Phenoloxidase Activity In Hemolymph Research Articles

Effects of a supplemented diet containing 7 probiotic strains (Honeybeeotic) on honeybee physiology and immune response: analysis of hemolymph cytology, phenoloxidase activity, and gut microbiome

BackgroundIn this study, a probiotic mixture (Honeybeeotic) consisting of seven bacterial strains isolated from a unique population of honeybees (Apis mellifera ligustica) was used. That honeybee population was located in the Roti Abbey locality of the Marche Region in Italy, an area isolated from human activities, and genetic contamination from other honeybee populations. The aim was to investigate the effects of this probiotic mixture on the innate immunity and intestinal microbiome of healthy common honeybees in two hives of the same apiary. Hive A received a diet of 50% glucose syrup, while hive B received the same syrup supplemented with the probiotics, both administered daily for 1 month. To determine whether the probiotic altered the immune response, phenoloxidase activity and hemolymph cellular subtype count were investigated. Additionally, metagenomic approaches were used to analyze the effects on gut microbiota composition and function, considering the critical role the gut microbiota plays in modulating host physiology.ResultsThe results revealed differences in hemocyte populations between the two hives, as hive A exhibited higher counts of oenocytoids and granulocytes. These findings indicated that the dietary supplementation with the probiotic mixture was safe and well-tolerated. Furthermore, phenoloxidase activity significantly decreased in hive B (1.75 ± 0.19 U/mg) compared to hive A (3.62 ± 0.44 U/mg, p < 0.005), suggesting an improved state of well-being in the honeybees, as they did not require activation of immune defense mechanisms. Regarding the microbiome composition, the probiotic modulated the gut microbiota in hive B compared to the control, retaining core microbiota components while causing both positive and negative variations. Notably, several genes, particularly KEGG genes involved in amino acid metabolism, carbohydrate metabolism, and branched-chain amino acid (BCAA) transport, were more abundant in the probiotic-fed group, suggesting an effective nutritional supplement for the host.ConclusionsThis study advocated that feeding with this probiotic mixture induces beneficial immunological effects and promoted a balanced gut microbiota with enhanced metabolic activities related to digestion. The use of highly selected probiotics was shown to contribute to the overall well-being of the honeybees, improving their immune response and gut health.

Higher temperature accelerates the aging-dependent weakening of the melanization immune response in mosquitoes.

The body temperature of mosquitoes, like most insects, is dictated by the environmental temperature. Climate change is increasing the body temperature of insects and thereby altering physiological processes such as immune proficiency. Aging also alters insect physiology, resulting in the weakening of the immune system in a process called senescence. Although both temperature and aging independently affect the immune system, it is unknown whether temperature alters the rate of immune senescence. Here, we evaluated the independent and combined effects of temperature (27°C, 30°C and 32°C) and aging (1, 5, 10 and 15 days old) on the melanization immune response of the adult female mosquito, Anopheles gambiae. Using a spectrophotometric assay that measures phenoloxidase activity (a rate limiting enzyme) in hemolymph, and therefore, the melanization potential of the mosquito, we discovered that the strength of melanization decreases with higher temperature, aging, and infection. Moreover, when the temperature is higher, the aging-dependent decline in melanization begins at a younger age. Using an optical assay that measures melanin deposition on the abdominal wall and in the periostial regions of the heart, we found that melanin is deposited after infection, that this deposition decreases with aging, and that this aging-dependent decline is accelerated by higher temperature. This study demonstrates that higher temperature accelerates immune senescence in mosquitoes, with higher temperature uncoupling physiological age from chronological age. These findings highlight the importance of investigating the consequences of climate change on how disease transmission by mosquitoes is affected by aging.

Identification of an essential role against shrimp pathogens of prophenoloxidase activating enzyme 1 (PPAE1) from Fenneropenaeus merguiensis hemocytes

