Hearnpv

THE
HELICOVERPA ARMIGERA NPV

PRODUCTION
MANUAL
D Grzywacz, R J Rabindra,
M Brown, K A Jones
& M Parnell
TABLE OF CONTENTS
Page
1
Executive Outline
1.1
Using the manual
Insect Viruses - an Introduction
2.1
Baculoviruses
2.2
Nucleopolyhedroviruses (NPV)
2.3
Granuloviruses (GV)
2.4
Non-occluded Baculoviruses
2.5
Cytoplasmic Polyhedrosis Viruses
2.6
The Life Cycle of Baculoviruses
10
2.7
Introduction to H. armigera NPV
13
2.8
Commercial Products
13
2.9
References
14
Insect Culture for Virus Production and Testing
15
3.1
Importance of Clean Culture, Sanitation and Hygiene
15
3.2
Insectary Design
15
3.2.1
Quarantine room
17
3.2.2
Entry preparation room
17
3.2.3
Adult moth room
17
3.2.4
Larval holding room
18
3.2.5
Egg handling room
18
3.2.6
Diet preparation room
18
3.2.7
Washing area
18
3.2.8
Sterilisation room
18
3.3
Equipment
19
3.4
Insect culture in the laboratory
19
3.4.1
Establishment of a culture from the wild
20
3.4.2
Quarantine and colony clean up
20
3.5
3.6
Diet
23
3.5.1
Procedure for larval diet
24
3.5.2
Adult diet
24
Insect Rearing Protocol
25
3.6.1
Egg production
25
3.6.2
Larval rearing
25
3.6.3
Rearing of late stage larvae
26
3.6.4
Selection for vigour
27
ii
3.6.5
Supply of Insects for Virus Production
28
3.8
Problems in Rearing Larvae
28
3.8.1
Mould on diet caused by Aspergillus niger
29
3.8.2
Phorid flies
29
References
29
The Production of Insect Viruses
30
4.1
Introduction
30
4.2
Tissue Culture Production
30
4.3
Production in Whole Insects
30
4.4
Insects
31
4.5
Inoculation
32
4.6
Inoculum Purity
32
4.6.1
Protocol for purifying inocula
33
4.6.1.1 Primary processing
33
4.6.1.2 Gradient centrifugation
34
4.7
Incubation and rearing
38
4.8
Sanitation of production facilities
38
4.9
Harvesting
39
4.10
Model protocol for the production of Helicoverpa armigera NPV
4.11
4.12
27
3.7
3.9
Pupation
In multicell trays
40
4.10.1
Preparation of production trays
40
4.10.2
Inoculation of production trays and loading larvae
42
4.10.3
Harvesting infected larvae
43
4.10.4
Processing of infected larvae
44
Processing insect viruses for use as biopesticides
44
4.11.1
Homogenising and filtering
45
4.11.2
Centrifugation
46
Storage of insect viruses
48
4.12.1
Freeze-drying
48
4.12.2
Spray drying
52
4.13
Summary of Main Points
56
4.14
References
56
Quality Control in NPV Production
57
5.1
The Importance of Quality Control
57
5.1.1
57
The importance of routine protocols
5.2
Counting NPV
57
5.3
Larval Equivalents
58
5.4
Quality Control Techniques
58
iii
5.4.1
Observation
58
5.4.2
Microscopy
59
5.4.3
Bioassay
59
5.4.4
DNA identification
60
Microscopic examination of NPV & GV
61
6.1
Microscopes and choice of illumination
61
6.2
Counting baculoviruses and the use of standards
61
6.3
Microscopic staining techniques
62
6.3.1
Sikorowski quick stain for CPV polyhedra (& NPV)
62
6.3.2
Method for differential Giemsa staining of occluded insect

Viruses and other pathogenic micro-organisms in smears of
Insect tissues
6.3.3
63
Giemsa stain with acid hydrolysis for nucleopolyhedrovirus

(granulovirus) and cytoplasmic polyhedrosis virus inclusions
6.4
65
Counting NPV using the improved Neubauer haemocytometer or

Counting chamber
66
6.5
Identifying contaminants under the microscope
70
6.6
References
70
Bioassay Techniques and Microbial Pesticides
72
7.1
Introduction
72
7.1.1
Bioassay
72
7.1.2
Lethal dose50 and lethal concentration50
72
7.1.3
Types of assay
74
7.1.4
Summary of main points
7.2
7.3
7.4
7.5
7.6
74
st
Surface Dose Bioassay to Determine LC50 in 1 Instar Larvae
74
7.2.1
Introduction
74
7.2.2
Procedure
76
Droplet Bioassay
76
7.3.1
Introduction
76
7.3.2
Sample preparation
77
7.3.3
Administration of dose
78
7.3.4
Artificial diet for polypots
81
Diet Plug Bioassay to Determine LD 50
82
7.4.1
Introduction
82
7.4.2
Procedure
83
Leaf Dip Bioassay
83
7.5.1
Introduction
83
7.5.2
Procedure
84
Analysis of bioassay data

7.6.1
85
Graphical method for estimation of the median lethal

Concentration (LC50)
85
iv
7.6.2
7.7
Transformation of percentages to probits
References
86
86
Identification of Insect Viruses with Restriction

Endonucleases
88
8.1
Introduction
88
8.2
DNA Profiling
88
8.3
DNA Extraction and Identification Procedure
89
8.3.1
Extraction and dissolution of virus
89
8.3.2
Phenol extraction of DNA
90
8.3.3
Dialysis
91
8.3.4
Ethanol extraction as an alternative to dialysis
92
8.3.5
Digestion
92
8.3.6
Electrophoresis
93
8.3.7
Preparation of solutions
95
8.3.8
Additional Notes
96
Microbiological examination of viral pesticides
98
9.1
The Problem
98
9.2
Counting Bacteria and Other Contaminants
98
9.3
Equipment
99
9.4
Sampling and Dilution
99
9.5
Total Viable Count
100
9.6
Pathogen Screening
102
9.7
Coliforms, Shigella and Salmonella
102
9.7.1
Motility determination
104
9.7.2
Oxidase test
104
9.7.3
Catalase test
104
9.8
Staphylococcus aureus
104
9.9
Bacillus Species
105
9.10
Yeasts
106
9.11
References
106
EXECUTIVE OUTLINE
This manual details the methods and techniques required to produce Helicoverpa armigera
nucleopolyhedrovirus (NPV) for controlling H. armigera on crops. This virus is a highly virulent
natural pathogen of the insect. When applied to crops in the field in sufficient quantity the virus acts
as a natural pesticide infecting and killing H. armigera larvae and protecting the crop. The virus is a
pathogen specific to H. armigera and a few closely related lepidoptera and will therefore not harm
other insects, animals or man. It is therefore extremely safe and is compatible with all other
biological control techniques using insect parasites, predators or pathogens.
The virus can be produced both in live insects or tissue culture but the latter is currently not
economic for mass production so that only production methods based upon live insects are detailed
here. Production is achieved by feeding larvae with a small quantity of NPV, and then rearing them
for a period. During this time the larvae grow, the infection develops and the virus multiplies. When
the larvae are harvested the virus has increased 10,000-fold over the initial dose. These infected
larvae are then processed to release the virus and as such are used as the basis for the biopesticide.
The high efficiency of this technique of virus replication means that the equivalent of 250-500
infected larvae can be used successfully to protect one hectare of crop.
This production process for viral biopesticides can be carried out using relatively simple techniques
and low capital cost equipment. It is, therefore, appropriate for small scale production or for
production where technological resources are limited. Using these systems the cost of such
biopesticides can be comparable or cheaper than conventional chemical alternatives. In addition,
unlike chemical or bacterial insecticides, no significant history of resistance development to viral
biopesticides has occurred.
However while production is, in principle, simple it does in practice require considerable care and
strict adherence to quality control standards to achieve a consistent product. Failure to adhere
strictly to production procedures can result in loss of production efficiency and contamination by
micro-organisms. Contamination of the insect production facilities by other viruses, bacteria and
insect parasites is the most common cause of production failure. Heavy contamination in batches of
NPV biopesticide results invariably in reduced pesticidal action and failure to control the pests when
used in the field. This manual details the range of procedures needed to maintain and monitor the
quality of the virus.
1.1
USING THE MANUAL
This manual describes a number of procedures used to produce H. armigera NPV. However this
manual does not detail all the possible modifications and adaptations that may be required for
specific local production situations and should not be taken as a replacement for good product
development. The availability of local ingredients, equipment and resources may determine that
some modifications, however minor, will almost always be required in practice. Each modification
will need to be validated, specifically, before being implemented. This information in this manual
should be used as a guide and to assist local product development not as a replacement for good
applied research.
While we have endeavoured to describe as clearly and comprehensively as possible all the
procedures detailed in this manual, many of them do require a level of expertise and skill that is best
acquired only through hands-on training and experience with someone already competent in the
techniques. Actual training alongside experienced staff in such procedures is always preferable to
attempting to replicate procedures from this manual alone. The authors are always willing to offer
advice on suitable training opportunities if users have difficulty in locating appropriate training.
Many of these procedures can be used in the production of other insect viruses either unchanged or
with some modification and it is hoped that the information contained here will stimulate and assist
workers developing other viruses as pest control agents. The authors welcome enquiries and
feedback on modifications or new procedures for producing other viruses.
INSECT VIRUSES - AN INTRODUCTION
Over 600 viruses have been reported as infecting insects, these are drawn from 15 families of
viruses. Important characteristics used to differentiate these viruses are:
(a)
(b)
(c)
(d)
The nature of the genetic material (RNA or DNA) and its form (single or double
stranded).
The size and shape of the virus particles.
Whether or not the virus particle is enveloped by a membrane.
Whether or not the virus particle is occluded within a proteinaceous inclusion
body.
Table 1. Families of insect viruses and their characteristics.

