Dirofilaria immitis: effect on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against microfilariae

[35S]Methionine-labeled hemolymph polypeptides from adult, female Aedes aegypti Liverpool strain mosquitoes inoculated with the microfilariae of the filarial nematode Dirofilaria immitis were compared with those from saline-inoculated and uninoculated controls by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. SDS-PAGE analysis of cell-free hemolymph collected via perfusion at 6, 12, 24, 48, 72, and 96 hr postinoculation (PI) detected the enhanced expression of an 84-kDa polypeptide. This polypeptide, expressed constitutively in the hemolymph of all three groups of mosquitoes, increased considerably in inoculated mosquitoes as time progressed as compared with uninoculated controls. Moreover, the 84-kDa polypeptide was expressed at higher levels in D. immitis-inoculated mosquitoes than in saline-inoculated controls. This stimulation of de novo biosynthesis of the 84-kDa polypeptide in inoculated mosquitoes may play a role in the immune response of mosquitoes. Since it is likely that the wound healing response in insects involves many of the same chemical processes as occur in melanotic encapsulation reactions of mosquitoes against filarial worms, the preferential expression of the 84-kDa polypeptide in saline-inoculated mosquitoes seen in this study may reflect a wound healing response. The greater increase in synthesis of this protein in D. immitis-inoculated mosquitoes may reflect production of melanotic material required for parasite destruction as well as for wound healing.     

Immune competence of Aedes trivittatus hemocytes as assessed by lectin binding

Hemocytes were perfused from uninoculated, saline-inoculated, and Dirofilaria immitis microfilariae-inoculated Aedes trivittatus and assessed for their capacity to bind wheat germ agglutinin (WGA). Approximately one-fourth of perfused hemocytes were WGA positive in all groups of mosquitoes at all time periods tested. Results suggest A. trivittatus has a larger inherent population of immune-competent hemocytes as compared with Aedes aegypti and, therefore, is faster and more effective in killing microfilariae by melanotic encapsulation reactions.     

Hemocyte cell surface changes in Aedes aegypti in response to microfilariae of Dirofilaria immitis

This study involved the assessment of surface changes on hemocytes of Aedes aegypti black-eyed Liverpool strain in association with the melanization response against intrathoracically inoculated Dirofilaria immitis microfilariae (mff). Surface changes on hemocytes were identified using fluorescein-labeled wheat germ agglutinin (WGA). In mosquitoes eliciting a melanization response against inoculated mff, there was a 5-fold increase in the percentages of hemocytes exhibiting WGA binding compared with saline inoculated controls. Relationships of this hemocyte activation in relation to cell-mediated melanization responses of adult mosquitoes against mff are discussed.     

Hemocyte population changes during the immune response of Aedes aegypti to inoculated microfilariae of Dirofilaria immitis

Ultrastructural and lectin-binding studies have established that the melanotic encapsulation reaction of Aedes aegypti Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) is a hemocyte-mediated reaction. Total hemocyte counts from mff-inoculated (= immune-activated), saline-inoculated, and uninoculated female A. aegypti were determined using a hemocoel perfusion technique. Total hemocyte populations in uninoculated mosquitoes were significantly larger in younger mosquitoes, but no significant change was noted as mosquitoes aged beyond 14 days. Hemocyte populations in immune-activated mosquitoes increased from 1 to 3 days postinoculation (PI) and decreased on days 4 and 5 PI. Hemocyte populations at 1 to 4 days PI were significantly elevated in mff-inoculated A. aegypti as compared with saline-inoculated controls. Saline-inoculated mosquitoes displayed little change in total hemocyte numbers from 1 to 5 days PI, and their hemocyte populations were similar to those seen in uninoculated insects of the same age. Experiments involving the inoculation of [3H]thymidine along with mff or saline alone and studies involving the administration of colchicine suggest that increased hemocyte populations in immune-activated A. aegypti are a result of mitotic division of circulating blood cells.     

Quantitative analysis of hemolymph monophenol oxidase activity in immune reactive Aedes aegypti

A hemocyte-mediated melanotic encapsulation reaction is elicited in adult Aedes aegypti in response to intrathoracically inoculated microfilariae of Dirofilaria immitis. The activity of monophenol oxidase in cell-free hemolymph collected from uninoculated, microfilariae-inoculated and saline-inoculated control mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of l-[3,5-³H]tyrosine to dopa. Enzyme activity in immune reactive hosts examined 2 days postinoculation was aproximately twice as high (96–206 nmol/min per mg protein) as in uninoculated or saline inoculated insects (34–80 nmol/min per mg protein). The augmented activity of the enzyme coincides in time with the early development of melanotic capsules around the microfilariae. The possible involvement of hemocytes in the activation and/or generation of monophenol oxidase in response to infection, and the metabolism of catecholamines in relation to insect immune responses are discussed.     

