A Cytoplasmic Polyhedrosis Virus Isolated from the

. (2007), 17(4), 632–637

J. Microbiol. Biotechnol

A Cytoplasmic Polyhedrosis Virus Isolated from the Pine Processionary Caterpillar, Thaumetopoea pityocampa INCE, IKBAL AGAH, ISMAIL DEMIR, ZIHNI DEMIRBAG, AND REMZIYE NALCACIOGLU* Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey

Received: October 10, 2006 Accepted: November 28, 2006

Abstract A cytoplasmic polyhedrosis virus (CPV) was

isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 µm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae. Keywords: Thaumetopoea pityocampa, cytoplasmic polyhedrosis virus, Cypovirus, anaphylactic shock, biological control Thaumetopoea pityocampa (the pine processionary moth)

is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East, and North Africa [31]. The caterpillars cause severe damage to pine plantations, especially in warm districts and low altitudes. Young pine plantations are the most susceptible, and may be completely destroyed if the attack is severe enough. Less severe larval feeding damage can pave the way for harmful secondary pests and pathogens. Mature trees may suffer reductions in growth but are rarely killed outright by the pest. The caterpillars are highly social. At *Corresponding author

Phone: 90-0-462-377-3554; Fax: 90-0-462-325-3195; E-mail: [email protected]

first, they are nomadic, spinning, and abandoning a series of flimsy shelters constructed by enveloping a few needles in silk, but in the third instar, they initiate the construction of a permanent nest and settle down to become central place foragers. By that way, they form the processionaries on pine tree. Controlling this type of moth is an important part of an integrated pest management system. So far, efforts to control T. pityocampa have mainly involved the use of chemical insecticides, particularly insect growth inhibitors. However, these agents can have undesirable side-effects on humans, plants and other animal species, particularly predators and parasites of T. pityocampa. Therefore, it is necessary to find alternative and environmentally friendly control methods, such as the utilization of viruses. Caterpillars of T. pityocampa not only cause significant damage to forest trees but are also responsible for dermatitis, ocular lesions, and, more rarely, respiratory signs and anaphylactic reactions in humans and animals [5, 32]. Airborne urticating hairs from these caterpillars can be detected using techniques designed for airborne microorganism and pollen collection [33]. An IgE-mediated mechanism of hypersensitivity to this caterpillar has been demonstrated in a study [34], and anaphylactic shock in a pine-forest worker was described by Vega et al. [32]. Cytoplasmic polyhedrosis viruses (CPVs) belong to the genus Cypovirus in the family Reoviridae [18, 22]. CPV virions are occluded by the viral polyhedrin protein, forming inclusion bodies (polyhedra) within the host cell cytoplasm [26]. These icosahedral, cubic, or sometimes irregular polyhedra dissolve in the insect gut, infecting the midgut cells of a wide range of insect species and have been considered as potential biological control agents for harmful insects. CPVs are commonly isolated from insects belong to Lepidoptera and occasionally Diptera or Hymenoptera but only rarely Coleoptera or Neuroptera [2, 3, 6, 13, 20]. The CPV genomes usually consist of 10 double-stranded RNA (dsRNA) segments (Seg-1 to Seg-10), although for

A CPV FROM

some viruses, a small eleventh segment (Seg-11) has been reported [7, 9]. Each dsRNA segment is composed of a plus-strand mRNA and its complementary minus strand, in an end-to-end base-paired configuration, except for a protruding 5' cap on the plus strand. On the basis of electrophoretic migration patterns of the dsRNA segments in agarose or acrylamide gels, CPVs have been classified into 16 different species [9, 16-18, 21-23] by the International Committee for the Taxonomy of Viruses (ICTV) with five further proposed types (see http://www.iah.bbsrc.ac.uk/ dsRNA_virus_proteins/CPV-RNA-Termin.htm). The occurrence of polyhedral inclusion bodies from Thaumetopoea pityocampa have been reported [4, 10, 11, 24, 25, 27-29]. A cytoplasmic polyhedrosis virus was the first agent to be mass produced and used to control Thaumetopoea pityocampa in field conditions in 1959 at France [15]. However, no further detailed studies of these viruses were subsequently published. Here, we report the isolation and characterization of a CPV isolated recently from Thaumetopoea pityocampa larvae in Turkey. The structure of this virus, which we have called TpCPV, was analyzed by light and electron microscopies. The RNA genome was analyzed electrophoreticaly and has a migration pattern by agarose gel electrophoresis (AGE) that shows some similarity to that of CPV-5 [21]. Bioassays were performed to study the pathogenicity of this virus and evaluate its potential as a biocontrol agent.