Family
Particle morphology
Shape
Size (nm)
Isometric
18-26
Icosahedral
125-300
Envelope
Parvoviridae
Iridoviridae
Nucleic
acid
SsDNA
DsDNA
Occl'n.
body
-
Baculoviridae
Poxviridae
Polydnaviridae
DsDNA
DsDNA
DsDNA
Ascoviridae
DsDNA
40-60 x 200-400
165-300 x150-470
150x350
85x330
130x400
+
+
+
+
+
+
+
-
Nodaviridae
Picornaviridae
Tetraviridae
Reoviridae
Birnaviridae
Rhabdoviridae
SsRNA
SsRNA
SsRNA
DsRNA
DsRNA
SsRNA
29 diam
22-30 diam
35-39 diam
55-69 diam
60 diam
50-95 x 130-380
+
-
Togaviridae
Flaviviridae
Bunyaviridae
SsRNA
SsRNA
SsRNA
Bacilliform
Ovoid
Ovoid
Nucleocapsid
Allantoid/
Bacilliform
Icosahedral
Spherical
Icosahedral
Icosahedral
Icosahedral
Bullet shaped/
Bacilliform
Spherical
Spherical
Spherical/Oval
60-65 diam
35-45 diam
90-100 diam
+
+
+
For the purposes of pest control, interest has concentrated on those families of viruses that do not
infect mammals, for obvious safety reasons. Of the 15 families infecting insects only three, the
baculoviruses, the polydnaviruses and the ascoviruses are not suspected of having mammalian hosts.
The baculoviruses have been the most studied as these are both highly infectious by ingestion and
show good horizontal transmission (from adult to egg). They also have the advantage of infecting
many important species of lepidopteran and other insect pests. The ascoviruses are considered
poor candidates as they have a low infectivity. The polydnaviruses are also poor candidates as they
are known to replicate only in the ovaries of parasitic wasps.
2.1
BACULOVIRUSES
Biopesticide development is concerned almost exclusively with members of one family, the
Baculoviridae (the Baculoviruses). The two groups of viruses found in this family are the
nucleopolyhedroviruses (NPV) and the granuloviruses (GV). The baculoviruses infect >600 species
of insect, mainly Lepidoptera, including many important pest species. Insect species in other orders
infected by baculoviruses include Hymenoptera (31 species), Diptera (27) and Coleoptera (5).
Baculoviruses also infect a few other species of arthropods such as prawns and other crustacea, but
do not infect any non-arthropod species. This specificity is one attraction of baculoviruses as
biopesticides; they are known to be completely safe to man, animals and important beneficial insects
such as bees, predatory insects and parasitoids.
The baculoviruses are rod-shaped (baculo = rod) particles containing double stranded DNA
enclosed by a lipoprotein envelope.
Both the NPV and GV are found occluded within
proteinaceous inclusion bodies which provide some environmental protection.
Over 20 species of baculovirus have been developed or registered as commercially available
insecticides and over 30 different products, based upon NPV or GV, have been registered as
commercial insecticides. This text will concentrate mainly on the Helicoverpa (Heliothis) armigera
NPV (HaNPV).
Another important reason for the interest in NPV as potential insect control agents is that, unusually
for viruses, they are relatively easy to see and count using a light microscope. Almost all other
viruses are too small to be seen except with an Electron Microscope, and so are much more difficult
to identify and isolate.
NPV attracted the attention of pest control scientists, interested in looking for an alternative to
chemical pesticides, because they can cause highly infectious disease that kills in 5-7 days. These
viruses attack some of the most important Lepidopteran crop pests, including species of Heliothis,
Helicoverpa and Spodoptera. Some of the related GV species are also highly infectious, including
Cydia pomonella (apple codling moth) GV (CpGV) and Plutella xylostella (diamond back moth)
GV (PxGV). However not all GV are as fast acting as NPV and some such as Spodoptera
littoralis (cotton leafworm) GV (SlGV) produce chronic infections that may only kill after an
extended period 20-30 days and thus are much less suitable for biopesticide use.
The other important characteristic of baculoviruses is that most kill only target pest species. This can
be seen as an advantage or a drawback. This specificity and safety to most other insects means it is
completely compatible with other biological control options. The virus has no effect on beneficial
insects, predators or parasites, so that its use does not produce any of the secondary pest problems
encountered when broad spectrum chemicals are used. Its specificity also means it is safe to
humans and can be sprayed onto crops right up to time of harvesting with no residue problems.
2.2
NUCLEOPOLYHEDROVIRUSES (NPV)
These are the viruses most studied and used commercially in pest control. They infect over 400
species of insect and are well known to cause major lethal epizootics in important lepidopteran pest
species. They are commonly isolated from insects in the field and being visible with light microscopy
can be easily detected if present. They also have, for viruses, a relatively fast mode of action, killing
infected insects within 4-7 days.
NPV are rod shaped double stranded DNA viruses of the family Baculoviridae which infect a wide
range of insect species, chiefly Lepidoptera but also some members of the Hymenoptera and
Diptera. These viruses are the cause of a highly infectious lethal disease in the larvae of susceptible
species. The NPV are highly specific and most infect only a few species of closely related insects.
The viruses are named after the insect in which they were first isolated and identified.
The NPV are seen in dead or dying larvae as bright irregular crystals called occlusion bodies (OB)
or polyhedral inclusion bodies (PIB) (Plate 1). These are protein crystals normally 1-7m across
that show up at x400 as bright refractive crystals, especially under phase contrast. These crystals
are many sided (polyhedra in Latin) and are composed of a protein with a molecular weight of 2530 kilodaltons called polyhedrin. The whole OB crystal is surrounded by a calyx or membrane of
protein.
Plate 1
Helicoverpa armigera NPV. Phase contrast
illumination at X1000 magnification. NPV
arrowed.
Within this PIB are found embedded up to 200 virus particles or virions. These virions are the
actual virus infective particles and are composed of a rod-shaped DNA/protein structure called a
nucleocapsid (approximately 50 x 250-400nm), inside a membrane envelope (Plate 2). The circular
double stranded DNA is 88-200 kilobase pairs (kbp) in length and 50-100 megadaltons in weight.
The nucleocapsids may be found either enveloped singly (SNPV) by the lipoprotein membrane
envelope or, in some species, multiple (MNPV) nucleocapsids are enveloped within each envelope.
Between 20 to 200 virions are embedded in the polyhedrin matrix depending upon species, e.g. in
HaNPV there are commonly up to 30 virions in each OB.
These OB are the infective stage of the virus, designed to transmit the infection from insect to insect.
The polyhedrin crystal helps to protect the vulnerable virions from inactivation by environmental
factors. Baculoviruses in this form are extremely stable and can retain infectivity for many years, if
not exposed to UV light or high temperatures(>50C).
For more detail on the biology of the Baculoviruses the reader is recommended to refer to one of
the standard works such as Tanada & Kaya (1993), Adams & McClintock (1991), Hunter-Fujita
et al (1998) or the two volume work by Granados and Federici (1986).
The virus infects the insect in the form of a polyhedral inclusion body (PIB). When the polyhedra
enters the mid gut of an insect it dissolves under the alkaline conditions (pH 9-11) releasing the
virions. These virions enter the cells of the mid gut and proceed to multiply in the nucleus. From this
initial infection new virions are produced which proceed to spread the infection to other body tissues
such as haematocytes, tracheal cells, fat body cells and hypodermis. It is in these tissues during the
later stages of the infection that polyhedra are produced in which virions become embedded. When
the insect dies it ruptures releasing these polyhedra to infect other insects. Insects killed by NPV will
commonly contain up to 100 million PIB. In the wild, infection occurs through the ingestion of PIB.
Insects infected with NPV show few symptoms for the first 2-4 days after ingestion of the virus.
The larvae then progressively cease to feed and become less active. During advanced stages of the
infection, as the epidermis is infected, the skin becomes very fragile and ruptures easily. The larvae
become wilted and the body contents become a fluidised mass of decomposed tissues and
polyhedra. Just prior to death infected larvae often climb to the highest parts of the substrate they
are located, e.g. tops of plants and attach themselves by their prolegs. On death they hang in a
characteristic V-shape (Plate 3).
Plate 2 Electron micrograph of NPV. Virions arrowed.
Plate 3
Classic symptoms of an H.armigera larva killed by NPV infection
2.3
GRANULOVIRUSES (GV)
The GV are slightly different to NPV in that there is normally one virion (rarely two) in each
protective inclusion crystal. The crystals, called granules or capsules, are much smaller than the
polyhedra and are rod-shaped with rounded ends (Plate 4). Each granule measures 0.2-0.5m x
0.05m and are just visible under the light microscope at x1000 (Plate 5). The single virion within
each granule is surrounded by a crystalline protein matrix very similar to that of the polyhedra of
NPV. In GV this protein is called granulin. It is chemically very similar to polyhedrin being a 25-30
kilodalton (Kda) polypeptide. The genome of GV is double stranded DNA of 90-160 kbp.
Plate 4
Electron micrograph of Granulovirus (R. Bateman)
Plate 5
Granulovirus. Dark field at X1000
Over 150 species of insect, mainly lepidoptera, are known to be susceptible to GV. These species
are principally the Noctuidae (approx. 50 species) and Tortricidae (20 species).
GV usually infect the insects through the midgut epithelia cells, similar to the action of NPV. The
capsules dissolve under the alkaline conditions of the midgut releasing the virions which then invade
the midgut epithelium cells. The virus replicates in these cells and the virions produced then go on to
infect the major target organ, the fat body. Some species of GV may also attack the hypodermal
and tracheal tissues.
Where the fat body is the major organ invaded, the main symptom of infection is reduced growth
rate. In some species growth may continue beyond the normal larval life time, even producing larvae
which are eventually larger than uninfected larvae. These larvae do eventually die after becoming
progressively weaker and sluggish, and showing mottling or other colour changes (Plate 6). These
infections are much slower than with NPV, often taking 20-30 days to kill.
In those GV species where tissues other than the fat body are attacked, the symptoms resemble
those of NPV with death resulting in 4-7 days. The larvae, once infected, become wilted with the
skin being very fragile and tending to rupture in the later stages.
Plate 6
Spodoptera littorals larva infected with GV
There is one GV, affecting the grape leaf skeletoniser (Harrisinia brillians), in which the infection
and virus replication is confined to the midgut tissue alone. These larvae become wilted, and often
are shrunken and darken in colour. A diahorreal discharge from the gut, containing many infective
GV capsules, is common.
Thus, it is only those GV which attack tissues other than the fat body which produce rapid death and
are, therefore, of interest as microbial insecticides. Of these, the Cydia pomonella GV has already
been commercialised for control of the apple codling moth Cydia pomonella. Other GV of