Prophenoloxidase (proPO) activating enzymes, known as PPAEs, are pivotal in activating the proPO system within invertebrate immunity. A cDNA encoding a PPAE derived from the hemocytes of banana shrimp, Fenneropenaeus merguiensis have cloned and analyzed, referred to as FmPPAE1. The open reading frame of FmPPAE1 encompasses 1392 base pairs, encoding a 464-amino acid peptide featuring a presumed 19-amino acid signal peptide. The projected molecular mass and isoelectric point of this protein stand at 50.5 kDa and 7.82, respectively. Structure of FmPPAE1 consists of an N-terminal clip domain and a C-terminal serine proteinase domain, housing a catalytic triad (His272, Asp321, Ser414) and a substrate binding site (Asp408, Ser435, Gly437). Expression of the FmPPAE1 transcript is specific to hemocytes and is heightened upon encountering pathogens like Vibrio parahaemolyticus, Vibrio harveyi, and white spot syndrome virus (WSSV). Using RNA interference to silence the FmPPAE1 gene resulted in reduced hemolymph phenoloxidase (PO) activity and decreased survival rates in shrimp co-injected with pathogenic agents. These findings strongly indicate that FmPPAE1 plays a vital role in regulating the proPO system in shrimp. Furthermore, upon successful production of recombinant FmPPAE1 protein (rFmPPAE1), it became evident that this protein exhibited remarkable abilities in both agglutinating and binding to a wide range of bacterial strains. These interactions were primarily facilitated through the recognition of bacterial lipopolysaccharides (LPS) or peptidoglycans (PGN) found in the cell wall. This agglutination process subsequently triggered melanization, a critical immune response. Furthermore, rFmPPAE1 exhibited the ability to actively impede the growth of pathogenic bacteria harmful to shrimp, including V. harveyi and V. parahaemolyticus. These findings strongly suggest that FmPPAE1 not only plays a pivotal role in activating the proPO system but also possesses inherent antibacterial properties, actively contributing to the suppression of bacterial proliferation. In summary, these results underscore the substantial involvement of FmPPAE1 in activating the proPO system in F. merguiensis and emphasize its crucial role in the shrimp's immune defense against invading pathogens.

The immune response of Locusta migratoria manilensis at different times of infection with Paranosema locustae.

Paranosema locustae is an entomopathogenic microsporidia with promising potential for controlling agricultural pests, including Locusta migratoria manilensis. However, it has the disadvantage of having a slow insecticidal rate, and how P. locustae infection impacts the host immune response is currently unknown. The present study investigated the effect of P. locustae on the natural immune response of L. migratoria and the activities of enzymes that protect against oxidative stress. Infection with P. locustae increased the hemocytes and nodulation number of L. migratoria at the initial stage of infection. The hemocyte-mediated modulation of immune response was also affected by a decrease in the number of hemocytes 12 days postinfection. Superoxide dismutase activity in locusts increased in the early stages of infection but decreased in the later stages, whereas the activities of peroxidase (POD) and catalase (CAT) showed opposite trends may be due to their different mechanisms of action. Furthermore, the transcription levels of mRNA of antimicrobial peptide-related genes and phenoloxidase activity in hemolymph in L. migratoria were suppressed within 15 days of P. locustae infection. Overall, our data suggest that P. locustae create a conducive environment for its own proliferation in the host by disrupting the immune defense against it. These findings provide useful information for the potential application of P. locustae as a biocontrol agent.

Evaluation of haemolymph phenoloxidase activity from the grub of Zophobas morio as a predictor of immune response.

In insects, enzyme phenoloxidase plays a critical role in cuticular sclerotisation and defensive functions. In the present investigation, haemolymph phenoloxidase activity from the grub of Zophobas morio was attempted to evaluate as a reliable predictor of insect's immunological response. Among the various substrates tested, L-DOPA was chosen as an appropriate substrate due to its high oxidation. The optimum pH and temperature for haemolymph PO activity was found to be 8 and 30°C, respectively. The optimum substrate concentration of L-DOPA was found to be 7.5mM for subsequent PO enzymatic characterisation. Among the various chemical inhibitors and copper chelators, PO activity was significantly reduced in the case of PMSF and thiourea. Preincubation of haemolymph with non-self-molecules showed enhancement of PO activity in the case of LPS from Serratia marcescens. In addition, exogenous proteases like α-chymotrypsin enhanced the PO activity of haemolymph and an increase in PO activity was demonstrated when haemolymph was preincubated with the anionic detergent, SDS and cationic detergent, cetyl pyridium chloride. Alteration of PO activity was observed under agonising conditions of starvation, ligation and microplastics injection at different time intervals. Interestingly, there were no correlation between PO and insect defence under live challenge of microbes. SDS protein profile revealed a significant increase in the 85kDa and 55kDa polypeptides in all the experiments over control after 24h, 48h and 96h. Mass spectrophotometric analysis of the polypeptides revealed their homology to antimicrobial peptides for 55kDa protein and 85kDa protein. A significant increase in 85kDa polypeptide was observed in the haemolymph of the grubs after 72h in the case of starved and microplastics injected groups only. These results demonstrated that PO may not be a reliable benchmark of immunological response in this insect.

