Phenoloxidase Activity Research Articles

Combined Effects of Temperature and Salinity on Antioxidants in the Immune System of the Pearl Oyster Pinctada fucata

A water environment can impact many physiological processes of aquatic animals. The antioxidant response of immune system of the pearl oyster to temperature and salinity is of great significance to health. This study analyzed the physiological changes and immune responses under different temperature and salinity levels (temperature: 20, 25, and 30 °C; salinity: 23, 28, and 33‰) in the short term (7 d) in the hepatopancreas and other tissues of 405 pearl oysters. The combined effects of temperature and salinity on antioxidants in the immune system of the pearl oyster were evaluated via response surface methodology and Box–Benhnken design (BBD) under laboratory conditions. The secondary effects of salinity on the activities of glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) were significant, and CAT and SOD showed an inverted U-shaped trend with the increase in salinity. Temperature significantly impacted GSH-PX, CAT, glutathione (GSH), and SOD in primary and secondary effects, and the maximum values of CAT, GSH, and SOD were observed. The phenoloxidase (POX) and alkaline phosphatase (AKP) activities increased with the increasing temperatures. The interaction of temperature and salinity was significant on CAT and SOD, but was not significant on GSH-PX. The interaction between salinity and temperature on AKP was significant, which was contrary to the results of AKP in the hemolymph. The expression levels of antioxidant genes varied between tissues, and the expression levels of different genes in the same tissue were different. Appropriate immunity and antioxidant index models were established under the combined temperature and salinity conditions. The optimal combination of temperature and salinity was 24.95 °C and 28.11‰, respectively, and the desirability was 0.803. This study provides theoretical reference points for the pearl oyster to respond to temperature and salinity changes and can be used to establish an index model for shellfish aquaculture.

The genotoxic, cytotoxic and growth regulatory effects of plant secondary metabolite β-caryophyllene on polyphagous pest Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Use of secondary metabolites as an alternative to organic pesticides is an eco-friendly and safe strategy in pest management. β-caryophyllene [(1R,4E,9S)-4,11,11-trimethyl-8-methylene bicyclo [7.2.0]undec-4-ene], a natural sesquiterpene is found as an essential oil in many plants like Syzygium aromaticum, Piper nigrum, Cannabis sativa. The present study aims at exploring the insecticidal, genotoxic and cytotoxic potential of β-caryophyllene against common cutworm Spodoptera litura (Fab.), a major polyphagous pest. S. litura larvae were fed on different concentrations (5, 25, 125, 625 and 3125 ppm) of β-caryophyllene. Results revealed delay in larval and pupal period with increase in concentration. Larval mortality increased and adult emergence declined significantly with increase in concentration. Higher concentrations of β-caryophyllene caused pupal and adult deformities. A negative impact of β-caryophyllene was also seen on the nutritional physiology of S. litura. Parameters such as relative growth rate, relative consumption rate, efficiency of conversion of ingested food, efficiency of conversion of digested food and approximate digestibility showed a significant reduction in a dose dependent manner. DNA damage assessed using comet assay revealed significant genotoxic effects at LC30 and LC50 concentrations. There was an increase in tail length, percent tail DNA, tail moment and olive tail moment. Phenol oxidase activity was suppressed at LC50 concentration with respect to control. Total hemocyte count also declined significantly at LC30 and LC50 concentrations as compared to control. β-caryophyllene induced genotoxic and cytotoxic damage affecting the growth and survival of S. litura larvae. Our findings suggest that β-caryophyllene has the potential to be used for the management of insect pests.

Effect of feeding different types of β-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) on growth performance and immunity of banana shrimp (Penaeus merguiensis)