commercial interest include Pieris rapis GV and Plutella xylostella GV.
2.4
NON-OCCLUDED BACULOVIRUSES
There was until recently a third group of baculoviruses, the non-occluded baculoviruses (NOB), in
which no occlusion bodies had been observed. In these viruses only virions are found. Three
species have been studied in any detail, these include NOB affecting the rhinoceros beetle (Oryctes
rhinoceros) and Heliothis zea. The O. rhinoceros NOB has been used as an agent for control of
O. rhinoceros in Melanesia. However now these viruses have been removed from the baculovirus
group and re-designated as a separate family of non-affiliated viruses.
2.5
CYTOPLASMIC POLYHEDROSIS VIRUSES
There is one group of viruses that under the light microscope can be confused with NPV, and these
are the cytoplasmic polyhedrosis viruses (CPV). These are from a completely different family of
viruses, the Reoviridae. These differ from baculoviruses in having RNA as their genetic material
and the infectious particles are icosahedral (many sides, all equal in shape) of 55-70nm in diameter
rather than rod-shaped, as in the baculoviruses. The genetic material si composed of double
stranded RNA found in 9-10 distinct segments of total molecular weight 13-18 Kda and 20-30 kbp
in length.
However these icosahedral CPV virus particles are also embedded in a large crystal protein
protective crystal similar to OB, though they can be larger than the OB of NPV at 3-15m. The
biology and life cycle of CPV are different to that of NPV as these viruses infect only certain
tissues, producing chronic infections of the gut and are much slower to kill and therefore less useful
as insecticides than baculoviruses (although they may be effective as classical biological control
agents). CPV are important as they can infect the same species as NPV, can become contaminants
in the production of NPV and may be a problem in insect rearing. Distinguishing the OB of CPV
from NPV is not easy under a light microscope (Plate 7) and correct identification of CPV requires
special staining techniques, nucleic acid analysis or an electron microscope. As the name suggests
these viruses multiply in the cytoplasm of infected cells. While there are some superficial similarities
with NPV in that the polyhedra dissolve in the midgut to release the virions and these then infect the
midgut epithelial cells, in CPV the infection is confined to the midgut alone and does not spread to
other tissues. CPV do not kill the insect quickly but give rise to chronic infections in which insects
continually shed infective polyhedra in the faeces.
Plate 7
CPV at X1000 magnification
As a result of the slow speed of kill there has been little interest in developing them as insect control
agents. They are, however, commonly found infecting lepidoptera including the Heliothis species.
2.6
THE LIFE CYCLE OF BACULOVIRUSES
An appreciation of the life cycle of baculoviruses like NPV and its mode of replication is essential
for an understanding of virus production dynamics. Baculoviruses infect insects when the OB are
ingested by a susceptible species along with the food. It must be emphasised that these viruses have
no contact effect and cannot infect an insect unless eaten. In the alkaline conditions of the insect
midgut the protective polyhedral crystal dissolves releasing the infectious virions into the gut. The
infective dose for NPV varies with the age of the larvae, in some species we know that a single
polyhedra contains sufficient virions to infect a newly hatched neonate larva. In older, larger larvae
the dose needed to produce an infection increases and for later instars up to 10,000 OB may be
required.
The released virions pass through the peritrophic membrane of the gut and fuse with the microvilli on
the columnar epithelial cells. This releases the nucleocapsids, the DNA/protein rods contained in the
virions, which enter the cell and migrate to the nucleus, entering through the nuclear pores. This
initiates the first replication cycle of the virus that is restricted to the cells in the midgut. Here the
nucleocapsid unwinds, the DNA is exposed and virus replication begins. Like all viruses, NPV and
GV need to use a living insect cell to replicate. Within as little as one hour post infection (PI), virus
replication can begin. During this time the virus must halt the host cells normal pattern of activity,
enabling it produce virally encoded proteins and replicating the viral DNA.
The nucleus of the infected cell becomes swollen and enlarged at 8 hours PI as progeny virus start to
be produced. These progeny virus particles are called extra cellular virus (ECV) or budded virus
(BV) and consist of naked nucleocapsids. These migrate to the outside of the cell, where they
acquire an envelope and protein structures, called peplomers, of a particular 64K protein at one end
10
of the virion before leaving the cell. Some production of polyhedra may also occur in the first 48
hours PI but these OB are usually small and defective containing no virions.
At this point the first cycle of viral replication is complete and the second phase of infection in the
other body tissues follows. Just as the OB is the form of the virus designed to carry the infection
from insect to insect, the ECV or BV is the form in which the virus spreads from the initial site of
infection in the midgut to the other tissues of the body of the insect.
It is in the midgut tissue that the host insects are sometimes able to contain and halt the infection by
destroying or shedding infected cells before the first cycle is completed. The immune system of the
host insects can detect infected cells and then try to destroy these cells before viral replication is
complete thus halting the infection. However, some NPV are known to have the genes for proteins
that block this infected cell recognition system thus helping the virus defeat the hosts immune
system. Even in successfully infected insects the destroyed midgut tissues are replaced in a day or
two after initial infection so the gut recovers and the insect resumes feeding.
After the midgut cycle the BV particles spread throughout the body in the haemolymph and infects,
in turn, the haematocytes, and cells of the fat body, trachea and hypodermis. It is in these tissues
that a second cycle of infection occurs and that the OB are produced. If the infection gets
established and this second cycle of virus replication occurs, it is often found that 90% or more of
susceptible cells may become infected. In these the nuclei become swollen and nucleocapsids are
produced, but unlike in the first cycle large amounts of polyhedrin are also synthesised and condense
to form crystals in which the nucleocapsids become embedded, to form new OB and so complete
the cycle. Cells containing large numbers of OB burst and disintegrate. This OB production in a
species such as H. armigera starts around 5 days after infection, peaking at 7-8 days PI. The
massive destruction of body tissue that accompanies the production of OB eventually kills the insect.
The virus also produces in the last stages various protease enzymes that aid the breakdown of insect
cells, thus releasing the OB to help them spread and infect other insects.
In a typical nucleus 10-50 OB are produced giving 109-1010 OB per insect. On death these OB
may comprise in total 10% of the insects weight. This astonishing high productivity of NPV in
larvae is another reason for their attraction as biopesticides and is unmatched by any other type of
virus. Successfully infected insects secrete NPV during the later stages of infection and move about
the host plants spreading virus extensively before dying. OB also remain active if eaten and passed
through predators and the activities of birds, mammals and other larval predators may be important
in spreading NPV epidemics.
In some GV, such as CpGV or PxGV, the cycle is similar but in others, such as SlGV, a smaller
range of tissues is infected and instead of rapidly destroying the insect they produce chemicals to
prolong its larval life. In these viruses an infected insect produces virus at a much slower rate than in
NPV, but as the larvae may live twice as long as normal before dying this can also be a successful
strategy for spreading the GV widely.
The OB are the persistent form of the virus with the crystal protecting the vulnerable nucleocapsids.
Under the right conditions, for example in soil, these OB can remain active for years. However
while OB are relatively resistant to heat, desiccation and some other environmental factors, they are
11
quickly denatured and inactivated by UV light which rapidly inactivates the DNA, without destroying
the OB.
NPV and GV have strictly limited host ranges and most species successfully infect only a few related
species of host insect. HaNPV for example can replicate in H. armigera, H. zea and H. virescens
as can HzNPV. NPV and GV are named after the insect species they were first described from,
and also on the basis of whether one (singly enveloped) or more than one (multiple enveloped)
nucleocapsids are contained in a single envelope, e.g. H. armigera Singly enveloped
Nucleopolyhedrovirus, HaSNPV.
Apart from the multiple or singly enveloped feature, NPV or GV cannot be identified visually from
either light or EM microscopy. To identify viruses beyond the grouping into GV or NPV you need
to look at the DNA sequence using restriction enzymes or molecular probes. Examination of the
DNA using these techniques has shown that many variants of a species may exist. The existence of
these genetic variants with different biological activities may have important implications for the
development of biopesticides, both in the possibility to select better naturally occurring strains and as
a source of material for genetic manipulation.
2.7
INTRODUCTION TO H. ARMIGERA NPV
The Helicoverpa armigera nucleopolyhedrovirus (HaNPV) is a baculovirus that infects and kills H.
armigera (podborer, gram podborer, American boll worm, tomato fruit worm). The pest is
endemic to Africa, Southern Europe, Asia and Australia, and the HaNPV has also been isolated
from all of these areas. H. armigera is susceptible to both the H. armigera NPV (HaNPV),
originally isolated from it, and also the Heliothis zea NPV. The HaNPV virus itself also infects both
H. virescens and H. zea. This characteristic specificity confers both advantages and limitations. It
means the NPV are harmless to man, domestic animals and other wildlife making them extremely
safe and environmentally acceptable. They will not harm domestic insects such as bees or
silkworms or beneficial insects such as parasitoids and predacious insects, and they are ideal
candidates for use within an Integrated Pest Management (IPM) strategy. The limitation is that a
NPV capable of killing H. armigera will not infect other pest insects present on the same crop, even
other Lepidoptera such as Spodoptera spp., Erias spp., or Agrotis spp.
2.8
COMMERCIAL PRODUCTS
A number of commercial products based upon NPV have been developed, mainly for controlling
lepidopteran pests (Plate 8). These have included the Heliothis virus product Elcar, the first one
registered as a true commercial NPV product in 1975, but since discontinued. More recently a new
generation of baculovirus-based products have appeared, including another Heliothis NPV product
Gemstar and Spod-X, based upon Spodoptera exigua NPV. Other similar products are expected
to appear in due course.
12
Plate 8
Commercial NPV products produced in Thailand and India
2.9
REFERENCES
Adams, J R & McClintock, J T (1991) Nucleopolyhedroviruses of insects. In: Atlas of

