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J. Microbiol. Biotechnol.

(2007), 17(4), 632–637

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 first, they are nomadic, spinning, and abandoning a series
isolated from the larvae of Thaumetopoea pityocampa and of flimsy shelters constructed by enveloping a few needles
shown to cause an infection of midgut cells. This viral infection in silk, but in the third instar, they initiate the construction of
revealed several important diagnostic symptoms, including a permanent nest and settle down to become central place
discoloration of the posterior midgut, reduced feeding, and foragers. By that way, they form the processionaries on
extended development time of the larvae. The virus infection pine tree. Controlling this type of moth is an important part
is lethal to Thaumetopoea pityocampa, and with the increasing of an integrated pest management system. So far, efforts to
doses kills the larvae within 4-5 days post infection. Electron control T. pityocampa have mainly involved the use of
microscopy studies showed typical cytoplasmic polyhedral chemical insecticides, particularly insect growth inhibitors.
inclusion bodies that are icosahedral, and ranged from 2.4 to However, these agents can have undesirable side-effects
5.3 µm in diameter. Electrophoretic analysis of the RNA on humans, plants and other animal species, particularly
genome showed that the virus has a genome composed of 10 predators and parasites of T. pityocampa. Therefore, it is
equimolar RNA segments with the sizes of 3,907, 3,716, necessary to find alternative and environmentally friendly
3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, control methods, such as the utilization of viruses.
respectively. Based on morphology and nucleic acid analysis, Caterpillars of T. pityocampa not only cause significant
this virus was named Thaumetopoea pityocampa cytoplasmic damage to forest trees but are also responsible for dermatitis,
polyhedrosis virus (TpCPV), and belongs to the genus ocular lesions, and, more rarely, respiratory signs and
Cypovirus, family Reoviridae. anaphylactic reactions in humans and animals [5, 32].
Keywords: Thaumetopoea pityocampa, cytoplasmic Airborne urticating hairs from these caterpillars can be
polyhedrosis virus, Cypovirus, anaphylactic shock, biological detected using techniques designed for airborne microorganism
control 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].
Thaumetopoea pityocampa (the pine processionary moth) Cytoplasmic polyhedrosis viruses (CPVs) belong to the
is one of the most important pine pests in the forests of genus Cypovirus in the family Reoviridae [18, 22]. CPV
Mediterranean countries, Central Europe, the Middle East, virions are occluded by the viral polyhedrin protein,
and North Africa [31]. The caterpillars cause severe damage forming inclusion bodies (polyhedra) within the host cell
to pine plantations, especially in warm districts and low cytoplasm [26]. These icosahedral, cubic, or sometimes
altitudes. Young pine plantations are the most susceptible, irregular polyhedra dissolve in the insect gut, infecting the
and may be completely destroyed if the attack is severe midgut cells of a wide range of insect species and have
enough. Less severe larval feeding damage can pave the been considered as potential biological control agents
way for harmful secondary pests and pathogens. Mature for harmful insects. CPVs are commonly isolated from
trees may suffer reductions in growth but are rarely killed insects belong to Lepidoptera and occasionally Diptera or
outright by the pest. The caterpillars are highly social. At Hymenoptera but only rarely Coleoptera or Neuroptera
[2, 3, 6, 13, 20].
*Corresponding author

Phone: 90-0-462-377-3554; Fax: 90-0-462-325-3195; The CPV genomes usually consist of 10 double-stranded
E-mail: remziye@ktu.edu.tr RNA (dsRNA) segments (Seg-1 to Seg-10), although for
A CPV FROM T. PITYOCAMPA 633