Host defense mechanisms of cephalopods

Humoral and cellular mechanisms of defense have been described for cephalopods, a relatively advanced group of mollusks. Typical of other mollusks, cephalopod agglutinins are the most documented component of humoral immunity. Lectins, which have agglutinating properties, have been described and characterized from octopuses. Agglutinins from cephalopod hemolymph have also been shown to agglutinate a variety of vertebrate red blood cells, as well as potential bacterial pathogens. Hemocytes are the primary component of cellular immunity. Although the hemocyte role in phagocytosis has been extensively studied in other mollusks, the mechanisms of phagocytosis have not been described extensively for cephalopods. Cephalopod hemocytes have phagocytic capabilities and may function in encapsulation and neutralization of foreign substances; however, the effects of environmental factors and the full extent of phagocytic capabilities of cephalopod hemocytes have not been reported. Hemocytes from cephalopods have a role in wound healing and inflammation which have been reported in detail by several investigators.     

Effect of gamma irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.     

N-acetyl-D-glucosamine in crustacean hemocytes; possible functions and usefulness in hemocyte classification

Abstract. The lectin wheat-germ agglutinin (WGA) selectively binds N-acetyl-D-glucosamine. Fluorescence and electron microscopy were used to show that WGA stains the cytoplasmic granules in the granulocytes, but not the hyaline cells, of two decapods, the ridgeback prawn Sicyonia ingentis and the American lobster Homarus americanus. Using fluorescence microscopy, two intermediate stages in granulocyte maturation were observed. Cells smaller than typical small-granule hemocytes were observed with 5 or fewer granules, which in previous studies using brightfield and phase optics were probably counted as hyaline cells. Also, some granulocytes were observed containing both small and large granules, supporting the suggestion that small and large granule hemocytes represent stages in the maturation of one cell line. Granules in the single type of hemocyte in the branchiopod Artemia franciscana did not stain with WGA. The possible roles of N-acetyl-D-glucosamine in wound healing, pathogen encapsulation, and maintenance of normal crustacean connective tissues are discussed.     

Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.     

Infection of the European chafer,Amphimallon majalis,by Bacillus popilliae: Ultrastructure

Electron microscopical studies indicate that midgut alterations observed in larval Amphimallon majalis during Bacillus popilliae invasion were due to infection of epithelium, wound repair, and hemocytic encapsulation. Hemocytes bearing angular cytoplasmic granules formed the capsule which adhered to the basal lamina subjacent to the mesenteronic lesion. Exocytosis of hemocyte granules into bacilli-containing endocytic invaginations of plasma membranes and vacuoles was observed. These hemocytes as well as mesenteronic cells exhibited bactericidal capabilities.     

Secretory protein biosynthesis in snail hemocytes: in vitro modulation by larval schistosome excretory-secretory products

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti

Hemocytes are an essential component of the mosquito immune system but current knowledge of the types of hemocytes mosquitoes produce, their relative abundance, and their functions is limited. Addressing these issues requires improved methods for collecting and maintaining mosquito hemocytes in vitro, and comparative data that address whether important vector species produce similar or different hemocyte types. Toward this end, we conducted a comparative study with Anopheles gambiae and Aedes aegypti. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery, we concluded that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Significantly more hemocytes were recovered from An. gambiae females than Ae. aegypti. However, granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The same hemocyte types were collected from larvae, pupae and adult males albeit the absolute number and proportion of each hemocyte type differed from adult females. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. Two antibodies tested as potential hemocyte markers (anti-PP06 and anti-Dox-A2) also exhibited alterations in staining patterns following immune challenge with the bacterium Escherichia coli.     

Ultrastructure of the melanization response of Aedes trivittatus against inoculated Dirofilaria immitis microfilariae

Scanning and transmission electron microscopy revealed that intrathoracically-inoculated microfilariae (mff) of Dirofilaria immitis elicited a rapid and effective immune response in the hemocoel of Aedes trivittatus mosquitoes. Hemocyte lysis and melanization of inoculated mff began immediately following exposure to the hemolymph environment. Initial melanin accumulation occurred at any site along the surface of mff and rapidly increased in thickness. Hemocyte encapsulation generally described for insects did not occur, but hemocytes might be necessary for activation of the melanization response. Although intact hemocytes were never abundant, those that were present seemed to show an active secretion of membrane-bound vacuoles directed toward mff. Activated hemocytes were in close association, but never in direct contact with the parasite, and were most commonly seen in various stages of lysis. Numerous cell remnants were noted throughout the developing melanin capsule. Parasites were completely melanized by 24 hr postinoculation (PI). By about 3 days PI, a membrane began to form around deposited melanin and hemocyte remnants. This developed into a double membrane-like structure of 25-30 nm thickness and resulted in the enclosure and isolation of the mff, melanin deposits, and cellular remnants from hemolymph components. It is suggested that this membrane functions as a boundary to isolate the melanized parasite and prevents additional hemocyte involvement.     

