The antioxidants dimethylsulfoxide and dimethylthiourea affect the immediate adhesion responses of larval haemocytes from 3 lepidopteran insect species

Antioxidants, dimethylsulfoxide (DMSO) and dimethylthiourea (DMTU), at concentrations not affecting the viability of blood cells (haemocytes) from the larval stage of 3 lepidopteran insects - Galleria mellonella, Lymantria dispar, and Malacosoma disstria - differed in their influence on the innate binding of haemocytes to glass, bacteria to haemocytes, and on humoral responses to alien materials. In vitro DMSO had little effect, whereas DMTU substantially impaired the adhesion of the haemocyte types, the plasmatocytes and granular cells, to slides as well as the attachment of Bacillus subtilis to these haemocytes. Although both antioxidants increased lysozyme and phenoloxidase activities, there was no correlation of enzyme activity and haemocyte adhesion responses, possibly reflecting sequestered radicals. Nitric oxide and hydroxyl radicals offset the DMTU effect. In the absence of antioxidants, inactivate protein kinases A (PKA) and C (PKC) enhanced haemocyte aggregation. In general, DMSO, as opposed to DMTU, did not alter the effects of PKA and PKC activators and inhibitors on haemocyte aggregation or of PKC and PKA activities. High concentrations of DMSO and all levels of DMTU, although inhibiting PKA and PKC, inhibited haemocyte adhesion to slides. Comparable results occurred for DMTU-treated haemocytes incubated with B. subtilis. In vivo DMSO, unlike DMTU, did not impair plasmatocyte or granular cell responses to foreign materials, including bacterial removal from the haemolymph and nodulation.     

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells

Experiments indicate that the prophenoloxidase activating system, which is responsible for melanin production, is also involved in immunorecognition in insects. Using haemocyte monolayer preparations of Blaberus craniifer, Galleria mellonella and Leucophae maderae, it was shown that laminarin, a β 1,3-glucan extracted from fungal cell walls and an activator of the prophenoloxidase system, enhanced the phagocytosis of test bacteria.Scanning electron microscopy of haemocyte monolayers showed that incubation of test bacteria with laminarin significantly increased the number of microorganisms attached to both the plasmatocytes and the granular cells. Furthermore with the granular cells, these bacteria became entrapped in an amorphous matrix. This material probably consists of the “sticky” proteins previously reported to be produced by crustacean haemocytes following prophenoloxidase activation. Pretreatment of haemocytes with laminarin abolished the stimulatory effect on ingestion, indicating that these “sticky” proteins are opsonic, since they would have been discharged from the haemocytes onto the glass monolayer leaving few molecules available for subsequent coating of the test particles.Preliminary biochemical studies on the G. mellonella prophenoloxidase system demonstrated that it was activated by trypsin, laminarin and laminarin G, a highly purified β 1,3-glucan, but not by dextran. Serine protease activities were also enhanced by adding laminarin to a haemocyte lysate supernatant, suggesting that the stimulatory mechanism may involve the proteolytic activity of such enzymes.     

Haemocytes of larval Malacosoma disstria (Lepidoptera: Lasiocampidae) and factors affecting their adhesion to glass slides

Abstract.  Although haemocytes of the forest pest lepidopteran, Malacosoma disstria (L.) have been studied, the physico-chemical factors and signalling components affecting their non-self activities have not been examined. Both the ameboid and stellate forms of plasmatocytes and the granular cells from fifth-instar larvae adhere best to glass slides with phosphate-buffered saline (PBS), with maximum granular cell binding within a pH range of 6.0–7.0 and plasmatocyte binding at pH 6.0. The divalent cations, calcium and magnesium, do not affect granular cell attachment. However, calcium in Galleria -anticoagulant and PBS and, to a lesser extent, magnesium in the anticoagulant, increase plasmatocyte-glass contact. Based upon the use of selective type I protein kinase A inhibitor (Rp-8-Br-cAMPS) and activator (Sp-8-Br-cAMPS), active protein kinase A inhibits the adhesion of both haemocyte types. Similarly, protein kinase C inhibited by Gö 6976 enhances haemocyte adhesion whereas the enzyme activator, phorbol-myristate-acetate, impairs attachment.     

