Tyrosine metabolism in carbidopa and phenylthiourea administered Corcyra cephalonica

The effects of carbidopa and phenylthiourea on dopa decarboxylase and phenoloxidase in relation to their involvement in sclerotization were assessed after injection of these compounds to late last instar Corcyra cephalonica larvae. Carbidopa, on administration, inhibited dopa decarboxylase activity but served as a substrate of phenoloxidase, leading to the development of yellow coloured pupal cuticle. The larvae, injected with phenylthiourea, had reduced phenoloxidase activity, and a prolonged larval period. The control mechanisms of tanning in these insects have been discussed with reference to tyrosine metabolism.     

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β-sclerotization in the puparia of flesh fly Sarcophaga bullata

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o-diphenolic compounds. While catechol, 3,4-dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 3,4-dihydroxyphenyl glycol, 3,4-dihy-droxymandelic acid, and N-acetyldopamine were oxidized to their side-chain oxygenated products. In addition, the enzyme-catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β-sclerotization. Radioactive trapping experiments failed to support the participation of 1,2-dehydro-N-acetyldopamine as a freely formed intermediate during phenoloxidase-mediated oxidation of N-acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7-³H]N-acetyldopamine and not from either [8-³H] or [ring-³H]N-acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme-catalyzed N-acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2-dehydro-N-acetyldopamine are the reactive intermediate of β-sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.     

The toxic effects of phenoloxidase from the haemolymph of tenebrionid beetles

The injection of haemolymph originating from several species of tenebrionid beetles into blowfly larvae caused a gradual paralysis accompanied by colour changes in the haemolymph of the injected test insects. It was found that the lethal effect of the haemolymph of the beetle Blaps sulcata was due to phenoloxidase. The enzyme was activated by the exposure and incubation of the haemolymph at room temperature.The identity between the toxic factor and phenoloxidase in the beetle's haemolymph was demonstrated by the following data: (1) A correlation between the rate of lethal and phenoloxidase activities during the activation process of the toxic haemolymph. (2) Phenylthiourea, a well-known inhibitor of phenoloxidase, inhibited both the enzymatic and the toxic action of the beetle's haemolymph. (3) A commercial preparation of phenoloxidase (originating from mushrooms) imitated the lethal effects and the accompanying symptoms of the toxic haemolymph. (4) Sephadex G-100 column separation of the Blaps haemolymph revealed a complete overlap between the enzymatic and lethal regions of the elution pattern.The possible effects of phenoloxidase on the haemolymph of the injected insects are discussed.     

Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony

Microinjection of radioactive tyrosine, dopa, and dopamine into mature larvae of Drosophila revealed that the sclerotization pathway is similar but not identical to that in Calliphora: (a) tyrosine is converted to tyrosine-o-phosphate and not to dopa, and (b) the substrate N-acetyldopamine does not accumulate.Larvae of the mutant ebony appear to be similar to the wild type with respect to tyrosine, dopa, and dopamine utilization. About the time of eclosion, however, ebony has twice as much dopamine as normal. Some implications of this are discussed with reference to the mutant phenotype.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

Phenoloxidase activity in the haemolymph of parasitized and unparasitized Heliothis virescens

Hymenopterous parasitoids of Heliothis virescens are able to evade the immune mechanisms of their host, especially the ability of the host to encapsulate foreign objects within a haemocyte capsule. This encapsulation is often accompanied by melanization. Our present findings show that hosts parasitized by Microplitis croceipes, Cardiochiles nigriceps and Campoletis sonorensis show changes both in the amount of melanin formed when their haemolymph is exposed to air and in the total protein as determined by the Folin phenol reagent. Furthermore, these changes are promoted by each of the three parasitoids in a different way. However, none of these parasitoids was able to alter the levels of host phenoloxidase as measured by the ability of the haemolymph to convert 4-methylcatechol to its quinone. The hymenopteran egg must therefore escape encapsulation in Heliothis in a way other than through inhibition of phenoloxidase, since the melanization reactions in the host's haemolymph are apparently unimpaired, particularly during the egg stage, by either the egg or venom of any one of the three parasitoids.     

Adipokinetic hormone enhances laminarin and bacterial lipopolysaccharide-induced activation of the prophenoloxidase cascade in the African migratory locust,Locusta migratoria

Lom-AKH-I enhances the activation in vivo of prophenoloxidase in the haemolymph of the African migratory locust, Locusta migratoria, in response to challenge with laminarin. AKH does not influence the speed or initial magnitude of the phenoloxidase response to laminarin, but prolongs the period of activation of the enzyme in a dose-dependent manner. Injections of preparations of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase in vivo, but co-injection of Lom-AKH-I with commercial preparations of LPS from Klebsiella pneumoniae, Escherichia coli, or Shigella flexneri (but not one from Pseudomonas aeroginosa) results in dose-dependent increases in the levels of phenoloxidase that persist in the haemolymph for several hours. It is argued that the effects of AKH on phenoloxidase activation in locusts described here are, at least in part, related directly to changes in lipid metabolism brought about by the hormone.     

Silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in Anopheles gambiae

The production of melanin is a complex biochemical process in which several enzymes may play a role. Although phenoloxidase and serine proteases are clearly key components, the activity of other enzymes, including dopa decarboxylase and dopachrome conversion enzyme may also be required. We tested the effect of knockdown of gene expression for these two enzymes on melanization of abiotic targets in the mosquito, Anopheles gambiae. Knockdown of dopa decarboxylase and dopachrome conversion enzyme resulted in a significant reduction of melanization of Sephadex beads at 24 h after injection. Knockdown of a third enzyme, phenylalanine hydroxylase, which is involved in endogenous production of tyrosine, had no effect on bead melanization. Quantitative analysis of gene expression demonstrated significant upregulation of phenylalanine hydroxylase, but not the other two genes, following injection.     

Egg chorion tanning in Aedes aegypti mosquito

The biochemical pathway of egg chorion tanning in the mosquito, Aedes aegypti, is described and compared with chorion protein crosslinking in Drosophila and silkmoths and the biochemical pathways of cuticular tanning in insects. Phenol oxidase, dopa decarboxylase and tyrosine are critical components involved in egg chorion tanning in A. aegypti. Tanning of the mosquito egg chorion is initiated following activation of phenol oxidase, which then catalyzes the hydroxylation of tyrosine to dopa and further oxidizes dopa and dopamine to their respective o-quinones. Because intramolecular cyclization is much slower in dopaminequinone than dopaquinone, the chance to react with external nucleophiles to participate in protein crosslinking reactions also is much greater in dopaminequinone than dopaquinone. This might partly explain the necessity for the involvement of dopa decarboxylase in mosquito chorion tanning. Intramolecular cyclization of dopaquinone and dopaminequinone to form dopachrome and dopaminechrome, respectively, the structural rearrangement of these aminochromes to produce 5,6-dihydroxyindole, and the subsequent oxidation of 5,6-dihydroxyindole by phenol oxidase also lead to melanin formation during egg chorion tanning.     

Effect of dopa-decarboxylase inhibition on Aedes aegypti eggs: evidence for sclerotization

Newly deposited fertilized and unfertilized Aedes aegypti eggs are soft and white. Within a short time they darken and harden. Injection of a potent dopa decarboxylase inhibitor (dl)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid (α-MDH) into females, subsequent to a blood meal, results in oviposited eggs which are pale in colour. Moreover, such fertilized eggs do not hatch. The severity of both effects seems to be positively correlated and is dependent upon the time of α-MDH injection.Extracts of mature ovaries are capable of converting dopa to dopamine in the absence of a pretreatment with α-MDH. Mature ovaries obtained from females who had been previously injected with α-MDH could not accomplish this conversion. The inhibitor does not seem to have any effect on dopa oxidase activity and subsequent melanization. We conclude that dopamine is synthesized by blood-fed females via decarboxylation of dopa by dopa decarboxylase and propose that the normal darkening and hardening of A. aegypti eggs is a result of sclerotization.     

The effect of Habrobracon hebetor venom on the activity of the prophenoloxidase system, the generation of reactive oxygen species and encapsulation in the haemolymph of Galleria mellonella larvae

The cellular and humoral immune reactions in haemolymph of the wax moth Galleria mellonella larvae naturally injected by venom of ectoparasitic wasp Habrobracon hebetor were analyzed. A strong decline of phenoloxidase (PO) activity in the haemolymph and the number of haemocytes with PO activity of envenomated wax moth was observed. In addition, it has been shown that the rate of capsule melanization in the envenomated larvae was half that of the control. Also production of reactive oxygen species (ROS) in the haemolymph of envenomated larvae decreased. The obtained data casts light on the suppression of the main immune reactions in G. mellonella larvae during natural envenomation by H. hebetor.     