invertebrate viruses, J R Adams & J R. Bonami (eds.), CRC Press, Boca Raton
Copping, L G (1993) Baculoviruses in crop protection. Agrow Business report, DS 85, PJB
Publications Ltd.
Granados, R R & Federici, B A (eds.) (1986) Biology of Baculoviruses, Volume 1: Biological
properties and molecular biology, and Volume 2: Practical Applications for Insect Control,
CRC Press, Boca Raton, Florida.
Hunter-Fujita, F R, Entwistle, PF, Evans, H F and Crook, N E (eds.) (1998) Insect Viruses and
Pest Management. Wiley & Sons, Chichester.
King, L A & Possee, R D (1992) The Baculovirus expression system. Chapman Hall, London.
OReilly, D R; Miller, L K & Luckow, V A (1994) Baculovirus expression vectors: a laboratory
manual. Oxford University Press, New York.
Shuler, M L; Wood, H A; Granados, R R & Hammer, D A (eds.) (1995) Baculovirus expression
systems and biopesticides, Wiley Liss, New York.
13
Tanada, Y & Kaya, H K (1993) Insect Pathology, Academic Press, San Diego.
14
INSECT CULTURE FOR VIRUS PRODUCTION AND

TESTING
A supply of healthy, disease-free insects is the first requirement of any programme to either produce
or test insect viruses. In practice the establishment of such a culture, even with ideal facilities, can be
a surprisingly long process and often at least three months is required to achieve this, even if no
serious problems are encountered. Where conditions are not ideal, i.e. where strict quarantine
facilities for acclimating field collected insects are not available, it can take much longer or become
impossible. Even with an apparently vigorous and successfully widespread insect pest such as H.
armigera, it can prove difficult to establish a culture in the laboratory. While the major enemies of
culture establishment are disease, fungus and parasites, phenomena such as diapause and infertility
can also be serious problems.
3.1
IMPORTANCE OF CLEAN CULTURE, SANITATION AND HYGIENE
The establishment and production of a healthy colony of insects is only possible if basic facilities are
available and rigorous sanitation and hygiene protocols are established and followed. The
production facility must be maintained in very clean conditions. All working surfaces, flooring and
plastic ware must be surface sterilised with 0.5 % sodium hypochlorite daily.
Glassware and metal ware can be sterilised in a hot air oven at 180C for 2 hours. Plastics, if
autoclavable, can be sterilised by autoclaving for 30 minutes at 121C and 12 bar.
Various types of plastic disposable rearing vessels are available. These range from individual cups
(approximately 25ml) to multi-cavity trays with suitable lids. Disposables may be expensive in
developing countries, but have an advantage of making disease control in the colony very much
easier. If reusable containers are used, they should be sterilised, washed thoroughly in a separate
room and then sterilised again prior to re-use.
All waste from the insectary or virus production unit should be autoclaved or burned before
disposal.
3.2
INSECTARY DESIGN
The insectary for the production of host insects should be designed in such a way as to avoid
contamination of the colony with pathogenic diseases. The rearing area should be well isolated with
restricted access. A plan of a model insectary with dimensions is shown in Figure 1.
15
Figure 1. Basic plan of a model Insectary for Helicoverpa armigera
10
10
16
STERILIZATION
STORE
WASHING
DIET PREPARATION
EGG HANDLING
LARVAL HOLDING
MOTH ROOM
10
10
16
ENTRY
PREPARATION
QUARANTINE
OFFICE
14
14
While a smaller less dedicated facility may have to serve, especially when a research or production
programme is in its early days, as far as possible insectaries should try to incorporate the isolation
characteristics and protocols of the dedicated facility as described below.
16
The requirements of the different sections are as follows.
3.2.1
Quarantine room
Laboratories used for establishing new colonies must have a separate special quarantine facility. The
greatest potential source of disease is from insects newly introduced from the wild. No insects
should be introduced into the main culture until they have undergone quarantine to determine that
they are disease-free, or a selection process has been carried out to eradicate any disease. Wild
colonies are held here for observation and examination of insects dying in the process of rearing, and
later on for the examination of reproducing female moths to check for microsporidia infection1.
Staff working in the quarantine room should not enter any other part of the facility. The office as
well as the quarantine area should be isolated from the main rearing facility.
3.2.2
Entry preparation room
Access to the main insect culture facilities should be through a room where the workers can wash
their hands and change to sterile overall clothing before entering the rearing area. If full changes of
clothing are impractical then the wearing of clean laboratory coats and shoe covers or special indoor
footwear should be adopted as routine. An air shower may also be built into this part of the facility
for the staff to pass through after changing and before entering the rearing facilities. Shower facilities
may also be included, particularly where insects that are known to produce allergenic reaction in
humans are reared.
3.2.3
Adult moth room
This room should have temperature and humidity control that maintains optimum conditions for the
host insect; for H. armigera and most important noctuid species these are 24 + 2C and a relative
humidity of 90 + 5%). Air coolers with humidistats may be used to regulate the humidity. The filters
in the air coolers trap the moth scales and can be periodically removed and cleaned. A suitable air
circulation and filtration system can also be installed to trap moth scales which may otherwise pose
health hazards to workers. Allergy to insect scales and hairs do occur, and are most pronounced
with those insect species with urticating hairs. However, even such species as H. armigera that are
not associated with urticating hairs may initiate dermal or pulmonary allergic responses in staff. This
can be overcome by the use of particle masks and regular cleaning of air filters to reduce scale and
hair problems. In some cases though, it may be necessary for sensitised staff to wear full protective
suits and wear air filter apparatus.
Insects are infected with microsporidia through ingestion of contaminated food, however, microsporidia can be
transmitted transovarially and is therefore very difficult to eradicate from a colony once infection is established.
Infection is normally chronic and results in reduced longevity and fecundity. This is one of the most common
problems in insect rearing systems; eradication normally requires re-establishment of the colony from single,
uninfected breeding pairs (see section 3.4.2).
17
3.2.4
Larval holding room
This room should be maintained at a temperature of 24 + 2C and a low relative humidity of 45

5%. It should be large enough to hold larval holding trays arranged in racks (see Section 3.6).
3.2.5
Egg handling room
This facility should be isolated from the moth room. The temperature should be 24
Humidity is not critical.
3.2.6
2C.
Diet preparation room
This is where the diet is prepared, cooked blended and stored. This room should have a balance
for weighing out the diet components, a stove or microwave to cook the diet and a blender of
suitable size to mix the diet. Annexed to this room should be the store for the diet materials, and
should contain a refrigerator for storing items such as vitamins and antibiotics. A large refrigerator
for holding prepared diet prior to use is also advisable.
3.2.7
Washing area
This should be an isolated area to wash the larval trays, moth cages, oviposition cages, and all other
rearing appliances.
3.2.8
Sterilisation room
This facility for sterilisation of equipment should be located next to the wash area.
18
3.3
EQUIPMENT
The following items should be considered as essential, or at least highly desirable, equipment in any
insectary. More specialised, industrial scale equipment may also be used for large-scale commercial
production.
Equipment
Purpose
1. Research microscope with phase contrast Examination of tissues for disease diagnosis.
and
dark field facility x400 - x1000
2. Horizontal laminar hood(s)
Clean, contaminant-free air during cooling of
diet and infesting the diet with eggs/larvae.
3. Electronic balance(s)
0.1g to 300g for weighing diet ingredients;
0.001g to 30g for weighing larvae/pupae.
4. Gas stove/microwave oven
Cooking diet.
5. Refrigerator
For storing larval diet and adult feed;
Vanderzant vitamin mix and antibiotics.
6. Water distillation \ osmosis unit
Distilled water for diet.
7. Hot air oven
Sterilisation of glass and metal ware.
8. Autoclave
Sterilisation of autoclavable rearing materials.
9. Room air conditioners
Temperature control in rearing rooms.
10. Room air coolers/humidifiers
Humidity control in rearing room.
11. Thermohygrographs or electronic
Monitoring conditions in moth and rearing
temperature/humidity monitors with data rooms.
logger
12. Blender
Mixing diet components.
3.4
INSECT CULTURE IN THE LABORATORY
Great care should be taken while establishing a colony of H. armigera. There are two ways of
initiating a colony.
Acquiring insects from a well established insectary.
Starting a new colony from wild populations.
19
3.4.1
Establishment of a culture from the wild
In most developing countries, starting a culture by obtaining insects from a disease-free established
colonies may not be possible. The only other option is to initiate the colony from wild populations.
H. armigera is polyphagous, attacking several crops and can be collected in sufficiently large
numbers on chickpea, pigeon pea and sunflower. Populations collected from the early broods can
be expected to be largely free from diseases, particularly microsporidiosis. In India, late broods
collected in the months of January and February can often be infected with Vairimorpha spp.
Ideally isolated breeding pairs should be kept separately, so that the origin of all eggmasses are
known and source of disease isolated and destroyed (see next section).
3.4.2
Quarantine and colony clean up
Field-collected insects should be reared in isolation in the quarantine room following the routine
protocols (see Section 5: Quality Control), and closely watched for any disease symptoms.
Larvae which show symptoms of disease should be removed from the colony and destroyed by
autoclaving. Strict sanitation should be maintained to avoid infection and the spread of the disease.
Slow-growing and stunted larvae, as well as malformed pupae and adults, should be destroyed by
autoclaving prior to disposal (caution: Never discard directly into the waste bin without
autocalving). Forceps used for collection of diseased insects should also be sterilised. All the spent
moths should be examined for microsporidian infection and only the progeny from disease-free
moths should be selected for further rearing, and the rest should be destroyed by autoclaving.
The microsporidia (Plate9), particularly Vairimorpha spp., commonly occur in laboratory colonies
of H. armigera and weaken the insects, leading to reduced fecundity. The production of NPV in
microsporidian infected larvae can be reduced substantially. In order to maintain the vigour and
productivity of the colony, it is essential that this pathogen is kept out of the colony. Since the
pathogen can be transmitted vertically to the next generation through the eggs, the utmost care
should be taken to select insects free from microsporidiosis. This is achieved through maintaining
separate breeding groups (or, alternatively, single breeding pairs) and careful microscopic
examination of smears of moths after egg laying.
Pupae are segregated in groups of 20 and kept in separate emergence cages with identification
numbers. The progeny groups are maintained separately and the abdomen of spent moths are
pooled, macerated in distilled water (l0ml), filtered through muslin, and a droplet smeared on a slide
and examined under a phase contrast research microscope (Figure 2). The spores of the
microsporidian are elliptical measuring about 2 x 4.5m. All progeny from infected moths should be
discarded. Progeny eggs and larvae obtained from moths free from microsporidians are reared very
carefully and the second generation moths screened again for microsporidians. The selection
process is repeated with the third generation moths, and pupae from the clean colony are moved to
the regular rearing facility after surface sterilisation with 0.5% sodium hypochlorite.
20
Plate 9
Microsporidia (arrowed), a common contaminant.
21
Figure 2. Selection of microsporidian-free H. armigera population

Moth
groups
Egg
Batches
Spent
Moth
groups
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
na
nb
nc
nd
ne
Pool abdomens from each group separately and prepare smears in distilled water
Examine under Phase Contrast microscope (x45 objective)
Vairimorpha
spores*
*If present (+),
the subcolony
must be
destroyed.
Select microsporidian-free groups for

further rearing
REPEAT SCREENING - II GENERATION
SELECT
REPEAT SCREENING - III GENERATION
SELECT FOR COLONY
22
3.5
DIET
Larvae can be reared on either natural host plants or artificial diet. Development times are often
reported as shorter for insects reared on host plants but in most insectaries artificial diets are
preferred for convenience, standardisation and hygiene reasons.
The larvae can be grown on a wide variety of semi-synthetic diets, most of which are variants on the
original semi-synthetic diets of Vanderzant et al (1962). Diets based upon wheat germ have been
used very successfully but as this item is often not available in developing countries a diet based
upon a more generally available alternative such as chickpea is preferable. The composition of one
such diet used for many generations in India is given in Table 1, below.
Table 1.
Composition of semi-synthetic diet for H. armigera
COMPONENT
Chickpea flour
Yeast
Wesson's salt mix
Methyl Paraben2
Sorbic acid
Ascorbic acid
Agar
Vanderzant vitamin solution
Streptomycin sulphate
Carbendazim
Formalin
Water
*
**
***
QUANTITY
100 g*
30 g
7g
2g
1g
3g
13 g
8 ml**
40 mg
675 mg
2 ml***
720 ml
Whole chickpea seeds can also be used (soak in distilled water overnight).
28% solution in distilled water.
Not included in diets used for inoculation of larvae with virus and post-inoculation rearing.
Other bases for diet have been tested including haricot bean flour, soybean flour and sorghum.
Chickpea has often been found to be the best while sorghum has been reported as tending to
produce deformed pupae.
4-methyl parahydroxy benzoate
23
3.5.1
Procedure for larval diet
1. Boil agar in 360ml of water, until completely dissolved.