Immune Profile of Litopenaeus vannamei in Monoculture and IMTA Ponds System

Integrated Multi Trophic Level (IMTA) is identified as the integrating culture of multi-species in one area. The effectiveness of space and feed is the main aspect from this point. The disease and health of shrimp management on shrimp production including immune boosters plays a critical aspect. The aim of this research is to determine and analyze the Litopenaeus vannamei immune system in different culture systems, namely polyculture (IMTA) and monoculture. Alginate was applied as supplementation diet by oral administration. The cellular and humoral immune parameters ie. Total hemolymph Count (THC), Phagocyte Activity (PA), Phagocyte Index (PI), Phenoloxidase activity (PO), and Superoxide dismutase activity (SOD) were assessed, monthly. A completely randomized design (CRD) with two treatments (IMTA and monoculture, and four replications in 500 m2 pond area was conducted. In the third month, THC and PA of shrimp from the monoculture pond resulted in significant differences (p&lt;0.05) compared to polyculture (IMTA). SOD activity from monoculture is significantly higher (p&lt;0.05) at every sampling period. In PO enzyme activity of shrimps from monoculture pond produced higher results compared to IMTA ponds in the 2nd and 3rd months. It is concluded that L. vannamei monoculture ponds produced higher immune parameters compared to IMTA ponds. The food competition in terms of feed and space from IMTA ponds may affect this fact. Improving the feeding technique is recommended.

RNAi-based knockdown of candidate gut receptor genes altered the susceptibility of Spodoptera frugiperda and S. litura larvae to a chimeric toxin Cry1AcF.

A multitude of Cry toxins (secreted by Bacillus thuringiensis or Bt) has been deployed globally either via transgenic mean or bio-pesticidal formulations in order to manage insect pests. However, Bt resistance development in insects is emerging as a major concern. To avoid this problem, multiple gene pyramiding or protein-engineered chimeric toxin-based strategy has been analyzed. In the present study, one such chimeric toxin Cry1AcF (contain the swapped domains of Cry1Ac and Cry1F) was used to investigate its in vivo pathogenesis process in lepidopteran pests Spodoptera frugiperda and S. litura. A number of biochemical and molecular analysis were performed. Oral ingestion of Cry1AcF caused greater toxicity in S. frugiperda than S. litura with larvae displaying increased hemolymph melanization. Histopathology of the midgut transverse sections exhibited Cry1AcF-induced extensive gut damage in both the test insects followed by cytotoxicity in terms of reduced hemocyte numbers and viability. Elevated hemolymph phenoloxidase activity indicated the immune-stimulatory nature of Cry1AcF. In order to analyze the role of gut receptor proteins in Cry1AcF intoxication in test insects, we performed RNAi-mediated silencing using bacterially-expressed dsRNAs of individual receptor-encoding genes including CAD, ABCC2, ALP1 and APN. Target-specific induced downregulation of receptor mRNAs differentially altered the insect susceptibility to Cry1AcF toxin in our study. The susceptibility of ALP1 and APN dsRNA pre-treated S. frugiperda was considerably decreased when treated with Cry1AcF in LD50 and LD90 doses, whereas susceptibility of CAD and ABCC2 dsRNA pre-treated S. litura was significantly reduced when ingested with Cry1AcF in different doses. CAD/ABCC2-silenced S. frugiperda and ALP1/APN-silenced S. litura were vulnerable to Cry1AcF alike of control larvae. In conclusion, our results indicate ALP1/APN and CAD/ABCC2 as the functional receptor for Cry1AcF toxicity in S. frugiperda and S. litura, respectively.

Serpin-4 Facilitates Baculovirus Infection by Inhibiting Melanization in Asian Corn Borer, Ostrinia furnacalis (Guenée).