β-glucans are produced by many organisms and could be used as supplementary feed to enhance immunity and growth in some aquatic animals. This study aimed to compare the effectiveness of β-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) as growth promoters and immunity enhancers in banana shrimp (Penaeus merguiensis). Shrimp were divided into 3 groups: the control group was fed without β-glucan; the second and the third group were fed with 2 g kg−1 of β-glucan derived from C. muelleri and T. weissflogii, respectively. Shrimp were fed over a 30-day period to determine growth performance (final weight, weight gain, average daily gain (ADG), and feed conversion ratio (FCR)) at day 15 and day 30, respectively. The immune parameters determined were total hemocyte count (THC), phenoloxidase activity (PO) and immune gene expression. Survival rates were measured after 14 days of the feeding trial and Vibrio parahaemolyticus infection (6, 24, 48 h post infection). There was no significant difference (P > 0.05) for growth stimulation of shrimps between the two types of β-glucans (C. muelleri or T. weissflogii). Notably, shrimps fed with β-glucans had a higher final weight, weight gain, and ADG (P < 0.05) than shrimps fed with the control diet, while FCR of shrimps fed with both β-glucans was lower when compared to the control diet. Immune parameters, THC, PO, and gene expression of anti-lipopolysaccharide factor (ALF) and crustin were significantly higher (P < 0.05) in shrimps fed with β-glucans, especially with β-glucans from C. muelleri than the control group both before and after V. parahaemolyticus infection. Expression of penaeidin 3 and peroxiredoxin genes was significantly higher in shrimps fed with β-glucans after bacterial infection. Histopathology of hepatopancreas revealed an increase in blasenzellen hepatopancreatic epithelial cells (B cells) after 14 days of feeding which remained higher following infection with V. parahaemolyticus. The survival rate of shrimps fed with the diet containing β-glucan derived from either C. muelleri (82.2%) or T. weissflogii (77.8%) after V. parahaemolyticus infection was significantly higher than for the control group (51.1%) (P < 0.05). In conclusion, we propose that feeding banana shrimps with β-glucans derived from marine diatoms either C. muelleri or T. weissflogii at a 2 g kg−1 diet can significantly improve their growth performance and immunity.

The colonisation of saline waters is associated with lowered immune responses in aquatic beetles

Abstract 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.

Impact of aluminum exposure on oxidative stress, intestinal changes and immune responses in red swamp crayfish (Procambarus clarkii)

Aluminum (Al) is an abundant metal that has been classified as a threatening pollutant due to indiscriminate use and anthropogenic activities. This study aimed to evaluate the impacts of Al on crayfish (Procambarus clarkii), including biochemical change, histological alteration, gut microbial community diversification, and immune changes. The bioaccumulation of Al was detected in the hemolymph and intestine of crayfish after Al exposure at different time points. Results showed that Al exposure significantly induced oxidative stress and caused pathohistological changes on intestinal barrier structures in crayfish. It was found that the intestinal microbiota was affected by retained Al and the intestinal community diversity was changed after Al treated in the crayfish. Furthermore, Al exposure affected the immunity in crayfish, by altering the expression of a set of immune-related genes, as well as reducing the phenoloxidase and lysozyme activities. Moreover, Al exposure promoted hemocytes apoptosis and impaired hemophagocytic capacity against Vibro parahamolyticus, resulting in higher mortality of crayfish upon bacterial infection. Taken these results together, we conclude that excessive Al exposure caused adverse effects on multiple biological processes of crayfish and Al pollution is a potential threat to crayfish culture.

Acute thermal stress and endotoxin exposure modulate metabolism and immunity in marine mussels (Perna canaliculus)

Mass mortalities of New Zealand Green-lipped mussels (Perna canaliculus) are thought to be associated with increased water temperatures and immune challenges from opportunistic pathogens. However, the combined effects of acute thermal stress and immune stimulation on mussels are poorly understood. To investigate these responses, adult mussels were exposed to different temperatures (26 °C [thermal stress] vs 15 °C [ambient]) and a bacterial-derived endotoxin injection (with vs without) to mimic a pathogen infection. Various immunological and metabolic parameters were measured over two days via enzyme staining reactions, flow cytometry, and metabolomic profiling. None of the treatments impacted total and differential haemocyte counts, haemocyte viability or production of reactive oxygen species. Acid phosphatase and phenoloxidase activities were detected only within granulocytes (not in hyalinocytes), although their relative expressions also were not affected. Conversely, metabolite profiling exposed impacts of thermal stress and endotoxin exposure at a metabolic level, indicative of physiological changes in energy expenditure and partitioning. At the higher water temperature, free fatty acid and amino acid constituents increased and decreased, respectively, which supports an elevated energy demand and higher metabolic rate due to thermal stress. Ultimately, energy production is being sustained via multiple routes including the glycolysis pathway, TCA cycle, and β-oxidation. Additionally, branched-chain amino acids, the urea cycle and the glutathione pathway were affected by the higher temperature. The metabolic response of mussels exposed to endotoxin exposure resulted in increased metabolite response largely linked to protein and lipid degradation. After 5 days of exposure, survival data confirmed a severe physiological impact of the higher temperature through incidences of mortality. However, the thermal challenge in combination with the specific endotoxin treatment applied did not lead to a synergistic effect on mortality. These findings provide new insights into the relationship between thermal stress and immunity to better understand the immune defence system in mussels.

Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrioparahaemolyticus

Vibrio parahaemolyticus (Vpara) is the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS) in shrimp. Shrimp, like other invertebrates, lack an adaptive immune system and depend solely on innate immunity against invading pathogens. To better understand the defense mechanisms of shrimp to this problematic pathogen, we evaluated the changes in hematology, immunology and biochemical values of the hemolymph from shrimp challenged with V. parahaemolyticus up to 8 days post-challenge. Thirty-six shrimp (12 g) were distributed in 9 tanks (75 L), divided into three groups (non-challenged, challenged with 5 × 102 cfu/shrimp and challenged with 1 × 103 cfu/shrimp) in triplicate. Pacific white shrimp, Litopenaeus vannamei, were administered an inoculum of V. parahaemolyticus under the shell between the 5th and 6th abdominal segment to assess cellular and humoral immune responses. Total hemocyte count (THC) significantly decreased in shrimp challenged with Vpara at 6 h, 12 h and 24 h-post infection. Hemocyte lysate phenoloxidase (PO) activity in Vpara-challenged shrimp at 48 h post challenge was significantly increased compared to that of control shrimp. No significant differences were observed in total plasma protein between plasma from control and Vpara-challenged shrimp. However, shrimp challenged with 5 × 102, and 1 × 103 cfu/shrimp had significantly lower hemocyanin at 6 h and 48 h sampling point, respectively. At 24 h post-challenge, the ≥140 kDa and 70 kDa bands from SDS-PAGE of hemocyanin-concentrated hemolymph lysate samples showed a higher and lower intensity, respectively, in Vpara-challenged group than those of the control group. Plasma from Vpara-challenged shrimp at 6 h and 12 h-post infection significantly suppressed V. parahaemolyticus growth. However, significantly less bacterial growth suppression was observed in plasma of shrimp challenged with higher dose compared to control shrimp at the 192 h post-challenge point. Plasma chemistry parameters did not significantly differ among treatments. The changes observed in hemolymph parameters may be useful indicators of the health status of shrimp.

Effects of Organic Amendments on Phenol Oxidase, Peroxidase, Urease, and Nitrogen Mineralization: A Laboratory Incubation Study

Mitigating climate change and enhancing fertility in agricultural systems require the adoption of more sustainable fertilizer management practices. Applications of recycled organic materials, such as animal and green wastes, can promote soil carbon stabilization via changing extracellular enzyme activities while providing the necessary nitrogen (N) for plant growth. The goals of this study were to quantify the effects of compost type (cow manure, green manures, mixtures of green and cow manure at various proportions, and inorganic fertilizers) on (1) enzyme activity (phenol oxidase, peroxidase, and urease), and (2) mineralized N under laboratory incubation at 30 °C over an eight-week period. The lowest oxidative enzyme activities (phenol oxidase and peroxidase) were found in the soil treated with a mixture of 50% cow manure and 50% green manure (2.45 μmol h−1 g−1) and a mixture of 30% cow manure and 70% green manure (3.21 μmol h−1 g−1) compared to all other fertilizer treatments. The highest phenol oxidase activity was found in soils amended with green manures (3.52 μmol h−1 g−1), while the highest peroxidase activity was found in soils amended with a mixture of 70% cow manure and 30% green manure (5.68 μmol h−1 g−1). No significant differences were found in total net mineralized N content among all organic fertilizer treatments, but these were significantly lower than total net mineralized N in soil treated with inorganic fertilizers. These results indicate similar effects of organic amendments and mixtures in providing plant-available N, but different effects on lignin-degrading enzyme activities, which may lead to differences in soil organic carbon cycling and long-term C storage depending on which organic amendment is utilized.