2. Cook chickpeas in 360ml of water for 10 minutes and then transfer to a mixer.
3. Add the agar to the chickpea flour and mix.
4. The Wessons salt mix, methyl paraben, sorbic acid and yeast are added together and
homogenised for 5 minutes.
5. After the diet has cooled to about 70C, add the vitamin mix, ascorbic acid, carbendazim,
formalin and streptomycin sulphate, and mix the diet well for 5 minutes.
6. While still warm, dispense the diet into the rearing vessels, place (preferably) in a laminar flow
hood and leave to cool and solidify.
7. Cover the diet with clingfilm and store in a refrigerator until use.
3.5.2
Adult diet
The natural food of adult moths is nectar. In the laboratory they are commonly fed on a 10% sugar
or honey solution, however a syrup such as that detailed below in Table 2, containing vitamins and a
preservative is better. Adult diet pots should be changed every day, because if pots are left the diet
ferments.
Table 2.
Composition of adult diet
Sucrose
Methyl paraben
Absolute alcohol
Vanderzant vitamin solution
(28% in distilled water)
Distilled water
50g
1g*
1ml*
10ml
500ml
*Dissolve methyl paraben in absolute alcohol before mixing with water.

This syrup should be stored in a refrigerator.
24
3.6
INSECT REARING PROTOCOL
3.6.1 Egg production

1. Place the pupae in a dish of about 200ml capacity, filled with damped, autoclaved vermiculite (or
sand) inside netlon emergence cages (40 x 40 x 40cm).
2. Dispense the adult diet in to suitable sterile glass vials containing sterile absorbent cotton; then
place these inside the emergence cages.
3. Select 10 adult males and 10 females from the emerged moths. The females have a brownish
tinge and from batches of pupae of the same age will emerge first, followed by the males which
have a greenish tinge. To keep a continuous culture, egg batches from at least 20-30 pairs are
required. In the absence of humidity controlled rooms the cages can be held in large plastic tents
with open pots containing water pots with wicks. With small cages, similar water pots can be
placed inside the cages.
4. Release the 10 pairs of moths into each of the egg laying cages. These are plastic containers (20
x 30cm) and covered with a fine muslin cloth. Adult feed is again provided inside in two glass
vials.
5. Check the cages daily and remove the cloth on which the eggs are laid, replace it with a fresh
one. Also replace the diet pots with fresh ones.
6. After five days of egg laying the moths are generally spent and should be discarded.
7. After collection, store the egg cloths in humid plastic containers (>95% RH).
8. After 24 hours, surface sterilise the eggs by immersing them in 10% formalin for 10 minutes.
Then wash the cloth in running tap water for 15 minutes before hanging it up to dry. Do not wash
too vigorously or the eggs will be washed off. As an alternative to the use of formalin immersion
of eggs in 0.1% freshly prepared sodium hypochlorite is also effective in sterilising eggs.
9. Replace the cloth in the humidified storage box. Normally the eggs hatch on the third day at 24 +
2C.
In-breeding has been reported as a problem in cultures and efforts should be made to avoid sibling
matings. Bartlett (1985) gives guidelines for achieving this. To improve breeding success some
authorities recommend only maintaining adults as pairs, singly to a cage, i.e. one pair to a cage.
However, the multiple pair cage system described above has been used very successfully in NRI,
India and Thailand.
3.6.2
Larval rearing
25
Larvae can be grown in groups (Plate 10) until the early third instar stage at about 7 days post
hatching. After this they turn cannibalistic and must be reared separately.
Plate 10
Group rearing of H. armigera larvae
1. After hatching starts, transfer the egg cloth with the neonate larvae to boxes (30 x 20cm)
containing a 4 blocks of diet.
2. Close with a tight fitting lid and keep on the bench.
3. Alternatively cut up the cloth into parts with 25-50 neonate larvae on, put one-to-a-tub in a
200ml tubs with a block of diet (20x20mm) at the bottom and store as above. The larvae will
climb on to the diet and settle to feed.
4. Allow the larvae to grow until the third instar stage.
3.6.3
Rearing of late stage larvae
The containers carrying the third instar larvae should be carefully examined for any disease incidence
and even if a single larva is found infected, the entire lot of larvae in the container should be
destroyed and the container either sterilised carefully or disposed of.
1. Transfer Larvae from disease free batches individually to the preferred individual rearing
containers.
26
2. Hold the larvae in their individual containers at 24

cycle and pupate.
2C and 45 + 5% RH to complete their life
There are a number of options for individual rearing containers. Glass penicillin or freeze drying vials
of 10ml or 25ml capacity, containing about 5g of diet and sealed tightly with sterile absorbent cotton
are very effective and have the advantage of being autoclavable. Or, 25ml plastic cups with clip-on
lids are also widely used (Plates 11 & 12). These are often disposed of after use but can be
sterilised chemically and re-used. Another alternative is to use plastic multicell rearing trays specially
made for bioassay and insect rearing.
Plates 11 & 12
Individual rearing of late stage larvae
3.6.4
Selection for vigour
Apart from regular examination of the colony for infectious diseases, the larvae should be subjected
to selection for vigour. All larvae being reared on diet for breeding purposes should be examined at
the fourth and fifth instar stages, and only the healthy, vigorously growing larvae should be selected
to continue. Any slow growing or stunted larvae should be discarded.
3.6.5
Pupation
The larvae usually pupate inside the diet and if there is excess humidity, pupation will be affected.
Hence the humidity should be maintained at around 45%. In plastic pots the addition of a piece of
filter paper fixed between the lid and the pot can help control excess moisture.
27
1. Discard larvae pupating late. (The slow growing insects usually harbour infectious diseases)
2. Four days after pupation, collect the pupae but discard all cracked, malformed or undersized
pupae.
3. Wash pupae selected for further culturing in running tap water and surface sterilise them by
immersing in 0.5% sodium hypochlorite solution, containing 0.1% Teepol.
4. Wash pupae thoroughly in water to remove the hypochlorite and keep them on filter paper to
dry.
5. Place pupae inside the emergence cages and continue the culturing as described above (Plates
13 & 14).
Plates 13 & 14
Pupae prepared for emergence and rearing cages for adult H. armigera
3.7
SUPPLY OF INSECTS FOR VIRUS PRODUCTION
Contamination of the colony with NPV can destroy the cultures and lead to serious set back in the
production programme. One of the main sources of contamination is the movement of materials and
staff between the Helicoverpa insectary and the virus production facility. Hence it is essential that
two separate set of personnel are employed for the two programmes. Movement of staff and reusable materials between the two facilities should be avoided.
It is best to supply eggs to the virus production unit where an isolated clean space may be created
for culturing the insects until it they are inoculated with the virus for large scale production at third
instar stage.
28
3.8
PROBLEMS IN REARING LARVAE
Under tropical conditions, apart from infectious diseases such as NPV and granulovirus, two other
major problems often encountered when mass rearing insects are mould on the diet and phorid flies.
3.8.1
Mould on diet caused by Aspergillus species
If carbendazim is not added to the diet Aspergillus species can start growing on the faecal pellets
and subsequently spread to the whole diet surface interfering with the larval growth and pupation. It
is presumed that the mould inhibitors (Methyl paraben and sorbic acid) are digested in the insect gut
making the faecal pellets amenable for the growth of the fungus. The incidence can sometimes be as
high as 50-60%. Addition of carbendazim has be shown to give significant control of the mould
(Kennedy and Rabindra, unpublished data). The fungicide did not affect the larval development,
pupation, adult emergence, fecundity and fertility.
3.8.2
Phorid flies
Phorid flies are attracted to the diet and the flies drop their eggs on the diet if it is exposed. Multicavity trays with holes in the lids are particularly vulnerable for fouling by the phorid fly. The flies
can drop their eggs through even minute holes and the larvae can spoil the diet and make it unfit for
H. armigera. This problem can be solved by using glass vials plugged with cotton. In commercial
scale production this may not be cost-effective and hence the entire insectary should be made flyproof. However, one should remember that the flies are very small and can sneak through the
smallest of holes. UV fly traps installed inside the insectary are helpful in reducing the intensity of the
problem. These phorid flies also attack the pupae and hence, pupal cages should be phorid flyproof.
3.9
REFERENCES
Armes, A J; Bond, G S & Cooter, R J (1992) The laboratory culture and development of
Helicoverpa armigera. NRI Bulletin No.57. Natural Resources Institute, Chatham
Maritime, Kent. ME4 4TB.
Nagarkatti, S & Prakash, S (1974) Rearing Heliothis on artificial diet. Tech. Bull. of the
Commonwealth Institute of Biological Control, Bangalore. 17, 169-173.
Singh, P & Moore, R F (1985) Handbook of Insect Rearing. Elsevier, Amsterdam.
Vanderzant, E S; Richardson, C D & Fort, S W (1962) Rearing of bollworm on artificial diet. J.
Econ. Entomol. 55, pp. 140.
29
30
THE PRODUCTION OF INSECT VIRUSES
4.1
INTRODUCTION
Insect viruses, like all other viruses, can only replicate by infecting a living cell (see Section 2.6:
The Life Cycle of Baculoviruses). In this they differ from other biopesticides like Bacillus
thuringiensis (Bt) or fungi that can be mass-produced by fermentation on simple media.
Both the nucleopolyhedroviruses (NPV) and the granuloviruses (GV) that have been developed as
pest control agents can, therefore, only be produced in living insect tissues. These can either be in
whole insects (in vivo) or in isolated insect cell culture lines (in vitro).
4.2
TISSUE CULTURE PRODUCTION
Tissue culture of insect cells is still only at the research and development stage and while an
important research tool it is not yet either reliable or economic enough for large-scale production of
viruses for use as pesticides. However, tissue culture may provide a useful tool for production of
inocula that is subsequently used for in vivo production. The cell culture of NPV is the most
developed with a number of well-established cell lines capable of supporting the replication of
Spodoptera and Heliothis NPV. The cell culture of GV is less advanced and only a few cell lines
capable of replicating these viruses are as yet established.
Currently tissue culture has the drawbacks that it needs expensive media that mimic insect blood and
the problems of maintaining oxygen levels in large vessels (>250 litres) without killing the delicate
insect cells have not yet been overcome. Also there is a problem sustaining occlusion body (OB)
production in tissue culture. With prolonged tissue culture the virus has a tendency to revert to
budded virus (BV) only mutants and loose the capacity to produce occluded virus. Considerable
efforts are underway in USA & Europe to develop large industrial scale systems of >20,000 litres
for pesticide production but achievement of this goal seems some way off yet.
For information on techniques for tissue culturing NPV two excellent sources of detailed information
are the books by King and Possee (1992) and OReilly et al. (1994).
4.3
PRODUCTION IN WHOLE INSECTS
All industrial production of NPV for use as biopesticides is therefore currently done in vivo by
infecting, rearing and harvesting whole insects. Systems range from simple home made pesticides,
involving farmers using field collected larvae, to computer controlled robot operated mass
production facilities with capacity of 0.25 - 1 million ha per annum. Examples of such modern mass
production facilities have been built in France, Canada and the USA.
31
The procedure for in vivo production is very simple in principle:

1.
2.
3.
Infect larvae of a susceptible species.

