Phenoloxidase Levels Research Articles

Are immune responses gender-related in Carabus lefebvrei (Coleoptera: Carabidae)?

The “live hard, die young” theory predicts the evolution of gender differences in immunocompetence, with males having a weaker immune system than females. To test thishypothesis in Carabus lefebvrei, total and basal phenoloxidase (PO) activities and lysozyme-like enzyme activity were compared among males and females of different reproductive status. The sexual dimorphism occurred only in reproductively active adults and for total and basal PO levels, while no significant differences were recorded between sexes in virgin adults. Differences were not recorded for lytic activity between sexes. Basal PO and lytic activities decreased in both males and females after mating, while the total PO value increased in males and decreased in females. Thus, resources seem to be invested to increase the humoral response in pre-reproductive phase forming a barrier against pathogens and preserving the fecundity and longevity of both sexes. Males preserve their survivorship in reproductive phase by increasing enzymatic levels in hemolymph to avoid fitness reduction due to the increased exposure to pathogen as result of mating. Females shift resources from PO and lytic activity to other physiological systems involved in reproduction in order to maximize their fitness.

Phenoloxidase activity in the infraorder Isoptera: unraveling life-history correlates of immune investment.

Within the area of ecological immunology, the quantification of phenoloxidase (PO) activity has been used as a proxy for estimating immune investment. Because termites have unique life-history traits and significant inter-specific differences exist regarding their nesting and foraging habits, comparative studies on PO activity can shed light on the general principles influencing immune investment against the backdrop of sociality, reproductive potential, and gender. We quantified PO activity across four termite species ranging from the phylogenetically basal to the most derived, each with their particular nesting/foraging strategies. Our data indicate that PO activity varies across species, with soil-dwelling termites exhibiting significantly higher PO levels than the above-ground wood nester species which in turn have higher PO levels than arboreal species. Moreover, our comparative approach suggests that pathogenic risks can override reproductive potential as a more important driver of immune investment. No gender-based differences in PO activities were recorded. Although termite PO activity levels vary in accordance with a priori predictions made from life-history theory, our data indicate that nesting and foraging strategies (and their resulting pathogenic pressures) can supersede reproductive potential and other life-history traits in influencing investment in PO. Termites, within the eusocial insects, provide a unique perspective for inferring how different ecological pressures may have influenced immune function in general and their levels of PO activity, in particular.

Aging in personal and social immunity: do immune traits senesce at the same rate?

How much should an individual invest in immunity as it grows older? Immunity is costly and its value is likely to change across an organism's lifespan. A limited number of studies have focused on how personal immune investment changes with age in insects, but we do not know how social immunity, immune responses that protect kin, changes across lifespan, or how resources are divided between these two arms of the immune response. In this study, both personal and social immune functions are considered in the burying beetle, Nicrophorus vespilloides. We show that personal immune function declines (phenoloxidase levels) or is maintained (defensin expression) across lifespan in nonbreeding beetles but is maintained (phenoloxidase levels) or even upregulated (defensin expression) in breeding individuals. In contrast, social immunity increases in breeding burying beetles up to middle age, before decreasing in old age. Social immunity is not affected by a wounding challenge across lifespan, whereas personal immunity, through PO, is upregulated following wounding to a similar extent across lifespan. Personal immune function may be prioritized in younger individuals in order to ensure survival until reproductive maturity. If not breeding, this may then drop off in later life as state declines. As burying beetles are ephemeral breeders, breeding opportunities in later life may be rare. When allowed to breed, beetles may therefore invest heavily in "staying alive" in order to complete what could potentially be their final reproductive opportunity. As parental care is important for the survival and growth of offspring in this genus, staying alive to provide care behaviors will clearly have fitness payoffs. This study shows that all immune traits do not senesce at the same rate. In fact, the patterns observed depend upon the immune traits measured and the breeding status of the individual.

Temporal Variation in Immune Components of the White Grub Phyllophaga polyphylla (Bates) (Coleoptera: Melolonthidae).

