Phenoloxidase Activity In Hemolymph Research Articles

Influence of fungal infection on the DOPA–semiquinone and DOPA–quinone production in haemolymph of [formula omitted] larvae

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA–semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA–quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.

Comparative study of the strategies evolved by two parasitoids of the genus Asobara to avoid the immune response of the host, Drosophila melanogaster

Asobara tabida and Asobara citri are two braconid endoparasitoids of Drosophila melanogaster larvae. We studied and compared the strategies evolved by these two species to avoid the immune reaction of their host. A. tabida has no negative impact on host cellular defenses and its eggs avoid encapsulation by adhering to host tissues. At the opposite, we found that A. citri, whose eggs are devoid of adhesive properties, affects the host encapsulation abilities, hemolymph phenoloxidase activity and concentrations of circulating hemocytes. Some of these effects could directly rely on a severe disruption of the hematopoietic organ anterior lobes observed in parasitized larvae. This is the first report of the immune suppressive abilities of a parasitoid from the Asobara genus. Results are presented and discussed with respect to the strategies of virulence evolved by other parasitoids to counteract the D. melanogaster immune system.

Heritability of immune function in the caterpillar Spodoptera littoralis.

Phenoloxidase (PO) is believed to be a key mediator of immune function in insects and has been implicated both in non-self recognition and in resistance to a variety of parasites and pathogens, including baculoviruses and parasitoids. Using larvae of the Egyptian cotton leafworm, Spodoptera littoralis, we found that despite its apparent importance, haemolymph PO activity varied markedly between individuals, even amongst insects reared under apparently identical conditions. Sib-analysis methods were used to determine whether individuals varied genetically in their PO activity, and hence in one aspect of immune function. The heritability estimate of haemolymph PO activity was high (h(2) = 0.690 +/- 0.069), and PO activity in the haemolymph was strongly correlated with PO activity in both the cuticle and midgut; the sites of entry for most parasites and pathogens. Haemolymph PO activity was also strongly correlated with the degree to which a synthetic parasite (a small piece of nylon monofilament) was encapsulated and melanized (r = 0.622 +/- 0.142), suggesting that the encapsulation response is also heritable. The mechanism maintaining this genetic variation has yet to be elucidated.

Effects of shell disease syndrome on the haemocytes and humoral defences of the edible crab, Cancer pagurus

Crustaceans affected by shell disease syndrome display characteristic black-spot lesions in regions of exoskeletal degradation. Vogan et al. [Dis. Aquat. Org. (2001)] have shown that the severity of shell disease in the edible crab, Cancer pagurus, correlates with an increase in haemocoelic bacterial infections and may therefore serve as an external marker for the internal health of the animal. Therefore, this present study examined if the same crabs with shell disease lesions and varying degrees of bacterial septicaemia also displayed differences in the haemogramme, haemolymph phenoloxidase activity, total protein, copper and urea, as well as haemolymph-derived antibacterial activity, compared to uninfected individuals. A strong correlation was found between the severity of shell disease and a reduction in serum protein, which was further indicative of haemocoelic infection. Levels of copper, urea, phenoloxidase, and antibacterial activity in the haemolymph showed no correlations with the proportion of exoskeletal lesion cover. However, the degree of melanisation of haemolymph samples taken from shell-diseased individuals was less than that seen in disease-free crabs. Total haemocyte counts were unaffected by the disease, although some minor changes were found in the differential counts. Overall, despite having intrahaemocoelic infections, shell disease-affected individuals displayed few changes in the cellular or humoral defence parameters examined.

Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus.

Melanization is an effective defence reaction used by mosquito hosts to kill malarial and filarial worm parasites. Although phenoloxidase (PO) has long been considered to be the key enzyme in the biosynthesis of melanotic material in insects, there is no direct evidence verifying its role in parasite melanization. To elucidate the role of PO in the melanization of microfilariae (mf) by mosquitoes, a double subgenomic Sindbis (dsSIN) recombinant virus was used to transduce Armigeres subalbatus mosquitoes with a 600 base antisense RNA targeted to the highly conserved copper-binding region of an Ar. subalbatus PO gene. Compared with controls, haemolymph PO activity in mosquitoes transduced with antisense RNA was significantly reduced. When these mosquitoes were challenged with Dirofilaria immitis mf, the melanization of mf was almost completely inhibited. These data verify that PO is an essential component of the biochemical pathway required for the melanization of parasites, and that the dsSIN expression system represents a useful tool in the functional analysis of endogenous gene expression in mosquitoes.