some viruses, a small eleventh segment (Seg-11) has been 10 min. The pellets containing polyhedra were further purified
reported [7, 9]. Each dsRNA segment is composed of a by Percoll density gradient centrifugation at 12,000 ×g for
plus-strand mRNA and its complementary minus strand, in 20 min. A nine-to-one ratio of Percoll to PBS was employed
an end-to-end base-paired configuration, except for a for this purpose. The resulting band containing purified
protruding 5' cap on the plus strand. On the basis of polyhedra was washed three times with PBS and finally
electrophoretic migration patterns of the dsRNA segments suspended in TE [12].
in agarose or acrylamide gels, CPVs have been classified into
16 different species [9, 16-18, 21-23] by the International Light and Electron Microscopies
Committee for the Taxonomy of Viruses (ICTV) with five Different organs of T. pityocampa were dissected and
further proposed types (see http://www.iah.bbsrc.ac.uk/ investigated under bright field microscopy. Differantial
dsRNA_virus_proteins/CPV-RNA-Termin.htm). Giemsa staining method was used to stain polyhedral
The occurrence of polyhedral inclusion bodies from inclusion bodies in squash preparations [35]. A suspension
Thaumetopoea pityocampa have been reported [4, 10, 11, of purified polyhedra was air-dried on coverglass, coated
24, 25, 27-29]. A cytoplasmic polyhedrosis virus was with gold, and examined in a JSM 6400 scanning electron
the first agent to be mass produced and used to control microscope operated at 15 kV, and photographed. For
Thaumetopoea pityocampa in field conditions in 1959 at electron microscopy, sections of midgut were fixed in
France [15]. However, no further detailed studies of these 2.5% glutaraldehyde and 0.14 M NaCl in 0.2 M phosphate
viruses were subsequently published. buffer (pH 7.4) followed by a secondary fixation for 1 h at
Here, we report the isolation and characterization of a room temperature, with 2% OsO4 and 1.25% (w/v) sodium
CPV isolated recently from Thaumetopoea pityocampa larvae bicarbonate at pH 7.4. The primary fixation solution was
in Turkey. The structure of this virus, which we have called changed several times. Between the primary and secondary
TpCPV, was analyzed by light and electron microscopies. fixations, the specimens were washed several times with
The RNA genome was analyzed electrophoreticaly and has 0.2 M phosphate buffer containing 0.3 M NaCl. Subsequently,
a migration pattern by agarose gel electrophoresis (AGE) they were rinsed in phosphate buffer and dehydrated in a
that shows some similarity to that of CPV-5 [21]. Bioassays graded series of ethanol-acetone, infiltrated, and embedded
were performed to study the pathogenicity of this virus and in Epon resin. Thin sections were stained with 5% acidic
evaluate its potential as a biocontrol agent. uranyl acetate and Reynold’s lead citrate and examined
under a JEOL 100 CX transmission electron microscope at
50 kV.
MATERIALS AND METHODS
Isolation of Total Genomic RNA
Field Collections After dissolving the purified polyhedral in alkali with trizol
Dead Thaumetopoea pityocampa larvae were collected reagent (Gibco, BRL) according to the manufacturer’s
from pine trees during 2004-2005 field studies in Samsun, instructions. Genomic RNA was extracted from purified
located in the middle of the Blacksea Region of Turkey. polyhedra by a standard guanidium isothiocyanate method
Fifty processionaries were checked for infected virus. All [10]. RNA was then separated in 1% agarose gel in Tris-
dead larvae in these processionaries were stored individually phosphate buffer. RNA segments were visualized on the
and screened for virus infection by light microscopy. ethidium bromide stained (final concentration of 0.5 mg/ml)
The ones that have polyhedral inclusions were kept at gel. Size markers (Bio Basic Inc. Canada) of 1,000 bp
-20oC. were used to determine segment sizes.
Virus Production and Purification of Polyhedral Bodies Pathogenicity Test
Third instar T. pityocampa larvae were used for virus Third instar T. pityocampa larvae were used for the
production. They were put in cups without food overnight, bioassay. Experiments were performed with 20 larvae per
and then exposed to polyhedral inclusion bodies (PIB)- dose and were replicated three times with the control group.
positive, homogenized T. pityocampa cadavers in phosphate- Larvae were exposed to four different concentrations of
buffered saline (PBS) with fresh pine leaves as food, at virus (2×104, 2×105, 1×106, 2×106 PIBs). They were starved
10oC. Larvae were incubated for 7 days and than examined overnight and then fed with pine leaves contaminated with
under a light microscope for patent infection. The ones that one of the four virus concentrations and reared separately
have PIB were stored at -20oC. CPV polyhedra were for 10 days. After they consumed the entire contaminated
purified from the infected T. pityocampa midgets. Infected leaves, they were given fresh leaves. The larvae were
midguts were first dissected from the insect body, homogenized incubated at 10oC. The mortalities of larvae were recorded
in PBS, and purified first by filtration through cheesecloth every 24 h, with all dead larvae removed from the containers.
to remove the debris and then centrifuged at 3,500 ×g for Data were evaluated using Abbott’s formula [1].
634 INCE et al.

