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This study investigated the total phenol content (TPC) and DPPH inhibition of ulvan from Ulva lactuca, along with its supplementation effects on the specific growth rate (SGR) and phenoloxidase (PO) activity of Litopenaeus vannamei. Ulva lactuca samples were processed to obtain four ulvan extracts using different methods such as P-HWE (Polysaccharide-Hot Water Extract), O-HWE (Oligosaccharide-Hot Water Extract), P-A-HWE (Polysaccharide-Acid-Hot Water Extract), and O-A-HWE (Oligosaccharide-Acid-Hot Water Extract). The U. lactuca powder was treated with 80% ethanol overnight, then centrifuged and dried. Depigmented U. lactuca was extracted with water (65°C, 2 hours), and polysaccharides were precipitated with 99% ethanol, yielding P-HWE. P-HWE was heated at 145°C for 4.5 hours to yield O-HWE. Another extraction after depigmentation, using water containing H2O2 and ascorbic acid (65°C, 2 hours), precipitated P-A-HWE, which was dried and heated to yield O-A-HWE. FTIR analysis provided insights into the chemical composition and structural characteristics of ulvan extracts. TPC and DPPH inhibition were measured spectrophotometrically, and PO activity using a colorimetric assay with L-DOPA. Ulvan supplementation in shrimp feed was evaluated for growth and immune response. Results showed significant differences in TPC, DPPH inhibition, and PO activity among extracts, with O-A-HWE having the highest phenol content and DPPH inhibition. Ulvan supplementation significantly influenced the SGR of L. vannamei, with the highest SGR in the O-A-HWE treatment. This study suggests that ulvan extracts, especially O-A-HWE, could be effective natural immunostimulants for enhancing the health and growth of L. vannamei, warranting further research to optimize extraction methods and understand the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Background: Invasion of microorganisms into the gut of insects triggers a cascade of immune reactions accompanied by increased synthesis of effectors (such as antimicrobial peptides, cytokines, and amino acids), leading to changes in the physiological state of the host. We hypothesized that even an inactivated bacterium can induce an immune response in an insect. The aim of this study was to compare the roles of reactive oxygen species (ROS) formation and of the response of detoxification and antioxidant systems in a Colorado potato beetle (CPB) larval model in the first hours after invasion by either an inactivated or live bacterium. Methods: The influence of per os inoculation with inactivated entomopathogenic bacterium Bacillus thuringiensis var. tenebrionis (Bt) on the survival and physiological and biochemical parameters of CPB larvae was assessed as changes in the total hemocyte count (THC), activity of phenoloxidases (POs), glutathione-S-transferases (GSTs), nonspecific esterases (ESTs), catalase, peroxidases, superoxide dismutases (SODs) and formation of reactive oxygen species (ROS). Results: A series of changes occurred within the hemolymph and the midgut of CPBs inoculated with inactivated Bt at 12 h after inoculation. These physiological and biochemical alterations serve to mediate generalized resistance to pathogens. The changes were associated with an increase in the THC and a 1.4–2.2-fold enhancement of detoxification enzymatic activities (such as GST and EST) as well as increased levels of antioxidants (especially peroxidases) in hemolymph in comparison to the control group. Suppressed EST activity and reduced ROS formation were simultaneously detectable in the larval midgut. Inoculation of beetle larvae with active Bt cells yielded similar results (elevated THC and suppressed PO activity). A fundamental difference in the immune activation processes between larvae that ingested the inactivated bacterium and larvae that had consumed the active bacterium was that the inactivated bacterium did not influence ROS formation in the hemolymph but did reduce their formation in the midgut. At 24 h postinfection with active Bt, ROS levels went up in both the hemolymph and the midgut. This was accompanied by a significant 5.7-fold enhancement of SOD activity and a 5.3-fold suppression of peroxidase activity. The observed alterations may be due to within-gut toxicity caused by early-stage bacteriosis. The imbalance in the antioxidant system and the accumulation of products toxic to the "putative" pathogen can activate detoxification mechanisms, including those of an enzymatic nature (EST and GST). The activation of detoxification processes and of innate immune responses is probably due to the recognition of the "putative" pathogen by gut epithelial cells and is similar in many respects to the immune response at early stages of bacteriosis. [ABSTRACT FROM AUTHOR]

Resource subsides can link aquatic and terrestrial ecosystems. Animals that emerge from aquatic ecosystems can transport nutrients that are scarce in terrestrial environments, like long-chain polyunsaturated fatty acids (LC-PUFA), which are synthesized by algae in aquatic environments. LC-PUFAs have critical roles in animal physiology and are precursors to eicosanoids, which help mediate immune responses. We previously found that wolf spiders in wetland ecosystems had higher levels of LC-PUFA and stronger immune responses than nearby upland spiders. However, we could not determine if the stronger immune responses were because of eicosanoids derived from LC-PUFA subsidies or because they had a larger resource pool from aquatic prey subsidies. Here, we use 2 immune assays, 1 affected by eicosanoids and 1 not affected, to differentiate between those 2 possibilities. We fit generalized linear mixed models and found that LC-PUFA subsidies are responsible for the stronger immune responses in wetland spiders. We also found evidence that wolf spiders maintain some immune responses across reproductive states and increase other immune responses when they are gravid. Our results demonstrate that LC-PUFA subsidies can alter immune function in recipient consumers because LC-PUFAs are preferentially stored rather than used as energy. This alteration of immune function could have consequences for disease transmission and pathogen abundance in linked aquatic and terrestrial ecosystems. [ABSTRACT FROM AUTHOR]

The adulteration of wax foundation is, for many reasons, a growing problem of modern beekeeping not only in Europe but also around the world. Wax foundation contaminated with stearin addition leads to a brood die-off, while paraffin addition negatively affects the strength of combs. It is tenable that such adulterated wax foundation reduces bees' immunity. The aim of the study was to determine the activities of two bee immune enzymes, lysozyme and phenoloxidase, in the haemolymph of worker bees which had emerged from combs with wax foundations contaminated with stearin or paraffin. Combs built with stearin- or paraffin-adulterated wax (both adulterants at concentrations of 10%, 30% or 50%) or pure wax (0% adulterated) foundations were placed in the colonies, one for each adulterant and percentage. The workers were marked upon emergence from these combs and those bees were introduced into one strong colony per adulterant and percentage. Phenoloxidase and lysozyme activities were determined in the haemolymph of 1-, 7- and 14-day-old workers. The higher the concentrations of stearin and paraffin in the wax foundation, the lower the phenoloxidase activities were. These activities increased with the bee age. In contrast, the trends in lysozymes were opposite. Paraffin seems to be less toxic than stearin. Adulteration of wax foundation with even a small amount of stearin or paraffin has negative effects on the functioning of the bee. [ABSTRACT FROM AUTHOR]