Parasitism of Lacanobia oleracea (Lepidoptera) by the ectoparasitoid, Eulophus pennicornis, is associated with a reduction in host haemolymph phenoloxidase activity

When haemolymph from fifth instar Lacanobia oleracea was incubated in vitro, rapid melanization occurred. Similar levels of melanization occurred in haemolymph from larvae that had been experimentally injected with venom from the ectoparasitic wasp, Eulophus pennicornis. In contrast, haemolymph from larvae parasitized by this wasp melanized more slowly and less extensively. Phenoloxidase assays indicated that enzyme activity was present in haemocyte lysate supernatants, serum and plasma from L. oleracea and that on day 5 post-parasitization, fractions prepared from parasitized larvae had significantly less phenoloxidase activity than similar fractions from untreated or experimentally envenomated larvae. In addition, no PO activity was detectable in wasp venom, and the venom had no effect on L. oleracea plasma phenoloxidase activity in vitro. These results indicate that parasitism of L. oleracea by E. pennicornis suppresses host haemolymph phenoloxidase activity and that this suppression is not induced by adult wasp venom. The results are discussed with reference to the survival advantages of suppressing the activity of this host enzyme, and to the possible source(s) of putative suppressive factors.

Baculovirus resistance in the noctuid Spodoptera exempta is phenotypically plastic and responds to population density

Parasite resistance mechanisms can be costly to maintain. We would therefore predict that organisms should invest in resistance only when it is likely to be required. Insects that show density-dependent phase polyphenism, developing different phenotypes at high and low population densities, have the opportunity to match their levels of investment in resistance with the likelihood of exposure to pathogens. As high population densities often precipitate disease epidemics, the high-density form should be selected to invest relatively more in resistance. We tested this prediction in larvae of the noctuid Spodoptera exempta. Larvae reared at a high density were found to be considerably more resistant to a nuclear polyhedrosis virus than those reared in isolation. A conspicuous feature of the high-density phase of S. exempta and other phase-polyphenic Lepidoptera is cuticular melanization. As melanization is controlled by the phenoloxidase enzyme system, which is also involved in the immune response, this suggests a possible mechanism for increased resistance at high population densities. We demonstrated that melanized S. exempta larvae were more resistant than non-melanized forms, independent of rearing density. We also found that haemolymph phenoloxidase activity was correlated with cuticular melanization, providing further evidence for a link between melanization and immunity. These results suggest that pathogen resistance in S. exempta is phenotypically plastic, and that the melanized cuticles characteristic of the high-density form may be indicative of a more active immune system.

Sex Differences in Susceptibility of House Crickets,Acheta domesticus,to Experimental Infection withSerratia liquefaciens

Fragile X syndrome (FXS), caused by a mutation in the Fragile X Mental Retardation 1 (FMR1) gene, is a common form of inherited mental retardation. Mutation of the gene leads to a loss of the gene product Fragile X Mental Retardation Protein (FMRP). While a loss of FMRP has been primarily associated with neural and cognitive deficits, it has also been reported to lead to immune system dysfunction in both humans and flies. We used the Acheta domesticus transcriptome to identify a highly conserved cricket ortholog of FMR1 (adfmr1). We cloned a partial cDNA of adfmr1, used systemic RNA interference (RNAi) to knockdown adfmr1 expression, and examined the impact of this knockdown (KD) on the cellular and humoral responses of the insect innate immune system. Following RNAi, both male and female crickets exhibited an increase in the number of circulating hemocytes, a decrease in total hemolymph phenoloxidase (PO) activity, and an increase in fat body lysozyme expression. Despite similar changes in these immune parameters in both sexes, male and female crickets responded differently to an immune challenge. Most KD males failed to survive an intra-abdominal injection of bacterial lipopolysaccharide, while KD females were just as likely as control females to survive this challenge. Our results support that decreased fmr1 expression can alter the cellular and humoral defenses of the insect innate immune system, and may lead to a decrease in male, but not female, immunocompetence.

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca 2+. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p′-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, β-nitrophenyl p′-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.

Phenoloxidase Activity Independent of Calmodulin and Calcium in Hemolymph of Heliothis virescens (Lepidoptera: Noctuidae) Larvae

Using a calmodulin-affinity column, it was determined that phenoloxidase activity in hemolymph of Heliothis virescens (F.) larvae is not regulated by the calciumbinding protein calmodulin. Because the enzymatic activity of phenoloxidase was not affected by calcium or ethylene glycol-bis (β-aminoethylether) N, N, N’, N’ -tetraacetic acid, it was concluded that phenoloxidase activity is not regulated by calmodulin nor is calcium required for its activity.

