Dosage and frequency effects of the microalgae Dunaliella sp. on the diet of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

Carotenoid sources in shrimp diets have shown to be effective for improving survival, growth, reproductive capacity, stress resistance, and also for diminishing disease. Dunaliella sp. is known to have high levels of ß-carotenes, which works as pro-vitamin A, enhancing the immune response in shrimp. However, the administration of Dunaliella sp. in shrimp diet needs to be evaluated to determine the appropriate dose and frequency of administration needed to optimize performance in cultured white shrimp. Diets with three different concentrations of Dunaliella sp. flour (1.5, 2 and 3%) were tested, and each one was administered at three different time frequencies: daily, and at 3- and 7-days intervals. Shrimp fed for 20 days were then infected with Vibrio parahaemolyticus (1 × 106 CFU/mL). Hemolymph parameters including protein, glucose, lactate, cholesterol and triglycerides were analyzed to evaluate shrimp stress status. Additionally, L. vannamei innate non-specific immune response was examined by evaluating the activity of prophenoloxidase (proPO), phenoloxidase (PO) and superoxide dismutase (SOD) in hemolymph; shrimp survival was also recorded. Survival after infection with V. parahaemolyticus was higher for shrimp fed with diets consisting of 2% Dunaliella sp. administered every 3 and 7 days. Shrimp fed a diet consisting of 2% or 3% Dunaliella sp. administered every third day showed positive physiological and immune responses to infection. A decrease in lipid oxidation in plasma triglycerides was observed at 48 h post inoculation in shrimp fed at all diets regimes due to Dunaliella sp. antioxidant action. Experimental results suggest the importance of Dunaliella sp. dosage and feeding frequency in L. vannamei diet to improve the survival and immune response.

A novel type of prophenoloxidase from the kuruma prawn Marsupenaeus japonicus contributes to the melanization of plasma in crustaceans.

Melanization is one of the major immune responses in arthropods. Prophenoloxidases (proPOs) catalyze the oxidation of mono- or o-diphenols, a reaction that is the key initial step of melanin formation. Well-characterized proPOs from crustaceans are synthesized in haemocytes and are released into plasma in response to microbial attack. However, PO activity does exist in the plasma of haemolymph without pathogenic infections. Here, we demonstrate that a novel type of proPO contributes to such PO activity in the plasma fraction of haemolymph of crustaceans. The novel enzyme, which was purified from the plasma of the kuruma prawn (Marsupenaeus japonicus), possessed strong and specific monophenol and o-diphenol oxidation activity compared with that of known haemocyte-type proPO. Amino acid sequence analyses indicated that this enzyme was distinct from the known proPO. The cDNA sequence and deduced amino acid sequence of this enzyme has a putative binuclear copper center, and showed approximately 30% and 20% identity with the primary structures of reported proPO and haemocyanin sequences of the kuruma prawn, respectively. Reverse transcription PCR analysis showed that this enzyme was synthesized in the hepatopancreas rather than in haemocytes. Although the primary structure and enzymatic properties of this novel enzyme suggested that it is a phenoloxidase, its biogenesis, tissue distribution, and oligomeric state resemble those of haemocyanin, which belongs to the same protein family (type III copper protein). This novel proPO enzyme may share a role with the already characterized version, itself a major component of the innate immune system in crustaceans.

Identification of the gene encoding pro-phenoloxidase A(3) in the fruitfly, Drosophila melanogaster.

The fruitfly, Drosophila melanogaster, has three proPO genes (DoxA1, CG8193 and CG2952). DoxA1 has been shown to encode proPO A(1), one of the two proPO isoforms (A(1) and A(3)). However, which of CG8193 or CG2952 encodes proPO A(3) has so far remained elusive. In Northern analysis, CG8193 expression was strong during the larval stage, yet expression of CG2952 was not detected at any stage. Immunoblot analyses with specific antibodies detected CG8193 in the larval hemolymph at the mobility of the endogenous proPOA(3), though no signal for CG2952. These results indicate that the expression of CG2952 is very low and that CG8193 is the gene that encodes proPO A3. Processing of A(1)and A(3) isoforms in adult homogenate and activity of recombinant proPOs were also investigated.

Changes in hemocytes of Plutella xylostella after parasitism by Diadegma semiclausum.

