venom application elicited total neuromuscular paralysis in firebugs, but the co-application of venom and Pyrap-AKH significantly reduced paralysis (up to 3.2 times) compared to the application of venom only. Although the mechanisms of their action are unknown, both agents might affect neuromuscular junctions. Venom application significantly increased the expression of both P. apterus Akh genes (Pyrap-Akh 5.4 times and Peram-Cah-II 3.6 times), as well as the level of AKHs in the central nervous system (2.5 times) and haemolymph (3.0 times). In the haemolymph, increased AKH levels might have led to the mobilization of stored lipids, which increased 1.9 times, while the level of free carbohydrates remained unchanged. Total metabolism, monitored by carbon dioxide production, significantly declined in paralysed P. apterus individuals (1.4 times and 1.9 times, 24 and 48h after the treatment, respectively), probably because of a malfunction of the muscular system. The results suggest an active role of AKH in the defence mechanism against the stress elicited by neuromuscular paralysis, and the possible involvement of this hormone in neuronal/neuromuscular signalling.

Adipokinetic hormone activities in insect body infected by entomopathogenic nematode.

The role of adipokinetic hormone (AKH) in the firebug Pyrrhocoris apterus adults infected by the entomopathogenic nematode (EPN) Steinernema carpocapsae was examined in this study. It was found that co-application of EPN and AKH enhanced firebug mortality about 2.5 times within 24h (from 20 to 51% in EPN vs. EPN+AKH treatments), and resulted in metabolism intensification, as carbon dioxide production in firebugs increased about 2.1 and 1.6times compared to control- and EPN-treated insects, respectively. Accordingly, firebugs with reduced expression of AKH receptors showed a significantly lower mortality (by 1.6 to 2.9-folds), and lower general metabolism after EPN+AKH treatments. In addition, EPN application increased Akh gene expression in the corpora cardiaca (1.6times), AKH level in the corpora cardiaca (1.3times) and haemolymph (1.7times), and lipid and carbohydrate amounts in the haemolymph. Thus, the outcomes of the present study demonstrate involvement of AKH into the anti-stress reaction elicited by the nematobacterial infection. The exact mechanism by which AKH acts is unknown, but results suggested that the increase of metabolism and nutrient amounts in haemolymph might play a role.

Acp70A regulates Drosophila pheromones through juvenile hormone induction.

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. Here we demonstrate that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed.

Peripheral regulation by ecdysteroids of olfactory responsiveness in male Egyptian cotton leaf worms, Spodoptera littoralis.

Physiological and behavioral plasticity allows animals to adapt to changes in external (environmental) and internal (physiological) factors. In insects, the physiological state modulates adult behavior in response to different odorant stimuli. Hormones have the potential to play a major role in the plasticity of the olfactory responses. To explore if peripheral olfactory processing could be regulated by steroid hormones, we characterized the molecular, electrophysiological, and behavioral response to changes in endogenous hormone levels in adult male Spodoptera littoralis. The expression of the receptor complex (EcR/USP) was localized by in situ hybridization in the olfactory sensilla of antennae. Injections of 20-hydroxyecdysone (20E) induced an ecdysteroid signaling pathway in antennae and increased expression of the nuclear receptors EcR, USP and E75. Diacylglycerol kinase (DGK) and CaM expression were also up-regulated by 20E. Taken together, these molecular, electrophysiological, and behavioral results suggest a hormonal regulation of the peripheral olfactory processing in S. littoralis.

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration.

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes.