Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

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.

No evidence for an up-regulation of female immune function in response to elevated risk of sexual conflict.

Sexual conflict is widespread among sexually reproducing organisms. Phenotypic plasticity in female resistance traits has the potential to moderate the harm imposed by males during mating, yet female plasticity has rarely been explored. In this experiment, we investigated whether female seed beetles invest more in immunocompetence, measured as phenoloxidase (PO) capacity, when exposed to cues signalling a greater risk of sexual conflict. Risk perception was manipulated by housing focal individuals alone or with a companion as developing larvae, followed by exposure to a mating-free male- or female-biased social environment when adults. We predicted that females exposed to cues of increased sexual conflict would have increased PO capacity. However, PO capacity did not differ between either larval or adult social treatments. Our results suggest that females may not perceive a risk to their fitness on the basis of increased male presence or are unable to adjust this aspect of their phenotype in response to that risk.

Zinc oxide-Ulva lactuca nanocomposite is a robust dietary immunostimulant in the red swamp crayfish (Procambarus clarkii).

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.

Infection with a non-lethal fungal parasite is associated with increased immune investment in the ant Myrmica scabrinodis.

Social insects, such as ants, are preferred host organisms of pathogens and parasites because colonies are densely populated, and the number of potential hosts is high in the same place and time. Within a colony, individuals are exposed differentially to risks according to their function and age. Thus, older individuals forage and are therefore the most exposed to infection, predation, or physical stress, while young workers mostly stay inside the sheltered nest being less exposed. Immune investment is considered to be dependent on an individual's age and pathogen pressure. Long-term exposure to a parasite could affect the immune activity of individuals in an intriguing way that interferes with the age-dependent decline in immunocompetence. However, there are only few cases in which such interferences can be studied. The myrmecopathogenic fungus Rickia wasmannii, which infects entire colonies without killing the workers, is a suitable candidate for such studies. We investigated the general immunocompetence of Myrmica scabrinodis ant workers associated with non-lethal fungal infection by measuring the levels of active phenoloxidase (PO) and total PO (PPO) (reflecting the amount of both active and inactive forms of the enzyme) in two age classes. The level of PO proved to be higher in infected workers than in uninfected ones, while the level of PPO increased with age but was not affected by infection. Overall, these results indicate that a long-term infection could go hand in hand with increased immune activity of ant workers, conferring them higher level of protection.

In vitro immune analysis of serum from the hemolymph of the anomuran crab Albunea symmysta (Linnaeus, 1758) displayed diversified reactions.

The present investigation aims to substantiate that serum from the hemolymph of anomuran crab Albunea symmysta encompasses multiple immunological reactions in in vitro condition. The serum highly agglutinated human O erythrocytes in the presence of Ba2+. Distinct and unique sugar binding capacity of serum towards laminarin, N-acetyl sugars and higher binding specificity towards a glycoprotein, fetuin was inferred. In vitro enhancement of melanin synthesis due to enhanced oxidation of 3, 4-dihydroxy-dl-phenylalanine (dl-DOPA) by preincubation of nonself molecules with serum phenoloxidase (PO) was documented. Similarly, dl-DOPA oxidation by serum PO was reduced when preincubated with chemical inhibitors and copper chelators. Further, the crab serum lysed the vertebrate erythrocytes with maximum hemolysis against chicken and it unveiled dependency on divalent cation, serum concentration, ionic strength, pH, temperature and time interval. Occurrence of maximum hemolysis at a concentration of 30 µl, pH 8.0, temperature 37 °C and time interval of 60 min in the presence of Ba2+ were documented. Interestingly, serum hemolysis was reduced by different osmoprotectants suggesting a colloid-osmotic mechanism involving in hemolysis. It was observed that A. symmysta serum had antimicrobial activity against Gram-positive Staphylococcus aureus and fungal pathogen Candida albicans. The serum showed higher glycan content, potent lysozyme and free radical scavenging activity suggesting the existence of potential immune molecules of therapeutic use. These results clearly demonstrated the diversified immunogenicity of A. symmysta serum confirming a highly conserved non-specific immunity of crustaceans.