Morbidity and mortality in Norway lobsters, Nephrops norvegicus: physiological, immunological and pathological effects of aerial exposure

A multivariate approach has been used to study progression in the post-capture condition of trawl-caught Nephrops norvegicus destined for the live transport market. A range of biochemical (L-lactate, glucose, glycogen), endocrinological (crustacean hyperglycaemic hormone), immunological (total haemocyte counts, phenoloxidase), microbiological and pathological measures of condition were utilised. During prolonged periods of aerial exposure N. norvegicus experience large disruptions to the carbohydrate profile, with increases in haemolymph L-lactate and crustacean hyperglycaemic hormone concentrations, and corresponding fluctuations in haemolymph pH; the severity of this disruption increases with the temperature of aerial exposure. This in turn impacts on the immune competence of the lobsters, with significant reductions in the number of circulating haemocytes and phenoloxidase levels observed as well as increases in the degree of bacteraemia of the haemolymph. Utilising evidence obtained during histological and other studies, possible causes of the immuno-suppression and subsequent meat spoilage are discussed. The information obtained should help to identify critical periods in the post-capture period that promote poor stock condition and mortality. Such data may be used to generate an internationally accepted Code of Practice for the capture, handling and transport of commercially exploited decapod crustaceans.     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

DOPA and tyrosine decarboxylase activity in tissues of Periplanet a americana in relation to cuticle formation and ecdysis

DOPA decarboxylase activity in haemolymph and integument was low in last instar and early pharate adult Periplaneta americana, but began to increase shortly before ecdysis. Decarboxylation rates of l-DOPA, about 10 times the larval level by the start of ecdysis, reached a peak about 6 hr afterward, coinciding with the main period of cuticular sclerotization. Activity decreased rapidly during the next 18 hr, then decreased gradually for several days. Haemolymph DOPA decarboxylase activity was about four times greater than the integument, based on tissue dry weights. The fat body and gut tissues had low DOPA decarboxylase activity in all ages tested, and this did not increase at ecdysis. Tyrosine decarboxylase activity was significant only in the haemolymph and at consistently low levels.DOPA decarboxylase, therefore, apparently plays a major rôle in production of catecholamine derivatives for cuticular sclerotization in P. americana, while tyrosine decarboxylation is minor. Both haemolymph and integument appear to be important sites of dopamine biosynthesis.     

Control mechanisms of tanning in Corcyra cephalonica

Localisation of a prophenoloxidase in the cytoplasm of plasmatocytes was histochemically demonstrable in Corcyra cephalonica, by incubating the blood with different phenolic substrates. The activation of this latent enzyme was found to be controlled by a protein factor present in the cuticle. The haemolymph prophenoloxidase could be activated in vitro by prior incubation of the blood sample at 0°C. The treatment with NaCl, EDTA, or detergents did not cause any activation. The exposure of blood samples to gamma-rays, repeated freezing and thawing, or addition of the cuticular activator, brought about rapid disruption of the haemocytes causing a release of phenoloxidase and acceleration of melanization of the blood. The administration of α-ecdysone to the last instar larvae induced premature pupation and accentuated the activation of phenoloxidase without altering the level of enzyme activity. The possible regulatory mechanisms of tanning during the development of Corcyra have been discussed.     

Activation of prophenoloxidase and inhibition of melanization in the haemolymph of immune Galleria mellonella larvae

After immunization of Galleria larvae with bacterial lipopolysaccharide, inhibition of haemolymph melanization developed parallel with antibacterial immunity. Failure of melanization was correlated with significantly decreased amounts of active cell-associated phenoloxidase (PO). In normal, nonimmune haemolymph, cellular PO originated from plasma proPO activated by, and largely attached to, the haemocytes; activation was maximal by 150 min. With immune haemolymph, such cellular activation of plasma proPO did not occur. Immune plasma proPO was activated by homogenization, but not by freeze-thawing. Normal plasma proPO was activated by freeze-thawing, but only to a very slight extent by homogenization. Mixing immune plasma with the freeze-thaw activated PO in normal plasma (1:2, v:v) caused a 37%, average reduction in PO activity. The results suggest that inhibition of melanization in immune Galleria haemolymph is caused by plasma factor(s) inhibiting the activation of plasma proPO by haemocytes.     

The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata, undergoing development in vitro

During the postembryonic development of Sarcophaga bullata, two large peaks of dopa decarboxylase activity were observed. These were associated with the sclerotization (hardening) of the puparium and the adult cuticle, respectively. A small peak of activity 5.5–6.5 days after pupariation was possibly associated with the sclerotization of the prothoracic spiracles.A premature increase in enzyme activity was observed in young, third-instar larvae injected with 20 μg of β-ecdysone. However, the advantage of studying the effect of the hormone on enzyme activity in vitro led to an attempt to induce² dopa decarboxylase in cultured wing discs.In the presence of β-ecdysone, wing discs underwent evagination and a substantial increase in dopa decarboxylase activity was observed in these discs. The enzyme activity began to appear after the rupture of the peripodial membrane and reached a maximum about the time disc evagination ceased. We suggest that this enzyme activity was responsible for the slight sclerotization of a fine cuticle secreted by the discs. The cultured imaginal discs underwent changes that are very similar to those which occur in intact animals. Therefore, this system appears promising for further studies on the role in differentiation of the hormonal control of enzyme activity.