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THE EFFECT OF A POLYDNAVIRUS OF APANTELES GALLERIAE ON THE HEMOLYMPH PROTEINS OF HOST GALLERIA MELLONELA

Abstract

The presence of a polydnavirus in the ovary of the microhymenoptera Apanteles galleriae was detected. This wasp parasites the larvae of the wax moth Galleria mellonella. To study the possible viral effects on the total protein of G.mellonella, hemolymph extracts of the ovary calyxes of parasitized A. galleriae were injected into 6th and 7th instar cells of G.mellonella larvae. Protein profiles on polyacrilamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) of host cell extracts showed a suppression of several protein bands when infection occurred in the 6th instar. However, in the 7th instar, parasitism caused an amplification in the synthesis of most proteins with some changes in the electrophoretic profile.

microhymenoptera; wax moth; parasitism; polydnavirus; SDS-PAGE; Apanteles galleriae; Galleria mellonela


Original paper

THE EFFECT OF A POLYDNAVIRUS OF APANTELES GALLERIAEON THE HEMOLYMPH PROTEINS OF HOST GALLERIA MELLONELA

M.R. BROCHETTO-BRAGA

CORRESPONDENCE TO: M.R. BROCHETTO-BRAGA - Departamento de Biologia, Instituto de Biociências - UNESP, Caixa Postal 199 - CEP 13.506-900 - Rio Claro, São Paulo, Brasil. , M.S. PALMA , J.C. RIBEIRO , N. GOBBI2,3

1 Department of Biology of the Institute of Biosciences of Rio Claro - UNESP, State of São Paulo, Brazil, 2 Department of Ecology of the Institute of Biosciences of Rio Claro - UNESP, State of São Paulo, Brazil, 3 Center for the Study of Venoms and Venomous Animals - CEVAP - UNESP, State of São Paulo, Brazil.

ABSTRACT. The presence of a polydnavirus in the ovary of the microhymenoptera Apanteles galleriae was detected. This wasp parasites the larvae of the wax moth Galleria mellonella. To study the possible viral effects on the total protein of G.mellonella, hemolymph extracts of the ovary calyxes of parasitized A. galleriae were injected into 6th and 7th instar cells of G.mellonella larvae. Protein profiles on polyacrilamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) of host cell extracts showed a suppression of several protein bands when infection occurred in the 6th instar. However, in the 7th instar, parasitism caused an amplification in the synthesis of most proteins with some changes in the electrophoretic profile.

KEY WORDS: microhymenoptera, wax moth, parasitism, polydnavirus, SDS-PAGE, Apanteles galleriae, Galleria mellonela.

INTRODUCTION

It has been recognized that all females of several species of parasitoid wasps carry virus in their ovaries for successful host parasitization. Polydnaviruses (PDV) replicate into the ovaries of braconid and ichneumonid endoparasitoid wasps and are injected into their hosts during oviposition with a venom fluid (1,11,16,17).

It has been shown that in lepidopteras parasitized by endoparasitoid wasps, with the polydnavirus in their reproductive tract, viral gene expression is detected inducing immunosuppression and altering host development(4,6,9,18). These physiological and developmental alterations have been accompanied by major alterations in the protein content of host larval hemolymph beginning a few hours after host parasitization (1,5,7). These alterations may also occur in the late stages of wasp development inside the host(2), or they may increase 4h post-parasitism and the expression continues for six more days(18).

In the present study, we detected the presence of a PDV in the ovary of the microhymenoptera Apanteles galleriae which parasites the wax moth larva of Galleria mellonela. By using polyacrilamide gel electrophoresis, (SDS-PAGE) we compared the profile of hemolymph protein synthesis of G.mellonella in the virus presence and absence.

MATERIAL AND METHODS

Caterpillars of the host G.mellonella (Lepidoptera, Pyralidae) were reared in plastic boxes at 28°C (with 14-hour light/10-hour dark photoperiod) and fed with an artificial diet(10). Adults of A. galleriae were obtained from caterpillars parasitized in laboratory and the ovary calyxes were dissected on the same day of emergence.

Calyxes extracted from 23 female A. galleriae were kept in insect physiological solution during the experiment. The calyx extract was obtained by sonication and centrifugation at 6000g for 15 minutes at 4°C for removal of cell remains. The supernatant was used for microinjection (5 µl) in the 4th abdominal segment of 6th and 7th instar G. mellonela. A microinjection of physiological solution (5 µl) was given as control.

Eighteen and 36 hours later, the caterpillars were killed by freezing and the 3rd, 4th and 5th abdominal segments were macerated to obtain the cell extract which was also submitted to sonication and centrifugation, as described above. Total protein determination of calyx extracts and abdominal segments was carried out by the method of Sedmak & Grossberg (13).

The calyx extracts, including those of virus-infected hosts, and the control extracts were submitted to SDS-PAGE at 12.5% acrylamide according to Laemmli(12). Bovine albumin (66.0 kDa), ovalbumin (45.0 kDa) and ribonuclease (14.5 kDa) were used as molecular weight (MW) patterns. After electrophoresis, gel was stained with Coomassie Blue G-250 in methanol: acetic acid: water (5:2:5) and de-stained with the same mixture without the dye. In all cases, the same amount of total protein (20 µg) was applied in gel lanes.

