Observations on the Presence of the Peritrophic Membrane in LarvalTrichoplusia niand Its Role in Limiting Baculovirus Infection

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

72, 57–62 (1998)

IN984759

Observations on the Presence of the Peritrophic Membrane in Larval Trichoplusia ni and Its Role in Limiting Baculovirus Infection Ping Wang*,† and Robert R. Granados† *Department of Entomology and †Boyce Thompson Institute, Cornell University, Ithaca, New York 14853 Received July 24, 1997; accepted January 20, 1998

Baculovirus enhancins are a group of proteins encoded and carried by some baculoviruses. They are known to be capable of significantly increasing the efficacies of virus infection in insects (see review by Corsaro et al., 1993). To date, the mechanism(s) for increased larval infection due to enhancin are still being determined and several modes of action for enhancins have been proposed at various steps during virus–host interactions. These proposed modes of action include that: (1) enhancin is a binding molecule mediating the binding of the baculovirus to host cells (Tanada, 1985; Uchima et al., 1988, 1989), (2) enhancin increases the fusion of the baculovirus envelope to the host cell plasma membrane (Kozuma and Hukuhara, 1994), and (3) enhancin degrades proteins of the midgut peritrophic membrane, disintegrating the protective peritrophic membrane and facilitating the passage of virus from the midgut lumen to the intestinal epithelial cells (Derksen and Granados, 1988; Wang et al., 1994; Wang and Granados, 1997a). Studies with enhancin from Trichoplusia ni granulosis virus (TnGV) have shown that the enhanced infections were correlated with the degradation of PM proteins (Derksen and Granados, 1988; Wang et al., 1994). However, enhancement of larval infection did not correlate with specific binding of TnGV enhancin to the brush border membranes from four noctuid species tested (Wang et al., 1994). Furthermore, recent studies (Wang et al., 1997) on the binding and fusion of AcMNPV to SF 21 cultured cells showed that TnGV enhancin did not have an effect on these two events. Enhancin was determined to be a metalloprotease (Lepore et al., 1996). Three enhancin genes have been cloned and sequenced (Roelvink et al., 1995) and an enhancin expressed from a recombinant AcMNPV was active against PM proteins (Lepore et al., 1996). Recently, a high-molecular-weight PM protein from T. ni was identified as the first invertebrate intestinal mucin (Wang and Granados, 1997a). This mucin is the major protein component of the PM and is designated as insect intestinal mucin (IIM). The IIM has similar biochemical characteristics to vertebrate mucins (Wang

Light microscopical examinations of dissected and stained peritrophic membranes (PMs) were conducted to determine the presence or absence of this protective structure in larvae of Trichoplusia ni, prior to and through ecdysis. Observations of fourth- and fifthinstar larvae of T. ni from two independent rearing colonies showed that PMs were present and lined the midgut prior to, during, and immediately after ecdysis in both instars. Western blot analysis of insect intestinal mucin (IIM), a major protective protein in the T. ni PM, indicated that synthesis of IIM occurred during T. ni embryonic development, or more precisely, that IIM synthesis started approximately 4 h prior to hatching. These results demonstrated that the neonate T. ni midgut is lined with a protective mucinous layer at hatching. A baculovirus enhancin from T. ni granulosis virus (TnGV) enhanced per os viral infections of budded viruses (BVs) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and T. ni single nuclear polyhedrosis virus (TnSNPV) in neonate, fourth-, and fifth-instar larvae of T. ni. These results provided further evidence that the PM may serve as a partial barrier to viruses in the midgut lumen and that enhancins can facilitate the infection process. r 1998 Academic Press

Key Words: Autographa californica MNPV; baculovirus; budded virus; enhancin; peritrophic membrane; Trichoplusia ni SNPV.