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.     

Characterization of hemocytes from the yellow fever mosquito,Aedes aegypti

Mosquitoes are the most important arthropod disease vectors, transmitting a broad range of pathogens that cause diseases such as malaria, lymphatic filariasis, and yellow fever. Mosquitoes and other insects are able to mount powerful cellular and humoral immune responses against invading pathogens. To date, most studies have concentrated on the humoral response. In the current study we describe the hemocytes (blood cells) of the yellow fever mosquito, Aedes aegypti, by means of morphology, lectin binding, and enzyme activity and immunocytochemistry. Our light and electron microscopic studies suggest the presence of four distinct hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. We believe granulocytes and oenocytoids are true circulating hemocytes, but adipohemocytes and thrombocytoids are likely adhered to fixed tissues. Granulocytes, the most abundant cell type, have acid phosphatase and alpha-naphthyl acetate esterase activity, and bind the exogenous lectins WGA, HPA, and GNL. Phenoloxidase, an essential enzyme in the melanotic encapsulation immune response, was detected inside oenocytoids. This is, to our knowledge, the first report that has detected phenoloxidase inside mosquito hemocytes at the ultrastructural level. These results have begun to form a knowledge base for our ongoing studies on the function of Ae. aegypti hemocytes, and their involvement in controlling infections.     

Calcium dynamics of hemocytes of the gastropod Biomphalaria glabrata: effects of digenetic trematodes and selected bioactive compounds

Abstract. Two fluorescent calcium indicators, Calcium Green AM (CG) and Fura Red AM (FR), were used in conjunction with confocal microscopy to monitor hemocyte calcium dynamics following exposure to digenetic trematode larvae or relevant bioactive compounds. Changes in intracellular calcium levels, as measured by fluctuations in the CG/FR ratio, were correlated with hemocyte morphological changes. Hemocytes exposed to culture medium remained spread and had few calcium transients. However, following exposure to sporocysts, sporocyst secretory-excretory products, or small rediae of Echinostoma paraensei in culture medium, significantly more hemocytes both rounded up and exhibited calcium transients, though some hemocytes showed one response or the other but not both. Hemocytes did not respond significantly to large rediae, to sporocysts of another digenean ( Schistosoma mansoni ), or to bacterial lipopolysaccharides. Exposure to either zymosan particles or mannose BSA provoked responses similar to those seen with sporocysts of E. paraensei Caffeine caused rounding but no calcium transients, and phorbol myristate acetate provoked calcium transients but no rounding. The results show that sporocysts and small rediae of E. paraensei have pronounced effects on hemocyte rounding and calcium dynamics, and that these two events can occur independently of one another. This suggests that parasites may influence hemocytes in at least two separate ways.     

Excretory-secretory products of Echinostoma paraensei sporocysts mediate interference with Biomphalaria glabrata hemocyte functions.

Miracidia of Echinostoma paraensei were cultured in medium containing 14C-labeled amino acids, allowed to transform into sporocysts, and their excretory/secretory products (E-S) were collected and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Effects of E-S on hemocytes of Biomphalaria glabrata were also assessed. E-S collected during day 1 of culture (E-S1) contained several polypeptides, none of which were labeled, suggesting that E-S1 are largely preformed. E-S1 significantly depressed the ability of hemocytes to phagocytose sheep red blood cells (SRBC), but otherwise had little effect on hemocyte structure or behavior. E-S released by sporocysts in day-2 cultures (E-S2) and in older cultures generally were similar and also contained several polypeptides, many of which were labeled, indicating active synthesis of E-S in vitro. E-S2 strongly inhibited hemocyte uptake of SRBC. Also, hemocytes pretreated with E-S2 assumed a spherical shape and failed to spread normally. E-S obtained through 10 days of culture mediated this effect. Active components of E-S2 were greater than 100 kDa in their native configuration, were heat- and trypsin-labile, and were bound by anti-E-S antibodies. Both greater than 200- and 80-kDa bands were prominent in anti-E-S immunoprecipitates. Hemocytes derived from snails of the 13-16-R1 strain of B. glabrata (a strain resistant to infection with Schistosoma mansoni), when pretreated with E-S2, bound to sporocysts of S. mansoni but lost their ability to damage such sporocysts. E-S2 interfered with hemocyte functions in ways inferred from earlier classic in vivo studies of trematode-snail interactions.