Phenoloxidase (PO)–catalyzed melanization is a vital immune response in insects for defense against pathogen infection. This process is mediated by clip domain serine proteases and regulated by members of the serpin superfamily. We here revealed that the infection of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) significantly inhibited the PO activity in Ostrinia furnacalis hemolymph and induced the expression of O. furnacalis serpin–4. Addition of recombinant serpin-4 protein to O. furnacalis hemolymph resulted in a great increase of AcMNPV copies. Serpin-4 significantly suppressed the PO activity and the amidase activity in cleaving colorimetric substrate IEARpNA (IEARase activity) of hemolymph. Further experiments indicated it formed covalent complexes with three serine proteases (SP1, SP13 and SP105) and prevented them from cleaving their cognate downstream proteases in vitro. Altogether, O. furnacalis melanization restricted AcMNPV replication and serpin-4 facilitated AcMNPV infection by inhibiting serine proteases, SP1, SP13, and SP105 which were all involved in the melanization response.

KPI5 Is Involved in the Regulation of the Expression of Antibacterial Peptide Genes and Hemolymph Melanization in the Silkworm, Bombyx mori.

Kunitz-type protease inhibitors (KPIs) are ubiquitously found in many organisms, and participate in various physiological processes. However, their function in insects remains to be elucidated. In the present study, we characterized and functionally analyzed silkworm KPI5. Sequence analysis showed that KPI5 contains 85 amino acids with six conserved cysteine residues, and the P1 site is a phenylalanine residue. Inhibitory activity and stability analyses indicated that recombinant KPI5 protein significantly inhibited the activity of chymotrypsin and was highly tolerant to temperature and pH. The spatio-temporal expression profile analysis showed that KPI5 was synthesized in the fat body and secreted into the hemolymph. In vivo induction analysis showed that the expression of KPI5 in the fat body was significantly upregulated by pathogen-associated molecular patterns (PAMPs). Binding assays suggested that KPI5 can bind to pathogens and PAMPs. In vitro pathogen growth inhibition assay and encapsulation analysis indicated that KPI5 can neither kill pathogenic bacteria directly nor promote the encapsulation of agarose beads by silkworm hemocytes. Recombinant protein injection test and CRISPR/Cas9-mediated knockdown showed that KPI5 promotes the expression of antimicrobial peptides (AMPs) in the fat body. Moreover, the survival rate of individuals in the KPI5 knockdown group was significantly lower than that of the control group after pathogen infection. Phenoloxidase (PO) activity assays showed that KPI5 significantly inhibited the hemolymph PO activity and melanization induced by PAMPs. These findings suggested that KPI5 plays a dual regulatory role in innate immunity by promoting the expression of antimicrobial peptides in the fat body and inhibiting hemolymph melanization. Our study furthers the understanding of the function of insect KPIs and provides new insights into the regulatory mechanism of insect immune homeostasis.

Consuming sunflower pollen reduced pathogen infection but did not alter measures of immunity in bumblebees.

Certain diets can benefit bee health by reducing pathogens, but the mechanism(s) driving these medicinal effects are largely unexplored. Recent research found that sunflower (Helianthus annuus) pollen reduces the gut pathogen Crithidia bombi in the common eastern bumblebee (Bombus impatiens). Here, we tested the effects of sunflower pollen and infection on two bee immune metrics to determine whether sunflower pollen diet drives changes in host immunity that can explain this medicinal effect. Bees were infected with C. bombi or not and given either sunflower or wildflower pollen. Subsequently, bees received a benign immune challenge or were left naive to test the induced and constitutive immune responses, respectively. We measured haemolymph phenoloxidase activity, involved in the melanization cascade, and antibacterial activity. Sunflower pollen reduced C. bombi infection, but we found no significant pollen diet effect on either immune measure. Phenoloxidase activity was also not affected by C. bombi infection status; however, uninfected bees were more likely to have measurable constitutive antibacterial activity, while infected bees had higher induced antibacterial activity. Overall, we found that sunflower pollen does not significantly affect the immune responses we measured, suggesting that the mechanisms underlying its medicinal effect do not involve these bee immune parameters. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Interaction of Penaeus vannamei hemocyanin and α2-macroglobulin modulates the phenoloxidase activity

Prophenoloxidase (proPO)-activating system is a critical innate immune defense in invertebrates. However, the mechanisms involved in regulating the phenoloxidase (PO) activity in shrimp hemolymph remain ill-defined. Our previous studies showed that Penaeus vannamei hemocyanin (HMC) and α2-macroglobulin (α2M), two key regulators of proPO-activating system in plasma, might interact with each other, indicating that this interaction could be implicated in controlling PO activity. Herein, we further confirmed that HMC specifically bind to α2M using Pull down and Far-Western blot analyses. Further studies demonstrated that HMC could directly interact with the receptor binding domain of α2M. In addition, HMC and α2M followed similar expression pattern upon Vibrio parahaemolyticus infection, suggesting the interaction of HMC and α2M might have a role in immune response. Finally, we found that α2M, as a broad-spectrum proteinase inhibitor, suppressed the serum PO activity in vitro, while hemocyanin could partially restore this inhibitory effect. In sum, the present data indicate that HMC interacts with α2M and therefore modulates the PO activity. This finding contributes to better understanding of stable state maintenance of PO activity in shrimp.