Ecological immunology assumes that the host immune efficiency is correlated with its survival after pathogen challenge. To test this hypothesis, we challenged Phyllophaga polyphylla (Bates) larvae with the naturally occurring fungus Metarhizium pingshaense on two consecutive years (2011 and 2012). In each year, we injected the blastospores of M. pingshaense and then used levels of prophenoloxidase (proPO), phenoloxidase (PO) and total haemolymph serum protein as indicators of immune efficiency. Larvae were injected with (1) phosphate buffered saline (PBS) + Tween and viable blastospores of M. pingshaense, (2) PBS + Tween and non-viable blastospores of M. pingshaense, (3) PBS + Tween, or (4) non-manipulated. Overall, levels of PO, proPO and total haemolymph serum protein in larvae after 12h were similar amongst treatments within each year of collection. However, larvae collected in 2011 showed higher PO and proPO activity but lower total haemolymph serum protein compared with larvae collected in 2012. A survival study injecting viable blastospores showed that larvae collected in both years died within 48h; however, when non-viable blastospores were injected, which were still toxic to larvae, mortality was greater in larvae collected in 2011 compared with larvae collected in 2012. Altogether, these results indicate that PO, proPO and total haemolymph serum protein do not predict immune strength of P. polyphylla against blastospores of M. pingshaense, but higher values of PO and proPO were correlated with higher survival rates against non-infective but toxic agents. The possible role of some abiotic factors over the differences observed for immune components of P. polyphylla in different years of collection is discussed.

Differential Expression of Immune-Related Genes in Larval and Adult Apis mellifera Honeybees Challenged with Korean Sacbrood Virus

Recently, the Korean sacbrood virus (KSBV) caused great loss of honeybee colonies in Korean Apis mellifera. Three antimicrobial peptide genes (AMPs) (abaecin (ABA), defensin (DEF) and hymenoptaecin (HYM)) and two immune-related enzyme genes (phenol-oxidase (PO) and lysozyme (LYS)) were investigated using qRT-PCR as a measure of the defense reactions of honeybee larvae and adults challenged with KSBV in vitro. Transcript levels of three AMPs were upregulated in adults exposed to KSBV, compared to control adults. ABA and DEF transcripts were 22.7 and 21.9 fold higher (P<0.05), respectively, after 24h exposure while HYM transcript did not change significantly. At this time point, PO and LYS mRNA levels were 18.08 and 16.03 fold higher, respectively, in KSBVinfected adult bees compared to control adults (P<0.05). By contrast, the larvae infected with KSBV had downregulated expression of AMPs and slight up-regulation of PO and LYS compared to the controls. These results suggest that the differential expression of the genes studied could indicate their involvement in defense processes against KSBV in adult and larva bees.

Oxidative damage of hepatopancreas induced by pollution depresses humoral immunity response in the freshwater crayfish Procambarus clarkii

Previous studies provide evidences for the possible oxidative damage of toxic environmental pollutants to tissue protein in fish and amphibian, but little information is available about their effects on immunity response in crustacean. In the present study, we evaluated the relationship between oxidative damage and immune response induced by both typical pollutants (viz. copper and beta-cypermethrin), by exposing the freshwater Procambarus clarkii to sub-lethal concentrations (1/40, 1/20, 1/10 and 1/5 of the 96 h LC50) up to 96 h. Five biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and protein carbonyl in hepatopancreas, and two immune factors, i.e. phenoloxidase (PO) and hemocyanin in haemolymph were determined. The results indicated that there was a significant increase (P < 0.05) in the contents of ROS, MDA and protein carbonyl accompanied by markedly decreased (P < 0.05) PO and hemocyanin levels in a dose and time dependent manner. The significant and positive correlation (P < 0.01) between protein carbonyls induction and MDA formation was observed in crayfish hepatopancreas at 96 h. The production of these protein carbonyls could significantly depress (P < 0.01) the levels of phenoloxidase and hemocyanin in hemolymph. Higher contents of ROS enhanced the risk of lipid peroxidation, protein carbonylation and immunosuppression of crayfish, and hepatopancreas might play an important role in immune system of crustaceans. Protein oxidation may be one of the main mechanisms for pollution-induced immunotoxicity in P. clarkii.