We examined the changes of hemocytes in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), due to parasitism by the endoparasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae). Necrosis of prohemocytes in different stages was observed while cell death was absent in the mature hemocytes in the parasitized larvae, which was related to the declined total hemocyte count per microliter (THC). THC in the host hemolymph declined sharply by 12 h post-parasitization and then remained at a low level. When hemocytes of the parasitized larvae were cultured in vitro, encapsulation ability was suppressed coincidently with the inhibited spreading ability; however, such effects were transient. Simultaneously, activation of the prophenoloxidae from the hemocytes was inhibited. Unlike the results of previous studies, the decrease in hemocytes, which was due to the necrosis of the prohemocytes instead of the mature hemocytes in our study, was not responsible for the impaired encapsulation. Our studies suggest that parasitism by D. semiclausum have some effects on hematopoietic regulation and on hemocyte immune reaction of P. xylostella larvae.

Dietary Protein and Carbohydrates Affect Immune Function and Performance in a Specialist Herbivore Insect (Manduca sexta).

Nutrition structures ecology and evolution across all scales of biological organization. It is well known that nutrition can have direct effects on performance and fitness, but indirect effects on physiological systems that mediate biotic interactions have been studied less frequently. Here, we focus on the interaction between nutrition, performance, and the immune system in a specialist herbivorous insect, Manduca sexta. We used a conceptual framework in nutritional ecology (the geometric framework) to examine how changes in diet quality affect aspects of the immune system used for defense against parasitoids. We raised caterpillars throughout their entire larval development on five different experimental diets that varied in protein and carbohydrate content and measured five aspects of the immune system: encapsulation, phenoloxidase activity, prophenoloxidase activity, total hemolymph protein, and hemocyte density. Overall, different parts of the immune function varied in response to interactions between carbohydrates, protein, and intake, but protein reductions had the largest impacts-mostly detrimental. In addition, our data suggest that diet quality mediates the relationship between performance (growth and survival) and immune function, as well as trade-offs among different components of immune function. Our work is the first to examine the interplay between nutrition, performance, and immune function with the geometric framework in a specialist insect herbivore.

The role of hemocytes, serine protease inhibitors and pathogen-associated patterns in prophenoloxidase activation in the desert locust, Schistocerca gregaria.

The prophenoloxidase-activating system is an important component of the innate immune response of insects, involved in wound healing and melanotic encapsulation. In this paper we show that in the desert locust, Schistocerca gregaria, hemocytes, challenged with microbial elicitors, are indispensable for the limited proteolytic activation of prophenoloxidase (proPO) in plasma. In addition, we assessed the influence of serine protease inhibitors on the induction of PO-activity in plasma. While soybean Bowman-Birk inhibitor (SBBI) inhibited the PO activation by laminarin-treated hemocytes, the endogenous pacifastin-related inhibitors, SGPI-1 (S. gregaria pacifastin-related inhibitor-1) and SGPI-2 did not affect the PO-activity under similar conditions. On the other hand, real-time PCR analysis revealed that the transcripts, encoding SGPI-1-3, were more abundant in the fat body of immune challenged animals, as compared to control animals.

Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris).

ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.

Serum tyrosinase in malignant disease, its activity, and the electrophoretic patterns of the enzyme as carried by immunoglobulins'.

Serum tyrosinase activity in many persons with metastatic diseases was found to be significantly higher than activity in normal persons. The highest activity was observed in melanoma and breast carcinoma. The electrophoretic patterns of serum tyrosinase, resolved by electrophoresis of a serum tyrosinase fraction followed by incubation of the gel sample with L-dopa, and represented as sets of RF's of melanin bands, were characteristically different in melanoma, breast carcinoma, and certain other diseases. The RF's of melanin and protein bands in the serum enzyme preparations from melanoma patients were concisely defined. Further, some potent serum fractions inhibiting tyrosinase melanogenic activity have been obtained, and the presence of tyrosinase inhibitors in the serum enzyme preparation has also been demonstrated. More detailed exploration of these serum tyrosinase parameters may provide more specific and sensitive detection for certain malignant diseases.

Purification and characterization of a novel human red cell enzyme with diphenol oxidase activity.

A new enzyme protein, diphenol oxidase, has been isolated and purified from human red cells and catalyzes the in vitro formation of adrenochrome from epinephrine and of melanin from dihydroxyphenylalanine. Some immunological and physicochemical properties of this protein have been studied.

[Diphenoloxydase (DPOx) in red cell membranes]. / Etude des diphénoloxydases (DPOx) dans les membranes de globules rouges.