Antarctic krill (Euphausia superba) is a vital species in the Antarctic ecosystem, boasting substantial reserves and high nutritional value. It has emerged as a significant commercial fishery resource in the Southern Ocean. However, because of its exceptionally high autolytic enzyme activity, it is susceptible to autolysis and blackening during processing, storage, and transportation, resulting in a decline in the sensory quality and nutritional value of raw materials. Shrimp melanosis is caused by a series of enzymatic reactions mediated by phenoloxidase (PO) within the body. PO facilitates the oxidation of phenolic substances, leading to the formation of highly unstable quinone compounds, which further react to produce melanin. The accumulation of melanin significantly affects the appearance of Antarctic krill, thereby reducing its commercial value. On account of their cost-effectiveness and potent anti-blackening effects, sulfites are commonly used as shrimp anti-black agents. Sodium metabisulfite is one of the compounds whose use is allowed for shrimp preservation under GB 2760-2014 regulations. It not only inhibits PO activity, but also reduces quinone compounds to prevent blackening. However, excessive sulfite use can lead to discomfort and health issues such as gastrointestinal problems, liver disturbances, dyspnea, vomiting, diarrhea, and headaches. Therefore, GB 2760-2014 regulations stipulate that the residual SO2 content should not exceed 0.1 g/kg. Currently, the use of sodium metabisulfite in production is widespread but lacks standardized requirements, with limited guidance available on how to regulate its usage. Therefore, to assess the impact of sodium metabisulfite on blackening control in Antarctic krill and refine the treatment process, experiments were conducted to measure the changes in SO2 residue and PO activity in Antarctic krill subjected to various concentrations of sodium metabisulfite solutions and soaking times. Furthermore, observations and analyses were conducted to monitor the blackening of Antarctic krill during cold storage and frozen storage. The results demonstrated a positive correlation between the SO2 residue in Antarctic krill and the concentration of sodium metabisulfite solution. A concentration of 1 g/L sodium metabisulfite solution resulted in a SO2 residue of 0.065 g/kg in Antarctic krill, which is below the national standard limit of 0.1 g/kg. However, treatment with a 2 g/L solution led to a SO2 residue of 0.166 g/kg in shrimp, exceeding the national standard limit. Treatment with a 3 g/L solution resulted in a SO2 residue of 0.254 g/kg in shrimp, which significantly surpassed the national standard limit. Consequently, we recommended using 1 g/L or 2 g/L sodium metabisulfite solutions to prevent blackening in Antarctic krill while maintaining the SO2 residue below 0.1 g/kg. Under these conditions, the treatment time for a 1 g/L sodium pyrosulfite solution could be extended to 300 s, with a relative PO activity of 21.2%. For a 2.0 g/L sodium pyrosulfite solution, a treatment time of 30 s was suitable, yielding a relative PO activity of 34.5%. During storage, the control group exhibited noticeable blackening after storage at 4 ℃ for 24 h and frozen storage for 6 months, predominantly in the head, chest, abdomen, and tail sections, and slight variations were observed in the blackening process among individuals. Compared with the control group, the sodium metabisulfite treatment group displayed significantly lower blackening scores during storage (P < 0.05). Specifically, the 1 g/L sodium metabisulfite solution with 300 s treatment group and the 2 g/L sodium metabisulfite solution with 30 s treatment group showed obvious blackening at 48 h and 72 h of cold storage, respectively, and after 12 months of frozen storage, indicating that higher concentrations of sodium metabisulfite solution during treatment led to a better control effect on the blackening of Antarctic krill during storage. Notably, PO activity is just one of the factors influencing the melanosis process. Although the 1 g/L sodium metabisulfite solution with treatment for 300 s could better inhibit PO activity, the anti-blackening effect of this group was not as pronounced as that of the 2 g/L sodium metabisulfite solution with treatment for 30 s during storage. In summary, soaking in 1 g/L sodium metabisulfite solution for 300 s and 2 g/L sodium metabisulfite solution for 30 s resulted in relative PO enzyme activities of 21.2% and 34.5%, respectively. Simultaneously, the SO2 residues in shrimp were measured at 0.094 g/kg and 0.096 g/kg, respectively, meeting the GB 2760-2014 limit of SO2 residue not exceeding 0.1 g/kg. Compared with the control group, sodium metabisulfite treatment effectively delayed the blackening process of Antarctic krill during cold storage and frozen storage, with the 2 g/L sodium metabisulfite solution soaking for 30 s exhibiting superior anti-blackening effects. This suggests that a high concentration of sodium metabisulfite solution and a short treatment period had a better blackening inhibition effect. These findings offer a theoretical foundation and technical guidance for maintaining the quality of Antarctic krill during storage and transportation.

Pathogen infection and conspecific density may considerably affect key life‐history traits of organisms. For naturally aggregating species, even low concentrations of pathogens or high larval densities may have detrimental effects. However, the detailed influence of these factors, particularly their interaction effect, is often overlooked in ecological and life‐history studies. To investigate the effects of conspecific density and pathogen infection on life‐history traits, we explored the influence of larval density (1 and 5 larvae cm−2, i.e., low and high density, respectively) on phenoloxidase (PO) activity, body mass, and development time of Hermetia illucens L. (Diptera: Stratiomyidae) larvae in the presence of two strains of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.‐Criv.) Vuill. We observed higher PO activity in EPF‐treated larvae than in the untreated control and a pronounced difference in PO activity between the two EPF‐strain treatments. Larvae reared at high density and treated with EPF showed higher PO activity than untreated larvae at low density. The EPF‐treated larvae and larvae reared at high density had longer larval periods than untreated larvae and larvae reared at low density, respectively. Larvae reared at high density also achieved reduced prepupal and pupal masses compared to conspecifics at low density. Interestingly, untreated larvae only achieved higher prepupal and pupal masses at low density, whereas at high density, the pattern was reversed (treated individuals had higher pupal masses). Overall, our results demonstrate that high density and fungal pathogens both induce a higher immune response compared to low density and pathogen‐free environments, but this comes with a cost of a longer larval period and reduced body mass. [ABSTRACT FROM AUTHOR]

Simple Summary: Varroa destructor is a mite that parasitises honeybee hives, weakening colonies and transmitting viruses. Beekeepers apply acaricide treatments to bee colonies to limit the spread of Varroa and the resulting negative effects on bee health. Oxalic acid treatment is the most widely applied, but little is known about its potential effects on the physiology of bees, particularly their immune system. This study investigated the short-term effects of oxalic acid treatment on the immune and antioxidant systems of house bees (i.e., glucose oxidase, phenoloxidase, glutathione S-transferase, catalase activities, and vitellogenin content). Residual concentrations of oxalic acid on the bees' external body and in the haemolymph were also measured. The results showed that the treatment did not affect the concentration of oxalic acid in the haemolymph, in which it was constitutively present. Residues of oxalic acid remained on the outer body of the bees for up to 48 h after treatment. Both glucose oxidase activity and vitellogenin concentration were affected by the treatment, increasing significantly after 48 and 24 h, respectively. No effects were observed on the other parameters investigated (phenoloxidase, glutathione S-transferase, and catalase activities). Honeybees (Apis mellifera L.) have to face many challenges, including Varroa destructor infestation, associated with viral transmission. Oxalic acid is one of the most common treatments against Varroa. Little is known about the physiological effects of oxalic acid, especially those on honeybees' immune systems. In this study, the short-term effects (0–96 h) of oxalic acid treatment on the immune system components (i.e., glucose oxidase, phenoloxidase, glutathione S-transferase, catalase activities, and vitellogenin contents) of house bees were preliminarily investigated. Oxalic acid contents of bee bodies and haemolymphs were also measured. The results confirm that oxalic acid is constitutively present in bee haemolymphs and its concentration is not affected by treatment. At 6 h after the treatment, a maximum peak of oxalic acid content was detected on bees' bodies, which gradually decreased after that until physiological levels were reached at 48 h. In the immune system, the oxalic acid treatment determined a peak in glucose oxidase activity at 48 h, indicating a potential defence response and an increase in vitellogenin content at 24 h. No significant changes were recorded in phenoloxidase, glutathione S-transferase, and catalase activities. These results suggest a time-dependent response to oxalic acid, with potential immune system activation in treated bees. [ABSTRACT FROM AUTHOR]

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth‐blocking peptide (GBP) is a member of the stress‐responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity‐related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens. [ABSTRACT FROM AUTHOR]

Sweet sagewort (Artemisia annua) is a species that has been used for many years to treat high fever due to its artemycin content. Artemycin is the active ingredient in antimalarial drugs. Additionally, the plant's leaves are consumed as tea or aqueous extract. Invertebrate model organism Galleria mellonella is preferred in immune studies due to its ease of administration, natural immunity shared with mammals, including humans, and rapid determination of results. In this research, we systematically assessed the impact of A. annua extract on the immune system of animals by employing the G. mellonella model organism. We meticulously examined key facets of innate immune responses, including total hemocyte count, encapsulation-melanization, and phenoloxidase activity. Furthermore, we delved into the effects on antioxidant enzyme activity, specifically superoxide dismutase, catalase, and lipid peroxidase. The hemocyte count showed a statistically significant decrease across all given doses of the A. annua extract when compared to the control groups. The A. annua extract had no effect on the catalase activity, malondialdehyde amount, phenoloxidase activity, or melanization response of G. mellonella larvae, according to the findings of our study. Nonetheless, it enhanced SOD activity. In comparison to the control groups, it decreased the strong encapsulation response, which is a cell-mediated immune response, at all doses. While the A. annua extract had little effect on enzymatic processes in animals, it reduced cell-mediated immune responses by altering cell behaviours and count with cytotoxic and genotoxic effects. [ABSTRACT FROM AUTHOR]

Controlling the worldwide invasive pest Drosophila suzukii remains a challenge. One promising biological method for managing this pest is the use of larval and pupal parasitoids. Unfortunately, most of the larval parasitoids fail to successfully parasitise D. suzukii larvae in laboratory experiments due to the high immunity of the pest. So far, only the summer phenotype (summer morph) of D. suzukii has been tested for parasitisation. However, the winter phenotype (winter morph) is the dominant form of D. suzukii throughout the year in the northern hemisphere. Therefore, this study investigates the immunity during parasitisation for both phenotypes using the larval parasitoid Asobara japonica and the pupal parasitoid Trichopria drosophilae. It is the first to compare across all life stages the immunity of the winter phenotype to the summer phenotype of not only D. suzukii but also D. melanogaster. Our results indicate differences in the immunity between the two phenotypes for larvae, pupae, and adults. However, the degree and direction of these differences were inconsistent across the different life stages of D. suzukii. The findings have important implications for the integrated pest management (IPM) of D. suzukii. [ABSTRACT FROM AUTHOR]