MATERIALS AND METHODS Field Collections

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10 min. The pellets containing polyhedra were further purified by Percoll density gradient centrifugation at 12,000 ×g for 20 min. A nine-to-one ratio of Percoll to PBS was employed for this purpose. The resulting band containing purified polyhedra was washed three times with PBS and finally suspended in TE [12].

Light and Electron Microscopies

Different organs of T. pityocampa were dissected and investigated under bright field microscopy. Differantial Giemsa staining method was used to stain polyhedral inclusion bodies in squash preparations [35]. A suspension of purified polyhedra was air-dried on coverglass, coated with gold, and examined in a JSM 6400 scanning electron microscope operated at 15 kV, and photographed. For electron microscopy, sections of midgut were fixed in 2.5% glutaraldehyde and 0.14 M NaCl in 0.2 M phosphate buffer (pH 7.4) followed by a secondary fixation for 1 h at room temperature, with 2% OsO4 and 1.25% (w/v) sodium bicarbonate at pH 7.4. The primary fixation solution was changed several times. Between the primary and secondary fixations, the specimens were washed several times with 0.2 M phosphate buffer containing 0.3 M NaCl. Subsequently, they were rinsed in phosphate buffer and dehydrated in a graded series of ethanol-acetone, infiltrated, and embedded in Epon resin. Thin sections were stained with 5% acidic uranyl acetate and Reynold’s lead citrate and examined under a JEOL 100 CX transmission electron microscope at 50 kV.

Isolation of Total Genomic RNA

Dead Thaumetopoea pityocampa larvae were collected from pine trees during 2004-2005 field studies in Samsun, located in the middle of the Blacksea Region of Turkey. Fifty processionaries were checked for infected virus. All dead larvae in these processionaries were stored individually and screened for virus infection by light microscopy. The ones that have polyhedral inclusions were kept at -20oC.

After dissolving the purified polyhedral in alkali with trizol reagent (Gibco, BRL) according to the manufacturer’s instructions. Genomic RNA was extracted from purified polyhedra by a standard guanidium isothiocyanate method [10]. RNA was then separated in 1% agarose gel in Trisphosphate buffer. RNA segments were visualized on the ethidium bromide stained (final concentration of 0.5 mg/ml) gel. Size markers (Bio Basic Inc. Canada) of 1,000 bp were used to determine segment sizes.

Virus Production and Purification of Polyhedral Bodies

Pathogenicity Test

Third instar T. pityocampa larvae were used for virus production. They were put in cups without food overnight, and then exposed to polyhedral inclusion bodies (PIB)positive, homogenized T. pityocampa cadavers in phosphatebuffered saline (PBS) with fresh pine leaves as food, at 10oC. Larvae were incubated for 7 days and than examined under a light microscope for patent infection. The ones that have PIB were stored at -20oC. CPV polyhedra were purified from the infected T. pityocampa midgets. Infected midguts were first dissected from the insect body, homogenized in PBS, and purified first by filtration through cheesecloth to remove the debris and then centrifuged at 3,500 ×g for

Third instar T. pityocampa larvae were used for the bioassay. Experiments were performed with 20 larvae per dose and were replicated three times with the control group. Larvae were exposed to four different concentrations of virus (2×104, 2×105, 1×106, 2×106 PIBs). They were starved overnight and then fed with pine leaves contaminated with one of the four virus concentrations and reared separately for 10 days. After they consumed the entire contaminated leaves, they were given fresh leaves. The larvae were incubated at 10oC. The mortalities of larvae were recorded every 24 h, with all dead larvae removed from the containers. Data were evaluated using Abbott’s formula [1].

