Virus and Virus-Like
Particles Found in Southern Pine Beetle Adults in Mississippi and Georgia
P.P. Sikorowski
Professor, Entomology
Department of Entomology and Plant Pathology
Mississippi State University |
A.M. Lawrence
Senior Research Assistant, Entomology
Department of Entomology and Plant Pathology
Mississippi State University |
T.E. Nebeker
Professor, Entomology
Department of Entomology and Plant Pathology
Mississippi State University |
T.S. Price
Forest Entomologist
Georgia Forestry Commission
Macon, Georgia |
Published by the Office of Agricultural Communications (Publications),
Division of Agriculture, Forestry, and Veterinary Medicine, Mississippi State
University. Edited by Keith H. Remy, Senior Publications Editor. Cover designed
by George Taylor, Chief Illustrator.
Contents
Introduction
The
Southern Pine Beetle (SPB), Dendroctonus frontalis Zimmermann (Coleoptera:
Scolytidae), is the most destructive insect pest of pine forests in the southeastern
United States and in parts of Mexico and Central America (Payne, 1980). It usually
is endemic, but appears periodically in epidemic outbreaks (Blackman, 1949).
During
1995, it was estimated that in Mississippi more than $15 million in timber was
lost to the SPB (Nebeker, unpublished), and losses in South Carolina were estimated
at more than $100 million (Mike Remion, personal communication). From 1960-1990,
U.S. timber losses to the SPB have been estimated at $901.8 million (Price et
al., 1990).
Current
methods of minimizing SPB losses, according to Lorio (1980), include these:
(1) salvage, (2) cut and leave, (3) fell and spray with insecticides, or (4)
pile and burn. Silvicultural treatments, such as thinning, are recommended to
prevent or reduce the impact of SPB should an attack occur (Nebeker et al.,
1985). Little attention has been given to biological control methods, which
may eventually eliminate or complement conventional methods.
The
use of biological agents, such as viruses, to regulate SPB populations will
require much research before practical applications can be made. Dissemination
of self-propagating pathogens of SPB (viruses, bacteria, fungi, protozoa, or
nematodes) would provide the first step toward the use of naturally occurring
host-specific microorganisms in biological control and would reduce pesticide
pollution with subsequent benefits to man and the environment.
There
is a general lack of understanding of the interrelationships of insect parasites,
predators, and pathogens with the SPB populations. Berisford (1980) presented
a general review of the natural enemies of the SPB including pathogens. Sikorowski
et al. (1979) found that pathogens of the SPB may play an important role in
regulating populations of the SPB. Pabst and Sikorowski (1980) found that under
laboratory conditions, three entomophagous fungi were pathogenic to SPB larvae,
with Beuveria bassiana [(Balsamo); Deuteromycotina] being most virulent.
Thus
far, viruses of SPB are unknown and have not been investigated in previous studies.
A review of the literature revealed that viruses have been found in coleopterous
insects. Oryctes baculovirus, nonoccluded virus (subgenus C), was described
by Huger (1966, 1972) as a viralpathogen of the larvae of the Indian rhinoceros
beetle, Oryctes rhinoceros (L.), Dynastidae.
Several
entomopox viruses (Entomopoxvirinae) affecting coleopterous insects have been
reported from Scarabaeidae by Vago et al. (1968); Bergoin et al. (1968); Goodwin
and Filshie (1969); and Goodwin et al. (1991). Entomopox viruses affecting coleopterous
insects have been reported from Europe, North and South America, Madagascar,
and Australia.
Thomas
and Gouranton (1975) studied development of virus-like particles in the crystal-containing
nuclei of the midgut cells of Tenebrio molitor (L.), Tenebrionidae. Arnold
and Barson (1977) reported occurrence of virus-like particles in midgut epithelial
cells of the elm bark beetle (Scolytus scolytus [F.], Scolytidae). Longworth
and Archibald (1975) isolated a virus of black beetle Heteronychus arator,
F., Scarabaeidae) and Dearing et al. (1980) isolated a small RNA virus from
the grass grub (Costelytra zealandica [White], Scarabaeidae) (L.).