INTRODUCTION

Baculoviruses represent the largest group and most broadly studied insect viruses. It has long been believed that baculoviruses are potentially useful as safe biological control agents, and in some cases they have been used successfully to control insect pests (Huber, 1986; Wood and Granados, 1991). However, the overall use of baculoviruses for biological control is limited, compared to other pest control means (Wood and Granados, 1991; Wood, 1996). Improving the potency and efficacy of baculoviruses is one focus of the development of modern baculovirus pesticides (Miller, 1995; Wood, 1996). 57

0022-2011/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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and Granados, 1997a,b), which are well known for their protective functions in vertebrate intestines (Forstner and Forstner, 1994). TnGV enhancin shows proteolytic activity to IIM both in vitro and in vivo, and the extent of in vivo degradation of IIM was correlated with the level of increased virus infection (Wang and Granados, 1997a). Although these findings further demonstrated the site of action of enhancin on the PM, Washburn et al. (1995) suggested that the PM in larvae of T. ni provided little protection against AcMNPV infection, based on their observation of the absence of a PM in newly molted fourth-instar larvae of T. ni. In this paper, we report our observations which show the presence of a PM prior to, during, and immediately after ecdysis in two T. ni larval instars in independent rearing colonies. Baculoviruses have two phenotypes, occluded virus (OV) and budded virus (BV), which have distinctly different functions in pathogenesis (Blissard and Rohrmann, 1990). AcMNPV BV for example, is more than 10,000-fold less infectious per os compared to per os infection with OV (Keddie and Volkman, 1985). Prior studies on the enhancement of NPV infections to insect larvae have been restricted to the use of NPV occlusion bodies (OBs) containing the OV phenotype (Tanada, 1985; Goto, 1990; Gallo et al., 1991; Wang et al., 1994). There are some reports suggesting that some NPV OBs may contain an endogenous enhancing factor as well (Derksen and Granados, 1988; Bischoff and Slavicek, 1997). However, BVs are virus particles which bud through the cell plasma membrane and are assumed to be free of any enhancing factor which might be present in the OBs. Therefore, the determination of enhancin activity on per os infection by BVs can rule out the possible interference resulting from a potential endogenous AcMNPV enhancing factor carried by the OB inoculum and thus may provide more precise information on understanding the mode of action for enhancins. MATERIALS AND METHODS

Insects A laboratory colony of T. ni was reared on high wheat germ artificial diet and used for all bioassays (Gallo et al., 1991). To determine the presence of PMs, larvae from our Boyce Thompson Institute laboratory colony, and larvae obtained from Entopath, Inc. (Easton, PA), were examined. The larvae obtained from Entopath were reared under the same conditions as the Boyce Thompson Institute colony. Viruses An AcMNPV isolate, strain 1A (Wood, 1980), was used in this study. AcMNPV was amplified in SF21 cells

in TNM-FH medium containing 10% fetal bovine serum. AcMNPV BV containing infectious medium was collected at 36 h postinfection (p.i.) before cell lysis occurred. Virus OBs and cells were removed by lowspeed centrifugation. BV infectious medium, following centrifugation, was microscopically determined to be free of OBs and visible cell debris, and subsequently titered in SF21 cells. The BV titer was determined to be 3.0 3 108 plaque forming units (pfu)/ml. This infectious medium was used as inoculum for bioassays. TnSNPV was produced in BTI-Tn5B1–4 cells (Granados et al., 1994) in TNM-FH medium containing 10% fetal bovine serum. The infectious medium was collected at 3 days p.i. and prepared as described above for AcMNPV BV. The TnSNPV BV inoculum had an infectivity titer of 6.7 3 107 pfu/ml as determined with BTI-Tn5B1–4 cells. TnGV and TnGV enhancin were prepared following the procedures described by Wang et al. (1994). Observation of PMs in Larvae of T. ni during Ecdysis Larvae of T. ni were reared on artificial diet at 28°C. As the larvae reached particular stages of ecdysis (i.e., from third instar to fourth instar and from fourth instar to fifth instar), their transition was closely monitored. At stages shortly before and immediately after ecdysis, the larvae were carefully dissected in water under a dissecting microscope. The PMs were carefully dissected from the midgut and stained with 0.1% Congo Red. Detection of IIM Synthesis during T. ni Embryonic Development Trichoplusia ni eggs were collected on Parafilm over a 1-h period at peak egg laying times for T. ni adults and incubated at 28°C. At time points of 0, 12, 24, 36, 48, 60, and 64 h after egg collection, 50 eggs or neonate larvae (from the 64-h collection) were collected and placed at 270°C. For posthatching larval samples, neonate larvae were placed on artificial diet and incubated at 28°C. At 4 and 24 h after feeding on artificial diet, 50 larvae were collected and stored at 270°C. IIM synthesis during embryonic development was determined by Western blot analysis using a rabbit anti-IIM antiserum (Wang and Granados, 1997a). For Western blot analysis, eggs or first-instar larvae collected at different developmental stages were homogenized in 100 µl of SDS–PAGE sample buffer (0.0625 M Tris-HCl, pH 6.8, 2% SDS, 5% b-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) and boiled for 5 min. Five microliters of the supernatants of these embryo/