Immune response and disease resistance of shrimp fed biofloc grown on different carbon sources

The objective of this study was to document the immunological effects of growing shrimp in biofloc systems. The experiment consisted of four types of biofloc systems in which bioflocs were produced by daily supplementation of four different carbon sources, i.e. molasses, tapioca, tapioca-by-product, and rice bran, at an estimated C/N ratio of 15 and a control system without any organic carbon addition. Each biofloc system was stocked with Pacific white shrimp (Litopenaeus vannamei) juveniles that were reared for 49 days. The use of tapioca-by-product resulted in a higher survival (93%) of the shrimp as compared to the other carbon sources and the control. The highest yield and protein assimilation was observed when tapioca was used as the carbon source. After 49 days, phenoloxidase (PO) activity of the shrimp grown in all biofloc systems was higher than that of the shrimp from the control system. Following a challenge test by injection with infectious myonecrosis virus (IMNV), the levels of PO and respiratory burst (RB) activity in the shrimp of all biofloc treatments were higher than that of the challenged shrimp from the control treatment. An increased immunity was also suggested by the survival of the challenged shrimp from the experimental biofloc groups that was significantly higher as compared to the challenged shrimp from the control treatment, regardless of the organic carbon source used to grow the bioflocs. Overall, this study demonstrated that the application of biofloc technology may contribute to the robustness of cultured shrimp by immunostimulation and that this effect is independent of the type of carbon source used to grow the flocs.

Is Sexual Dimorphism in the Immune Response of Gryllodes sigillatus Related to the Quality of Diet?

Whereas some authors have proposed that sexual dimorphism in the immune response is fixed, others pose that it is dynamic and depends on diet. The aim of the present study was to explore the second hypothesis. Immunocompetence differences between females and males can be linked to resource availability. We tested this idea by providing a low or high quality diet to two groups of Gryllodes sigilatus during their developmental period. Then, at the adult phase half of each group was challenged with LPS from Serratia marcescens. The size, phenol oxidase (PO), and lytic activity were compared between groups according to diet, sex, and immune challenge. Results show that diet quality favor size in both sexes. However, the overall immune response did not seem to be significantly different based on diet, but instead on sex. Females showed greater phenol oxidase levels than males, but the opposite was found with lytic activity. Perhaps in G. sigillatus the differences in the pathogens commonly confronted by each sex in the distinct habitat of each could explain the differences on PO and lytic activity.

Bioactivity of &lt;I&gt;Piper hispidinervum&lt;/I&gt; (Piperales: Piperaceae) and &lt;I&gt;Syzygium aromaticum&lt;/I&gt; (Myrtales: Myrtaceae) Oils, With or Without Formulated Bta on the Biology and Immunology of &lt;I&gt;Spodoptera frugiperda&lt;/I&gt; (Lepidoptera: Noctuidae)

The combination of essential oils and Bacillus thuringiensis Berliner may represent an interesting control strategy. Thus, the study tested the following hypothesis: the combination of long pepper oil (Piper hispidinervum L.) and clove (Syzygium aromaticum L.) oils in two concentrations with Xentari WG (Bta) yields a more effective control of Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) affecting biological and reproductive parameters and leading to changes in the levels of phenoloxidase and nitric oxide in the hemolymph of the pest. The results demonstrate that only long pepper oil, at the highest concentration with Xentari WG (Bta), promotes reduced larval survival. However, both oils with or without the insecticide interfere in the biology and humoral immunity of S.frugiperda. All treatments caused a decrease in the amount of eggs, except for the clove oil at both concentrations without Bta. Therefore, the use of these oils is a promising alternative for the integrated management of S. frugiperda; however, its association with Bta demonstrated no significant increase in their efficiency.