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RESULTS Field Collection

The first TpCPV-infected T. pityocampa larvae were found in March 2004 in a field-collected processionary. Dead or diseased larvae were frequently found in the field, suggesting an epizootic was effecting the pest populations. We observed 40% infection among 50 examined processionaries and 90 percent infection of T. pityocampa larvae in one processionary. A similar result was also observed in 2005. The infected larvae were smaller, not feeding and not moving, and became moribund. Many of the diseased T. pityocampa larvae were observed to be hanging down by their prolegs immediately prior to death. Since this behaviour has previously been observed in insect larvae infected with nuclear polyhedrosis viruses [8], these T. pityocampa larvae were checked for the presence of such viruses. However, the analysis failed to detect any NPVs.

Light and Electron Microscopy Studies

Polyhedra were observed by light microscopy in midgut cells from tissue smear samples of field-collected dead pine processionary larvae. Differential Giemsa staining showed a typical cytoplasmic polyhedrosis virus infection. Whereas the first and the second zones stained colourless and purple, respectively, the third zone was black, correlating with the CPV staining in the literature (data not shown). Scanning electron microscopy (Fig. 1A) demonstrated that the occlusion bodies were of irregular shape and ranged from 2.4 to 5.3 µm in diameter. This was confirmed by transmission electron microscopy studies, which showed typical cytoplasmic polyhedral inclusion bodies (Fig. 1B).

Electrophoretic Analysis of dsRNA

An initial analysis of the TpCPV genome by 1% agarose gel using a 14-cm gel at 75 V for 4 h generated seven RNA bands, some of which stained more intensely and appeared to contain more than one genome segment each (the first intense band contained segments 1, 2, and 3 and the fourth band contained segments 6 and 7). Analysis using a longer agarose gel with a lower voltage resolved the first band into three bands and the fourth band into two single bands, confirming that the genome contains a total of 10

Electron micrographs of virus samples from Thaumetopoea pityocampa. A. Scanning electron micrograph showing inclusion bodies (×5,000). B. Fig. 1.

Transmission electron micrograph of virions exhibiting surface spikes (×15,000).

segments. Approximate sizes of segments were estimated with size markers as follows: Seg-1, 3,907 bp; Seg-2, 3,716 bp; Seg-3, 3,628 bp; Seg-4, 3,249 bp; Seg- 5, 2,726 bp; Seg-6, 1,914 bp; Seg-7, 1,815 bp; Seg-8, 1,256 bp; Seg-9, 1,058 bp; Seg-10, 899 bp.

Pathogenicity Test

In comparison to an unifected control group, TpCPV infection affected the development, feeding, and behaviour of the larvae, correlating with the virus dosage. The posterior midgut also appeared white (due to the presence of large numbers of polyhedra) in infected caterpillars.

Mortality rate of TpCPV on Thaumetopoea pityocampa exposed as third instar larvae and examined 5 days and 15 days post inoculation. After 5 days After 15 days Dosage (PIBs/larva) % feeding larvae % cessation of feeding % larval death % feeding larvae % cessation of feeding % larval death Control 100.0 00.0 00.0 95 00.0 05.0 2×104 070.0 30.0 00.0 00 40.0 60.0 2×105 057.2 33.3 09.5 00 33.3 66.6 1×106 040.0 50.0 10.0 00 15.0 85.0 2×106 010.0 60.0 30.0 00 10.0 90.0

Table 1.

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Electrophoretic separation of TpCPV:1 total genome in 1% agarose gel. Fig. 2.

Lane 1: DNA molecular weight marker (1 kb); lane 2: TpCPV genome segments. The arrows indicated that viral segments are 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp. in size, respectively.

Patent infections developed by 4-5 days post inoculation (p.i.). Infected larvae stopped feeding and some deaths were observed from 5 days p.i. onwards, with an increasing percentage of infected individuals observed with increasing doses of virus (Table 1). At the end of the 15th day, the percentages of the mortality at higher and lower doses used were 90% and 60%, respectively. Percent mortality of the T. pityocampa increased with increasing dose of the virus. At lower doses, the development of the larvae was very slow. The mortality of larvae in the control group was 5%. These larvae died at the end of the 15th day.