Hydrolysable tannins are polyphenolic molecules contained in plant foodstuffs, potentially toxic for animals. Since tannins have been found in some bee products such as bee pollen, propolis, and honey, this work aimed to test the effects of tannins dietary assumption on bees' health. Results showed that supplementation of honey bees diets with 0.05% (w/w), as naturally amount reported in the honey, and 0.5% (w/w) of tannins significantly reduced bees' survival in a dose-dependent manner. In honey bees fed with a 0.5% tannins-enrichment diet a significantly decreased phenoloxidase activity than those fed with a 0.05% tannins-enrichment diet and with only syrup was recorded. Conversely, glucose oxidase activity significantly increased in honey bees fed with 0.5% tannins-enrichment diet than those with a 0.05% tannins-enrichment diet and only syrup. No differences were detected in glutathione S-transferase activity between honey bees fed with tannins-enrichment diets and syrup. Therefore, a naturally amount of hydrolysable tannins in foods can weakly affect bee wellness, whereas high levels of tannins can have a negative impact on honey bees' immunity and metabolism leading to a reduction of lifespan. [ABSTRACT FROM AUTHOR]

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Background Invasion of microorganisms into the gut of insects triggers a cascade of immune reactions accompanied by increased synthesis of effectors (such as antimicrobial peptides, cytokines, and amino acids), leading to changes in the physiological state of the host. We hypothesized that even an inactivated bacterium can induce an immune response in an insect. The aim of this study was to compare the roles of reactive oxygen species (ROS) formation and of the response of detoxification and antioxidant systems in a Colorado potato beetle (CPB) larval model in the first hours after invasion by either an inactivated or live bacterium. Methods The influence of per os inoculation with inactivated entomopathogenic bacterium Bacillus thuringiensis var. tenebrionis (Bt) on the survival and physiological and biochemical parameters of CPB larvae was assessed as changes in the total hemocyte count (THC), activity of phenoloxidases (POs), glutathione-S-transferases (GSTs), nonspecific esterases (ESTs), catalase, peroxidases, superoxide dismutases (SODs) and formation of reactive oxygen species (ROS). Results A series of changes occurred within the hemolymph and the midgut of CPBs inoculated with inactivated Bt at 12 h after inoculation. These physiological and biochemical alterations serve to mediate generalized resistance to pathogens. The changes were associated with an increase in the THC and a 1.4–2.2-fold enhancement of detoxification enzymatic activities (such as GST and EST) as well as increased levels of antioxidants (especially peroxidases) in hemolymph in comparison to the control group. Suppressed EST activity and reduced ROS formation were simultaneously detectable in the larval midgut. Inoculation of beetle larvae with active Bt cells yielded similar results (elevated THC and suppressed PO activity). A fundamental difference in the immune activation processes between larvae that ingested the inactivated bacterium and larvae that had consumed the active bacterium was that the inactivated bacterium did not influence ROS formation in the hemolymph but did reduce their formation in the midgut. At 24 h postinfection with active Bt, ROS levels went up in both the hemolymph and the midgut. This was accompanied by a significant 5.7-fold enhancement of SOD activity and a 5.3-fold suppression of peroxidase activity. The observed alterations may be due to within-gut toxicity caused by early-stage bacteriosis. The imbalance in the antioxidant system and the accumulation of products toxic to the “putative” pathogen can activate detoxification mechanisms, including those of an enzymatic nature (EST and GST). The activation of detoxification processes and of innate immune responses is probably due to the recognition of the “putative” pathogen by gut epithelial cells and is similar in many respects to the immune response at early stages of bacteriosis.

Nodulation is the first immune defence mechanism related to melanisation in response to microbial infections in insects. Adult parasitoid insects have been hypothesised to produce nodules with melanisation in response to viral infections and, eicosanoids, to mediate nodulation reactions and phenoloxidase (PO) activation in this type of infections. To test this hypothesis, endoparasitoid Pimpla turionellae adults were first inoculated with a novel generation nonsteroidal anti-inflammatory drug (NSAID) dexketoprofen trometamol (DT) (5 μg/adult), which is a selective cyclooxygenase-1 (COX-1) inhibitor. These adults were then immediately injected with intrahaemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1) as a model insect-virus interaction. Additionally, adults were fed on artificial diet with increasing concentrations of DT (0.001, 0.01, or 0.1 g/100 ml diet) per os prior to intrahaemocoelic injection of BHSV-1 (2 × 103 PFU/adult) and nodulation and PO activity were recorded at 2 h post inoculation (PI). BHSV-1-treated newly emerged adults fed with inhibitors showed low levels of nodulation and increased PO enzyme activity. DT-treated Pimpla adults produced significantly fewer nodules (approximately nine nodules/adult), whereas viral infection provoked nodules (approximately 33 nodules/adult) in comparison with needle (vehicle)-treated controls (approximately five nodules/adult). Increasing dietary dexketoprofen trometamol concentrations decreased nodulation (by 12-fold at the highest concentration) and increased PO reactions (by approximately 3-fold at the highest concentration) to BHSV-1 injection. Compared with control adults, adults orally fed on the lowest DT concentration (0.001 %) significantly increased PO activity (1.22 ± 0.23–2.74 ± 0.31 unit/min/mg protein) while nodules significantly decreased (43.19 ± 4.26–17.84 ± 2.19) in response to virus infections. These findings suggest that eicosanoid biosynthesis, at least in the context of prostaglandins (PGs) formed by COX-1, mediates nodulation reactions and PO activation in response to viral infection in adults of this endoparasitoid. This is the first demonstration that the immune response of P. turionellae adults to viral pathogens is modulated by DT, which initiates haemolymph PO activation.

In insects, melanism, a fundamental pigmentation process, is of significant importance in evolutionary biology due to its complex genetic foundation. We investigated the role of the RNA‐binding gene Musashi (msi) in melanism in Laodelphax striatellus, a Hemiptera species. We identified a single L. striatellus msi homolog, Lsmsi, encoding a 357 amino acid protein with 2 RNA recognition motifs. RNA interference‐mediated knockdown of LsMsi resulted in complete body melanism and increased cuticular permeability. Additionally, we found the involvement of G protein‐coupled receptor A42 and tyrosine hydroxylase (Th) in L. striatellus melanism. Knockdown of LsTh lightened the epidermis, showing dehydration signs, while LsA42 knockdown enhanced LsTh expression, leading to melanism. Surprisingly, Lsmsi knockdown decreased both LsA42 and LsTh expression, which was expected to cause whitening but resulted in melanism. Further, we found that Lsmsi influenced downstream genes like phenoloxidase homolog LsPo and dopa decarboxylase (Ddc) homolog LsDdc in the tyrosine‐mediated melanism pathway. Extending to Nilaparvata lugens and Sogatella furcifera, we demonstrated the conserved role of msi in melanism among Delphacidae. Given MSI proteins’ roles in cancer and tumors in vertebrates, our study is the first to link msi in insects to Delphacidae body color melanization via the tyrosine‐mediated pathway, offering fresh perspectives on the genetic basis of insect melanism and msi functions. [ABSTRACT FROM AUTHOR]

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments. [ABSTRACT FROM AUTHOR]

Excessive use of synthetic insecticides has resulted in environmental contamination and adverse effects on humans and other non-target organisms. Entomopathogenic fungi offer eco-friendly alternatives; however, their application for pest control requires significant advancement owing to limitations like slow killing time and effectiveness only when applied in higher amounts, whereas exposure to UV radiation, high temperature, and humidity can also reduce their viability and shelf-life. The nanoparticles synthesized using fungal extracellular extracts provide a new approach to use fungal pathogens. Our study focused on the synthesis of Metarhizium anisopliae–based silver nanoparticles (AgNPs) and evaluation of their efficiency on various physiological and behavioral parameters of the mosquito Aedes aegypti. The synthesis, size (27.6 d.nm, PDI = 0.209), zeta potential (− 24.3 mV), and shape of the AgNPs were determined through dynamic light scattering, scanning and transmission electron microscopic, and UV-visual spectroscopic analyses (432 nm). Our results showed significantly reduced survival (100% decrease in case of 3.2 and 1.8 μL/cm2 volumes, and 60% decrease in case of 0.8 μL/cm2 volume), phenoloxidase activity (t = 39.91; p = 0.0001), and gut microbiota, with increased oxidative stress and cell apoptosis in AgNPs-challenged mosquitoes. Furthermore, the AgNPs-exposed mosquitoes presented a concentration-specific decrease in flight locomotor activity (F = 17.312; p < 0.0001), whereas no significant changes in antifungal activity, self-grooming frequencies, or time spent were found. These findings enhance our understanding of mosquito responses to AgNPs exposure, and offer a more efficient mosquito control strategy using entomopathogenic fungi. [ABSTRACT FROM AUTHOR]