Comparing Natural Parasitism and Resistance with Proxies of Host Immune Response in Lestid Damselflies

Commonly used proxies for measuring immune responses in invertebrates include the amount of melanin deposited on nylon inserts and assays of activity of the enzyme phenoloxidase (PO) in the haemolymph. We used these proxies to estimate immunity in unpar- asitized individuals from four Lestid damselfly species from popula- tions with different levels of water mite parasitism. Levels of para- sitism and resistance by hosts were population level estimates from published papers. These parasitism levels were not correlated posi- tively with immune response measured by proxies in the current study. The species with the strongest melanization response to the inserts and the highest PO levels was the one that currently experienced no mite parasitism. The species with the weakest response to the inserts and the lowest PO levels had low current levels of parasitism. The two species that are heavily parasitized had an intermediate response. Natural resistance levels were also not correlated with the response measured by proxies, but the species with a strong response had high levels of resistance in the past. This finding is supported by earlier work done with Lepidoptera in which the most well-defended species currently experience no natural parasitism.

Acorn Consumption Improves the Immune Response of the Dung Beetle Thorectes lusitanicus

Thorectes lusitanicus, a typically coprophagous species is also actively attracted to oak acorns, consuming, burying them, and conferring ecophysiological and reproductive advantages to both the beetle and the tree. In this study, we explored the possible relation between diet shift and the health status of T. lusitanicus using a generalist entomopathogenic fungus (Metarhizium anisopliae) as a natural pathogen. To measure the health condition and immune response of beetles, we analysed the protein content in the haemolymph, prophenoloxidase (proPO) content, phenoloxidase (PO) activity and mortality of beetles with diets based on either acorns or cow dung. Protein content, proPO levels and PO levels in the haemolymph of T. lusitanicus were found to be dependent on the type of diet. Furthermore, the beetles fed with acorns developed a more effective proPO-PO system than the beetles fed with cow dung. Furthermore, a significant decrease in mortality was observed when infected individuals were submitted to an acorn-based diet. In addition to enhancing an understanding of the relevance of dietary change to the evolutionary biology of dung beetles, these results provide a more general understanding of the ecophysiological implications of differential dietary selection in the context of fitness.

Density-dependent prophylaxis in crowded Beet Webworm, Loxostege sticticalis (Lepidoptera: Pyralidae) larvae to a parasitoid and a fungal pathogen

Transmission of parasites and pathogens is generally positively density-dependent: as an insect population's density increases, the risk of an individual becoming attacked or infected also increases. In some insect species, individuals experiencing crowded conditions are more resistant to natural enemies than those experiencing low density conditions, and they are predicted to divert resources to increase resistance. This phenomenon is called density-dependent prophylaxis. Here, possible expression of prophylaxis in fifth-instar larvae of Beet Webworm, Loxostege sticticalis, to biocontrol agents was investigated under rearing densities of 1, 10, and 30 larvae per jar (650 mL). Larvae reared at the moderate density and those reared in isolation displayed the greatest and lowest resistance, respectively, to an entomopathogenic fungus and a parasitoid. Moreover, larvae from the moderate density treatment exhibited elevated phenoloxidase, total haemocyte count and antibacterial activity in the haemolymph, whereas phenoloxidase levels in the midgut were not affected. The results suggest that larval rearing density significantly affects the immune system, and they provide evidence for density-dependent prophylaxis of larval L. sticticalis against its biocontrol agents. These results have implications for understanding the population dynamics and biocontrol of beet webworm.

Costs of immunity in insects: an induced immune response increases metabolic rate and decreases antimicrobial activity

Summary 1. Life-history theory predicts that immune responses have evolved in the context of costs and benefits. However, our understanding of the costs of mounting an immune response is limited. 2. Using four species of insects, we tested for metabolic costs of immunity by inducing a short-term immune response and/or wounding and measuring CO2 production. Inducing an encapsulation response and/or wounding raised resting metabolic rate by up to 28% with a strong positive correlation between individual encapsulation response and metabolic rate in Tenebrio molitor, Acheta domesticus, Cotinis nitida and Periplaneta americana. Interestingly, we found that haemolymph removal increased metabolic activity relative to only wounding, suggesting a cost to sampling haemolymph. 3. We also tested for how mounting an encapsulation response and/or wounding would affect other immune components. We found that inducing an encapsulation response led to increased levels of phenoloxidase and decreased levels of lysozyme, an antimicrobial protein. 4. Our results support the growing evidence that immune responses entail specific energetic and corresponding physiological costs.