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.     

Haemocyte population and ultrastructural changes during the immune response of the mosquito Culex quinquefasciatus to microfilariae of Wuchereria bancrofti

Abstract Haemocytes circulating in the haemolymph protect insects against pathogens that enter the haemocoel. Changes in haemocyte morphology and differences in haemocyte counts during the immune response of Culex quinquefasciatus Say (Diptera: Culicidae) to microfilariae of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) were investigated in the present study. The mean number of total haemocytes was significantly elevated in infected mosquitoes (P < 0.001), reaching a peak on the third day post‐infection. Differential counts show that mean numbers of prohaemocytes, plasmatocytes, granular cells and oenocytoids increased significantly after infection with microfilariae granulocytes compared to the control and näive groups of Cx. quinquefasciatus (P < 0.05). Changes in proportional counts of haemocytes were also analysed in haemolymph perfusates of Cx. quinquefasciatus infected with W. bancrofti. On the first day post‐infection, infected mosquitoes showed an increase in the proportion of prohaemocytes (18.8% compared to 9.6% for the control) and of oenocytoids (7.1% compared to 4.7% control); however, they exhibited lower levels of plasmatocytes (36.6% compared to 42.1% control) and granular cells (36.1% compared to 41.4% control). On day 14 post‐infection, similar changes were observed for these haemocyte types, except that the proportion of granular cells was significantly greater than the control (41.2% compared to 31.3% control). Although an enhancement of prohaemocyte numbers was observed, this cellular type did not show any ultrastructural alteration. On the other hand, granular cells, plasmatocytes and oenocytoids presented morphological alterations indicative of innate immunological activation in mosquitoes infected with W. bancrofti.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.     

Impact of insecticides on the haemogram of Rhynocoris kumarii Ambrose and Livingstone (Hem., Reduviidae)

Abstract:  The haemogram of Rhynocoris kumarii Ambrose and Livingstone comprises prohaemocytes, plasmatocytes, granular haemocytes, cystocytes and oenocytoids. The impact of five insecticides, viz. monocrotophos, dimethoate, methylparathion, quinalphos and endosulfan on the total haemocyte count (THC) and differential haemocyte counts (DHC) was studied. All of the insecticides except endosulfan initially reduced both prohaemocytes and plasmatocytes, increased the granular haemocytes, altered the percentage of cystocytes and oenocytoids and increased the total haemocyte count (THC). On the contrary, endosulfan initially increased the prohaemocytes and plasmatocytes, decreased the granular haemocytes and also the THC. The highest impact on the DHC and THC was caused by methylparathion and monocrotophos and the least impact by endosulfan. Hence, endosulfan is considered as the safest insecticide followed by dimethoate and quinalphos among these five insecticides to use with R. kumarii .     

Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses.     

The induction of an injury reaction in cultured haemocytes from saturniid pupae

The plasmatocytes of diapausing saturniid pupae are round or spindle-shaped cells floating free in the haemolymph. Upon injury to the pupa, these haemocytes become amoeboid and adhesive. A technique is described for the isolation and short-term culture of pupal haemocytes in their inactive state and for their conversion in vitro into the active, ‘injured’ form. The activation of ‘uninjured’ haemocytes was stimulated by fragments of epidermal tissue or by plasma from previously activated blood samples. A fraction capable of stimulating the activation of haemocytes was partially purified from both plasma and epidermal tissue and has been called haemokinin. Haemokinin from either source has a molecular weight in the range of 50,000. The intensity of the haemocyte injury reaction in vitro changes systematically during diapause; the nature and significance of the changes are discussed.     

The encapsulation of foreign tissue implants in Galleria mellonella larvae.

When implanted into the haemocoel of the wax moth, Galleria mellonella, fragments of nerve cord from the locust Schistocerca gregaria were shown to be encapsulated by host haemocytes, in a two phase system. The initial reaction to the implant was observed within 5 min of implantation and involved the contact and lysis of the granular haemocytes on the nerve cord, and the release of granular material around it which resulted in localised clot formation.The second phase involved plasmatocyte adhesion to the implant ca. 30 min after its introduction into the haemocoel, but only at sites of granular cell lysis. These cells continued to be added to the capsule until 60 to 72 hr after implantation, and during this time granular release continued, although no further granular cell lysis occurred. The complete capsule (72 hr) could be divided into three distinct regions of cells showing various modifications from free plasmatocytes.It is suggested that encapsulation in this species involves some chemotactic element which originates from the granular cells and acts specifically on the plasmatocytes.     