Cellular encapsulation associated with melanization is a crucial component of the immune response in insects, particularly against larger pathogens. The infection of a Drosophila larva by parasitoid wasps, like Leptopilina boulardi, is the most extensively studied example. In this case, the encapsulation and melanization of the parasitoid embryo is linked to the activation of plasmatocytes that attach to the surface of the parasitoid. Additionally, the differentiation of lamellocytes that encapsulate the parasitoid, along with crystal cells, is accountable for the melanization process. Encapsulation and melanization lead to the production of toxic molecules that are concentrated in the capsule around the parasitoid and, at the same time, protect the host from this toxic immune response. Thus, cellular encapsulation and melanization represent primarily a metabolic process involving the metabolism of immune cell activation and differentiation, the production of toxic radicals, but also the production of melanin and antioxidants. As such, it has significant implications for host physiology and systemic metabolism. Proper regulation of metabolism within immune cells, as well as at the level of the entire organism, is therefore essential for an efficient immune response and also impacts the health and overall fitness of the organism that survives. The purpose of this "perspective" article is to map what we know about the metabolism of this type of immune response, place it in the context of possible implications for host physiology, and highlight open questions related to the metabolism of this important insect immune response. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune‐related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection‐responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune‐related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double‐stranded RNA and bacterial infection. [ABSTRACT FROM AUTHOR]

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

Honeybees (Apis mellifera L.) have to face many challenges, including Varroa destructor infestation, associated with viral transmission. Oxalic acid is one of the most common treatments against Varroa. Little is known about the physiological effects of oxalic acid, especially those on honeybees’ immune systems. In this study, the short-term effects (0–96 h) of oxalic acid treatment on the immune system components (i.e., glucose oxidase, phenoloxidase, glutathione S-transferase, catalase activities, and vitellogenin contents) of house bees were preliminarily investigated. Oxalic acid contents of bee bodies and haemolymphs were also measured. The results confirm that oxalic acid is constitutively present in bee haemolymphs and its concentration is not affected by treatment. At 6 h after the treatment, a maximum peak of oxalic acid content was detected on bees’ bodies, which gradually decreased after that until physiological levels were reached at 48 h. In the immune system, the oxalic acid treatment determined a peak in glucose oxidase activity at 48 h, indicating a potential defence response and an increase in vitellogenin content at 24 h. No significant changes were recorded in phenoloxidase, glutathione S-transferase, and catalase activities. These results suggest a time-dependent response to oxalic acid, with potential immune system activation in treated bees.

Background: Bellamya bengalensisis an important bioresource of the freshwater ecosystem of India. This edible mollusc is filter feeder, indigenous diet of human, poultry and fishery. Natural habitat of this freshwater gastropod mollusc bears the risk of contamination by agricultural pesticide fenvalerate, a synthetic type II pyrethroid. Hemocytes are the immuno effector cells of molluscs which are affected by environmental toxins or pathogenic microorganisms. Hemocytes perform various types of immunological functions such as phagocytosis, cytotoxic response etc. Results: Experimental exposure of fenvalerate resulted in impairment of phagocytic efficacy and alteration in the generation of phenoloxidase in B. bengalensis. Conclusion: The alteration of phagocytic response and generation of phenoloxidase of hemocytes, exposed to experimental concentrations of fenvalerate under static laboratory condition have been determined to establish as biomarker of aquatic toxicity in toxin contaminated natural habitat.

Penaeus spp. are the most cultivated type of shrimp because they have rapid growth and good adaptation to farming conditions. Due to diseases that result in high mortality and a decreased product quality, the cultivation of these shrimp globally, and in Cuba in particular, comes with the risk of significant financial losses. This study examined the effect of fructooligosaccharide (FOS, 1-kestose) on the growth and immune response of shrimp, as well as the multitude of Vibrio spp. and Pseudomonas spp. in Penaeus vannamei post-larvae under culture conditions. Fructooligosaccharide was applied in a completely randomized manner at a concentration of 0.4%, in both experimental groups with seventeen tanks each. In the results of this investigation, animals of greater weight (control 6.8 ± 0.2 mg; FOS 9.5 ± 0.3 mg; p < 0.001), length (control 1.1 ± 0.1 mm; FOS 1.3 ± 0.1 mm; p < 0.001), and survival (control 61.7% (95% CI of median 54.2–70.0); FOS 76.6% (95% CI of median 72.1–84.2); p < 0.001) were obtained when administered FOS relative to the control. An increase in the activity of proteases (p < 0.001), enzymes of the innate immune system such as phenoloxidase (p < 0.001), and lysozymes (p < 0.001) was observed, as well as an increase in the number of lectins (p < 0.001). Changes in the microbiota could be observed, with a reduction in Vibrio spp. and Pseudomonas spp. (control 2.4 × 103 ± 0.5 × 103; FOS 1.1 × 102 ± 0.3 × 102; p < 0.001). FOS improves the quality of the post-larvae of P. vannamei as reflected in the length, weight, and survival of the animals. Moreover, FOS stimulates the P. vannamei immune system through the enzymatic activities of phenoloxidase, lysozyme, and a number of lectins. The reduction in the population load of Vibrio spp. and Pseudomonas spp. might be a consequence of the improvement in the quality and immune system of P. vannamei.

This is an open access article under the CC BY-NC-SA license Enterocytozoon hepatopenaei (EHP) is a spore-forming microsporidia intracellular parasite, which slough off the cells of the hepatopancreases of the shrimp. This study was intended to determine the effect of immersion duration and maintenance time to stimulate the immune system of shrimps by using a crude protein from Zoothamnium penaei to protect against EHP infections. The study used a completely randomized design where shrimps were divided into four groups each having five replicates. Groups P1, P2, P3 were immersed in immunostimulant for 10, 15, and 20 minutes respectively, except negative control. The phenoloxidase, phagocytosis activities, histopathology study were determined. Also, the molecular diagnosis of EHP was measured to confirm EHP infection in the shrimp samples. The phenoloxidase activity significantly increased after immersion for 20 minutes (P3) from 0.64-, 0.72-, and 0.78-units min-1 mil-1 of protein on the first week, second week and third week, respectively. The phagocytosis activities of the sample immersed for 20 minutes were significantly increased to 68% on the second week. The sample also showed improved hepatopancreases epithelial cells with only 26% necrotic cell, less EHP spore and vacuolation. There was an interaction between immersion duration and maintenance time of the L. vannamei immersed in crude protein from Zoothamnium penaei. Twenty minutes immersion were significantly found to stimulate the immune system of the shrimp against EHP. The present work revealed that, the application of crude protein from Z. penaei is effective against EHP in shrimp culture. [ABSTRACT FROM AUTHOR]

The immature fruit exocarp of Juglans mandshurica Maxim., also called "green walnut husks" (GWH), are usually discarded as food waste. Although GWH has been used as folk insecticides for many years, its bioactive ingredient has been rarely reported. Herein, using insect's phenoloxidase (PO) as selective target, the insecticidal effect of green walnut husks and its PO-related active basis was comprehensively researched. Firstly, the potential insecticidal efficiency of different GWH extracts was predicted and demonstrated on tyrosinase and Aphis gossypii G., Plutella xylostella L., respectively. In vivo, the strongest stomach toxicity were determined with the LC50 values of 0.022 g/L, 0.328 g/L against Aphis gossypii G. and Plutella xylostella L., respectively among different 70% ethanol GWH extracts. Subsequently, the insecticidal target and mechanism of GWH was further clarified by means of the insect origin PO inhibition analysis. In which, the strongest PO inhibitory effect was observed from methanol GWH extract with the IC50 of 0.921 g/L. Finally, based on the chemical ingredients profiling of different GWH extracts using UPLC-Q-TOF-MS, the different metabolites was precisely explored through partial least squared discriminant analysis (PLS-DA). As the result, 13 biomarkers related to phenoloxidase inhibitory and insecticidal bioactivity were characterized from GWH. Thus, we demonstrated the potential value of GWH in pest control agrochemicals development, as well as clarified its active ingredients and anti-insects mechanism. Meanwhile, the current work also determined the efficiency and provided a practical strategy of using insect's phenoloxidase as target in botanical insecticides discovering, which will facilitate similar pesticides screening from natural resource. [ABSTRACT FROM AUTHOR]