Dietary administration of zingerone to enhance growth, non-specific immune response, and resistance to Vibrio alginolyticus in Pacific white shrimp ( Litopenaeus vannamei) juveniles

Zingerone, one of the active components of ginger, is a phenolic alkanone with antioxidant and anti-inflammatory properties. The effects of zingerone supplementation on the growth, immunity, and disease resistance of Pacific white shrimp ( Litopenaeus vannamei) juveniles were studied. Four experimental diets, including a control diet (without zingerone enrichment) and 1, 2.5, and 5 mg zingerone (kg diet) −1 were used. After 56 days of culture, shrimp fed diets supplemented with 1, 2.5, and 5 mg zingerone (kg diet) −1 had significantly greater weight gain and feed efficiency than the controls. Furthermore, after 56 days of culture, shrimp fed all doses of the zingerone diet had higher survival rates compared to the controls after 24–72 h of challenge by the pathogen, Vibrio alginolyticus. Significantly increased phenoloxidase levels were found in shrimp fed the zingerone diets at all doses, and respiratory bursts, lysozyme and phagocytic activities of shrimp fed 2.5 and 5 mg zingerone (kg diet) −1 also significantly increased. Neither the total hemocyte count nor superoxide dismutase activity of the experimental and control groups revealed significant differences at any dose. The results indicate that zingerone can be recommended as a supplement to shrimp feed to increase growth, immunity, and disease resistance against the pathogen, V. alginolyticus. Use of zingerone as appetizer and immunostimulant in shrimp is promising.

Species-specific responsiveness of four enzymes to endosulfan and predation risk questions their usefulness as general biomarkers

General biochemical biomarkers are widely used in current ecotoxicology and may function as early warning signals. We have, however, poor knowledge on how ecologically similar species differ in their biomarker responsiveness and how predation risk may affect these biomarkers, potentially in an interactive way with pesticides. We evaluated this by exposing four corixid water bug species to combinations of endosulfan and predation risk and quantifying the activity of four general enzymatic biomarkers: acetylcholinesterase (AChE), phenoloxidase (PO), catalase (CAT) and superoxidedismutase (SOD). AChE activity was inhibited at an endosulfan concentration of 2μgl(-1) and this did not differ significantly among species. Predation risk inhibited AChE activity with the same magnitude as endosulfan in one species, S. striata. Reduction in the investment of immune function following pesticide exposure, as measured by the activity of PO, was only observed in C. coleoptrata at 8μgl(-1) while we observed an increase of PO levels in S. striata. Overall, PO was suppressed under predation risk at 8μgl(-1) endosulfan. For SOD we observed a pesticide-induced increase across all species under predation risk, while for CAT the pesticide-induced increase was only present without predation risk. These results indicate that even within this group of ecologically similar and closely related species opposing biomarker responses may exist, as observed for PO. Effects of predation risk on all four enzymes, at a similar magnitude as the pesticide effects, further question their usefulness as general biomarkers.

Genetic diversity, parasite prevalence and immunity in wild bumblebees.

Inbreeding and a consequent loss of genetic diversity threaten small, isolated populations. One mechanism by which genetically impoverished populations may become extinct is through decreased immunocompetence and higher susceptibility to parasites. Here, we investigate the relationship between immunity and inbreeding in bumblebees, using Hebridean island populations of Bombus muscorum. We sampled nine populations and recorded parasite prevalence and measured two aspects of immunity: the encapsulation response and levels of phenoloxidase (PO). We found that prevalence of the gut parasite Crithidia bombi was higher in populations with lower genetic diversity. Neither measure of immune activity was correlated with genetic diversity. However, levels of PO declined with age and were also negatively correlated with parasite abundance. Our results suggest that as insect populations lose heterozygosity, the impact of parasitism will increase, pushing threatened populations closer to extinction.