Comparative study of immunological biomarkers in the carpet shell clams (Ruditapes decussatus) from metal-contaminated sites in the South Lagoon of Tunis (Tunisia)

The South Lagoon of Tunis (Tunisia) is a Mediterranean lagoon adversely affected by industrial contaminants, harbour activity and untreated urban sewage. In this lagoon, the clam Ruditapes decussatus has been widely used as a biomonitor of seawater pollution through measurements of parameters related to oxidative stress and neurotoxicity. However, few studies have considered parameters of the immune system of this species in the South Lagoon of Tunis. Therefore, the aim of the present work was to evaluate several immune-related parameters in the cell-free haemolymph of carpet shell clams sampled during August and February from three polluted sites in the South Lagoon of Tunis (S1, S2 and S3) and one less polluted site as a reference site (RS) in order to identify suitable biomarkers for environmental quality assessments of this ecosystem. Concerning the immune-related parameters, seasonal factors modulated phenoloxidase, lysozyme, protease and esterase activity, with lower values measured for samples collected in August than for samples collected in February. In fact, bactericidal activity against two of the pathogenic bacteria tested and the activity of most immune-related enzymes were reduced in the cell-free haemolymph of clams collected from the most sampling sites in August compared to February one. In addition, values of abiotic parameters (temperature, salinity and pH) and metal (cadmium, copper, iron, lead and zinc) concentrations in the clams’ soft tissues, previously obtained and published by the authors, as well as the values of immune-related parameters were integrated using principal component analyses. Results indicated that the values of all measured immune-related parameters were negatively correlated with the temperature values and the variations most of these parameters highlighted that the chemical industrial area (S3) was the most impacted location within the South Lagoon of Tunis. The present study illustrates that the immune-related parameters measured in carpet shell clam cell-free haemolymph represent suitable biomarkers for environmental quality assessments because they provide effective seasonal and spatial discrimination.

Proteomic analysis of pharate pupal molting fluid from the tobacco hornworm, Manduca sexta

The insect cuticle is a key component of their success, being important for protection, communication, locomotion, and support. Conversely, as an exoskeleton, it also limits the size of the insect and must be periodically molted and a new one synthesized, to permit growth. To achieve this, the insect secretes a solution of chitinases, proteases and other proteins, known collectively as molting fluid, during each molting process to break down and recycle components of the old cuticle. Previous research has focused on the degradative enzymes in molting fluid and offered some characterization of their biochemical properties. However, identification of the specific proteins involved remained to be determined. We have used 2D SDS-PAGE and LC/MS-based proteomic analysis to identify proteins in the molting fluid of the tobacco hornworm, Manduca sexta, undergoing the larval to pupal molt. We categorized these proteins based on their proposed functions including chitin metabolism, proteases, peptidases, and immunity. This analysis complements previous reported work on M. sexta molting fluid and identifies candidate genes for enzymes involved in cuticle remodeling. Proteins classified as having an immune function highlight potential for molting fluid to act as an immune barrier to prevent infections during the cuticle degradation and ecdysis processes. Several proteins known to function in melanin synthesis as an immune response in hemolymph were present in molting fluid. We demonstrated that the bacterium Micrococcus luteus and the entomopathogenic fungus Beauveria bassiana can stimulate activation of phenoloxidase in molting fluid, indicating that the recognition proteins, protease cascade, and prophenoloxidase needed for melanin synthesis are present as a defense against infection during cuticle degradation. This analysis offers insights for proteins that may be important not only for molting in M. sexta but for insects in general.

Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeusvannamei

The codon-optimized anti-lipopolysaccharide factor (ALF) sequence was introduced into pPICZαA vector and transformed into Pichia pastoris GS115. The recombinant ALF yeast supernatant (rALF-mix) was freeze-dried and evaluated as a feed additive for Litopenaeus vannamei. It was found by antibacterial activity test in vitro that the rALF-mix had antibacterial activity under different pH and temperature conditions. The 0, 0.00375%, 0.0075%, 0.015%, 0.03% and 0.06% of rALF-mix were added respectively to make the six experimental diets. After a 10-week feeding trial with shrimps (2.36 ± 0.02 g), it was found that the weight gain rate (WGR) and protein efficiency ratio (PER) of shrimp in the groups with 0.0075%, 0.015% and 0.03% of dietary rALF-mix supplementation were significantly higher than those in the control group (P < 0.05). Dietary rALF-mix supplementation significantly increased the total haemocyte count, respiratory burst, phagocytic activity, total anti-oxidative capacity (T-AOC), phenol oxidase activity, nitric oxide synthase activity, lysozyme (LYZ) activity, serum antibacterial capacity in the hemolymph and the T-AOC, LYZ in the hepatopancreas of shrimps (P < 0.05). The malondialdehyde contents in hemolymph and hepatopancreas were significantly decreased (P < 0.05). Meanwhile, the expression levels of toll, immune deficiency, heat shock protein 70, crustin and lipopolysaccharide-β-glucan binding protein in the gill of shrimps were significantly increased (P < 0.05). After the challenge test, it was showed that dietary rALF-mix supplementation significantly improved the resistance of L. vannamei to Vibrio parahaemolyticus (P < 0.05). In conclusion, the rALF-mix can be used as a functional feed additive to improve the growth, immunity and disease resistance of shrimp. Based on the quadratic regression analysis for WGR, the optimal supplemental level of rALF-mix in diet for shrimp was estimated to be 0.02813%.

The honey bees immune memory

Invertebrates' immune priming or innate immune memory is an analogous response to the vertebrates' adaptive memory. We investigated if honey bees have immune memory. We compared survival and immune response between bees that were: 1) manipulated (Naïve), 2) challenged twice with the same pathogen Escherichia coli (Memory), 3) challenged twice with different pathogens (Staphylococcus aureus versus E. coli, Micrococcus lysodeikticus versus E. coli), or 4) with PBS (the diluent of bacteria) versus E. coli (heterologous challenge; Control). Results indicate better survival in the Memory than the Control group, and the Memory group showed a similar survival than Naïve insects. The Memory group had higher lytic activity but lower prophenoloxidase, phenoloxidase activity, and hemocyte count than the Control and Naïve groups. No differences were found in relative expression of defensin-1. This first demonstration of immune memory opens the questions about its molecular mechanisms and whether, immune memory could be used against natural parasites that affect honey bees, hence, if they could be “vaccinated” against some natural parasites.

Combination of Nigella sativa and Phyllanthus niruri as An Immunostimulant for The Prevention of White Spot Disease in Litopenaeus vannamei

Pacific whiteleg shrimp (Litopenaeus vannamei) is a very important commodity, which accounts for almost 70% of the total world shrimp production. However, this production is still threatened by disease attacks, one of which is caused by white spot disease (WSD). Herbs are widely used as immunostimulants as an effort to prevent diseases. Black cumin (Nigella sativa) and stone breaker (Phyllanthus niruri) have long been known as excellent immunostimulants in human. This study aimed to evaluate the potential of N. sativa and its combination with P. niruri as an immunostimulant in L. vannamei for preventing WSD. Tested shrimp (±3.0 g) were treated by N. sativa (N), P. niruri (P) and the combination of both (1N:1P, 2/3N:1/3P, 3/4N:1/4P, 1/3N:2/3, 3/5N:2/5P, 1/4N: 3/4P, 2/5N:3/5P) through feed for 28 days. They were then infected by white spot syndrome virus (WSSV) and observed for 7 days. The parameters observed included immune response and production performance. N. sativa was able to reduce the mortality of test shrimp infected with WSSV, with an RPS value of 71%. N. sativa can be combined with P. niruri in the right composition (1/2N:1/2P and 2/3N:1/3P), while the other combinations are antagonistic. The administration of the best treatments in this study, relatively did not affect the value of total hemocyte count, phenoloxidase activity, respiratory burst activity, hemolymph clotting time and production performance of tested shrimp.

Circadian rhythms mediate infection risk in Daphnia dentifera.