Alterations in the haemocytes of Pseudoplusia includens after parasitism by Microplitis demolitor

Parasitism by the braconid Microplitis demolitor suppressed the encapsulation response of its host, Pseudoplusia includens. Total haemocyte counts were higher in parasitized larvae than unparasitized larvae. Differential haemocyte counts did not differ between parasitized and unparasitized hosts, but the in vitro spreading behaviour of haemocytes was altered significantly by parasitism. Greater than 80% of plasmatocytes and granular cells from P. includens were unable to spread in vitro 2 h after oviposition by M. demolitor, and spreading of plasmatocytes remained suppressed through the course of parasitoid development. In addition, phenoloxidase activity in haemolymph was greatly reduced in parasitized larvae when compared to unparasitized larvae. Similar alterations in haemocyte spreading behaviour, phenoloxidase activity and encapsulation response were observed after injection of calyx fluid or purified polydnavirus from M. demolitor. The activity of polydnavirus was abolished by pretreatment with psoralen and ultraviolet irradiation which suggests the virus must be transcriptionally active to alter the host encapsulation response. Injection of venom did not affect haemocyte spreading or phenoloxidase activity, but dose-response studies indicated venom synergized the effects of calyx fluid. At equal doses, injection of calyx fluid plus venom had a greater and more persistent effect on plasmatocyte and granular cell spreading than injection of calyx fluid alone.

Ultrastructural and functional maturation of teratocytes of Apanteles kariyai

AbstractEggs and larvae of Apanteles kariyai avoid the host defense reactions of Pseudaletia separata due to the action of calyx and venom fluids injected by females during oviposition and the teratocytes originated from the embryonic serosal cells 3.5 day postoviposition. Phenoloxidase (PO) activity in host larvae was unaffected during early stages of parasitization (4–6 days postoviposition), relative to unparasitized larvae, but was greatly reduced to 25% during the late stage of parasitization (days 7–10). Hemolymph PO activity was not affected, in vitro, by calyx and venom fluids but was reduced in the presence of teratocytes. An apparent PO inhibitor was detected in older teratocyte cells. First instar parasitoid larvae implanted into unparasitized hosts, following transfer of either young teratocytes (4 day postoviposition) or old teratocytes (8 day post‐oviposition) with calyx and venom fluids resulted in avoidance of encapsulation only when calyx and venom fluids with young (4 day) teratocytes were injected. These results indicated that during early parasitization of the host, teratocytes just released from the embryonic serosal cells (4 day) function in conjunction with calyx and venom fluids injected into the host with the parasitoid egg to prevent its encapsulation by host hemocytes. During late parasitization, the older teratocytes (8 day) may also function in preventing host encapsulation by producing an PO inhibitor suppresses host hemolymph PO activity at the time of parasitoid egression.

Inhibition of host phenoloxidase activity by parasitoid hymenoptera

Partial or complete inhibition of hemolymph phenoloxidase activity occurred in host species attacked by some parasitoid wasps. In one system, inhibition of enzyme activity could be achieved by injection of a virus purified from parasitoid ovaries.

A mutant affecting the crystal cells inDrosophila melanogaster.

Black cells (Bc, 2-80.6±) mutant larvae ofDrosophila melanogaster have pigmented cells in the hemolymph and lymph glands. In this report we present evidence that these melanized cells are a mutant form of the crystal cells, a type of larval hemocyte with characteristic paracrystalline inclusions.Bc larvae lack crystal cells. Furthermore, the distribution pattern of black cells inBc larvae parallels that of experimentally-blackened crystal cells in normal larvae (phenocopy).InBc/Bc zygotes black cells appear during mid embryonic development but inBc +/Bc zygotes pigmented cells are not found until late in the first larval instar.Crystal cells are present in the heterozygous larvae until this time, and paracrystalline inclusions can be seen in some of the cells undergoing melanization in these larvae.The rate of phenol oxidase activity inBc +/Bc larval cell-free extracts is less than half that ofBc +/Bc +extracts whereas enzyme activity is undetectable inBc/Bc larvae. We propose that theBc +gene product is required for maintaining the integrity of the paracrystalline inclusions; inBc/Bc larvae either the product is absent or nonfunctional so an effective contact between substrate and enzyme results in melanization of the cells.Phenol oxidase itself is either destroyed or consumed in the melanization process accounting for the absence of enzyme activity inBc/Bc larvae. These studies confirm that the crystal cells store phenolic substrates and are the source of the hemolymph phenol oxidase activity in the larva ofD. melanogaster.