Overcoming insect immune response: The role of Pseudomonas aeruginosa alkaline protease in phenoloxidase inhibition

AbstractThe role of Pseudomonas aeruginosa alkaline protease, one of the important virulence factors of these bacteria, in the prophenoloxidase (proPO) system activation in Galleria mellonella haemolymph was investigated. Immunization of G. mellonella larvae with alkaline protease led to an increase in phenoloxidase (PO) activity in haemolymph 2–8 h after the injection. However, 15 h after the challenge, almost no PO activity was detected, in contrast to insects immunized with heat‐killed P. aeruginosa, where an elevated PO activity in haemolymph persisted at all the time points of the experiment (2–24 h). To test the effect of alkaline protease on already activated PO in vitro, non‐immune G. mellonella haemolymph with proPO system pre‐activated in vitro by heat‐killed P. aeruginosa was used. Subsequently, direct incubation of the protease with non‐immune haemolymph was carried out to test the effect of the alkaline protease on the proPO system activation. It can be postulated that P. aeruginosa alkaline protease affected not only the active PO, but also significantly inhibited proPO cascade activation in the haemolymph of G. mellonella.

A standardized protocol for measuring phenoloxidase in captive and wild Murray crayfish Euastacus armatus

Innate immunity in arthropods is achieved largely through melanization which is in turn the result of the prophenoloxidase (ProPO) activation cascade; a series of biochemical reactions triggered by the immune identification of pathogen-recognition proteins (PRPs). Within this activation cascade, inactive proPO is cleaved to form the reactive enzyme phenoloxidase (PO). Methods of detecting PO are used to assess an arthropod's ability to respond to immune challenges. These detection assays have been described for some arthropods, especially those of commercial value, but none are available for Euastacus, a genus within the superfamily Parastacoidea. This study is the first step in developing a standardized protocol for the detection and quantification of PO activity in wild or captive Murray crayfish Euastacus armatus. Hemolymph extracts from 49 crayfish were assessed for PO activity using an assay measuring the conversion of l-dopa (3,4-dihydroxy-l-phenylalanine) into dopachrome. Short periods (up to 15 min) out of water did not cause any measurable change in PO activity. Phenoloxidase activity was detected in captive (n = 24, stressed) and wild (n = 25, healthy) crayfish with captive crayfish showing lower levels of PO possibly indicating immunosuppression. The proven protocol is the first of its kind to propose a standardized methodology for the detection and quantification of PO activity in Murray crayfish hemolymph as a means of determining stress.

Immune toxicity of phenanthrene and its combined effects of white spot syndrome virus on the survival of kuruma shrimp (Penaeus Japonicus)

Shrimp inhabiting coasts that are frequented by humans are exposed to various pollutants. Additionally, viral infections that cause serious damage to shrimp populations have been observed in these environments. The present study sought to evaluate the immunotoxic effects of phenanthrene (Phe), a pollutant detected in coastal environments, on kuruma shrimp (Penaeus japonicus). We further examined the survival of shrimp following combined exposure to Phe (30 or 300 μg/L) and white spot syndrome virus (WSSV). Results show that exposure to Phe for seven days decreased immune system-related parameters, including total hemocyte count and phenoloxidase activity in hemolymph (p < 0.05). However, these effects were not detected after three days of exposure. Moreover, a combined exposure assay revealed that shrimp mortality increased following exposure to 300 μg/L Phe and infection with WSSV. The number of WSSV gene copies was also observed to increase in these co-exposed shrimp. Taken together, these results indicate that long-term Phe exposure impairs the immune system of P. japonicus, resulting in fatal proliferation of WSSV. Hence, considering that combined exposure to Phe and WSSV leads to increased mortality of shrimp, it is imperative that the detrimental effects elicited by multiple stresses be considered, and controlled, in areas inhabited by kuruma shrimp.