Biological rhythms mediate important within‐host processes such as metabolism, immunity, and behavior which are often linked to combating disease exposure. For many hosts, exposure to pathogens occurs while feeding. However, the link between feeding rhythms and infection risk is unclear because feeding behavior is tightly coupled with immune and metabolic processes which may decrease susceptibility to infection. Here, we use the Daphnia dentifera–Metschnikowia bicuspidata host–pathogen system to determine how rhythms in feeding rate and immune function mediate infection risk. The host is known to have a nocturnal circadian rhythm in feeding rate, yet we found that they do not exhibit a circadian rhythm in phenoloxidase activity. We found that the time of day when individuals are exposed to pathogens affects the probability of infection with higher infection prevalence at night, indicating that infection risk is driven by a host's circadian rhythm in feeding behavior. These results suggest that the natural circadian rhythm of the host should be considered when addressing epidemiological dynamics.

Cadmium and copper mixture effects on immunological response and susceptibility to Vibrio harveyi in white shrimp Litopenaeus vannamei

Cadmium (Cd2+) and copper (Cu2+) are considered immunotoxic metals and their presence in combination in the aquatic environment may cause effects on shrimp species as Litopenaeus vannamei. Thus, this research evaluates the combined effects of Cd2+ and Cu2+ on shrimp inoculated with Vibrio harveyi bacteria. The experiments were performed at 96-h of exposure to sublethal concentrations of both metals. No mortality was observed in organisms exposed to the sum of Criterion of Continuous Concentration (ΣCCC) in Cd + Cu mixture and those inoculated with V. harveyi. Higher clotting times were recorded in Cd + Cu + V. harveyi treatment at higher metal concentrations. No significant differences (P > 0.05) were recorded in hemocyanin content between shrimp exposed to metals and those experimentally infected. Significantly higher (P < 0.05) total hemocyte count (THC) was recorded at 96 h exposure in the ΣCCC and 10% treatments of Cd + Cu + V. harveyi experiment. Regarding Cd + Cu + V. harveyi bioassay, the highest phenoloxidase (PO) activity was recorded in shrimp inoculated with V. harveyi (0.326 ± 0.031 PO units/mg protein) at 96-h exposure. The lowest PO activity was observed in organisms exposed to Cd + Cu + V. harveyi. Regarding superoxide dismutase (SOD) activity, shrimp exposed to higher metal concentrations at 96 h showed the lowest hemolymph activity (6.03 ± 0.62 SOD units/mL). Protein decrease was observed in organisms exposed to metal mixture. The results showed that L. vannamei could be more susceptible to V. harveyi when exposed to Cd + Cu.

Differential Activity of the Extracellular Phenoloxidases in Different Strains of the Phytopathogenic Fungus, Microdochium nivale.

To cause plant diseases, phytopathogenic fungi use numerous extracellular enzymes, among which, the phenoloxidases (POs) seem underestimated for the pathogens of non-woody plants. Our study aimed to (1) compare extracellular PO activities (lignin peroxidase, Mn peroxidase, laccase, and tyrosinase) in differentially virulent strains (inhabiting winter rye in a single field) of the phytopathogenic species, Microdochium nivale; (2) check whether these activities are responsive to host plant metabolites; and (3) search for correlations between the activities, lignin-decomposing capacity, and virulence. All strains displayed all four enzymatic activities, but their levels and dynamics depended on the particular strain. The activities displayed the hallmarks of co-regulation and responsiveness to the host plant extract. No relationships between the virulence of strains and levels of their extracellular PO activities or lignin-degrading capacity were revealed. We consider that different strains may rely on different POs for plant colonization, and that different POs contribute to the “uniqueness” of the enzymatic cocktails that are delivered into host plant tissues by different virulent strains of M. nivale. Our study supports the hypothesis of the differential behavior of closely related M. nivale strains, and discusses an important role of POs in the interactions of phytopathogens with herbaceous plants.

Effect of amide protoporphyrin derivatives on immune response in Apis mellifera

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = − 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = − 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.

The Photodegradation of Lignin Methoxyl C Promotes Fungal Decomposition of Lignin Aromatic C Measured with 13C-CPMAS NMR.

