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.     

Studies on the in vivo cellular reactions of insects: Clearance of pathogenic and non-pathogenic bacteria in Galleria mellonella larvae

The fate of various doses of bacteria of different pathogenicities injected into Galleria mellonella larvae was monitored over time, from haemocyte and bacterial counts, phagocytic responses and the speed and extent of formation of melanized cell aggregates (nodules). An initial haemocytopenia was recorded in all larvae, probably as a result of wound healing, an increased stickiness of the haemocytes for host tissues and/or cell clump or nodule formation. The results also showed that phagocytosis is the primary cellular defence reaction of this insect for doses of bacteria below ca. 10³ μl^?1 haemolymph while above this level phagocytosis and bacterial clearance are usually rapidly augmented by nodule formation. The extent to which these processes are elicited depends greatly upon the nature of the bacteria injected. In general, the more pathogenic strains produced greater responses than the relatively non-pathogenic forms. This enhanced cellular reactivity was, however, soon overcome by the pathogens which rapidly induced a secondary bacteraemia, a huge drop in haemocyte numbers and death of the larvae. The relative importance of phagocytosis and nodule formation in dealing with various doses of bacteria of differing pathogenicities is discussed.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Insect immunity: Galleria mellonella and other lepidoptera have cecropia-P9-like factors active against gram negative bacteria

The humoral immune system of diapausing pupae of Hyalophora cecropia was recently found to contain two small, basic antibacterial proteins, P9A and P9B, which are active against several Gram negative bacteria (Hultmarket al., 1980). These factors could be separated by polyacrylamide gel electrophoresis at pH 4 and afterwards visualized by seeding the gel with viable Escherichia coli. We have now used this technique to demonstrate that immune haemolymph from larvae of Galleria mellonella contains antibacterial proteins acting against Gram negative bacteria. Immunization with either live Enterobacter cloacae or heat killed Pseudomonas aeruginosa induced a potent antibacterial activity against both E. coli and P. aeruginosa. Cation exchange chromatography of immune haemolymph from G. mellonella was used to separate two antibacterial factors (designated G4 and G5) from the lysozyme activity. Several properties were found to be similar for Galleria factors G4 and G5 and the proteins P9A and P9B from Cecropia. It is concluded that the humoral immune systems of H. cecropia and G. mellonella are very similar. Both forms of P9 as well as G4 and G5 were shown to act on P. aeruginosa. Antibacterial factors against E. coli were demonstrated also in six other species of Lepidoptera.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Inflammation in insects: The release of a plasmatocyte depletion factor following interaction between bacteria and haemocytes

Injection of a suspension of the Gram-positive bacterium Bacillus cereus into the haemocoel of Galleria mellonella larvae caused a rapid decrease in the number of circulating plasmatocytes. A similar phenomenon was observed following the injection of cell-free haemolymph collected from larvae previously challenged with bacteria, indicating that plasmatocyte depletion was mediated by a humoral factor released into the haemolymph. The release of plasmatocyte depletion factor could be effected in vitro by incubating adherent haemocytes with a suspension of B. cereus. The in vitro production of the factor did not require the presence of exogenous humoral components, was dose-dependent, was inhibited at low temperatures, and showed the same specificity with respect to bacterial species as observed in vivo. Activity of the plasmatocyte depletion factor destroyed by a 15-min incubation at 56°C. The release of the factor appears to be an early biochemical signal in the complex cellular response of this insect to bacterial infection.     

Uptake of homologous haemolymph protein by salivary glands of Chironomus thummi

Radioisotopically labelled homologous protein fractions were injected into the haemolymph of Chironomus larvae. Among four different protein fractions, fraction II appears to enter the salivary gland and accumulates in the glandular lumen. Lack of blood pigmentation in the gland and secretion lumen indicates that the haemoglobin is probably not secreted, but a portion of the haemoglobin molecule or a fraction co-precipitating with haemoglobin in fraction II is being transported by the gland. Since only a portion of fraction II is taken up and secreted, the specificity of the process suggests to us a molecular basis for the activity which must ultimately be reflected in the genes of the transporting tissue.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Factors affecting the ability of the wasp parasite Pseudeucoila bochei to inhibit tumourigenesis in Drosophila melanogaster.

The cellular immune reaction of Drosophila melanogaster larvae which results in encapsulation of internal metazoan parasites is similar to the autoimmune reaction made by certain tumourmutant stocks against abnormally developing host tissues. The wasp parasite Pseudeucoila bochei is capable of actively inhibiting the haemocytic encapsulation reaction of host larvae, and this activity can also inhibit the haemocytic response made by the bw tu mutant flies against abnormal fat body tissue. The ability of the parasite to suppress tumourigenesis is dependent on the age of the host at the time of infection, and on the amount of previous oviposition of the parasite. The earlier during host development parasitisation occurs the more tumour formation is inhibited. partially spent females are less able to inhibit tumours than are newly emerged parasites and unspent parasites of the same age. In some parasitised individuals the tumour response is incomplete and consists of small pigment particles instead of the large melanotic growths characteristic of the strain. The autoimmune response made against “alien” host tissues is specific and does not interfere with the successful development of the parasite.     

Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus

Campoletis sonorensis is a habitual parasitoid of 3rd-instar larvae of Heliothis virescens. C. sonorensis eggs and small glass rods were encapsulated in 5th-instar host larvae implanted in the absence of wasp calyx fluid; prior injection of calyx fluid into larvae suppressed the encapsulation response. Within 8 h of calyx fluid injection there was a removal of approx. 75% of the circulating capsule-forming haemocytes (plasmatocytes). The remaining subpopulation of plasmatocytes, in addition to being incapable of encapsulating targets in vivo, spread at a significantly reduced rate in vitro. Identical changes in plasmatocyte count and behaviour were observed after injection of virus purified from calyx fluid. Additionally, the activity of calyx fluid was abolished after ultraviolet irradiation. The onset of haemocytic abnormalities occurred more rapidly after natural parasitism of 3rd-instar host larvae. The cell-free haemolymph of calyx fluid-injected 5th-instar larvae also retarded the spreading of plasmatocytes from non-injected control larvae in vitro. We conclude that the abnormalities induced in H. virescens plasmatocytes by C. sonorensis virus contribute to the suppression of encapsulation.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.     

Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV)

We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.     

Regulation of the corpora allata in the black mutant of Manduca sexta

Regulation of corpus allatum activity in the black mutant strain of Manduca sexta was studied in vivo and in vitro. Allatectomy, denervation, and implantation studies demonstrated that black mutant corpus allatum activity remains low in both wild-type and black mutant host larvae. Attempts to distinguish humoral control mechanisms versus mechanisms dependent on intact allatal nerves indicated that intact allatal nerves were not required for the reduced black mutant corpus allatum activity in vivo. Incubation of corpora allata, using [1-¹⁴C]propionate as a juvenile hormone biosynthetic precursor and haemolymph as culture medium, confirmed that black mutant corpora allata are suppressed by a factor(s) in the haemolymph. Under identical conditions wild-type corpora allata were unaffected. Finally, the lowered black mutant corpus allatum activity in haemolymph in vitro correlates with the lowered juvenile hormone titre in black mutant larvae.     

Induced immunity in Antheraea assama Ww larvae against flacherie causing Pseudomonas aeruginosa AC-3

This study reports for the first time the induction of immunity in Antheraea assama Ww larvae against bacterial flacherie. In silkworms group of disease caused by bacteria are collectively called "flacherie." This refers to the flaccid condition of the larvae due to the infections of bacterial strains pathogenic to muga silkworm. Antibacterial activity against pathogenic Pseudomonas aeruginosa AC-3 causing flacherie, was induced by injection of heat-killed cells of the same strain. Experiments on larval survivability and viable cell count revealed peak immune response on third day. Comparison of the amount of food ingested, excreta produced and larval weight of the saline-injected control, live bacteria-challenged larvae and heat-killed bacteria-injected larvae "(vaccinated)" confirmed the development of immunity against bacterial infection in the "vaccinated" set. The haemolymph of A. assama larvae was analyzed for proteins associated with bacterial infection. Out of the total 32 detected proteins, eleven (A1-2, A15-20, A22-23, and A29) were constitutively synthesized in both the control and live bacteria-injected larvae. Four inducible proteins A4, A9-10, and A21 were detected in the haemolymph of the live bacteria-injected larvae. Synthesis of rest of the proteins varied between the control and their live bacteria-injected counterparts. General protein profile of "vaccinated" larvae injected with live bacteria were found to be similar to that of the saline-injected control.     

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.     

Antibacterial Immune Competence of Honey Bees (Apis mellifera) Is Adapted to Different Life Stages and Environmental Risks

The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.     

An increase in the immune system activity of the wax moth Galleria mellonella and of the Colorado potato beetle Leptinotarsa decemlineata under effect of organophosphorus insecticide

Several aspects of immune response in insects treated with an organophosphate (pirimiphosmethyl) have been assessed. Both humoral (phenoloxidase activity) and cellular (hemocyte count and encapsulation rate) immunity changes have been studied in larvae of the representatives of two insect orders, the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera, Chrysomelidae) and the wax moth Galleria mellonella (Lepidoptera, Pyralidae). A direct contact with sublethal and half-lethal doses of the insecticide results in stimulation of immune reactions: phenoloxidase activity and encapsulation rate are heightened, and hemocyte count increases.     

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC₅), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC₁₅), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.