Purification of phenoloxidase from Haliotis discus hannai and its anti-inflammatory activity in vitro.

Haliotis discus hannai, a food with a high protein content, is widely consumed in Asian countries. It is known to have antioxidant, anticancer, and antibacterial effects. Since the biological significance of H. discus hannai hemolymph has not been widely studied, the objective of the present study was to purify phenoloxidase (PO) and investigate its immunological effects on human colonic epithelial cells. PO was purified through ammonium sulfate precipitation and one step column chromatography. The molecular weight of the protein was about 270 kDa. When PO was mixed with Gram-negative bacteria-derived lipopolysaccharide (LPS) at various ratios (10:1-1:10, w/w), the amount of residual LPS was reduced. PO at concentrations up to 200 µg/mL was not cytotoxic to HT-29 cells. The inflammatory response induced by LPS in HT-29 cells was regulated when the concentration of PO was increased. With increasing concentration of PO, production levels of pro-inflammatory cytokines, cytokines associated with hyperimmune responses such as IL4, IL-5, and INF-γ, and prostaglandin 2 (PGE2) were regulated. It was thought that simultaneous treatment with PO and LPS anti-inflammatory effects in HT-29 cells showed by regulating the ERK1/2-mediated NF-κB pathway. Results of this study suggest that H. discus hannai hemolymph is involved in the regulation of Gram-negative bacteria-related inflammatory immune responses in human colonic epithelial cells.

Effects of Host Plants on Development and Immunity of a Generalist Insect Herbivore.

Secondary plant chemistry mediates a variety of communication signals among species, playing a fundamental role in the evolutionary diversification of communities and ecosystems. Herein, we explored diet-mediated host plant effects on development and immune response of a generalist insect herbivore. Vanessa cardui (Nymphalidae) caterpillars were reared on leaves of three host plants that vary in secondary metabolites, Plantago lanceolata (Plantaginaceae), Taraxacum officinale (Asteraceae) and Tithonia diversifolia (Asteraceae). Insect development was evaluated by larval and pupal viabilities, survivorship, and development rate. Immune response was measured as phenoloxidase (PO) activity. Additionally, chemical profiles of the host plants were obtained by liquid chromatograph-mass spectrometry (LC-MS) and the discriminant metabolites were determined using a metabolomic approach. Caterpillars reared on P. lanceolata exhibited the highest larval and pupal viabilities, as well as PO activity, and P. lanceolata leaves were chemically characterized by the presence of iridoid glycosides, phenylpropanoids and flavonoids. Taraxacum officinale leaves were characterized mainly by the presence of phenylpropanoids, flavones O-glycoside and germacranolide-type sesquiterpene lactones; caterpillars reared on this host plant fully developed to the adult stage, however they exhibited lower larval and pupal viabilities compared to individuals reared on P. lanceolata. Conversely, caterpillars reared on T. diversifolia leaves, which contain phenylpropanoids, flavones and diverse furanoheliangolide-type sesquiterpene lactones, were not able to complete larval development and exhibited the lowest PO activity. These findings suggested that V. cardui have adapted to tolerate potentially toxic metabolites occurring in P. lanceolata (iridoid glycosides), however caterpillars were not able to cope with potentially detrimental metabolites occurring in T. diversifolia (furanoheliangolides). Therefore, we suggest that furanoheliangolide-type sesquiterpene lactones were responsible for the poor development and immune response observed for caterpillars reared on T. diversifolia.

Co-application of entomopathogenic fungi with chemical insecticides against Culex pipiens.