DISCUSSION An occluded virus with a 10-segmented dsRNA genome was isolated from Thaumetopoea pityocampa larvae collected from pine trees in Samsun, Turkey. The characteristics of this

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virus, determined by light microscopy, electron microscopy, and electrophoresis identified it as a CPV, a member of the genus Cypovirus of the family Reoviridae. CPVs usually possess a 10-segmented dsRNA (Seg-1 to Seg-10) genome, although for some viruses, a small eleventh segment (Seg-11) has been reported [9, 22]. The pattern of size distribution of the RNA genome fragments after electrophoresis in 1% agarose or 3-5% polyacrylamide gel provided an original basis for classification of CPV isolates into different electropherotypes [9, 16-18, 21-23]. Different CPV types can also be distinguished by sequence analysis and comparison of the viral genome segments (for example, by comparisons of Seg-10, the polyhedrin gene) and the different electropherotypes are now recognized as distinct Cypovirus species. Sequencing studies have also demonstrated that in many cases the CPV genome segments are considerably larger than initially estimated from their electrophoretic mobility [36]. Sixteen Cypovirus species have now been formally recognized by the International Committee for the Taxonomy of Viruses [18] with a further five proposed species to date (see http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ CPV-RNA-Termin.htm). TpCPV shows some similarity in its electropherotype to CPV- 5 [21], although because it is not feasible to make a direct electrophoretic comparison with the initial isolate of CPV-5 on the same gel, a conclusive identification of the species to which TpCPV belongs will be made after sequence analysis of the genome in further studies. A cytoplasmic polyhedrosis virus from Thaumetopoea pityocampa was reported earlier in 1958 [29]; however, it has not been characterized at the structural and molecular levels. Until now, cypoviruses have only been isolated from arthropods [18]. Experiments conducted to infect vertebrates or vertebrate cell lines have been unsuccessful, and thus CPVs appear as safe biocontrol agents. Although more than 230 cypoviruses have been reported, mainly from Lepidoptera and some also from Diptera and Hymenoptera [18], members of the genus are not frequently regarded as potential biocontrol agents, as they generally produce only chronic diseases, generally without extensive larval mortality. However, different virus strains can vary significantly in virulence [30, 36], and TpCPV exhibits a high level of virulence, particularly with increasing virus concentrations. However, even at the low concentration used in the studies described here, rapid and extensive larval mortality was observed (Table 1). Another feature of TpCPV that has also been reported for other cypoviruses is the rapid cessation of larval feeding activity after infection. This behavioral change could significantly reduce the damage to trees before the insect is killed by the virus. Even if some of the infected individuals survive to adulthood (as observed with other cypoviruses), their reproductive potential may be significantly reduced.

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The pine processionary moth caterpillar causes heavy defoliations in various stands of pines. In Turkey, T. pityocampa occurs over a forest area of 1,500,000 ha [15]. Pine processionary moth caterpillar also causes health problems because of their urticating hairs [31]. Contact with caterpillars induces dermatitis and ocular lesions by a mechanic and toxic mechanism. Additionally, IgE-mediated hypersensitivity to this caterpillar has been demonstrated in two recent studies [32, 34]. Controlling this type of moth is an important key for an integrated pest management system. The results in this article will offer useful information for future studies on TpCPV molecular biology or implementation as an insecticide. We are currently intending to determine the genome sequence and physical map of TpCPV.

9. 10.

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Acknowledgments

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The authors thank Dr. Peter Mertens (Pirbright Laboratory, Pirbright, U.K.) and Monique van Oers (Virology Laboratory, Wageningen University, The Netherlands) for their critical reading and comments on an earlier draft of the manuscript. They also acknowledge the Medical Faculty of Karadeniz Technical University for providing the electron microscopy service. The project was financially supported by the Turkish State Planning Organisation and The Scientific and Technological Research Council of Turkey.

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