Two diphenoloxydases (DPOx) with similar molecular weights (158 000) are found in normal human red cell hemolysates. One of these enzymes appears to be tightly bound to the membranes while the other is not, or at most only weakly bound.

Morphological and biochemical characterization of Procambarus clarki blood cells.

Morphological and cytochemical analysis of Procambarus clarki hemocytes demonstrated three cell types: hyaline, semigranular, and granular. Hyaline cells showed a higher nuclear/cytoplasmic ratio with few small electron-dense granules in the cytoplasm. Semigranular cells presented numerous round or oval eosinophilic granules (0.40-0.78 micron). Granular cell contained large eosinophilic granules (1.79-3.05 microns). Ultrastructurally, all cells showed microtubules near the borders, a poorly developed Golgi complex, and secretory-type electron-dense particles. No mitotic figures were seen. Cell monolayers showed three morphologically distinct cell types (composed of flattened and well-spread cells) depending on the presence and size of granules (hyaline, semigranular, and granular). No sex-related differences could be documented in cell features or proportions. Cytochemical studies showed that the three cell types were positive for acid phosphatase. Granular and semigranular cells were also positive for nonspecific esterase. Phenoloxidase activity was localized only in granular and semigranular hemocytes, and peroxidase activity was observed only in the granular hemocytes. These results may suggest that the semigranular and granular hemocytes participate in the prophenoloxidase system and also in phagocytic or cytotoxic function.

Prophenoloxidase system activation in the crayfish Procambarus clarki.

The prophenoloxidase system (proPO) was studied in primary cultures of hemocytes of the crayfish Procambarus clarki. Both zymosan and lipopolysaccharide (LPS) separately induced rapid degranulation and lysis of semigranular hemocytes, with concurrent release of proPO. ProPO could be demonstrated in the hemocyte lysate supernatant (HLS) obtained by a freeze/thaw method, and was specifically activated by LPS and zymosan. Phenoloxidase activity was blocked by serine protease inhibitors, such as soybean trypsin inhibitor (STI), leupeptin, and phenylmethyl-sulphonylfluoride (PMSF), and substantially increased by cysteine protease inhibitors (N-methylmaleimide, N-ethylmaleimide, and iodoacetamide). This enhancement was observed only when the proPO system was activated. Incubation without activators or preincubation with STI prevented the induced enhancement. Electrophoretic analyses of HLS treated with zymosan or LPS showed that three bands at 41, 39, and 37 kDa were specifically modified when the system was activated. These results suggest that a serine protease is involved in the activation of the proPO system in P. clarki, and a mechanism susceptible to cysteine protease inhibitors could be related to its regulation.

Activation of prophenoloxidase in the plasma and haemocytes of the marine mussel Perna viridis Linnaeus.

Phenoloxidase activity was detected in plasma and haemocytes of the marine mussel Perna viridis. This enzyme exists as a proenzyme, prophenoloxidase (proPO), in both these haemolymph fractions and could be activated in vitro by exogenous proteases (trypsin and alpha-chymotrypsin) and a detergent (sodium dodecyl sulphate). In addition, laminarin (a polymer of beta-1,3 glucan) and bacterial lipopolysaccharides (LPSa) effectively triggered proPO activation in these haemolymph fractions. The activation of proPO by non-self molecules was dependent upon calcium ions at a low concentration. This activation process appeared to involve a limited proteolysis, since serine protease inhibitors (soybean trypsin inhibitor, benzamidine or p-nitrophenyl-p'-guanidinobenzoate) suppressed conversion of proPO to the active enzyme. This study demonstrates the selective response of plasma and haemocytic proPO to activation by different types of bacterial LPS tested and suggests that proPO system in both plasma and haemocytes of P. viridis serves an important function in non-self recognition and host immune reactions.

Haemolymph monophenol oxidase activity in Armigeres subalbatus infected with Brugia pahangi.

The activity of monophenol oxidase can be elicited in the haemolymph of Armigeres subalbatus by both blood and filaria-infected blood feeding. Haemolymph collected from both blood-fed and filaria-infected mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of L-[3,5-3H]tyrosine to dopa. Enzyme activity in filaria-infected mosquitoes was found to be significantly lower than that found in the blood-fed mosquitoes within 3 days post-ingestion, but still remained measurable 72 h post-ingestion. The decreased enzyme activity coincided in time with the development of capsules around the microfilariae. The consumption of monophenol oxidase by the melanization of migrating microfilariae in the haemocoel of filaria-infected mosquitoes and the effects of excretory and secretory products of developing larvae on monophenol oxidase activity are suggested.