Grow infected larvae for a time to allow the infection to develop and the virus to
replicate.
Harvest the larvae and extract the virus.
However, in practice maintaining a sustained production of virus has not been found to be easy. It is
only with well-trained staff, adequately developed production procedures, the appropriate
equipment and a high standard of process quality control that effective production can be both
attained and maintained.
Typical problems found with production include:
Reduced production rate per insect over time.

Contamination of the system by other competing pathogens reducing the NPV yield as
well as lowering the quality of the product.
Failure to maintain a supply of healthy insects.
The causes of problems are various but important factors are:
4.4
Poor quality insects.

Impure inocula.
Inappropriate dosing.
Poor rearing.
Unsuitable harvesting.
Poor sanitation.
INSECTS
Good quality NPV can only be produced in healthy, disease-free larvae. For this reason most
commercial production in USA, S. E. Asia and Europe uses insects cultured specifically for
biopesticides production. The costs are higher than in using field collected larvae but there are
advantages especially where sustained production is needed. A major advantage is that insects can
be bred disease-free and so, when infected, give maximum production of NPV without any other
contaminant. They can also be cultured all year round in contrast to many species of field collected
larvae which may be available only seasonally. Seasonal production can be a serious drawback
where capital investment is high or there is a need to retain trained staff.
A common problem in using poorly cultured or field collected insects is that they are often infected
with other pathogens such as GV, cytoplasmic polyhedrosis virus (CPV), microsporidia, fungi or
bacteria. When these insects are inoculated with NPV, under the stress of infection the other
32
pathogens can also multiply at the expense of NPV production and may also kill the larvae before
maximum NPV production is achieved.
33
A major advantage with cultured insects is that their growth is under close control so that one can
select just the right age and size of larvae for maximum production. This is important as there is
often a crucial window of larval size and age that produces the maximum amount of virus. If larvae
are too small, they die too quickly of infection and the NPV yield can be only 5-10% of the
optimum. If larvae are too large they need too great an inocula to produce an infection and may
pupate before full infection, so again production efficiency suffers. With field collected larvae it is
found that collectors tend to select larvae too large for efficient production as these are more
obvious and preferentially collected.
With Helicoverpa armigera NPV produced in H. armigera, it was found that optimum size is very
important in maximising yield. At optimum infection size, using III/IVth instar larvae of 40-70 mg in
weight, one can get maximum yield and high infection rate with a low dose of 105 OB per insect
(surface dosing). 3 x 109 OB per insect can be harvested. With early instars (<IIIrd), 20mg larvae
are easily infected with a small dose but tend to die small having produced few OB. In these
instances production per larvae struggles to reach 1 x 109 OB per insect. In contrast late instars
(IV/Vth) are difficult to infect and need very large inocula. Even then the rapid growth means that
many do not succumb to infection and again average yields rarely exceed 1 x 108 OB per larva.
4.5
INOCULATION
Most insects are surface dosed by placing uninfected larvae on trays of artificial diet surface-sprayed
with the correct inoculating dose of virus. It is possible to use diet in which virus has been
incorporated to a specific concentration but in most cases this is more wasteful of inocula, however,
it may be appropriate for larvae that burrow into the diet. In most systems larvae placed on sprayed
diet are then reared through on the same tray until harvesting. However some producers use virus
sprayed onto leaves on which larvae are allowed to feed for 24 hours and then transferred to clean
diet for rearing on until harvesting, the main objection to this is that it adds another stage and is
therefore more labour intensive.
The dose used for inoculation is important and should be optimised. Too much virus is wasteful and
inefficient. Also, overdosing will kill small larvae before they can grow big enough to produce
maximum virus. Too little will leave some larvae uninfected. Each virus/pest system will have its
own optimum dose and an important part of setting up production is carrying out trials to determine
the best dose and size of insect. In the HaNPV production system developed at the NRI, 105 OB
per 60mg IIIrd instar larva sprayed onto diet trays was found to be the best dose.
4.6
INOCULUM PURITY
It is extremely important that good quality inocula is used. Probably one of the major reasons for
failure in production is the use of poor, impure or low-activity inocula. Inocula should be highly
purified by sucrose gradient centrifugation to ensure it is free from contamination by other pathogens,
(such as GV, Microsporidia, Fungi, etc.). The practice of recycling virus from production, without
purifying, is particularly dangerous as contaminants can build up every time it is recycled until
eventually it may contain 90% other contaminants and 10% NPV. Using such an inocula is a
34
guarantee of poor production. Therefore virus should never be recycled without being checked and
purified.
If such purification equipment as centrifuges for gradient purification are lacking, this problem can
still be reduced if all the infected larvae to be used for the inocula are individually examined
microscopically and any showing signs of microsporidia, CPV, GV or other pathogens are
discarded.
4.6.1 Protocol for purifying inocula

Most purification procedures are based on centrifugation and the possession of a centrifuge should
be considered as essential to any properly equipped insect virus facility be it laboratory or
production plant. The procedure employed will depend on the nature of the sample, on the purity
required and on the equipment available. If high and low speed centrifugation are available the
following procedure for purification of NPV or GV from individual infected insects should be used.
4.6.1.1 Primary processing.

1. Place insect cadaver in 1-1.5ml 0.1% sodium dodecyl sulphate (SDS) in distilled water in a
microtube.
2. Homogenise thoroughly using a micropestle to disrupt the cuticle then vortex for
1 - 2 minutes.
3. In a microfuge, spin at 100g for just 5 - 10 seconds to pellet large debris such as pieces of
cuticle.
4. Decant supernatant into a clean tube, re-suspend the loose pellet in 0.1% SDS and repeat
the above step.
5. Combine supernatants and either re-suspend again or discard the pellet.
6. Centrifuge the supernatant at 2500g for 5 minutes (or 5000g for 10 minutes for GV) to
pellet the virus.
7. Discard the supernatant with the layer of fat above and re-suspend the pellet in
1-1.5ml of distilled water. The pellet may need to be vortexed to achieve complete resuspension.
8. Pellet virus again at 2500 - 5000g for 5 minutes (10 minutes for GV), discard the
supernatant, and re-suspend pellet in a small volume of distilled water, as above.
35
4.6.1.2 Gradient centrifugation

If NPV is destined for inoculation it is recommended that NPV be further purified to remove any
GV, bacteria, microsporidia or other parasites. To do this a long spin, using gradient centrifugation
is needed. The purification of NPV by this technique is wasteful and usually entails loss of 50-75%
of the NPV OB, so the procedure is only used where high purity is essential.
To carry out gradient centrifugation much higher g forces in the order of >50,000g for at least an
hour are needed. Such centrifuges are very expensive costing UK60-90,000 with, in addition,
special rotors that also cost at least UK20,000 each.
It can help also to sediment the semi-pure NPV through 10% sucrose prior to gradient purification
with a single long spin. The NPV is pelleted but lower density material left above the sucrose.
The principle of gradient centrifugation is to place the sample in a tube that contains liquid of different
densities and then subject it to a high g force over a prolonged period. The particles of different
density then move through the liquid column until they reach a layer of equal density at which point
they halt (Figure 3). The different density liquid layers can be produced by dissolving different
quantities of a cheap inert solute such as sucrose or glycerol in water. The gradient can be made up
of discrete bands of liquid on top of one another, e.g. 40%, 45%, 50%, 55% and 60% sucrose, or
it can be in the form of a continuous gradient down the tube with the lightest at the top, e.g. 40-60 %
sucrose.
Figure 3. Gradient separation of impure NPV
Pre- Centrifugation
Centrifugal force
Post centrifugation
Impure
virus
Bands of
virus, insect
debris &
contaminants
Density
Gradient
Gradient Centrifugation
B.D Hames pp 45-93. In Centrifugation: A Practical Approach Ed. D Rickwood (Second Edition, 1989)
Reprinted by permission of Oxford University Press.
Continuous gradients can be made using a gradient former that itself can be home made from two
plastic bottles and a connecting tube. The other items needed are a magnetic stirrer and peristaltic
pump (Figure 4). Glycerol can be used in the gradient but food grade sucrose is generally adequate.
36
If a gradient former is not used, a series of suspensions needs to be made up covering the range of
densities, e.g. 40, 45, 50, 55, 60 and 65% sucrose. Start with the densest and add a measure to
each tube, e.g. 10ml in 100ml tube. Then carefully add 15ml of each successively lighter fraction by
decanting the suspension carefully down the inside of the tube wall taking care not to disturb the
lower layers. The tube should then be left for an hour to allow the layers to diffuse.
Figure 4. A simple apparatus for the preparation of linear gradients (a gradient former)
Stirrer
Gradient
B.D Hames pp 45-93. In Centrifugation: A Practical Approach Ed. D Rickwood (Second Edition, 1989)
Reprinted by permission of Oxford University Press.
Loading the sample is crucially important to successful centrifugation. Overloading produces poor
separation of fractions and poor results. The best separation is obtained if the sample volume is
small compared to the gradient volume, so the sample volume should be 2-3% of the gradient
volume. This makes the cost of the process high, as a run with 6 x 150ml tubes will only process
20-30ml
of
semi-pure
NPV
with
a
concentration
of
no
more
than
1 x 1010 OB ml-1. The sample itself should be loaded carefully to avoid disturbing the gradient or
layers, again by slowly running it down the side of the tube.3
To get the NPV to move into and through the gradient, forces of 50,000g are required and runs are 1-3 hours
long. When NPV is put through a gradient it settles or bands out at 54-56% sucrose, at a density of 1.25 g ml -1.
To process large batches of NPV, a special rotor called a zonal rotor is used and this can purify 50-100ml of
sample per run. Gradients can also be used to purify GV which has a similar density to NPV. However, as the
particles are smaller, greater g forces (90,000g) are needed to obtain good bands.
37
PREPARATION OF GRADIENTS
A sucrose gradient is best generated using a gradient former. A simple gradient maker can be
prepared without having to purchase costly equipment.
For a 40 - 65% linear, continuous gradient two sucrose suspensions are prepared at 40% and 65%
w/w in 0.1% SDS in distilled water. The 65% sucrose is placed in the reservoir and the 40%
sucrose into the mixing chamber. A multichannel peristaltic pump is used to fill several tubes at the
same time. Light sucrose is fed first to the bottom of tubes via long needles and is then displaced
upwards by progressively heavier suspensions.
If a gradient maker is unavailable then a step gradient can be substituted with results that can be as
good. For a 40 - 65% gradient suspensions at 40, 45, 50, 55, 60 and 65% sucrose (w/w) are
prepared. Starting with the 65%, 5ml are added to each tube. Then using a digital pipette, each
layer is overlaid with 5ml of each successively lighter suspension by pouring the suspension down
inside of tube wall, carefully and slowly. The gradient is left to stand for an hour to allow the layers
to diffuse.
Step gradient stage
1. Prepare solutions of 50% and 60% (w/w) sucrose in 0.1% SDS.
2. Add 0.5ml of 60% sucrose to a microtube. Carefully overlay a similar volume of 50%
sucrose and ensure there is no mixing.
3. Take a pelleted sample (as described in 4.6.2) and re-suspend it in an equal volume of 20%
sucrose in 0.1% SDS.
4. Take 100 microlitres of the resulting sample and carefully place it on to the 50% sucrose in
the microtube. Carefully place the tubes into a microfuge and centrifuge at maximum speed
for at least 15 minutes (for GV, at least 30 minutes).
5. NPV and GV will band at the interface. Heavier material will pellet while light material will
remain in the sample layer.
6. Extract the band and dilute with distilled water. Centrifuge at high speed to pellet virus.
Discard the supernatant, re-suspend in distilled water and repeat the centrifugation. Discard
the supernatant and re-suspend in distilled water.
This process will provide greater purity than primary processing but for even greater purity a
continuous sucrose gradient can be used . For this you will need access to an ultracentrifuge with
capacity to reach a relative centrifugal force (RCF) in excess of 50,000g.
38
Continuous sucrose gradient purification