Simple Summary: Bees are often fed by beekeepers with supplemented artificial diets. The formulation of an integrative diet for honey bee colonies able to prevent nutritional deficiencies is yet to be found. In this work, the effects of fat-enriched diet (coconut oil) administration in newly emerged and forager bees were tested. Fat supplementation in the honey bee diet determines an increase in newly emerged bees' survival in the short term and an increase in fat content. Further investigations to evaluate the use of such a supplement food to prevent the fat loss of weak families during winter are desirable. Searching for artificial diets positively affecting the survival, immune and antioxidant systems of honey bees is one of main challenges occurring in beekeeping. Among nutrients, lipids play a significant role in insect nutrition as structural components in cell membranes, energy sources and reserves, and are involved in many physiological processes. In this context, the aim of this work was to investigate the effect of 0.5% and 1% coconut oil-enriched diet administration on newly emerged and forager bees survival rate, feed intake, immune system, antioxidant system and both fat and vitellogenin content. In newly emerged bees, supplementation with 1% coconut oil determined a decrease in feed consumption, an increase in survival rate from the 3rd to 14th day of feeding, a short-term decrease in phenoloxidase activity, an increase in body fat and no differences in vitellogenin content. Conversely, supplementation with 0.5% coconut oil determined an increase in survival rate from the 3rd to 15th day of feeding and an increase in fat content in the long term (i.e., 20 days). Regarding the forager bee diet, enrichment with 0.5% and 1% coconut oil only determined an increase in fat content. Therefore, supplementation with coconut oil in honey bee diets at low percentages (0.5 and 1%) determines fat gain. Further investigations to evaluate the use of such supplement foods to prevent the fat loss of weak families during winter are desirable. [ABSTRACT FROM AUTHOR]

Penaeus spp. are the most cultivated type of shrimp because they have rapid growth and good adaptation to farming conditions. Due to diseases that result in high mortality and a decreased product quality, the cultivation of these shrimp globally, and in Cuba in particular, comes with the risk of significant financial losses. This study examined the effect of fructooligosaccharide (FOS, 1-kestose) on the growth and immune response of shrimp, as well as the multitude of Vibrio spp. and Pseudomonas spp. in Penaeus vannamei post-larvae under culture conditions. Fructooligosaccharide was applied in a completely randomized manner at a concentration of 0.4%, in both experimental groups with seventeen tanks each. In the results of this investigation, animals of greater weight (control 6.8 ± 0.2 mg; FOS 9.5 ± 0.3 mg; p < 0.001), length (control 1.1 ± 0.1 mm; FOS 1.3 ± 0.1 mm; p < 0.001), and survival (control 61.7% (95% CI of median 54.2–70.0); FOS 76.6% (95% CI of median 72.1–84.2); p < 0.001) were obtained when administered FOS relative to the control. An increase in the activity of proteases (p < 0.001), enzymes of the innate immune system such as phenoloxidase (p < 0.001), and lysozymes (p < 0.001) was observed, as well as an increase in the number of lectins (p < 0.001). Changes in the microbiota could be observed, with a reduction in Vibrio spp. and Pseudomonas spp. (control 2.4 × 103 ± 0.5 × 103; FOS 1.1 × 102 ± 0.3 × 102; p < 0.001). FOS improves the quality of the post-larvae of P. vannamei as reflected in the length, weight, and survival of the animals. Moreover, FOS stimulates the P. vannamei immune system through the enzymatic activities of phenoloxidase, lysozyme, and a number of lectins. The reduction in the population load of Vibrio spp. and Pseudomonas spp. might be a consequence of the improvement in the quality and immune system of P. vannamei. [ABSTRACT FROM AUTHOR]

Highlight Research • The effect of immersion duration of shrimp to the immunostimulant from crude protein Zoothamnium penaei was determined. • 20-minute immersion duration in crude protein from Zoothamnium penaei was found to increase the phagocytosis activities, phenoloxidase, and improve hepatopancreases of the shrimp. • There is increase in phagocytosis activities, phenoloxidase and improved hepatopancreases cell within a three-week maintenance of shrimp with immersion after each seven days. • There is interaction between immersion duration and maintenance time of shrimp into the crude protein from Zoothamnium penaei. Abstract Enterocytozoon hepatopenaei (EHP) is a spore-forming microsporidia intracellular parasite, which slough off the cells of the hepatopancreases of the shrimp. This study was intended to determine the effect of immersion duration and maintenance time to stimulate the immune system of shrimps by using a crude protein from Zoothamnium penaei to protect against EHP infections. The study used a completely randomized design where shrimps were divided into four groups each having five replicates. Groups P1, P2, P3 were immersed in immunostimulant for 10, 15, and 20 minutes respectively, except negative control. The phenoloxidase, phagocytosis activities, histopathology study were determined. Also, the molecular diagnosis of EHP was measured to confirm EHP infection in the shrimp samplesThe phenoloxidase activity significantly increased after immersion for 20 minutes (P3) from 0.64-, 0.72-, and 0.78-units min-1 mil -1 of protein on the first week, second week and third week, respectively. The phagocytosis activities of the sample immersed for 20 minutes were significantly increased to 68% on the second week. The sample also showed improved hepatopancreases epithelial cells with only 26% necrotic cell, less EHP spore and vacuolation. There was an interaction between immersion duration and maintenance time of the L. vannamei immersed in crude protein from Zoothamnium penaei. Twenty minutes immersion were significantly found to stimulate the immune system of the shrimp against EHP. The present work revealed that, the application of crude protein from Z. penaei is effective against EHP in shrimp culture.

This study aimed to evaluate the nutritional behavior and some immunological criteria (encapsulation index and phenoloxidase – PO activity, the key enzyme for melanization) as well as to study the effect of protein to fat (P : F) diets on hypopharyngeal gland (HPG) protein content. Bees were restricted to consuming specific P : F diets varying in fat ratio under laboratory conditions. These diets included 25 : 1, 10 : 1, 5 : 1 (low-fat diet, LFD); 1 : 1 (equal-fat diet); 1 : 5, 1 : 10 (high-fat diet, HFD), and 1 : 0 (zero-fat diet) as a control. Bees preferred low-fat diets over high-fat diets, where it was 11.27 ± 0.68 μl · day–1 bee in 10 : 1 P : F, while it was 4.99 ± 0.67 μl · day–1 bee in 1 : 10 P : F. However, sucrose consumption was higher in high-fat diets where it was 25.83 ± 1.69 μl · day –1 bee in 10 : 1 P: F, while it was 30.66 ± 0.9 μl · day–1 bee in 1 : 10 P : F. The encapsulation index and phenoloxidase activity of bees were positively linked with the fat level they consumed during all 10 days. The maximum percentage of encapsulation index was 74.6 ± 7.2% in bees fed a high-fat diet, whereas the minimum percentage was 16.5 ± 3.6% in bees which consumed a lowfat diet. Similarly, phenoloxidase activity increased in the haemolymph with increasing fat consumed by bees (0.001 ± 0.0001 and 0.005 ± 0.0003 mM · min –1 · mg –1 at 25 : 1 and 1 : 10 P : F, respectively). The protein content of hypopharyngeal glands in bees which consumed HFD was double that of LFD. Overall results suggest a connection between a fat diet and bee health, indicating that colony losses in some cases can be reduced by providing a certain level of fat supplemental feeding along with sucrose and protein nutrition.

Teratocytes are specialized cells released by parasitoid wasps into their hosts. They are known for producing regulatory molecules that aid the development of immature parasitoids. We have recently reported the primary structures of cystine‐rich peptides, including some containing inhibitor cystine knot (ICK) motifs, produced by teratocytes of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae). ICKs are known for their stability and diverse biological functions. In this study, we produced four putative ICK peptides from the teratocytes of C. flavipes using solid‐phase peptide synthesis or recombinant expression in E. coli, and investigated their functions on host immune modulation as well their potential to impair the development of two lepidopterans after ingestion of the peptides. In addition, the peptides were assayed against pathogens and human cells. The peptides did not influence total hemocyte count but suppressed cellular immunity, detectable as a reduction of hemocyte encapsulation (CftICK‐I, CftICK‐II, CftICK‐III) and spread indexes (CftICK‐IV) in the host. None of the peptides influenced the activities of prophenoloxidase and phenoloxidase in the hemolymph of larval Diatraea saccharalis (Lepidoptera: Crambidae). CftICK‐I and CftICK‐II with previously unknown function showed antifungal activity against Candida albicans but were non‐toxic to human cells. CftICK‐I, CftICK‐II, and CftICK‐III increased larval mortality and reduced leaf consumption of D. saccharalis, a permissive host for C. flavipes. The CftICK‐III also increased larval mortality and reduced leaf consumption of Spodoptera frugiperda (Lepidoptera: Noctuidae), a non‐permissive host for C. flavipes. This study highlights biological functions and biotechnological potential of ICK peptides from the teratocytes of C. flavipes. [ABSTRACT FROM AUTHOR]