ENHANCEMENT OF PER OS INFECTIONS OF NPV BVs

larval extracts was separated by SDS–PAGE. Proteins from the gels were transferred to blotting membranes. IIM was detected using anti-IIM antiserum with a secondary antibody conjugated to alkaline phosphatase. Bioassays Trichoplusia ni neonate larval bioassays performed with AcMNPV BV in the presence or absence of TnGV enhancin, were conducted as previously described by Gallo et al. (1991). Briefly, 2 to 4-h-old T. ni neonate larvae were inoculated with a virus suspension by the droplet feeding method (Hughes et al., 1986). All neonate larvae imbibed approximately 10 nl of inoculum (Hughes et al., 1984). The larvae that ingested inoculum were selected and individually reared on artificial diet. Mortality from virus infection was recorded at 4 days p.i. Fourth- and fifth-instar T. ni larval bioassays for both AcMNPV BV and TnSNPV BV infections were performed following the procedure described by Wang et al. (1994). Briefly, virus inoculum was applied to artificial diet disks approximately 5 mm in diameter and 1 mm in height. Mid-fourth- or mid-fifth-instar larvae were then allowed to feed on the disks. Larvae that ingested the entire diet disks within 16 h were transferred to individual rearing cups containing fresh artificial diet and incubated at 28°C. Virus infections were scored when noninfected control larvae pupated. RESULTS

Presence of PM before and after Ecdysis Our observations on the presence of PMs in the midgut of larvae of T. ni clearly showed that PMs exist

59

pre and postecdysis, in larvae of T. ni molting from third to fourth instar and from fourth to fifth instar. At both time points, the dissected PMs were translucent and difficult to see under a dissecting microscope without proper illumination. Staining the translucent PMs with Congo Red, a dichrotic dye that is commonly used for PM staining (Peters, 1992), assisted in the identification of these structures. At each time point approximately 30 to 40 larvae were examined. Figure 1A shows a larva shortly before ecdysis from third to fourth instar. The larva at this particular stage contained a PM in the midgut (Fig. 1B); furthermore, a complete PM could be isolated from this larva (Fig. 1C). At a stage immediately following ecdysis from third to fourth instar (Fig. 1D), larvae of T. ni were found to contain a complete PM in the midgut as well (Figs. 1E and 1F). Dissections of larvae shortly before and immediately after ecdysis from fourth to fifth instar were similar to the observations illustrated in Fig. 1 (data not shown). No differences in the presence of a PM were observed between larvae from our laboratory colony and a colony obtained from Entopath, Inc. Synthesis of IIM during Embryonic Development Western blot analysis of extracts from T. ni eggs at different embryonic development stages (Fig. 2), showed that IIM synthesis started in 60-h-old embryos when the dark head capsules of the larvae could be seen in the eggs. Newly hatched neonates already had IIM in an amount comparable to that found in larvae feeding on diet for 4 h (Fig. 2). Dissection of first-instar larvae of T. ni and electron microscopical observation confirmed the presence of a PM in the midgut throughout the instar (data not shown).

FIG. 1. Presence of the peritrophic in the midgut of larvae of T. ni prior to and throughout ecdysis. (A) A T. ni larva at the stage prior to ecdysis into the fourth instar. (B) The partly opened midgut from the larva shown in (A) confirming the presence of a PM in the midgut. (C) The PM isolated from the larva shown in (A) and stained with Congo Red. (D) A T. ni larva immediately after ecdysis into the fourth instar. (E) Presence of PM in the midgut of the larva shown in (D). (F) The PM isolated from the larva shown in (D) and stained with Congo Red. (M, midgut; PM, peritrophic membrane; Ex, exuviae).