1. Pellet the virus sample and re-suspend in a small volume (0.5 - 1.0ml) of 10 - 20% sucrose
(w/w) in 0.1% SDS.
2. Prepare a linear 40 - 65% sucrose gradient in 30ml polyallomer centrifuge tubes.
3. Centrifuge for one hour at 50,000g (two hours at 90,000g for GV) in a swing-out rotor.
This technique of isopycnic density gradient centrifugation will provide NPV/GV of the
highest purity particularly if attention is paid to preliminary preparation and purification of the
sample before this stage.
4. Occluded virus will form a thick white band at their isopycnic density point at about 54 56% sucrose, equivalent to approximately 1.25g/ml.
5. Collect the NPV band using a long needle and syringe or pipette, and dilute with distilled
water.
6. Pellet the virus by centrifugation at 2500 - 5000g for 5 - 10 minutes. Re-suspend pellets in
distilled water and store at 4C or in the freezer.
7. After sucrose gradient centrifugation, purified PIB tend to aggregate easily. Sonication
should be used to disperse occlusion bodies.
It may be useful to centrifuge samples through a single concentration of 40 or 45% sucrose prior to
isopycnic centrifugation. Alternatively, rate zonal centrifugation can be used to give greater sample
purity prior to an isopycnic run. For large quantities, zonal rotors are preferred but are VERY
expensive. They have a high capacity and eliminate much of the time consuming preparation
required when using swing out rotors.
LOADING SAMPLES IN TUBES FOR SWING-OUT ROTOR
Sample loading is a critical step in centrifugation. Narrow sample zones relative to gradient length
produce optimum resolution in fractionation. Sample volume is a function of the cross-sectional area
of the tubes, but is usually about 2-3% of the gradient volume.
When loading the sample, the pipette containing the sample is touched against the meniscus at the
tube wall and the sample allowed to run slowly out. Tubes should be filled to within 3mm of the rim
to prevent collapse and should be precisely balanced.
UNLOADING BANDS
Unloading the NPV band requires both care and skill. Where a number of bands form, it is
advisable to unload each in turn, lightest first and then to examine them to determine which one
39
contains the NPV. Nothing is more frustrating than after all the effort taken to produce the virus, to
find out later you have harvested a band full of bacteria and discarded the NPV!
40
Commercial unloaders are available. Satisfactory unloading can be achieved with either a syringe
and large bore needle, or with a pipette. Layers above the required band can be drawn off first to
avoid unnecessary contamination.
After collecting the band of NPV, the sucrose/NPV suspension is mixed in 10 times its volume of
water, centrifuge and pelleted to get rid of the sucrose. This pure NPV should then be stored in a
freezer at -20C. It should be noted that pure NPV clumps easily so sonication is needed to
separate the sample when defrosting and taking sub-samples.
4.7
INCUBATION AND REARING
Conditions should be kept to the optimum so that infected insects are not stressed but left to feed,
grow naturally and develop the infection. Insects subjected to heat, high humidity and other stresses
tend to die early before the NPV can complete its cycle, and so contain few polyhedra.
Cannibalism is a common problem in the later stages of infection and can severely reduce
production. For this reason most production of Heliothines is carried out in multicellular, divided
containers. Trays are subdivided into individual compartments. Each may be 1 - 2cm x 1 - 2cm
and a tray may contain 150-500 such compartments. These trays are best fitted with removable
dividers so that the diet can be poured onto them and then sprayed with virus; after which the
dividers are pressed into the diet producing the compartments and the insects introduced. These
trays can be made out of commercially available stacking trays with insertable plastic dividers
adapted from such sources as light defuser grids in fluorescent lighting units. Alternatively, specially
made rearing trays can be designed. The placing of larvae in such trays is very labour intensive and
in advanced plants, robots and other machines are used to carryout this task.
4.8
SANITATION OF PRODUCTION FACILITIES
It is very important that good sanitation practice is carried out and all equipment sterilised before reuse and facilities cleaned regularly. Dirty plants do not produce clean products. Good sanitation is
the key in preventing and controlling the outbreaks of contaminants such as microsporidia, bacteria,
fungi and insect pests that can impede production. There should be set procedures with specified
routines of regular cleaning of all surfaces and rooms involved in insect rearing, diet production and
virus production.
Autoclaving or low pressure steaming of equipment is the most effective sterilising technique, but
simple domestic industrial hypochlorite disinfectants are highly effective. They must however, be
made up fresh to prescribed strength (5%) every day. If left, and especially if exposed to diet or
dead insects, this becomes inactive and then if used in washing becomes itself a major source of
contamination. Production equipment should be washed then sterilised before re-use.
Screening of windows and the use of insecticuters can be important in controlling flies, which can be
a real problem in some climates. These lay their eggs in the diet on production trays and their larvae
then compete with the virus host larvae for food, thus reducing virus production. UV tubes to
disinfect rooms are also useful but should not be relied upon alone.
41
Where insects are reared for virus production it is very important to keep insectary staff from going
into the production or cleaning areas and vice versa. The biggest source of potential contamination
to the insectary facility of a virus production plant is the virus production areas themselves. If virus is
allowed to contaminate the insectaries, production of larvae for virus infection will rapidly collapse
thus halting production.
4.9
HARVESTING
It would seem logical to leave the larvae to die naturally so that you are sure that the virus has
completed its cycle and maximum production has been achieved. This in principle is correct and is
done in some production systems. However it also creates problems. Dead larvae are very fragile
due to the effect of virally expressed proteases produced during the last stages of infection and on
handling easily burst and leak the body contents so that NPV is lost. Another problem is that after
death larval corpses are rapidly invaded and colonised by bacteria which multiply and begin to
decompose the larvae. The decomposition action produces the bad smell often associated with
impure NPV products and the multiplication of bacteria produces bacterial spores that resemble
NPV. In live larvae bacterial levels are relatively low with around 106 bacteria per insect and few
spores (<102). In dead larvae, bacterial totals can be very much higher at around 1x109 total
bacteria per larvae and in excess of 106 spores.
To remove these contaminants after harvesting and get rid of the unacceptable smell is difficult and
expensive. It can be done using sucrose or glycerol gradient centrifugation but this may involve
losses of up to 50% of the NPV content and increase the cost of the final product by 200%.
Interestingly much of the bad smell often found in poor quality aqueous NPV products probably
comes from decomposing fats in the insect fat body being broken down by bacteria. If the infection
is correctly established most of this fat is metabolised during the infection so the smell is often worse
in samples poorly infected with virus containing low titres of virus and few mature OBs.
For these reasons it can be better to use larvae harvested alive or before they die of virus and wash
them to remove the surface bacterial contamination. By this simple technique it is possible to get a
low contamination product which does not develop a bad smell. However, in live harvesting, timing
is crucial if you are to get good yields. Significant numbers of mature OBs do not appear until five
days post infection (PI). For optimum production there is a need to harvest before significant
mortality occurs but must be done when production of mature OBs has reached its maximum.
The determination of the best harvesting time has to be done individually in each host/virus system.
In any system the precise combination of conditions varies with inocula, temperature, larval age and
virus species. In temperate species with long life expectancies it can take a prolonged time, e.g. for
producing the NPV of Euproctis chrysorrhoea it takes up to 70 days. However in most tropical
species the time is much shorter, for example in HaNPV one can get 2.5 x 109 OB per larvae by
harvesting after seven days, when 80% larvae are still intact for harvesting. In some cases where it
is difficult to get good live harvesting a procedure known as post harvesting incubation can be useful
to ensure OB are mature. It is known that polyhedra harvested early from live larvae are often much
less infectious than those from larvae that have died and therefore are probably mature. If live larvae
42
are washed and held for a second incubation away from diet, faeces, etc., this can often improve the
infectivity of the OB after harvesting, without unduly increasing the bacterial burden.
4.10 MODEL PROTOCOL
FOR
THE
PRODUCTION
OF
HELICOVERPA
ARMIGERA
NPV IN MULTICELL TRAYS