A toxin complex protein from Photorhabdus akhurstii conferred oral insecticidal activity against Galleria mellonella by targeting the midgut epithelium

The nematode-bacterium pair Heterorhabditis indica-Photorhabdus akhurstii is a malleable model system to investigate mutualistic relations. A number of toxins produced by P. akhurstii allow the bacterium to kill the insect host. However, a few of these heterologously expressed toxins are orally active against different insects which possibly caused neglected attention to Photorhabdus toxins compared to Bt (Bacillus thuringiensis). In the current study, a functional subunit of orally active toxin complex (Tc) protein, TcaB (63 kDa), isolated from two strains of P. akhurstii namely IARI-SGHR2 and IARI-SGMS1, was tested for biological activity against Galleria mellonella. A force feeding-based administration of the toxin translated into LD50 values of 45.63–58.90 ng/g which was even lower compared to injection LD50 values (51.48–64.30 ng/g) at 48 h after inoculation. An oral uptake of 500 ng toxin caused extensive gut damage in G. mellonella during 6–24 h incubation period coupled with a gradual disruption of gut integrity leading to escape of TcaB into the hemocoel. This finding was supported by the cytotoxic and immune-stimulatory effect of TcaB in the insect hemocoel at 6–24 h after force feeding. The circulatory hemocyte numbers and cell viability was markedly reduced to 0.66−0.68 × 106 ml−1 and 49–52 %, respectively, in TcaB force fed insect at 24 h, compared to control (2.55 × 106 ml−1; 100 %). The hemolymph phenoloxidase (PO) activity was elevated by 10.2-fold in force fed larvae than control at 24 h. An in silico docking study revealed that TcaB putatively interacts with a number of G. mellonella receptor proteins in order to become a gut-active toxin. Present research reinforces the potential of gut-active Photorhabdus toxins for their inclusion in sustainable insect management tactics and strengthens the existing Bt-dominated management repository.

Innate immune system function following systemic RNA-interference of the Fragile X Mental Retardation 1 gene in the cricket Acheta domesticus

Fragile X syndrome (FXS), caused by a mutation in the Fragile X Mental Retardation 1 (FMR1) gene, is a common form of inherited mental retardation. Mutation of the gene leads to a loss of the gene product Fragile X Mental Retardation Protein (FMRP). While a loss of FMRP has been primarily associated with neural and cognitive deficits, it has also been reported to lead to immune system dysfunction in both humans and flies. We used the Acheta domesticus transcriptome to identify a highly conserved cricket ortholog of FMR1 (adfmr1). We cloned a partial cDNA of adfmr1, used systemic RNA interference (RNAi) to knockdown adfmr1 expression, and examined the impact of this knockdown (KD) on the cellular and humoral responses of the insect innate immune system. Following RNAi, both male and female crickets exhibited an increase in the number of circulating hemocytes, a decrease in total hemolymph phenoloxidase (PO) activity, and an increase in fat body lysozyme expression. Despite similar changes in these immune parameters in both sexes, male and female crickets responded differently to an immune challenge. Most KD males failed to survive an intra-abdominal injection of bacterial lipopolysaccharide, while KD females were just as likely as control females to survive this challenge. Our results support that decreased fmr1 expression can alter the cellular and humoral defenses of the insect innate immune system, and may lead to a decrease in male, but not female, immunocompetence.

JNK pathway plays a key role in the immune system of the pea aphid and is regulated by microRNA-184.

Different from holometabolous insects, the hemipteran species such as pea aphid Acyrthosiphon pisum exhibit reduced immune responses with the absence of the genes coding for antimicrobial peptide (AMP), immune deficiency (IMD), peptidoglycan recognition proteins (PGRPs), and other immune-related molecules. Prior studies have proved that phenoloxidase (PO)-mediated melanization, hemocyte-mediated phagocytosis, and reactive oxygen species (ROS) participate in pea aphid defense against bacterial infection. Also, the conserved signaling, Jun N-terminal kinase (JNK) pathway, has been suggested to be involved in pea aphid immune defense. However, the precise role of the JNK signaling, its interplay with other immune responses and its regulation in pea aphid are largely unknown. In this study, using in vitro biochemical assays and in vivo bioassays, we demonstrated that the JNK pathway regulated hemolymph PO activity, hydrogen peroxide concentration and hemocyte phagocytosis in bacteria infected pea aphids, suggesting that the JNK pathway plays a central role in regulating immune responses in pea aphid. We further revealed the JNK pathway is regulated by microRNA-184 in response to bacterial infection. It is possible that in common the JNK pathway plays a key role in immune system of hemipteran insects and microRNA-184 regulates the JNK pathway in animals.