Lomer
(1987) reported that adults of Strategus aloeus (L.), Scarabaeidae were
capable of supporting Oryctes baculovirus similar to that produced in
O. rhinoceros. Kitajima et al. (1985) found virus-like particles in the
cytoplasm of oocytes and eggs of the Mexican bean beetle (Epilachna varivestis
Mulsant, Coccinellidae), as well as embedded in the nuclei of sperm. They suggested
vertical transmission of the virus (vertical transmission; synonym: genetic
transmission; the passage of viral genome from one host generation to the next).
Shukla
et al. (1984) found a baculovirus of mango nut weevil (Sternochetus mangiferae
[F.], Curculionidae). This is the first record of a baculovirus in mango nut
weevil. Kim and Kitajima (1984) described nonoccluded baculovirus in the spotted
cucumber beetle (Diabrotica undecimpunctata Mannerheim, Chrysomelidae).
Recently,
Wegenstiener and Weiser (1995) reported a new entomopoxvirus in the bark beetle
(Ips typographus (L.), Scolytidae). Ips typographus is a primary
insect pest on spruce (Picea abies L.) in central and northern Europe.
Insect Viruses
Viral
diseases are among the most widely investigated infections in insects. The viral
particle (virus, virion) is composed of a protein shell (capsid) that surrounds
the nucleic acid. Each virus has only one type of nucleic acid, either deoxyribonucleic
acid (DNA) or ribonucleic acid (RNA). The nucleic acid together with the capsid
form the nucleocapsid. Their nucleic acid contains information necessary for
their replication in a susceptible host cell. However, the host cells may also
be required to supply some of the enzymes necessary for replication. Viruses
contain no energy-producing enzyme systems, no functional ribosomes, or other
cellular organelles; those are supplied by the cell in which they replicate
(Hull et al., 1989).
Viruses
are classified by the International Committee on Taxonomy of Viruses. Today,
insect viruses are placed into 12 families, but many remain unclassified (Tanada
and Kaya, 1993). Insect viruses may be divided into two groups: occluded and
nonoccluded.
Occluded
viruses are those in which the virions are occluded in a dense protein crystal
(visible in the light microscope). The occlusion body serves to stabilize the
virus for prolonged periods outside the host. The occlusion bodies range in
size from ~0.5 to 24 µm. Virus particles are released in vivo following
ingestion of the occlusion body by the insect and solubilization of the matrix
protein by combined effects of pH and gut enzymes (Hull et al., 1989). Three
families of insect viruses are in this group, Baculoviridae, Entomopoxviridae,
and Reoviridae.
Nonoccluded
viruses are those in which the virions are not occluded in a dense protein crystal.
Examples of these viral families are Iridoviridae, Parvoviridae, and Flaviviridae.
The
primary route of viral entrance to the host is via the alimentary tract (mainly
midgut) during feeding. Transmission via egg, on the egg surface (transovum)
or within the egg (transovarial), is less common. Transovum transmission is
more common than transovarial transmission. Transovum transmission may be used
to introduce a potential microbial agent such as a virus into the SPB populations.
The
objective of the study reported in this bulletin was to determine if there were
evidence for a viral presence within SPB populations. This is the first time
this type of an investigation on D. frontalis has ever been attempted.
Materials and Methods
Adults
of SPB were trapped in Georgia and Mississippi over the course of two summers
(1993-1994). A total of È100 collections were made. Adults of SPB collected
in pheromone traps were used in most of this study. Some adults were also obtained
from infested bark that had been placed in emergence chambers at Mississippi
State University. The study is based on the gross morphology of virus and virus-like
particles, and further studies would be required to characterize viral particles.
Light
microscopy was used for detection of virus-caused signs and symptoms and also
for detection of occluded viruses.
Two
methods of electron microscopy were used for detection of nonoccluded viruses:
(1) the phosphotungstic acid negative stain method and (2) the histopathological
method (tissue changes associated with disease).