Simple Summary: It is known that food nutrients affect hosts immune responses in response to infection. However, the results found in the literature show that the effect of the quantity and quality of foods on immune responses differ across insects. In the current study, we revealed that high protein-to-carbohydrate ratio diets have different impacts on phenoloxidase-mediated melanization and antimicrobial peptide production, but no effects on cellular immune responses in the oriental armyworm, Mythimna separata Walker. Nutrients can greatly affect host immune defenses against infection. Possessing a simple immune system, insects have been widely used as models to address the relationships between nutrition and immunity. The effects of high versus low protein-to-carbohydrate ratio (P:C) diets on insect immune responses vary in different studies. To reveal the dietary manipulation of immune responses in the polyphagous agricultural pest oriental armyworm, we examined immune gene expression, phenoloxidase (PO) activity, and phagocytosis to investigate the immune traits of bacteria-challenged oriental armyworms, which were fed different P:C ratio diets. We found the oriental armyworms that were fed a 35:7 (P:C) diet showed higher phenoloxidase (PO) activity and stronger melanization, and those reared on a 28:14 (P:C) diet showed higher antimicrobial activity. However, different P:C diets had no apparent effect on the hemocyte number and phagocytosis. These results overall indicate that high P:C diets differently optimize humoral immune defense responses in oriental armyworms, i.e., PO-mediated melanization and antimicrobial peptide synthesis in response to bacteria challenge. [ABSTRACT FROM AUTHOR]

Potentially harmful compounds including pharmaceuticals are commonly found in marine waters and sediments. Amongst those, antibiotics and their metabolites are detected worldwide in various abiotic (at concentrations as high as µg/L) and biotic matrices at ng/gram of tissue, posing a risk to non-target species exposed to them such as blue mussels. Amongst those, oxytetracycline (OTC) belongs to the most detected antibiotics in the marine environment. In this work, we concentrated on studying the potential induction of oxidative stress, activation of cellular detoxification processes (including Phase I and Phase II xenobiotic biotransformation enzymes) and multixenobiotic resistance pumps (Phase III) as well as changes in the aromatisation efficiency in Mytilus trossulus exposed to 100 μg/L OTC. Our results show that 100 µg/L OTC concentration did not provoke cellular oxidative stress and did not affect the expression of genes involved in detoxification processes in our model. Moreover, no effect of OTC on aromatisation efficiency was found. Instead, phenoloxidase activity measured in haemolymph was significantly higher in OTC exposed mussels than in those from the control (30.95 ± 3.33 U/L and 17.95 ± 2.75 U/L, respectively). OTC exposed mussels were also characterised by a tissue-dependant activation of major vault protein (MVP) gene expression (1.5 times higher in gills and 2.4 times higher in the digestive system) and a decreased expression of the nuclear factor kappa B-a (NF-κB) gene (3.4 times lower in the digestive system) when compared to those from the control. Additionally, an elevated number of regressive changes and inflammatory responses in tissues such as gills, digestive system and mantle (gonads) was observed underlining the worsening of bivalves' general health. Therefore, instead of a free-radical effect of OTC, we for the first time describe the occurrence of typical changes resulting from antibiotic therapy in non-target organisms like M. trossulus exposed to antibiotics such as OTC. [ABSTRACT FROM AUTHOR]

Background: Bellamya bengalensisis an important bioresource of the freshwater ecosystem of India. This edible mollusc is filter feeder, indigenous diet of human, poultry and fishery. Natural habitat of this freshwater gastropod mollusc bears the risk of contamination by agricultural pesticide fenvalerate, a synthetic type II pyrethroid. Hemocytes are the immuno effector cells of molluscs which are affected by environmental toxins or pathogenic microorganisms. Hemocytes perform various types of immunological functions such as phagocytosis, cytotoxic response etc. Results: Experimental exposure of fenvalerate resulted in impairment of phagocytic efficacy and alteration in the generation of phenoloxidase in B. bengalensis. Conclusion: The alteration of phagocytic response and generation of phenoloxidase of hemocytes, exposed to experimental concentrations of fenvalerate under static laboratory condition have been determined to establish as biomarker of aquatic toxicity in toxin contaminated natural habitat. [ABSTRACT FROM AUTHOR]

Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) was an important pest that is adapted to utilize numerous plant-feeding sources, it has recently a main pest on vegetable crops. Since it was initially recorded in Egypt, it has spread throughout all regions. While relatively little information on behaviour and population and damages is available for vegetables like pepper, eggplant, squash cantaloupe, cucumber, and snake-cucumber. The current study focused on studying the variability of this pest infestation on these tested crops throughout two summer seasons 2022 and 2023. The present data proved that the population density of F. occidentalis was recorded as a high infestation on the tested six host plants after mid-May through two seasons. Moreover, snake-cucumber harboured a high infestation of F. occidentalis (7.91 individuals) followed by cantaloupe (6.49 individuals), but both cucumber and pepper were exposed to the lowest infestation (4.69 and 4.92 individuals, respectively). The Solanaceae crops harboured lower damages caused by F. occidentalis individuals on flowers than cucurbitaceae crops (Squash, cantaloupe, cucumber, and snake-cucumber). Between two/or three peaks of F. occidentalis at flowering and fruiting stages were detected through May-June. The injurious caused by F. occidentalis were up to 27.6%. Among three colour sticky traps (Red, yellow, and blue), blue sticky trap appeared to be the most attractive of F. occidentalis and Thrips tabaci Lindeman (Thysanoptera: Thripidae) adults and can be used to enhance the effectiveness of tactics for thrips controlling in Solanaceae and Cucurbitaceae cultivations. Moreover, eggplant and pepper crops were less attractive to F. occidentalis individuals on flowers than cucurbit crops. The presented results indicated that the susceptibility degree of this tested crop to the F. occidentalis infestation was limited to susceptible and low resistance. The two investigated Solanaceae crops had a low resistant, but squash and cucumber in cucurbit crops was a low resistance to F. occidentalis infestation. While cantaloupe and snake-cucumber in cucurbit crops were susceptible to F. occidentalis infestation in both two tested seasons. Mirid bug predator, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) associated with F. occidentalis and T. tabaci was slightly higher on Solanaceae than Cucurbitaceae plants, in spite of, the presence of thrips was high in cantaloupe and snakecucumber. The level of flavonoids, alkaloids and phenoloxidase contents was the increase with increasing of F. occidentalis infestations as like a positive relation. [ABSTRACT FROM AUTHOR]

In this study, the expression of four reference genes (β-actin, EF1-α, GAPDH, and SubF0), two prophenoloxidase genes (proPO1 and proPO2), and the distribution of phenoloxidase (PO) activity in eight tissues (carapace, cuticle, hemolymph, hepatopancreas, muscle, pereiopods, pleopods, and uropods) of black tiger shrimp (Penaeus monodon) were evaluated. According to the geNorm and NormFinder algorithms, the most stable reference gene or the best combinations of reference genes for the evaluation of proPO1 and proPO2 expression are SubF0 for cuticle, muscle, and uropods, EF1-α for hemolymph and pereiopods, EF1-α + SubF0 for carapace, β-actin + SubF0 for hepatopancreas, and β-actin + GAPDH for pleopods. In the hemolymph and uropods, proPO1 expression was significantly stronger than that of proPO2 (433 and 5 times higher, respectively). However, in hepatopancreas, proPO2 expression was significantly stronger than proPO1 (36 times higher). Trypsin cleaves the proPO zymogen into active PO in most of the studied tissues, except hepatopancreas. PO activity (units/mg protein) following trypsin activation varied among the tissues in the following order: uropods > hemolymph > pleopods > pereiopods > hepatopancreas > carapace > cuticle > muscle. The results indicate that proPO1 is dominant in hemolymph, whereas proPO2 is dominant in hepatopancreas. Altogether, these results contribute to the elucidation of the phenoloxidase function in P. monodon. [ABSTRACT FROM AUTHOR]

Background: Penaeus vannamei suffers a number of high-mortality diseases that cause enormous economic losses. Aim. To stimulate the immune system of Penaeus vannamei shrimps following non-lethal heat stress (NLHS). Materials and methods: Two NLHS conditions (acute and chronic NLHS) were developed. The acute challenge lasted 30 minutes, along which samples are collected at different intervals (0,30min, 3, 6, and 12 h). The chronic challenge lasted 5 minutes and took seven days. The samples were collected (0 and 7d) to evaluate the immunological response and osmotic stress resistance. The shrimp at 28 °C made the control group. Results: An acute challenge ANOVA showed that NLHS in culturing conditions P. vannamei was 37°C. The ANOVA (double) and HSD Tukey detected an increase (P<0.05) in enzyme phenoloxidase at 6 and 12 h after NLHS (0.04593 and 0.05268 x 10-1 mg), when compared with 30 min and the control (0.0003037 and 0.001217 x 10-1 mg). The chronic NLHS showed high phenoloxidase stimulation (P<0.05) at 7 days (4.233 ±10-1 mg). Meanwhile, the control showed 1.392 ±10-1 mg. Survival against osmotic stress at 0pmm (T-Student) was high (85%) in the NLHS group (P=0.05), when compared with the control (20%). Conclusions: The results show that NLHS at 37 ℃ stimulates the immune system and increases survival against a salinity challenge at 0 ppm. [ABSTRACT FROM AUTHOR]