Solar radiation has been regarded as a driver of litter decomposition in arid and semiarid ecosystems. Photodegradation of litter organic carbon (C) depends on chemical composition and water availability. However, the chemical changes in organic C that respond to solar radiation interacting with water pulses remain unknown. To explain changes in the chemical components of litter organic C exposed to UV-B, UV-A, and photosynthetically active radiation (PAR) mediated by water pulses, we measured the chemistry of marcescent Lindera glauca leaf litter by solid-state 13C cross-polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR) over 494 days of litter decomposition with a microcosm experiment. Abiotic and biotic factors regulated litter decomposition via three pathways: first, photochemical mineralization of lignin methoxyl C rather than aromatic C exposed to UV radiation; second, the biological oxidation and leaching of cellulose O-alkyl C exposed to PAR and UV radiation interacts with water pulses; and third, the photopriming effect of UV radiation on lignin aromatic C rather than cellulose O-alkyl C under the interaction between radiation and water pulses. The robust decomposition index that explained the changes in the mass loss was the ratio of aromatic C to O-alkyl C (AR/OA) under radiation, but the ratio of hydrophobic to hydrophilic C (hydrophobicity), the carbohydrate C to methoxyl C ratio (CC/MC), and the alkyl C to O-alkyl C ratio (A/OA) under radiation were mediated by water pulses. Moreover, the photopriming effect and water availability promoted the potential activities of peroxidase and phenol oxidase associated with lignin degradation secreted by fungi. Our results suggest that direct photodegradation of lignin methoxyl C increases microbial accessibility to lignin aromatic C. Photo-oxidized compounds might be an additional C pool to regulate the stability of the soil C pool derived from plant litter by degrading lignin methoxyl and aromatic C.

Changes in immune responses, gene expression, and life table parameters of Helicoverpa armigera Hübner fed on a diet containing the saponin of tea plant, Camellia sinensis.

Saponins cause mortality in insects by reducing food absorption and movement in the gut, which may be used to exploit the control of insect pests. In the current study, tea seed saponin (TSS) was extracted and then added to the artificial diets of Helicoverpa armigera. Pre-ovipositional period of the TSS-treated individuals increased while longevity and fecundity decreased compared to control. There was a significant reduction of the treated individuals in the life table parameters of TSS-treated Individuals including net reproduction rate (R0 ), intrinsic rate of population increase (r), finite rate of increase (λ), and gross reproduction rate (GRR). Also, we found that saponin suppressed the immune system by reducing the total hemocyte count, immune-related gene expression, and phenoloxidase activity. Our results demonstrated a lower expression of cecropin gene in the treated larvae with TSS while no significant differences were observed in attacin gene. Our results clearly showed that feeding ofH. armigera larvae in the diet containing TSS significantly reduced demographic parameters, forced insects to obtain more time to complete one generation, and caused vulnerabilities against pathogens. These discrepancies alleviated nutrient uptake of the larvae and disrupted their feeding and growth. Hence, a proper formulation with a desirable concentration would be prepared and applied in the fields suffering H. armigera damage to monitor insecticidal efficiency of TSS.

Sperm competition in yellow dung flies: No consistent effect of sperm size.

The male competition for fertilization that results from female multiple mating promotes the evolution of increased sperm numbers and can impact sperm morphology, with theory predicting that longer sperm can at times be advantageous during sperm competition. If so, males with longer sperm should sire more offspring than competitors with shorter sperm. Few studies have directly tested this prediction, and findings are inconsistent. Here we assessed whether longer sperm provide a competitive advantage in the yellow dung fly (Scathophaga stercoraria; Diptera: Scathophagidae). Initially, we let brothers with different temperature-mediated mean sperm lengths compete - thus minimizing confounding effects of genetic background - and found no clear advantage of longer sperm. We then used flies from lines subjected to bidirectional selection on phenoloxidase activity that had shown correlated evolutionary responses in sperm and female spermathecal duct lengths. This experiment also yielded no main effect of sperm size on siring success. Instead, there was a trend for a shorter-sperm advantage, but only when competing in females with longer spermathecal ducts. Our data corroborated many previously reported findings (last-male precedence, effects of copula duration and body size), suggesting our failure to find sperm size effects is not inherently due to our experimental protocols. We conclude that longer sperm are not competitively superior in yellow dung flies under most circumstances, and that, consistent with previous work, in this species competitive fertilization success is primarily determined by the relative numbers of sperm competing.