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.

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.

Age- and density-dependent prophylaxis in the migratory, cannibalistic Mormon cricket Anabrus simplex (Orthoptera: Tettigoniidae).

As a result of the increased potential for disease transmission, insects are predicted to show an increased constitutive immunity when crowded. Cannibalistic aggressive interactions further increase the risk of wounding and pathogen transmission in crowds. Nymphal Mormon crickets Anabrus simplex Haldeman were collected in Montana and reared in the laboratory either solitarily or at densities similar to that experienced by Mormon crickets in migratory bands. As teneral adults, solitarily-reared Mormon crickets tended to have greater phenoloxidase activity than those reared in groups. Sampling enzyme activity a second time when the adults were nearing reproductive maturity, group-reared Mormon crickets had elevated levels of prophenoloxidase and encapsulated foreign objects faster than solitarily-reared insects. Rearing density did not have a significant effect on either the darkness of the cuticle or antibacterial activity. This is the first report of age-related responses of adult insect immunity to crowding.

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.

Edible mushroom (Flammulina velutipes) extract inhibits melanosis in Kuruma shrimp (Marsupenaeus japonicus).

This study compared the potential of an aqueous extract of an edible mushroom (Flammulina velutipes) to prevent melanosis in cultured Kuruma shrimp (Marsupenaeus japonicus) with other antimelanosic compounds in vivo. The mushroom extract contained 9.1 mg/mL ergothioneine (ESH). Immersion of live full-grown shrimp in a 0.5% w/v solution of mushroom extract significantly reduced PPO activity in shrimp hemolymph. In addition, expression of the prophenoloxidase (proPO) gene decreased in hemocytes, suggesting that the extract blocked the activation of the proPO cascade. Consequently, the development of melanosis in the treated shrimp was significantly suppressed during ice storage. Treatment with a 0.05% w/v solution of sodium ascorbate and 4-hexyl-1,3-benzenediol had the same effect. In vitro experiments showed that ESH effectively inhibited PPO activity and activation of the proPO cascade in hemocyte lysate supernatant. This study suggests that in vivo application of F. velutipes mushroom extract is an effective natural alternative to synthetic antimelanosic agents to inhibit postmortem melanosis in shrimp. Practical Application: The extract of an edible mushroom (F. velutipes) containing ergothioneine can be a promising natural alternative to synthetic antimelanosic agents used to prevent postharvest melanosis in shrimp and other crustaceans. Furthermore, utilization of the mushroom trimmings could also help address the growing concerns on the disposal of such agricultural wastes and instead use it into a novel purpose as a source of antimelanosic and antioxidants for food and industrial application.

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.

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.

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.

Behavioural and physiological effects of the trophically transmitted cestode parasite, Cyathocephalus truncatus, on its intermediate host, Gammarus pulex.

Some parasites with complex life-cycles are able to manipulate the behaviour of their intermediate hosts in a way that increases their transmission to the next host. Gammarids infected by the tapeworm Cyathocephalus truncatus (Cestoda: Spathebothriidea) are known to be more predated by fish than uninfected ones, but potential behavioural manipulation by the parasite has never been investigated. In this study, we tested the hypothesis that C. truncatus is able to manipulate the behaviour of one of its intermediate hosts, Gammarus pulex (Crustacea: Amphipoda). To assess if any behavioural change was linked to other phenotypic alterations, we also measured the immunity of infected and uninfected individuals and investigated the pathogenic effects of the parasite. Infected gammarids were significantly less photophobic than uninfected ones, but no effect of infection on the level of immune defence was found. The results on survival, swimming activity and oxygen consumption suggest that the parasite also has various pathogenic effects. However, the alteration in host phototaxis was not correlated to some of these pathogenic effects. Therefore, we propose that the modification in host reaction to light is a behavioural manipulation, explaining the previously observed increase of gammarid predation rate.

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense.

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.

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.

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.

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.

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.

A method for demonstrating prophenoloxidase after electrophoresis.

The demonstration of prophenoloxidase after electrophoresis is based on its activation by sodium dodecyl sulfate (SDS) or sodium oleate and staining the activated phenoloxidase with dopamine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). A rapid method is presented for demonstrating the presence of activated phenoloxidase using polyacrylamide gel electrophoresis followed by staining in the presence of SDS or sodium oleate.

Purification and properties of a catechol oxidase from blood cells of the ascidian Pyura stolonifera.

A catechol oxidase (EC 1.10.3.1) was purified to homogeneity from blood cells of the ascidian Pyura stolonifera using gel filtration on Sephadex G-50 and hydrophobic interaction chromatography on PhenylSuperose. Two peaks of activity were eluted from PhenylSuperose, one with a decreasing salt gradient and the other with nonionic detergent. The latter represents an aggregated form of the enzyme. The enzyme has a molecular weight of 56 kd and shows a preference for catechols with uncharged hydrophobic side chains (e.g., 4-t-butylcatechol) but does not hydroxylate free tyrosine. Inhibition of the enzyme by diethyldithiocarbamic acid and thiol reagents implicate copper at the active site. Sequence analysis of a peptide generated by incubation with Staphylococcus aureus V8 protease demonstrated considerable homology to one of the conserved copper binding regions of tyrosinases. This enzyme is found in the same cells as the dopa-containing protein ferreascidin. When ferreascidin is incubated with the enzyme, its spectrum changes rapidly, indicating that the catechol oxidase uses it as a substrate. The P. stolonifera enzyme differs from an enzyme involved in adhesion, isolated from the mussels, M. edulis and G. demissa: it is isolated as a soluble enzyme that does not appear to exist as a latent precursor.

Relationship of hemolymph phenol oxidase and mosquito age in Aedes aegypti.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.

The prophenoloxidase system and in vitro interaction of Trypanosoma rangeli with Rhodnius prolixus and Triatoma infestans haemolymph.

The presence of the prophenoloxidase (proPO) system in the haemolymph of Rhodnius prolixus and Triatoma infestans and the role played by Trypanosoma rangeli in the in vitro activation of proPO were tested. Both R. prolixus and T. infestans whole blood preparations showed a very active ProPO system. The proPO cascade of the two insect species were differentially activated by microbial-derived extracts: laminarin was a better activator of T. infestans haemolymph than of R. prolixus blood, and lipopolysaccharides from Shigella flexneri or Pseudomonas aeroginosa caused significant proPO activation of T. infestans haemolymph but not of R. prolixus preparations. For the two insect species, neither T. rangeli from culture nor parasite lysates were able to trigger proPO activation. The presence of the parasite in R. prolixus haemolymph/laminarin assays, however, significantly reduced the level of proPO activation to that of spontaneous activating controls. The immobilization of T. rangeli in vitro in haemolymph preparations occurred in both insect species and was dependent on the proPO activation intensity. Our results suggest that the susceptibility of R. prolixus to T. rangeli haemocoel infection may be explained, at least in part, by the suppression of the insect immune defence system i.e., inhibition of proPO in the presence of this protozoan parasite.