Light Microscopy
(LM)
Whole, decapitated SPB were fixed in Perfix®
(Fisher Biologicals) and dehydrated through a graded ethanol series. Dehydrated
tissues were then cleared in Hemo-De® (Fisher Biologicals) and embedded
in fresh Paraplast. Serial 5-µm sections were cut on a rotary microtome
(American Optical 820 Spencer Microtome), mounted on glass slides, stained with
aematoxylin/eosin, and examined with LM at 100-900x.
Transmission Electron
Microscopy (TEM)
Negative stains. To check for virus
infection, internal tissue samples of SPB from each collection were macerated
in sterile water. A drop of the suspension was placed on a formvar and carbon-coated
200-mesh copper grid and allowed to stand for 5 minutes. The liquid was gently
removed with a filter paper wick, stained immediately with 2% phosphotungstic
acid for 1 minute, and the grid was then examined with a JOEL TEM at 60 kV.
Histopathology.
For histopathological studies, samples of SPB from each collection were decapitated
and placed into Karnovsky's fixative in 0.1 M potassium phosphate buffer, pH
7.2, and rinsed with the same buffer. The tissue was then post-fixed in buffered
(0.1 M potassium phosphate, pH 7.2) 2% osmium tetraoxide for 2 hours, dehydrated
through a graded ethanol series, and embedded in low viscosity Spurr's resin.
Thick sections (1 mm) were cut with glass knives on an ultramicrotome (Reichert-Jung
Co., Leica, Inc.), mounted on glass slides, stained with borate-buffered-toluidine
blue, and examined by light microscopy. Areas of interest, such as decomposed
midgut epithelia and fat bodies, were selected and located on the corresponding
tissue blocks. Ultrathin sections (60-80 nm thick) were cut from the selected
areas, mounted on formvar-coated 200-mesh copper grids, stained with 0.5% ethanolic
uranyl acetate followed by lead citrate, and examined using TEM as for negative
stains.
Results
Results
of the study imply that viruses are present in the adult populations of the
SPB. Although our results are based only on the external morphology of the virus-like
particles (VLP), we decided, based on morphological differences of the VLP,
to separate them into five families. A brief description of each family accompanies
the appropriate electron micrograph. All VLP dimensions are given in nanometers
(1 nanometer = one billionth of a meter or 10-9 meter).
Family Picornaviridae
The
family Picornaviridae is divided into four genera: the Enteroviruses, the Cardioviruses,
the Rhinoviruses, and the Aphthoviruses. The virions are small isometric particles
22 to 30 nm in diameter. The type viruses of this family are Cricket paralysis
virus, Drosophila C virus, and Gonometa virus (Evans and Entwistle,
1987). Picorna-like virus was also reported in Microplitis croceipes
(Creeson) by Hamm et al., 1992. Relatively few picorna-like viruses of insects
have been studied in sufficient detail to be classified as picornaviruses by
the International Committee on Taxonomy of Viruses (Matthews, 1982). The virions
(the infectious unit of the virus) contain a single-stranded RNA. Based on present
evidence the replicate schemes of insect picornaviruses mirror those of their
mammalian counterparts (Matthews, 1982). The virus replicates in the cytoplasm
of cells of the epidermis, alimentary canal, and nerve ganglia. The feces contain
a large amount of virus through the rupturing of midgut cells (Tanada and Kaya,
1993).
Family Bunyaviridae
Bunyaviruses
are abundantly represented (150 spp.) in the family Bunyaviridae and are classified
into 13 subgroups. A number of bunyaviruses cause severe diseases such as encephalitis
or hemorrhagic fevers in man and animals (Bouloy, 1991).
All
members of the Bunyaviridae family share the same morphological features: the
virus particles are spherical (80 to 100 nm in diameter) and possess a membrane
envelope from which protrude polypeptide spikes of 5 to 10 nm in length. The
Bunyaviridae replicate in the cytoplasm of infected cells. The genome consists
of three single-stranded RNA segments. The life cycles of bunyaviruses involve
alternate replication in vertebrates and arthropods (Bouloy, 1991). These viruses
are omnipresent in the world and have been isolated from various arthropods,
mosquitoes, ticks, and sandflies, as well as from vertebrates, including man.