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larvae was increased from 2 to 23% and 27 to 52%, in two separate experiments (Table 2). Enhancement of TnSNPV BV per os Infection in Fourth-Instar Larvae of T. ni TnSNPV BV per os infection in larvae of T. ni demonstrated that larval mortality was increased when the TnSNPV BV was coinoculated with 1 µg/larva of TnGV enhancin (Table 3). Addition of 1 µg/larva of enhancin resulted in increases of TnSNPV BV per os infection from 72 to 94% at 3.3 3 105 pfu/larva, and from 43 to 93% at 1.3 3 105 pfu/larva (Table 3). FIG. 2. Detection of insect intestinal mucin (IIM) synthesis during T. ni embryonic development by Western blot analysis using anti-IIM antiserum. The left lane is purified IIM used as a control. The arrowhead indicates IIM.

Enhancement of AcMNPV BV per os Infection in Larvae of T. ni Per os inoculation of T. ni neonate larvae at virus concentrations of 1.3 3 108 pfu/ml and 2.4 3 108 pfu/ml, which were equivalent to dosages of about 1300 and 2400 pfu/larva, did not cause any infection (Table 1). However, in the presence of TnGV enhancin, these same virus doses showed viral enhancement and caused 13 and 17% larval infection, respectively (Table 1). Similar results with AcMNPV BV per os infections were observed in fourth- and fifth-instar larval bioassays (Table 2). AcMNPV BV per os inoculation of fourthinstar larvae of T. ni with 1.5 3 106 pfu/larva, resulted in 14 and 28% infection in replicate experiments. Addition of 1 µg/larva of TnGV enhancin to the same dose of virus resulted in increased infection of approximately 68% (Table 2). Bioassays using fifth-instar larvae also showed increased AcMNPV BV per os infection with the addition of enhancin. The percentage infection in the presence of TnGV enhancin at 1 µg/ TABLE 1 AcMNPV BV Infection in T. ni Neonate Larvae with TnGV Enhancin a Inoculum Experiment

Virus

Enhancin

No. of insects % P tested Infection value b

1

1300 pfu/larva 0 1300 pfu/larva 4.4 ng/larva

30 30

0 13

6

0.04

2

2400 pfu/larva 0 2400 pfu/larva 1 ng/larva

49 48

0 17

6

0.003

a AcMNPV, Autographa californica nuclear polyhedrosis virus; BV, budded virus; T. ni, Trichoplusia ni; TnGV, T. ni granulosis virus. b P values are derived from x2 tests.

DISCUSSION

Washburn et al. (1995) reported that larvae of T. ni do not have a PM immediately following ecdysis, but a PM is present 15 h after molting. In previous studies we have dissected PMs from over 10,000 fourth- and fifth-instar larvae of T. ni at time intervals ranging from 15 min to 36 h postecdysis (unpublished data), and these observations are consistent with part of the findings reported by Washburn et. al. (1995). However, our findings do not support their observation that larvae of T. ni do not have a PM immediately after ecdysis and their suggestion that the PM provides little protection from AcMNPV infection. Our observations, based on dissected and stained PMs, provided evidence for the presence of PMs prior to, during, and immediately after ecdysis of larvae of T. ni from two independent rearing colonies. Therefore, under our rearing conditions, the midgut of T. ni appears to be lined with a PM during the active period of larval development. If a PM-free period could be found during a larval stage, that stage would be an important window of opportunity for studying the role of the PM in the pathogenesis of baculoviruses. Unfortunately, that stage does not exist in the larvae of T. ni from the two colonies we have examined. We were not able to compare our results with those reported by Washburn et al. (1995) since their report did not include any details on the experimental procedure used to detect the PMs nor did they show any PM photographs from control stages (i.e., 15 h after molting). However, it is possible that differences in rearing conditions or insect biotypes may account for our divergent findings. To further investigate the presence of PMs as a protective lining in the midgut of newly hatched larvae, Western blot analysis of the major protein constituent of PM in T. ni, IIM was conducted in order to follow the synthesis of this protective protein during and after embryonic development. We have previously shown that IIM secretion was specific to the midgut epithelium (Wang and Granados, 1997b). The Western blot analysis shown in Fig. 2 clearly shows that IIM synthe-

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ENHANCEMENT OF PER OS INFECTIONS OF NPV BVs

TABLE 2 AcMNPV BV Infection in T. ni Fourth- and Fifth-Instar Larvae with TnGV Enhancin a Inoculum P value b