The discharge of domestic and industrial effluents into the oceans is one of the main forms of marine pollution. The impact of these effluents on the marine environment can be studied through the responses at different levels of biological organization within the ecosystem. The objective of this work was to evaluate biochemical (amylases, alkaline proteases, lipases, phenoloxidase and pyrogallol peroxidases activity) and ecological responses (abundance, size and population structure) of Boccardia proboscidea polychaetes to sewage environmental exposure, and to explore the relationship among them. The intertidial zone exposed to two sewage effluents with different characteristics were selected for the study: an intertidal effluent without previous treatment (Quequén) and a submarine outfall with primary treatment (Mar del Plata), both with their corresponding control areas. The results showed that no significant differences were found in the activity of digestive enzymes, nor of the pyrogallol-peroxidases of B. proboscidea. In contrast, phenoloxidase activity presented the highest values in the impacted area from Quequén. In turn, the highest population density of B. proboscidea was recorded there. On the other hand, the individuals in the control area from Quequén were longer than those from the rest of the areas. Regarding the frequency of age classes (larvae, juvenile and adult), in the impacted area from Quequén, the highest value of adults individuals was recorded. The relationship between the biochemical and ecological responses and the environmental variables showed that the total organic matter in sediments and density of B. proboscidea presented a positive linear correlation with the activity of phenoloxidase. Our results suggest that exposure to the Quequén sewage effluent would induce physiological changes in B. proboscidea, reflected by the high activity of the enzyme phenoloxidase, which in turn showed a positive relationship with the population density of this species. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the nutritional behavior and some immunological criteria (encapsulation index and phenoloxidase - PO activity, the key enzyme for melanization) as well as to study the effect of protein to fat (P: F) diets on hypopharyngeal gland (HPG) protein content. Bees were restricted to consuming specific P: F diets varying in fat ratio under laboratory conditions. These diets included 25: 1, 10: 1, 5: 1 (low-fat diet, LFD); 1: 1 (equal-fat diet); 1: 5, 1: 10 (high-fat diet, HFD), and 1: 0 (zero-fat diet) as a control. Bees preferred low-fat diets over high-fat diets, where it was 11.27 ± 0.68 µl · day-1 bee in 10: 1 P: F, while it was 4.99 ± 0.67 µl · day-1 bee in 1: 10 P: F. However, sucrose consumption was higher in high-fat diets where it was 25.83 ± 1.69 µl · day-1 bee in 10: 1 P: F, while it was 30.66 ± 0.9 µl· day-1 bee in 1: 10 P: F. The encapsulation index and phenoloxidase activity of bees were positively linked with the fat level they consumed during all 10 days. The maximum percentage of encapsulation index was 74.6 ± 7.2% in bees fed a high-fat diet, whereas the minimum percentage was 16.5 ± 3.6% in bees which consumed a lowfat diet. Similarly, phenoloxidase activity increased in the haemolymph with increasing fat consumed by bees (0.001 ± 0.0001 and 0.005 ± 0.0003 mM · min-1 · mg-1 at 25: 1 and 1: 10 P: F, respectively). The protein content of hypopharyngeal glands in bees which consumed HFD was double that of LFD. Overall results suggest a connection between a fat diet and bee health, indicating that colony losses in some cases can be reduced by providing a certain level of fat supplemental feeding along with sucrose and protein nutrition. [ABSTRACT FROM AUTHOR]

It is well established that host plants influence an insect's immune responses; however, most studies have been conducted on lepidopteran insects; little is known about hemipteran insects, particularly the pea aphid, Acyrthosiphon pisum. Pea aphids feed on host plants by ingesting phloem sap. The chemical composition and concentration of the sap vary depending on the host plant. These differences may impact aphid immune responses to pathogens. In this study, we examined the variations of the immune responses of pea aphids reared on broad beans (Vicia faba) and alfalfa (Medicago sativa). Pea aphids on these two host plants were challenged with bacteria Staphylococcus aureus and Escherichia coli and then the concentration of hydrogen peroxide (H2O2), phenoloxidase (PO) activity, and phagocytosis index were measured. We found that the H2O2 level and PO activity increased in the aphids on these two host plants in response to bacterial infection. In the infected condition, aphids reared on broad beans had higher H2O2 level than those reared on alfalfa, on the contrary, aphids reared on alfalfa had higher PO activity than those reared on broad beans. Furthermore, for both groups of aphids, haemocytes engulfed bacterial cells, and the capacity to phagocytize did not vary significantly between the haemocytes from these two groups of aphids. Our findings suggested that the immune responses of the pea aphid to pathogen are affected by their host plant specifically. [ABSTRACT FROM AUTHOR]

Polydnavirus (PDV) is a parasitic factor of endoparasitic wasps and contributes greatly to overcoming the immune response of parasitized hosts. Protein tyrosine phosphatases (PTPs) regulate a wide variety of biological processes at the post-transcriptional level in mammals, but knowledge of PDV PTP action during a parasitoid–host interaction is limited. In this study, we characterized a PTP gene, CvBV_12-6, derived from Cotesia vestalis bracovirus (CvBV), and explored its possible regulatory role in the immune response of the host Plutella xylostella. Our results from qPCR show that CvBV_12-6 was highly expressed in hemocytes at an early stage of parasitization. To explore CvBV_12-6 function, we specifically expressed CvBV_12-6 in Drosophila melanogaster hemocytes. The results show that Hml-Gal4 > CvBV_12-6 suppressed the phenoloxidase activity of hemolymph in D. melanogaster, but exerted no effect on the total count or the viability of the hemocytes. In addition, the Hml-Gal4 > CvBV_12-6 flies exhibited decreased antibacterial abilities against Staphylococcus aureus. Similarly, we found that CvBV_12-6 significantly suppressed the melanization of the host P. xylostella 24 h post parasitization and reduced the viability, but not the number, of hemocytes. In conclusion, CvBV_12-6 negatively regulated both cellular and humoral immunity in P. xylostella, and the related molecular mechanism may be universal to insects. [ABSTRACT FROM AUTHOR]

Until four decades ago, ascidian cytotoxic cells were considered too differentiated to exert any important function in the biology of the organisms. Their unique accepted roles were related to tunic synthesis and storing nutrients (trophocytes) useful to support other processes. Things changed starting from the beginning of the eighties of the last century when it progressively became clear that they are directly involved in inflammatory reactions. In the last decade, it was ascertained that these cells, once neglected, deeply influence ascidian biology, as they are the main source of cytokines modulating the activity of immunocytes, and of the majority of the complement components that assure correct immune responses. Moreover, they can also produce and release antimicrobial peptides that protect the organism from infections. [ABSTRACT FROM AUTHOR]

Maternally transmitted microbes are ubiquitous. In insects, maternal microbes can play a role in mediating the insect immune response. Less is known about how ecological factors, such as resource use, interact with maternal microbes to affect immunity.In the context of a recent colonization of a novel host plant by the Melissa blue butterfly Lycaeides melissa, we investigated the interaction between host plant use and vertically transmitted, extracellular egg‐associated microbes in determining the strength of the insect immune response.We reared larvae on two different host plant species: a native host Astragalus canadensis and a novel host Medicago sativa. Egg‐associated microbes were removed through a series of antimicrobial egg washes prior to hatching. Immune response was measured through three assays: standing phenoloxidase (PO), total PO and melanization.We detected strong effects of microbial removal. Egg washing resulted in larvae with an increased immune response as measured by total PO—contrary to reports from other taxa. The effect of washing was especially strong for larvae consuming the native host plant.This result may explain why consumption of the egg casing is not a universal behaviour in insects, due to negative effects on larval immunity. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Phenoloxidase (PO) is an important enzyme for various insect processes, such as defensive encapsulation and melanization of foreign organisms as well as wound healing. Here, we characterize phenoloxidase extracted from the hemolymph of the cotton leaf worm, Spodoptera littoralis, for the first time. We obtained a cDNA sequence of 600 bp as a new record, which has elevated homology with known PO sequences of other lepidopterous insects. Moreover, we evaluated the antibacterial activity of PO induced by synthesized antimicrobial peptides (AMPs) against Gram-positive Staphylococcus aureus and Enterococci, and Gram-negative Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumonia. Standardized concentrations (0.5 McFarland) of bacterial suspensions were used. The highest antibacterial activity expressed as the "growth-inhibition zone," was 2.0 ± 0.2 Cm observed against Staphylococcus. For the Gram-negative bacteria, a growth-inhibition zone (1.5 ± 0.15 Cm) was observed only for E. coli. Based on these results, future studies evaluating the effect of AMPs compared with existing antibiotics are warranted. [ABSTRACT FROM AUTHOR]