Phenoloxidase but not lytic activity reflects resistance against Pasteuria ramosa in Daphnia magna

The field of ecological immunology strongly relies on indicators of immunocompetence. Two major indicators in invertebrates, the activity of phenoloxidase (PO) and lytic activity have recently been questioned in studies showing that, across a natural range of baseline levels, these indicators did not predict resistance against a manipulated challenge with natural parasites. We confirmed this finding by showing that baseline levels of PO and lytic activity in the host Daphnia magna were not related to spore load of the parasite Pasteuria ramosa. Yet, PO levels in infected hosts did predict spore load, indicating PO activity can be useful as an indicator of immunocompetence in this model parasite-host system.

The effects of age and social interactions on innate immunity in a leaf-cutting ant

Both developmental and environmental factors shape investment in costly immune defences. Social insect workers have different selection pressures on their innate immune system compared to non-social insects because workers do not reproduce and their longevity affects the fitness of relatives. Furthermore, hygienic behavioural defences found in social insects can result in increased survival after fungal infection, although it is not known if there is modulation in physiological immune defence associated with group living vs. solitary living. Here we investigated whether physiological immune defence is affected by both age and the short-term presence or absence of nestmates in the leaf-cutting ant Acromyrmex octospinosus. We predicted that older ants would show immune senescence and that group living may result in prophylactic differences in immune defence compared to solitarily kept ants. We kept old and young workers alone or in nestmate groups for 48 h and assayed a key innate immune system enzyme, expressing phenoloxidase (PO) and its stored precursor (proPO), a defence that acts immediately, i.e. it is constitutive. Short-term solitary living did not affect PO or proPO levels relative to group living controls and we found no evidence for immunosenescence in proPO. However, we found a significant increase in active PO in older workers, which is consistent with two non-mutually exclusive explanations: it could be an adaptive response or indicative of immunosenescence. Our results suggest that future studies of immunosenescence should consider both active immune effectors in the body, such as PO, and the stored potential to express immune defences, such as proPO.

Intromission Induces and Insemination Reduces Female Immune Response in the Medfly

Sexual reproduction exposes females to immune threats. The immune response of females to these threats may conflict with male reproductive interests. We examine the hypothesis that in the Mediterranean fruit fly, Ceratitis capitata, female immune activity during and after copulation reflects this conflict, and its resolution. By comparing the temporal changes in immune status of females paired with normal males to virgin females of the same age, or to those paired with surgically altered males (that perform normal courtship without intromission), we determined the specific effects of courtship, intromission and insemination on female immune response. At the initiation of mating, levels of the immune enzyme Phenoloxidase (PO) were higher in mated females compared to virgin females. Females who copulated with normal males, compared to females paired with males lacking external genitalia, had significantly higher levels of PO. However, 2 h after the beginning of mating PO levels decreased significantly, and increased again 24 h later. These changes may be interpreted as a sequential resolution of the ongoing conflict of interest between males and females, with the advantage shifting from one sex to another.

Effects of oral recombinant VP28 expressed in silkworm (Bombyx mori) pupa on immune response and disease resistance of Procambarus clarkii

The envelope protein VP28 of white spot syndrome virus (WSSV) was overexpressed in the silkworm Bombyx mori, which was achieved by using a baculovirus (HyNPV) expression system and by making silkworm pupa as an alternative host, and then it was directly supplemented in diet at a dose of 20 g kg−1 without purification. During a 30 day feeding period, the levels of phenoloxidase (PO) and superoxide dismutase (SOD) in the haemolymph of the tested Procambarus clarkii increased greatly (P < 0.05) when compared to the control crayfish fed with wild-type HyNPV baculovirus-infected silkworms or normal silkworms. Compared with two controls, the crayfish which had been infected for 20 days showed a significantly lower (P < 0.05) mean cumulative mortality (15.6%), which respectively, resulted in relative percent survivals (RPS) of 83.7 and 84.4%. The efficacy to inhibition of viral infection was further studied by in situ hybridization with a WSSV-specific DNA probe. The high levels of PO and SOD might be important for developing resistance against WSSV in these crayfish.