Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): Basis of insecticidal activity

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.     

The tanning enzymes of Corcyra cephalonica

In Corcyra cephalonica, the enzymatic synthesis of N-acetyldopamine involves the hydroxylation of tyrosine to dopa, decarboxylation of dopa to dopamine and its subsequent acetylation to N-acetyldopamine. Dopa decarboxylase is mainly present in the epidermal tissues of the rice moth, and phenoloxidase in the haemolymph. These enzymes, along with their substrates, are present in highest concentrations just prior to pupation. An activator for haemolymph phenoloxidase can be detected in the cuticles of the larva and pupa. Some of the characteristics of dopa decarboxylase and diphenoloxidase of Corcyra are similar to that of Drosophila and Calliphora.     

Effects of hypoxia,hypercapnia, and pH on ventilation rate in Nauphoeta cinerea

Injecting 50 μl of a phosphate-buffered Ringer's solution with adjusted oxygen pressure (PO₂), Carbon dioxide pressure (PCO₂) or hydrogen ion concentration (pH) stimulates ventilation in the cockroach, Nauphoeta cinerea. When the pH of the buffered Ringer's is held at 7.08, the observed pH of our cockroach haemolymph, neither a reduced PO₂ nor an elevated PCO₂ produced responses that could account for the observed changes in ventilation in insects during exercise or when exposed to elevated ambient CO₂. Ventilation changes produced by altering the pH of the injected saline are comparable to those observed in active insects and in insects exposed to elevated ambient CO₂. We show that the effect of an elevated ambient CO₂ on ventilation can be accounted for by changes in haemolymph pH.     

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.     

Feeding causes the appearance of a factor in the haemolymph that stimulates protein synthesis

Soon after a locust (Locusta migratoria) begins to feed, an increase in protein synthesis can be detected in the animal. Isolation of fat body shows that this tissue synthesizes protein at a faster rate in recently fed animals than it does in fasting insects. Fasting locusts injected with haemolymph from fed insects increased protein synthesis when compared with locusts injected with haemolymph from fasting locusts. The factor causing this increase was present in the haemolymph within 5 min of feeding. Feeding or direct contact with the food was not essential to increase protein synthesis. Exposure of fasting locusts to feeding insects was sufficient to elevate the rates of protein synthesis in the fasting animals.The increase inprotein synthesis was not a result of general excitation or an increase in the concentration of tryptophan or isoleucine in the haemolymph. Ecdysteroid titres were uniformly low during the first ten days of adult life. Gel filtration of the fed haemolymph revealed a low molecular weight fraction (about 600 daltons) which stimulated protein synthesis upon injection into fasting locusts.     

In vivo inactivation of the insect neuropeptide proctolin in Periplaneta americana

Supranormal levels (0.13–31 μg) of proctolin injected into the cockroach, Periplaneta americana, disappear within minutes from the haemolymph. Treated insects exhibit a short-term catatonic state followed by full recovery. Using [¹⁴C-Tyr²]proctolin, it was shown that inactivation results from the removal of arginine yielding H-Tyr-Leu-Pro-Thr-OH. Tyrosine was produced in a subsequent step. The aminopeptidase responsible for this in vivo hydrolysis of proctolin differed from leucine aminopeptidase of porcine kidney in the relative rates of hydrolysis of proctolin and H-Tyr-Leu-Pro-Thr-OH. The latter was not detected as an intermediate in proctolin degradation by leucine aminopeptidase. This study indicates that proctolin cannot be transported via the haemolymph unless it is protected in some manner from enzymatic degradation.     

PO-CALC: A novel tool to correct common inconsistencies in the measurement of phenoloxidase activity

A broad range of physiological and evolutionarily studies requires standard and robust methods to assess the strength and activity of an individual’s immune defense. In insects, this goal is generally reached by spectrophotometrically measuring (pro-) phenoloxidase activity, an enzymatic and non-specific process activated after wounding and parasite infections. However, the literature surprisingly lacks a standard method to calculate these values from spectrophotometer data and thus to be able to compare results across studies. In this study, we demonstrated that nine methods commonly used to extract phenoloxidase activities (1) provide inconsistent results when tested on the same data sets, at least partly due to their specific sensitivity to the noise regularly present in enzymatic reaction curves. To circumvent this issue, we then (2) developed a novel, free and simple R-based program called PO-CALC and (3) demonstrated the robustness of its calculations for the different types of noises. Overall, we show that PO-CALC corrects overlooked though important inconsistencies in the measurement of phenoloxidase activities, and claim that its broad use would increase the significance and general validity of studies on invertebrate immunity.     

Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses

Proteolytic activity released within an organism by wounded tissues or invading pathogens can strongly impair the physiological homeostasis when it remains non-regulated. Thus, an efficient mechanism that enables recognition and inactivation of non-regulated proteolytic activity is essential to limit toxic effects. In larvae of the Greater wax moth Galleria mellonella we discovered that injection of bacterial thermolysin at a sublethal concentration mediates both acquired resistance against a subsequently injected lethal concentration of this metalloproteinase and stimulation of humoral immune response accompanied by the synthesis of an inducible metalloproteinase inhibitor (IMPI) which is released within the haemolymph. In search of a putative mechanism mediating recognition and regulation of released microbial metalloproteinases we determined that thermolysin-mediated hydrolysis of G. mellonella haemolymph proteins in vitro yields small (<3 kDa), heat-stable molecules which were discovered to represent potent elicitors of humoral immune responses when injected into untreated larvae. Obtained results allowed to design a model explaining for the first time regulation of released metalloproteinases within the haemolymph of insects. The determined coherence between regulation of released metalloproteinases by IMPI and the simultaneous induction of antimicrobial proteins provides a new insight into the mechanisms leading to expression of genes in course of humoral immune responses.     

Fate of glucose in haemolymph of the American cockroach, Periplaneta americana

The rate of removal of high concentrations of glucose (10 μg/μl haemolymph) from haemolymph of adult male cockroaches, Periplaneta americana, was studied in normal and ligated insects. More than 50% of the injected glucose is removed from the haemolymph of normal insects within 20 min of injection. A period of rapid trehalose synthesis occurs during the initial 10 min following injection of glucose into the haemocoele, and this is succeeded by a period of glycogen synthesis. The results are discussed in terms of earlier observations on ‘stress-induced hypertrehalosemia’ and the possible involvement of a glycogenic agent.     

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.     

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.     

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.     

Characterization of the Melanoplus sanguinipes hemolymph after infection with Beauveria bassiana or wounding

• 1.1. The phenoloxidase activity, protein and carbohydrate levels were studied for 24 hr in the hemolymph of the migratory grasshopper, Melanoplus sanguinipes after artificial wounding of the insect cuticle or the injection of Beauveria bassiana conidia.
• 2.2. Injection or wounding induced a primary response and phenoloxidase activity was found to increase within 10–60 min. The values for phenoloxidase activity in viable B. bassiana-injected insects exhibited a secondary response, i.e., an increase 24 hr after injection.
• 3.3. In wounded insects and those injected with inactivated conidia, the phenoloxidase activity receded after the initial increase and remained at low levels.
• 4.4. Protein concentrations in the hemolymph increased immediately after infection and wounding and returned to basal levels during the course of the experiment.
• 5.5. Injection of viable B. bassiana resulted in a gradual increase in the protein concentrations between 12 and 24 hr.
• 6.6. There was no apparent change in the carbohydrate levels in either B. bassiana-infected or wounded insects.
• 7.7. These results are discussed in relation to their possible role(s) and interrelationships in the immune response to infection or wounding. Furthermore, we suggest that a “factor” is released after mechanical injury of the integument.

     

More than a colour change: insect melanism,disease resistance and fecundity

A ‘dark morph’ melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

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.     

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.     

Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation

In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.     

Indirect plant-mediated effects on insect immunity and disease resistance in a tritrophic system

Host plant quality can significantly influence the growth and condition of phytophagous insects, and consequently their susceptibility to pathogens. This study examined the relationship between host plant quality, insect condition, immune responsiveness and resistance to pathogens in the cabbage looper, Trichoplusia ni. Two baseline and induced immune parameters were estimated, haemocyte numbers and haemolymph phenoloxidase (PO) activity, for larvae on two host plants, broccoli and cucumber. Haemolymph protein concentration was assessed as an indication of insect condition, and the susceptibility of larvae to T. ni single nucleopolyhedrovirus (SNPV) was used as a measure of disease resistance. T. ni growth, survival and condition was much higher on broccoli than cucumber. Haemocyte numbers were significantly higher in broccoli-reared larvae, whereas PO activity was not. An immune challenge induced significantly elevated numbers of haemocytes for larvae reared on both host plants, but did not affect PO activity or protein concentrations. Susceptibility to T. ni SNPV was markedly higher in larvae reared on cucumber than on broccoli. These results clearly indicate that host plant quality can affect both immune response and disease resistance of T. ni larvae and that bottom-up effects could be important in interactions between insects and entomopathogens.     

Influence of fungal infection on the DOPA-semiquinone and DOPA-quinone production in haemolymph of Galleriamellonella 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.