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 Fig. 1. Electron micrographs of virus samples from Thaumetopoea
with the CPV staining in the literature (data not shown). pityocampa.
A. Scanning electron micrograph showing inclusion bodies (×5,000). B.
Scanning electron microscopy (Fig. 1A) demonstrated that Transmission electron micrograph of virions exhibiting surface spikes
the occlusion bodies were of irregular shape and ranged (×15,000).
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). segments. Approximate sizes of segments were estimated
with size markers as follows: Seg-1, 3,907 bp; Seg-2,
Electrophoretic Analysis of dsRNA 3,716 bp; Seg-3, 3,628 bp; Seg-4, 3,249 bp; Seg- 5, 2,726 bp;
An initial analysis of the TpCPV genome by 1% agarose Seg-6, 1,914 bp; Seg-7, 1,815 bp; Seg-8, 1,256 bp; Seg-9,
gel using a 14-cm gel at 75 V for 4 h generated seven RNA 1,058 bp; Seg-10, 899 bp.
bands, some of which stained more intensely and appeared
to contain more than one genome segment each (the first Pathogenicity Test
intense band contained segments 1, 2, and 3 and the fourth In comparison to an unifected control group, TpCPV
band contained segments 6 and 7). Analysis using a longer infection affected the development, feeding, and behaviour
agarose gel with a lower voltage resolved the first band of the larvae, correlating with the virus dosage. The
into three bands and the fourth band into two single posterior midgut also appeared white (due to the presence
bands, confirming that the genome contains a total of 10 of large numbers of polyhedra) in infected caterpillars.

Table 1. Mortality rate of TpCPV on Thaumetopoea pityocampa exposed as third instar larvae and examined 5 days and 15 days post
inoculation.
Dosage After 5 days After 15 days
(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
A CPV FROM T. PITYOCAMPA 635

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
Electrophoretic separation of TpCPV:1 total genome in levels.
Fig. 2.
1% agarose gel. Until now, cypoviruses have only been isolated from
Lane 1: DNA molecular weight marker (1 kb); lane 2: TpCPV genome arthropods [18]. Experiments conducted to infect vertebrates
segments. The arrows indicated that viral segments are 3,907, 3,716, 3,628, or vertebrate cell lines have been unsuccessful, and thus
3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp. in size, respectively. CPVs appear as safe biocontrol agents. Although more
than 230 cypoviruses have been reported, mainly from
Patent infections developed by 4-5 days post inoculation Lepidoptera and some also from Diptera and Hymenoptera
(p.i.). Infected larvae stopped feeding and some deaths [18], members of the genus are not frequently regarded as
were observed from 5 days p.i. onwards, with an increasing potential biocontrol agents, as they generally produce only
percentage of infected individuals observed with increasing chronic diseases, generally without extensive larval mortality.
doses of virus (Table 1). At the end of the 15th day, the However, different virus strains can vary significantly in
percentages of the mortality at higher and lower doses used virulence [30, 36], and TpCPV exhibits a high level of
were 90% and 60%, respectively. Percent mortality of the virulence, particularly with increasing virus concentrations.
T. pityocampa increased with increasing dose of the virus. However, even at the low concentration used in the studies
At lower doses, the development of the larvae was very described here, rapid and extensive larval mortality was
slow. The mortality of larvae in the control group was 5%. observed (Table 1).
These larvae died at the end of the 15th day. 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
DISCUSSION could significantly reduce the damage to trees before the
insect is killed by the virus. Even if some of the infected
An occluded virus with a 10-segmented dsRNA genome individuals survive to adulthood (as observed with other
was isolated from Thaumetopoea pityocampa larvae collected cypoviruses), their reproductive potential may be significantly
from pine trees in Samsun, Turkey. The characteristics of this reduced.
636 INCE et al.

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