Host permissiveness to baculovirus influences time-dependent immune responses and fitness costs.

Insects possess specific immune responses to protect themselves from different types of pathogens. Activation of immune cascades can inflict significant developmental costs on the surviving host. To characterize infection kinetics in a surviving host that experiences baculovirus inoculation, it is crucial to determine the timing of immune responses. Here, we investigated time-dependent immune responses and developmental costs elicited by inoculations from each of two wild-type baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa zea single nucleopolyhedrovirus (HzSNPV), in their common host H. zea. As H. zea is a semi-permissive host of AcMNPV and fully permissive to HzSNPV, we hypothesized there are differential immune responses and fitness costs associated with resisting infection by each virus species. Newly molted 4th-instar larvae that were inoculated with a low dose (LD15 ) of either virus showed significantly higher hemolymph FAD-glucose dehydrogenase (GLD) activities compared to the corresponding control larvae. Hemolymph phenoloxidase (PO) activity, protein concentration and total hemocyte numbers were not increased, but instead were lower than in control larvae at some time points post-inoculation. Larvae that survived either virus inoculation exhibited reduced pupal weight; survivors inoculated with AcMNPV grew slower than the control larvae, while survivors of HzSNPV pupated earlier than control larvae. Our results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.

Use of a methanolic olive leaf extract (Olea europaea) against white spot virus syndrome in Penaeus vannamei: Comparing the biochemical, hematological and immunological changes

The main goal of this study was to investigate the biochemical, hematological, and immunological impacts of olive (Olea europaea) leaf extract (OLE) in Penaeus vannamei experimentally infected by white spot syndrome virus (WSSV). A total of 160 juvenile whiteleg shrimp (11.1 ± 0.2 g), obtained from a private sector in Hormozgan Province (Iran), were kept for two weeks to acclimatize to conditions in the facility aquarium. The health status of the shrimp was assured by random sampling before experiment action; PCR testing confirmed the absence of common shrimp diseases. Three groups, each consisting of 20 shrimp fed with three different doses of OLE (50, 100, 200 mg/kg of the shrimp biomass), and a positive control group (not feeding with OLE), were all orally subjected to a virus challenge two weeks later. For comparison, three negative control groups with 20 shrimp were fed three different doses of OLE, and another group was fed with a diet lacking OLE. The PCR test confirmed the presence of the WSS virus in the treatment and positive control groups, while the negative control groups did not show any infections. The hemolymph of shrimp was taken on days 1, 8, and 22 from all groups after challenge for evaluation of hemolymph total protein, glucose, oxyhemocyanin (OxH), superoxide dismutase (SOD), phenoloxidase (PO) activity, coagulation time, and total hemocyte count (THC). Results showed that shrimp survival significantly increased up to 65% when fed with high doses of OLE for seven days. The maximum hemolymph total protein and glucose levels significantly belonged to the treatment that was challenged by WSSV without receiving OLE. The present findings indicate WSS infection caused a significant decrease in OxH level compared to that of the control groups. Therapeutic feeding with OLE could significantly decrease the SOD and PO activities, and the hemolymph clotting time. We did not see no remarckable effects of OLE on the total hemocyte count. In conclusion, the various indices studied in this research showed that the olive leaf extract would be useful in the control and prevention of white spot syndrome.

Aspergillus niger α-1,3-glucan acts as a virulence factor by inhibiting the insect phenoloxidase system

Phenoloxidase (PO) is a key enzyme in the melanization process involved in elimination of pathogens in insects. The PO system is rapidly activated in response to pathogen recognition. Inhibition of PO activity can be a way to avoid immune response and increase infection effectiveness. In this study, the effects of inoculation of Galleria mellonella larvae with Aspergillus niger α-1,3-glucan and conidia on PO activity in hemolymph are analyzed in comparison with the effects of β-1,3/1,6-glucan inoculation. Our results indicate that α-1,3-glucan, a fungal cell wall polysaccharide, can play a role of a virulence factor involved in inhibition of the insect PO system.