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme.

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.

[Diphenoloxidases: presence in the membrane of human erythrocyte (author's transl)]. / Diphénoloxydases: présence dans les membranes de globules rouges

Diphenoloxidases, enzymes which accelerate the auto-oxidation of epinephrine and Dopa, have been described by one of us in blood platelets. Earlier we identified these enzymes in different animal species and particularly in human red blood cells. With the object of localising these enzymes and of understanding their function in vivo, we separated the ghosts of red blood cells according to the method described by Fairbanks G., Steck, T.L. and Wallach, D.F.H. (1971) (Biochemistry 1, 2606) and using the protease inhibitors diisopropylfuorophosphate (DFP) (10(-3) M) and 6-aminocaproic acid 10(-2) M in sodium phosphate buffer, 5 X 10(-3) M (pH 8). These ghosts, totally free of haemoglobin, were first of all pulverised in liquid nitrogen then treated ultrasonically. The supernant shows the presence of a band of diphenoloxidase activity on starch gel electrophoresis and two bands on isoelectrofocusing in polyacrylamide gel pH 5 to pH 8 after incubation with 0.02 M Dopa, 0.076 M Tris, 0.005 M citric acid, 0.004 M magnesium, pH 8.7 for 2 h at 37 degrees C. These enzymes differ from (Na, K)-ATPase in that they are neither inhibited by DFP (10(-1) M) nor by EDTA (10(-2) M) but are inhibited by lead acetate 10(-2) M. Like (Na, K)-ATPase diphenoloxidases are present at membranes level. The role in vivo of these diphenoloxidases in ATPase activity of red blood cells is discussed.

An animal model for the survival of tyrosinase isozymes in serum.

Two tyrosinase isozymes were purified from pigmented hamster melanoma and injected i.v. into rats. It was shown that while T1 (sialylated) isozyme survived in the circulation, native asialo (T2) isozyme and neuraminidase-desialylated T1 isozyme disappeared from the circulation in a few minutes. Desialylated fetuin had a marked inhibitory effect on the removal of asialo-T1 tyrosinase. These data indicate that the enzyme tyrosinase shares the common pattern of clearance from circulation known for the majority of serum glycoproteins. The electrophoretic pattern of tyrosinase isozymes partially purified from the sera of melanoma-bearing animals were compared with those from the soluble fraction of the tumors. In hamsters, melanoma tissue revealed both T1 and T2 isozymes while serum exhibited T1 and very weak T2, supporting the mechanism of clearance demonstrated in rats. In mice bearing Cloudman S-91 or B-16 melanomas, only T1 isozyme was seen in sera and in tumors.

Prophenoloxidase from brown shrimp (Penaeus californiensis) hemocytes.

Prophenoloxidase (proPO) was purified from blood cells of the brown shrimp Penaeus californiensis by ultracentrifugation and dye affinity chromatography. The isolated proPO is a 114-kDa monomeric protein as determined by SDS-PAGE. This protein can be hydrolyzed by proteinases, producing a 107-kDa active phenoloxidase (PO). The isoelectric point for both protein forms was 7.35. The PO reaction using L-DOPA as substrate, has an optimum pH of 8, and was poorly inhibited by sodium azide, thiourea and EDTA, but strongly inhibited by diethyl thiocarbamate. According to the substrate affinity and inhibition characteristics, this phenoloxidase was classified as a tyrosinase-like phenoloxidase. Purified proPO was not activated by bacterial lipopolysaccharides or beta-glucans.

Immunological determination of tyrosinase in sera of melanoma-bearing hamsters.

The quantitative ring immunodiffusion technique was adapted for the determination of the DOPA-oxidase activity of tyrosinase [E.C.1.14.18.1] in the sera of melanotic melanoma-bearing hamsters. The smallest amount of tyrosinase detectable in antibody-agar plates was about 0.5 micrograms/ml. The quantity of tyrosinase in the hamster sera depended on the grade of tumor development and rose to a value of 40 microgram/ml. There was no detectable activity in the sera of hamsters without tumor. The possibility of using this method to test sera of patients and horses with melanoma malignum is discussed.

Dopa oxidase activity and ceruloplasmin in the sera of hamsters with melanoma.

Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.