When
the virus infects vertebrate cell lines, it usually causes a cytopathic effect
leading to cell death. In vivo, the infection causes little or no histopathologic
changes in arthropods and the virus is passed into the next generation by transovarial
(inside the egg) and venereal transmission.
Family Baculoviridae
-- Nonoccluded Group
Thus
far, only one genus, Baculovirus, has been described. Based on morphological
properties, members of this genus are divided into three subgroups: (1) Nuclear
polyhedrosis viruses, (2) Granulosis viruses, and (3) Nonoccluded rod-shaped
nuclear viruses.
Nonoccluded baculoviruses
(NOBV)
Lately, many rod-shaped viruses or virus-like
particles of arthropods have been reported in literature that have general properties
or structural and other similarities to NOBV (Huger and Krieg, 1991). They also
reported that the pertinent hosts belonging to Coleoptera, Lepidoptera, Orthoptera,
Diptera, Siphonatera, Hymenoptera, Homoptera, Acarina, Araneina, and Crustacea
either suffer from acute infections or are only carriers of the virus without
overt signs and symptoms.
But,
the majority of the NOBV's described have not been investigated in detail and
have been categorized as probable or possible members of this group. At the
present time, only three NOBV's of insects have been studied in sufficient detail
to warrant their formal inclusion in NOBV's group: the palm rhinoceros beetle
virus, Hz-1 (corn earworm) virus, and cricket virus (Huger and Krieg, 1991).
Infectious Flacherie
Virus (IFV)
The
target of IFV is the goblet cell of the midgut epithelium and the virus multiplies
in the cytoplasm, eventually disintegrating the cell. The virus particles are
isometric (of equal dimensions, thus appear spherical) and È 27 + 2 nm in diameter.
The infectivity of IFV is reduced at pH 2 but unaffected at pH 3 to 12. The
virus genome consists of a single molecule of single-stranded RNA. At a late
stage of infection, the cytoplasm of the goblet cells is filled with virus particles
(Watanabe 1991).
Unknown (recently found)
RNA Virus
Many
small RNA viruses, with similarities to vertebrate picornaviruses, are ubiquitous
in nature and have a wide host range. Many of them cause chronic or inapparent
infections, but some are virulent pathogens.
Discussion
We
believe that this preliminary report of SPB-VLP is the first record of viruses
associated with SPB and Dendroctonus (Coleoptera: Scolytidae) in general.
Our basic hypothesis is and has been that viruses associated with SPB are important
natural control (regulating) agents, which are suitable for control of forest
insects. This study also demonstrates the need for additional investigations
that will include, in this search for viruses, all life stages of SPB (from
egg to adult). Many viruses kill the immature stages of the host, and their
association with the adults could be rare or nonexistent. However, demonstration
of their association with adults is tremendously encouraging and suggests the
need to continue with studies of other life stages.
All
VLP found in SPB should be classified and their suitability as biocontrol agents
and/or population suppressers established. We found several possible virus infections
in various adult tissues, but further study is needed to check tissue specificity
and morphology of virus (using the preparations of purified viruses). Also,
symptomology associated with infection by different viral infections is unknown.
Even
though insect pathogens overcome many of the problems associated with chemical
pesticides, they are not extensively used despite their many positive features.
The reasons for their lack of use vary, but among the more important ones are
that most insect pathogens, including viruses, are unknown and thought to be
slow killers; and the public is reluctant to accept biocontrol agents, and viruses
in particular, as safe pesticides to control insects. However, one must remember
that to date these are considered very host specific. They have excellent potential
for future use in pest management as technologies continue to improve for detection.
Again,
we emphasize this preliminary report of SPB virus-like particles is the first
record of viruses associated with SPB and Dendroctonus. We found several
possible virus infections in various adult tissues, but further study is needed
to determine host specificity and symptomology associated with viral infection.
However, we are of the opinion that viruses associated with SPB are important
natural regulating agents, which may be suitable for management of this insect.
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