Instar

Experiment

Virus

Enhancin

No. of insects tested

% Infection

Fourth Fourth

1

1.5 3 106 pfu/larva 1.5 3 106 pfu/larva

0 1 µg/larva

58 60

28 67

6

,0.001

Fourth Fourth

2

1.5 3 106 pfu/larva 1.5 3 106 pfu/larva

0 1 µg/larva

50 48

14 69

6

,0.001

Fifth Fifth

1

1.5 3 106 pfu/larva 1.5 3 106 pfu/larva

0 1 µg/larva

60 60

2 23

6

,0.001

Fifth Fifth

2

1.5 3 106 pfu/larva 1.5 3 106 pfu/larva

0 1 µg/larva

60 60

27 52

6

,0.001

a b

AcMNPV, Autographa californica nuclear polyhedrosis virus; BV, budded virus; T. ni, Trichoplusia ni; TnGV, T. ni granulosis virus. P values are derived from x2 tests.

sis starts 4 h prior to hatching, indicating that the midgut of a newly hatched neonate is already protected by a mucinous layer. These results indicate that the midgut in larvae of T. ni is protected by the PM throughout the entire active larval period. There are no reports addressing the interaction of baculovirus BVs with the PM or with the midgut epithelium. It is known that the efficiency of AcMNPV BV per os infection is generally low, compared to OVs (Volkman and Summers, 1977; Keddie and Volkman, 1985), although a preparation from a polyhedronnegative recombinant baculovirus had relatively high infectivity for larvae of T. ni via per os inoculation (van den Heuvel et al., 1993). In addition, Ignoffo et al. (1985) reported that Helicoverpa zea SNPV BV was highly infectious to Heliothis virescens larvae. Although BV per os infection is not the natural pathway of infection in the environment, in this study we used BVs as a tool to study the effect of enhancin on larval infectivity. By using BVs we attempted to avoid potential interference in the bioassays caused by a possible endogenous enhancing factor from the AcMNPV inoculum (see Introduction) and confirmed the correlation of TABLE 3 TnSNPV BV Infection in T. ni Fourth-Instar Larvae with TnGV Enhancin a Inoculum Experiment

Virus

No. of insects % P Enhancin tested Infection valueb

1

3.3 3 105 pfu/larva 0 3.3 3 105 pfu/larva 1 µg/larva

50 50

72 94

6

0.003

2

1.3 3 105 pfu/larva 0 1.3 3 105 pfu/larva 1 µg/larva

60 60

43 93

6

,0.001

a TnSNPV, Trichoplusia ni nuclear polyhedrosis virus; BV, budded virus; T. ni, Trichoplusia ni; TnGV, T. ni granulosis virus. b P values are derived from x2 tests.

PM degradation by enhancin and the increased virus infection in larvae. The concentrations of enhancin inocula used in our bioassays have always resulted in the alteration of the T. ni PM protein profile and degradation of the PM structural matrix (Corsaro et al., 1993; Wang et al., 1994; Lepore et al., 1996). Using a PM permeability apparatus (Adang and Spence, 1983) we recently determined that TnGV enhancin treatment could affect the permeability characteristics of dissected PMs from T. ni and Pseudaletia unipuncta fifth-instar larvae (unpublished data). We showed that PMs treated with enhancin allowed much greater passage of Dextran Blue particles (diam 5 40 nm) or fluorescent-labeled AcMNPV OV than untreated PMs. These studies provided in vitro evidence of the effect of enhancin on the permeability of two lepidopterous species. Our T. ni larval bioassay results showed that TnGV enhancin had significant enhancing activity in per os infections of BVs from both AcMNPV and TnSNPV. These bioassay studies, together with our demonstration of a PM in larvae of T. ni prior to and after ecdysis, provide further evidence that enhancin can facilitate the infection process of baculoviruses by potentially affecting the porosity of the larval PM. ACKNOWLEDGMENTS We thank K. A. McKenna for his critical review of the manuscript. This research was supported in part by grants from Biotechnology Research and Development Corp. (Peoria, IL) and the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under Agreement No. 95–37302–1889. REFERENCES Adang, M. J. and Spence, K. D. 1983. Permeability of the peritrophic membrane of the Douglas fir tussock moth (Orgyia pseudotsugata). J. Comp. Biochem. Physiol. 75, 233–238. Bischoff, D. S., and Slavicek, J. M. 1997. Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus. J. Virol. 71, 8133–8140.

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