Culex pipiens (Diptera: Culicidae) is a vector of many human and animal diseases. Its control is regarded as a preventative approach that is focused on effectively managing such diseases. In this context, dose response assays of two insecticides, bendiocarb and diflubenzuron were performed with two entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae against 3rd instar C. pipiens larvae. The most effective agents, combination experiments as well as enzymatic activities of phenoloxidase (PO) and chitinase (CHI) were also assessed. The results showed that diflubenzuron was more effective at low concentrations (LC50: 0.001 ppm) than bendiocarb (LC50: 0.174 ppm), whereas M. anisopliae was more effective (LC50: 5.2 × 105 conidia/mL) than B. bassiana (LC50: 7.5 × 107 conidia/mL). Synergistic interactions were observed when diflubenzuron was applied at 2- and 4-days post- exposure to M. anisopliae, with the highest degree of synergism observed when diflubenzuron was applied 2 days post-fungal exposure (χ2 = 5.77). In contrast, additive interactions were recorded with all other insecticide-fungal combinations. PO activities significantly (p ≤ 0.05) increased during 24 h after a single diflubenzuron treatment as well as when diflubenzuron was applied prior to M. anisopliae, whereas suppressed after 24 h when M. anisopliae applied prior to diflubenzuron as well as after 48 h from single and combined treatments. CHI activity increased 24 h after both single and combined treatments, the activity remained elevated 48 h after a single diflubenzuron treatment and when diflubenzuron was applied after M. anisopliae. Histological study of the cuticle by transmission electron microscopy revealed abnormalities following single and combined treatments. Germination of the conidia and production of the mycelium that colonizes the lysing cuticle was obvious when diflubenzuron was applied 48 h after M. anisopliae exposure. Overall, these results demonstrate that M. anisopliae is compatible with diflubenzuron at lower concentrations and that combined applications can improve C. pipiens management.

Interactions of alumina and polystyrene nanoparticles with the innate immune system of Galleria mellonella.

Nowadays, particularly metallic, and polymeric nanoparticles (NPs) are widely produced and used in many fields. Due to the increase in both their usage and diversity, their release and accumulation in the environment are also accelerating. Therefore, their interactions with cells, especially immune cells, and their health risks are not fully understood. The impacts of metallic alumina (Al) NPs and polystyrene (PS) NPs obtained after the polymerization of carcinogenic styrene on living organisms have not yet been elucidated. Galleria mellonella larvae can biodegrade plastics. While biodegradation and solving the waste problem have attracted much attention, the interactions of this distinctive property of G. mellonella larvae in the immune system and ecosystem are not yet completely understood. Al and PS NPs were applied to G. mellonella separately. Al NPs were purchased and PS NPs were prepared from PS by single-emulsion technique and characterized. Then LC50 values of these NPs on G. mellonella were determined. The interactions of these NPs with encapsulation, melanization, and phenoloxidase activity, which express innate immune responses in G. mellonella larvae, were revealed. NP exposure resulted in suppression of the immune response, probably because it affects the functions of hemocytes such as enzymatic activation, hemocyte division, and populations. In this context, our data suggest that Al and PS NPs induce toxic impacts and negatively alter the physiological status of G. mellonella. It is also shown that G. mellonella has the potential to be an impactful alternative model for biosafety and nanotoxicology studies.

Temperature-dependent melanism and phenoloxidase activity in the dimorphic sepsid fly Sepsis thoracica.

Climate is changing towards both higher average temperatures and more frequent and severe heat waves. Whereas numerous studies have investigated temperature effects on animal life histories, assessments of their immune function are limited. In the size- and colour-dimorphic black scavenger (or dung) fly Sepsis thoracica (Diptera: Sepsidae), we experimentally studied how developmental temperature and larval density influence phenoloxidase (PO) activity, a key enzyme in insect pigmentation, thermoregulation, and immunity. Flies from five latitudinal European populations were raised at three developmental temperatures (18, 24, 30 °C). PO activity increased with developmental temperature differently in the sexes and the two male morphs (black and orange), altering the sigmoid relationship between melanism, i.e. colouration and fly size. PO activity further positively correlated with larval rearing density, potentially because of higher risks of pathogen infection or greater developmental stress following stronger resource competition. Populations varied somewhat in PO activity, body size and colouration, however with no clear latitudinal pattern. Overall our results indicate that morph- and sex-specific PO activity, and thus likely immune function, in S. thoracica depends on temperature and larval density, modifying the underlying putative trade-off between immunity and body size. The strong dampening of the immune system of all morphs at cool temperatures suggests low-temperature stress in this warm-adapted species common in southern Europe. Our results also support the population density dependent prophylaxis hypothesis, which predicts higher investment in immunity when facing limited resource availability and increased pathogen infection probability.