RESULTS AND DISCUSSION

It has been shown that a number of physiological effects of endoparasitism might be induced in nonparasitized insects by injecting the calyx fluid of parasitoid ovary with PDV(1), so that the alterations may be better characterized. Thus, several electrophoretic studies have been carried out to detect alterations in parasitism-induced hemolymph protein composition using parasitoids Apanteles sp (1,3,11,14).

In the present work, by injecting ovary calyx extracts of A. galleriae in 6th and 7th instar G. mellonela, we analyzed endoparasitism effects using SDS-PAGE technique. The profiles obtained for parasitized G. mellonela extracts in comparison with nonparasitized control larvae are shown in Figure 1. Great physiological effects might have been seen through great alterations in the electrophoretic profiles or intensity of proteins bands. However, the results showed that, in relation to control, only slight modifications (observed as differences in intensity) occurred, and apparently, no specific viral protein was expressed. Such slight alterations in intensity were conventionally named here "Amplification" or" Supression" of protein synthesis, as the total protein content applied in all gel lanes was always the same.

FIGURE 1.
Protein profile on SDS-PAGE of extracts of larval segments (3rd, 4th and 5th) of 6th and 7th instar Galleria mellonela associated with parasitoid Apanteles galleriae for 18 and 36h (V): calyx extract containing polydnavirus (PDVs) used for microinjections: C6-18, C6-36, C7-18, C7-36, represent, respectively, extracts of 6th and 7th instar nonparasitized G.mellonella, after 18 and 36h; E6-18, E6-36, E7-18, E7-36 represent, respectively, the extracts of 6th and 7th instar parasitized G.mellonella for 18 and 36h. On the right side of the figure: MWs pattern. On the left side: MWs for proteins analyzed in the experiment.

In the 6th instar, we observed suppression of some protein bands at 18-hour parasitism, among them those of MWs 42.0 and 52.0 kDa are shown in Figure 1. In contrast, regarding to the 7th instar, an amplification of protein synthesis was observed mainly 36h after parasitism, the most intense being those for MWs 49.0, 45.0, 42.0 and 15.0 kDa. The lowest suppression intensity (6th instar), as well as the highest amplification intensity (7th instar) in the host 36h post-parasitism (Figure 1 and Table 1) might indicate different host response pattern during its development when the virus was present.

TABLE 1.
Analysis related to control experiment of some of the most marked results obtained with SDS-PAGE for suppression/amplification of protein synthesis.

Amplification of band proteins of the gel: (+++) strong; (++) moderate; (+) slight.

Supression of band proteins of the gel: (---) strong; (--) moderate; (-) slight.

* = presence; a = absence of protein band.

In this experiment, only the most intenses protein bands were analyzed. However, a comparison between control and experimental results obtained with SDS-PAGE, showed some differences regarding suppression or amplification patterns of protein synthesis during larval development (6th and 7th instars) associated with the virus, that is: a) some specific proteins were more intensily suppressed at 18h rather than 36h post-parasitism, such as 52.0 and 42.0 kDa proteins; b) the highest amplification intensity for 7th instar proteins occurred at 36h post-parasitism, as it was observed for MWs 49.0, 45.0, 42,0 and 15.0 kDa proteins.

The presence of a MW 42.0 kDa band, which was intensively suppressed in the 6th instar after 18 and 36h post-parasitism and then strongly induced in the 7th instar under the same conditions, might indicate the existence of two different proteins of equal MW, probably involved in specific biological events at each instar of larval development.

The patterns observed here for proteins of the extract of host segments with the virus corroborate with the early reports about the interaction between viral DNA and that of the host(8,20,21). If such interaction used to occur differently in the various stages of host development, then this could explain our different patterns of protein synthesis supression observed in the present research. Such suppression was slightly higher in the 6th instar, and this result agreed with the fact that G.mellonella development is disrupted by the virus in parazitized 5th instar larvae, in spite of the fact that the larva remained alive. On the other hand, this interaction also seemed to allow a lower level of amplification of protein synthesis in the 6th instar (18 and 36h post-parasitism) in contrast with that in the 7th instar (intensification of 1 band in the 6th instar versus 4 bands in the 7th instar), which could indicate that, probably in this stage virus influence on protein synthesis supression at the 6th instar is likely to be higher in the host. Furthermore, by the gel analysis (Figure 1), it was observed that MWs 52.0, 45.0, 49,0 and 42.0 kDa bands, in contrast with that of MW 15.0 kDa, were already present in 7th instar control larvae and were intensified by the virus, suggesting that these proteins might take place in viral infection.

Intensification of MWs 69.0, 66.0, 64.0 and 60.0 kDa bands, in those of 7th instar parasitized extracts (E7-18 and E7-36) was certainly due to the presence of the virus because it was observed that the calyx fluid extract presented these same protein bands (Figure 1 - lane V).

Our preliminary results provided additional evidence that polydnaviruses were capable of inducing significant physiological alterations in lepidoptera, which undoubtedly might be associated with suppression of host defense mechanisms, as suggested by several authors(3,14,15,18,19). In this paper, we have shown that some alterations may be monitored in the 6th and 7th instars of G.mellonella when associated with parasitoid wasps A.galleriae. However, for a better characterization of these alterations more detailed studies are needed.

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  • CORRESPONDENCE TO:
    M.R. BROCHETTO-BRAGA - Departamento de Biologia, Instituto de Biociências - UNESP, Caixa Postal 199 - CEP 13.506-900 - Rio Claro, São Paulo, Brasil.
  • Publication Dates

    • Publication in this collection
      08 Jan 1999
    • Date of issue
      1995
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