The current investigation was conducted to study the efficacy of natural diatomaceous earth (DE) alone and combined with two fungal pathogens: Metarhizium anisopliae and Beauveria bassiana, as a new formulation against Spodoptera littoralis third instar larvae under laboratory conditions. When M. anisopliae, B. bassiana, and DE were applied alone, the mortalities of larvae were concentration-dependent. The LC50 values were 299.2, 391.5, and 572 ppm, respectively. The combination of DE with M. anisopliae and with B. bassiana enhances the efficacy of both. The LC50 values were 133.77 and 189 ppm, respectively, indicating a toxic potentiation effect. The results demonstrated that the application of DE in a binary combination with fungi significantly reduced the immune enzyme activity (phenoloxidase, prophenoloxidase, lysozyme) and protein concentration compared to the control. These findings are consistent with the theory that diatomaceous earth increases the effectiveness of entomopathogenic fungi against insects by causing cuticle damage, which increases conidial attachment and increases the availability of nutrients for conidial germination. It concluded that adding DE to entomopathogenic fungi helps in improving their quality and efficiency in reducing insect outbreaks, and it suggested participating in sustainable pest management of S. littoralis. [ABSTRACT FROM AUTHOR]

The immune response represents a suite of evolved traits that can involve energetic and evolutionary trade‐offs with other energy‐demanding and fitness‐related processes. Here, we tested the hypothesis that aquatic beetles living in inland hypersaline habitats have lower immune capacity than freshwater congeners.Phenoloxidase activity, encapsulation response and antimicrobial peptide activity were compared in freshwater/hypersaline species pairs with differing osmoregulatory capacity and cuticular waterproofing properties in the genera Nebrioporus (Dytiscidae) and Enochrus (Hydrophilidae), independent evolutionary lineages that have colonised saline media separately.Hypersaline species (N. ceresyi and E. jesusarribasi) showed significantly lower phenoloxidase activity and antimicrobial peptide responses than their freshwater relatives (N. clarkii and E. salomonis). Encapsulation responses in freshwater species also appeared to be higher than in hypersaline relatives.Our results reinforce the complex nature of immune responses and suggest that adaptation to saline environments may have involved a trade‐off between osmoregulation and investment in immune defences, but also are consistent with relaxed selection pressures on basal immune responses resulting from lower microbial infection load in saline habitats. In addition, the more resistant cuticle of species occupying such habitats may protect against the entry of parasites, reducing selection pressure on immunity.Because the evolution of salinity tolerance is associated with reduced immune capacity, saline specialists may be particularly vulnerable to the dilution of saline waters and consequent changes in pathogen communities and load following colonisation by more generalist microorganisms. [ABSTRACT FROM AUTHOR]

Serine/threonine phosphatase calcineurin (CN) is a unique but confounding calcium/calmodulin- mediated enzyme, which is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). We cloned six transcripts for CNA named from NlCNA-X1 to NlCNA-X6, one CNB named NlCNB1 and one CNB homologous gene NlCNBH1 from Nilaparvata lugens. All of them are constitutively transcripted in various tissues and developmental stages. The primary structure of the six isoforms showed obvious differences in the length and composition of the amino acid sequence between the two binding domains of Ca2+/calmodulin (CaM) and CNB. Ca2+-binding EF-hand motifs were found in NlCNB1 and NlCNBH1. The specific gene silencing of NlCNA, NlCNB1 and NlCNBH1 respectively by RNAi resulted in drastical reduction in survival rate, female weight, eclosion rate and fecundity of N. lugens. These results showed that NlCNA, NlCNB1 and NlCNBH1 were required for N. lugens growth and reproduction. The negative effects of NlCNB1 silence on nymph mortality (97%), molting malformation (90%) and female sterile (50%) were more serious than those of NlCNA or NlCNBH1. qRT-PCR and enzyme-linked immunosorbent assay (ELISA) analyses indicated that the nymphs with silenced NlCNA, NlCNB1 or NlCNBH1 showed impaired hormone and energy metabolism. In nymphs, the contents of 20-hydroxyecdysone (20E) after NlCNB1 RNAi and phenoloxidase after NlCNA RNAi were particularly decreased. These results suggested that NlCNA is involved in immunity of N. lugens by regulation of phenoloxidase, while NlCNB1 may control the growth and development of N. lugens by 20E signaling pathway in addition to interact with CNA. Injection of 70 ng/μL dsNlCNB1 resulted in 77.0% down regulation of NlCNB1, and the nymph mortality was up to 57.9% at 10 d after injection. Therefore, NlCNB1 could be a potential candidate target used for strategy design in control of N. lugens. Our results revealed the importance of CN in the regulation of the growth and development of N. lugens, which provided a basis for further study of the molecular mechanism of CN.

Aquaculture industry suffers significant limitations such as low resistance to diseases and expensive feed. This study investigated the antibacterial and immunostimulatory activities of ZnO- Ulva lactuca nanocomposite (ZnO- Ul NC) in the Procambarus clarkii. Zinc oxide nanoparticles (ZnO NPs) and ZnO- Ul NC were synthetized and characterized by electron microscopies as well as Fourier transform infrared spectroscopy. ZnO NPs and ZnO- Ul NC inhibited the growth of the isolated species Citrobacter freundii and Enterobacter hormaechei. For immunostimulatory evaluation, six crayfish groups (control, U. lactuca, ZnO L, ZnO H, ZnO- Ul L, and ZnO- Ul H) were fed on commercial diet, Ulva lactuca powder, and low or high dose of ZnO NPs or ZnO- Ul NCs, respectively for 90 days. The highest levels of total hemocyte count, granular cells%, phenoloxidase (PO) activity in hemolymph, and NO, superoxide dismutase (SOD), and GSH in hepatopancreas were all reported in the ZnO- Ul groups. The expression of proPO , SOD , and lysozyme exhibited the highest upregulation in the ZnO- Ul H group. Taken together, dietary ZnO- Ul NC significantly improved the non-specific immunity and antioxidant milieu of the crayfish at the genomic and proteomic levels. ZnO- Ul NC is cost effective, easily synthesized, and a promising immunostimulant for Procambarus clarkii that could be used in the aquaculture. • ZnO NPs and ZnO- Ul NC were synthetized and characterized. • ZnO NPs and ZnO- Ul NC inhibited Citrobacter freundii and Enterobacter hormaechei. • ZnO- Ul elevated hemocytes, granular cells, phenoloxidase in hemolymph, and NO, SOD, lysozyme, and GSH in hepatopancreas. • ZnO- Ul NC significantly improved the non-specific immunity and antioxidant milieu. • ZnO- Ul NC is cost effective and promising immunostimulant in Procambarus clarkii. [ABSTRACT FROM AUTHOR]

Searching for artificial diets positively affecting the survival, immune and antioxidant systems of honey bees is one of main challenges occurring in beekeeping. Among nutrients, lipids play a significant role in insect nutrition as structural components in cell membranes, energy sources and reserves, and are involved in many physiological processes. In this context, the aim of this work was to investigate the effect of 0.5% and 1% coconut oil-enriched diet administration on newly emerged and forager bees survival rate, feed intake, immune system, antioxidant system and both fat and vitellogenin content. In newly emerged bees, supplementation with 1% coconut oil determined a decrease in feed consumption, an increase in survival rate from the 3rd to 14th day of feeding, a short-term decrease in phenoloxidase activity, an increase in body fat and no differences in vitellogenin content. Conversely, supplementation with 0.5% coconut oil determined an increase in survival rate from the 3rd to 15th day of feeding and an increase in fat content in the long term (i.e., 20 days). Regarding the forager bee diet, enrichment with 0.5% and 1% coconut oil only determined an increase in fat content. Therefore, supplementation with coconut oil in honey bee diets at low percentages (0.5 and 1%) determines fat gain. Further investigations to evaluate the use of such supplement foods to prevent the fat loss of weak families during winter are desirable.

Maximization of life-history traits is under constraints due to both, limitations of resource acquisition and the restricted pathways of resource allocation. Drosophila melanogaster has served as an excellent model organism to not only unravel various trade-offs among life history traits but also numerous aspects of host immune response. Drosophila larvae are semi-aquatic that live, feed and excrete inside the food source-often over-ripe fruits and vegetables that are rich in both commensal and pathogenic microbiota that can impact the larval survival. In this study, we have used six populations of D. melanogaster, three of which are selected for faster pre-adult development and extended adult longevity, and their three ancestral controls, to explore the impact of selection on the basal immune activity in the larval stage. The larvae from selected populations had nearly significantly upregulated plasmatocyte density, significantly higher percent phagocytosis, phagocytic index and higher transcript levels of Tep3, eater and NimC1. Selected populations also had significantly upregulated crystal cell number along with higher transcript of PPO2. Out of seven tested AMPs level, Drosomycin was significantly upregulated in selected populations while Drosocin was significantly higher in control populations. ROS levels were comparable in the selected and control populations. Our results strongly suggest that enhanced basal immune activity during larval stage manages the faster development and could be responsible for comparable larval survival of selected and control populations.