Behaviour in vitro of separated fractions of haemocytes of the locust Schistocerca gregaria

Summary The haemocytes of locusts (Schistocerca gregaria) have been partially separated, by centrifugation on discontinuous Percoll gradients, into 3 subpopulations of plasmatocytes of which the most dense (band 5) displays more than 95% morphological homogeneity. The cells of band 5 are very granular, adhere and spread on glass, and contain nearly all the phenoloxidase activity of the total haemocyte population, as shown by 1,3-glucan (laminarin)-stimulation of both haemocyte lysate supernatant and monolayers of living cells. Cells from band 5 show negligible endocytosis of fluorescent latex beads, whereas those of band 4, which are less granular, plasmatocyte-like cells, are actively endocytic in vitro. The majority of the haemocytes are found in band 3, which is a mixture of coagulocytes and agranular plasmatocytes, with negligible phenoloxidase activity. The in vitro locomotory behaviour of adherent cells from bands 3 and 5 was compared, and addition of laminarin-activated haemocyte lysate supernatant, in which the prophenoloxidase activation sequence had been initiated, stimulated chemokinesis in cells of band 5 but not band 3. The separated fractions thus show marked behavioural and biochemical differences.     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.     

Immunological analysis of apolipophorin-III in the haemolymph,ovaries, and testes of the fall webworm,Hyphantria cunea (Drury)

Apolipophorin-III (apoLp-III) was purified from the haemolymph of adult Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation, gel filtration (Sephadex G-100) and ion exchange chromatography (CM-52), and its characteristics, molecular weight, tissue distribution, and sites of synthesis were examined. Molecular weight of apoLp-III was estimated to be 18 kDa. By electrophoretic analysis on 10% gels of male and female haemolymph from diverse developmental stages, apoLp-III was shown to be present in all stages. Western blotting was carried out to show that purified free apoLp-III is identical to apoLp-III associated with adult lipophorin. Immunological analysis also showed that apoLp-III is present in the ovary and the testis and in the case of testis, apoLp-III is heavily accumulated in the cyst. ApoLp-III is synthesized in larval and adult fat body but not in adult testis. Autoradiography following incubation of [¹⁴C]apoLp-III with testis showed that apoLp-III was taken up into testis. © 1996 Wiley-Liss, Inc.     

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.     

Die phenoloxydase von Chironomus thummi und ihre beeinflussung durch parasitäre Mermithiden

A dihydroxyphenoloxidase is found in the haemolymph of Chironomus thummi. By means of a substrate-impregnated agar a localization of the activity could be shown inside and outside the haemocytes. Granular haemocytes rich in lysosomes which are able to undergo autolysis are probably responsible for activation of the enzyme. Parasitism by mermithide nematodes reduces the activity of the phenolase to one-third, the number of ‘granular transitional haemocytes’ is reduced (11·6 per cent), and the number of plasmatocytes is increased (11·4 per cent). The rôle of the enzyme in the defence reaction is discussed.     

Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella

Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph. The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity. Iron augmentation returned these activities to control levels. Whereas dead B. subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X. nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased. Haemocyte lysate lowered the concentrations of iron in both FeCl₃ solutions and deproteinized larval serum independent of serum lipids. Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X. nematophila. X. nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.     

Differential adhesion of the haemocytes of Leucophaea maderae (Blattaria) to a glass surface

A system for the study of insect haemocytes in vitro is described. The system was used to analyse the adhesive properties of the haemocytes of the cockroach, Leucophaea maderae. The two main types of haemocytes, plasmatocytes and granulocytes, showed considerable differences in adhesive properties, which allowed the production of nearly homogeneous monolayers consisting of either plasmatocytes or granulocytes. The much stronger adhesion of the plasmatocytes is discussed in relation to their role in phagocytosis and encapsulation.