Helicoverpa armigera NPV (HaNPV) is produced at NRI by inoculating third instar Helicoverpa
armigera larvae of 40-80mg weight and rearing them on artificial diet in production trays. Infected
larvae are harvested after a period of incubation. Using the virus production method, it is possible to
gain a 1x105 fold return on inoculum used.
It is important to note that the method described below applies only to the insect colony and NPV
strain mentioned. A different colony dosed with a different strain of HaNPV may give different
results due to variation in potency of strain and susceptibility of insect colony.
This protocol uses plastic trays into which a metal dividing grid is inserted to produce the required
individual rearing cells.
There are four stages to production of HaNPV:
1.
Preparation of production trays.
2.
Inoculation of production trays and loading larvae.
3.
Harvesting infected larvae from production trays.
4.
Treatment of infected larvae.
4.10.1 Preparation of production trays

The preparation of production trays involves pouring molten artificial diet into plastic trays of size
420mm x 300mm; the trays should be filled with diet to a depth of at least 1cm. The surface of the
diet should be completely flat and smooth. When the trays are filled, the diet must be left to set and
they are then ready to be inoculated with NPV. The volume of diet prepared below is sufficient to
make three production trays for 1500 larvae.
43
PREPARATION OF ARTIFICIAL DIET

Ingredients
2400ml distilled water
50g agar fine powder
290g stabilised wheat germ
270g dried active yeast
50g white table sugar
50g casein powder
1.2g cholesterol powder
10g sorbic acid
12g methyl-4-hydroxybenzoate
24l linoleic acid (24 drops)
Method of cooking
Cooking diet is best done using a microwave oven as this provides even cooking of the diet without
the need of constant stirring, however if one is not available, electric or gas rings can be used but
care must to taken to stir the diet frequently to prevent burning.
1.
Measure out the water and mix in the agar.
2.
Boil the mixture to fully dissolve the agar.
3.
While boiling, measure out the rest of the ingredients. When all have been weighed mix
them together while dry to avoid casein forming lumps when added to agar.
4.
When the agar has dissolved, mix the dry ingredients into it using an electric hand mixer
and boil gently for one hour.
5.
Cover the bowl with cling film (not if using gas/electric heating) to avoid the mixture drying
out, but make a small hole in the film to allow the pressure to escape.
6.
Once the diet has cooked it needs to cool to 60C, below this the diet may begin to
solidify causing problems when pouring into trays.
7.
On reaching 60C add the following vitamins and minerals and mix in thoroughly:
24g of Wesson's salt mix
24ml of Vanderzant vitamin mix
30g of ascorbic acid
2.4g of choline chloride
8.
Once mixed, pour the diet into the trays immediately to avoid lumps occurring due to
cooling and solidification of the diet.
44
45
4.10.2 Inoculation of production trays and loading the larvae

The production trays are inoculated with pure HaNPV sprayed onto their surface using a
chromatography sprayer operated with rubber bellows (Plate 15) (If a chromatography sprayer is
not available a small hand-held plant or perfume sprayer can be used). The dose is applied at a rate
of 2x105 PIB per larva. Approximately 500 larvae are placed per tray, therefore a total of 1x108
PIB of inoculum is used. The inoculum is delivered in 5ml of water.
INOCULATION OF THE PRODUCTION TRAYS
Procedure
1.
Autoclave the chromatography sprayer.
2.
Prepare the inoculum (1x108 PIB in 5ml of distilled water for a tray of 500 larvae).
3.
Place a production tray into a clean air cabinet (if available) at an angle of 60.
4.
Fill the sterile sprayer with the inoculum and spray half of the diet surface very evenly.
Then turn the tray the opposite way up and spray the other half in the same manner.
Repeat this procedure until the contents of the sprayer has been expelled.
5.
Lay the production tray flat and allow the inoculum to dry in the clean air cabinet.
Plate 15
Applying NPV inoculum to a virus production tray using a chromatography sprayer
46
LOADING THE LARVAE

To prevent cannibalism the larvae must be reared in isolation. Segregation is achieved by pressing
an aluminium honeycomb, called an aeroweb, into the diet. This creates individual cells exposing an
area of diet inoculated with approximately 2x105 PIB.
Procedure
1.
Press a sterile sheet of aeroweb into the artificial diet.
2.
Using featherlight forceps, place one third instar larvae into each cell (Plate 16).
3.
When all the cells are full, place a sterile sheet of woven polythene over the top of the
aeroweb. Then clamp a perforated aluminium lid over the whole assembly to prevent
escapees.
4.
Incubate the production trays at 26C.
Plate 16
A production tray loaded with H. armigera larvae
4.10.3 Harvesting infected larvae

On the fifth day, production trays should be examined. If the larvae are at maximum weight,
showing heavy infection all live larvae should be harvested. Only live larvae should be collected in
order to keep any bacterial contaminants to a minimum. The number of larvae recovered should be
47
noted to enable yield values to be calculated. If the larvae have not reached maximum weight and
no prominent signs of infection are apparent, then the lid should be replaced and the tray checked
the next day.
Procedure
1.
Remove the lid and polythene mesh.
2.
Remove the aluminium honeycomb from the diet and tip the larvae onto a tray
3.
Remove all live (infected) larvae from the diet surface, tray and honeycomb and place
them into a suitable container.
4.
Incubate the collected larvae for a further 24 hours at 26C to allow full expression of
NPV in infected larvae.
5.
Place infected larvae in to the freezer until processing can be done.
4.10.4 Processing of infected larvae

Procedure
1.
Thoroughly homogenise harvested infected larvae in an equal volume of sterile distilled

water using a commercial blender or food mixer.
2.
Slowly filter the resulting slurry through a double layer of muslin supported in a large funnel
and wash the residue twice with sterilised water.
3.
Finally, gather the muslin and gently squeeze the residue to extract the remaining liquid.
Discard the muslin. Avoid excessive squeezing as this will cause the residue to pass
through the muslin and into the filtrate.
4.
Deep freeze the slurry to await formulation or purification as required.
4.11 PROCESSING INSECT VIRUSES FOR USE AS BIOPESTICIDES

The processing of NPV or GV covers the turning of virus-infected larvae into a useable form ready
for formulation as a biopesticide. It involves various processes, typically filtration and centrifugation,
to remove unwanted contaminants and insect wastes while retaining the active ingredients, the NPV
or GV. Typically, the processing of NPV-infected insects involves reduction in water content,
removal of insect debris, reduction in bacterial numbers and the release of NPV from insect tissues
and cells. The product of processing is a paste or suspension rich in the NPV occlusion bodies
(OB). This is then stored in a suitable form for later formulation either as a paste in water, which
needs refrigeration or as a dried powder.
All processing stages involve some loss of polyhedra. Therefore there is always a need to balance
what you gain from each stage against the loss of activity. To obtain pure virus you typically loose
50-70% of your starting material during processing. It is important therefore not to carry out more
processing than is necessary.
48
When working with newly harvested insects or crude unpurified homogenates it is important that all
the processing steps are carried out at 4-10C. Keeping the process cool prevents the
multiplication of bacteria that cause decomposition. Also, it prevents the enzymes present in the
dead insects from aiding further the decomposition of the insect body contents.
When warm, (above 10C), the bacteria and the insects enzymes will breakdown the cell contents
of the insect, including fats, to produce a range of degradation products including those responsible
for the foul smell found in poor quality NPV. These decomposition products do not appear to
directly affect the NPV, which is protected in its OB crystals. However, until either the NPV is
finally purified or dried, it is essential that it is kept in a refrigerator or cool on ice at all times, if the
production of bad odours is to be avoided.
4.11.1 Homogenisation and filtering

The first step in processing involves homogenising or blending the larvae to disrupt all the cells
releasing the NPV, and help break up any clumps of NPV. The NPV are produced in the nuclei of
infected cells, so the tissues of the dead insects and the nuclei of the cells in them need to be
disrupted to release the NPV. This process is aided by protease enzymes that the virus produces
late in the infection cycle, that help to breakdown the cells thus allowing the OB to escape.
However, if larvae are harvested alive prior to the completion of the cycle, this process may take
longer.
This step can be carried out in scientific blenders such as a Waring blender, and the 5 litre capacity
models are excellent for processing large batches. However, normal domestic or industrial catering
blenders are adequate. All equipment should be cleaned between batches and disinfected to reduce
bacteria contamination and smell. The material should be blended when still cool from the
refrigerator. Blending of collected insects is normally done in two or three times their own volume of
water, preferably sterile distilled water that is cooled to 3 0r 4 C but this is not essential. Disruption
in water with a small amount of detergent added, (e.g. 0.1% sodium dodecyl sulphate or Teepol or
Tween), often helps to promote good homogenisation and does not affect the NPV, but again is not
essential. However, highly chlorinated tap water should not be used as the chlorine can inactivate
the NPV.
The next stage is to filter the homogenate through muslin to remove pieces of the skin, legs and other
hard parts of the insect that can block the nozzles of sprayers. Simple cotton muslin folded over to
twice thickness is adequate to filter out most of these larger fragments. The muslin is placed in a
large filter funnel and the homogenate allowed to drip through. At the end of this filtration process,
the residue is washed with distilled water to carry through any NPV present. Finally, the muslin is
gathered together from the top and gently squeezed to extract the remaining liquid. Caution here is
important as excessive squeezing will force the solid waste to pass through the muslin. After filtering,
the used cotton muslin is best discarded as it will begin to smell very quickly and if used again could
contaminate other batches. Again the liquid should be kept cold throughout to avoid any
decomposition during this process.
49
Keeping the mixture cold also helps to solidify any insect body fats so that they get filtered out. This
is useful because the decay or rancidity of insect body fats is believed to be a major source of some
of the strongly smelling compounds that give poorly prepared NPV such a bad smell. Also, when
these fats chemically decay they can produce superoxide radicals that can inactivate viral DNA.
Thus removing the insect fats can both improve the smell of NPV and its storage qualities.
4.11.2 Centrifugation
Centrifugation is the process of concentrating the solid particles in a suspension by spinning them in a
centrifuge, thus separating them from the liquid fraction. As NPV are particles this is used to
concentrate and separate them from the water, added to assist homogenisation, and the insect
waste. The virus also needs to be separated from the homogenisation liquid, which contains insect
proteins, enzymes, etc. Only good centrifugation carried out in cool conditions can help to remove
the bacteria, insect body fats and contaminants that are a major cause of bad smell. These fats
break down over time and produce a smell, and high concentrations of fat breakdown products,
such as organic acids may interfere with the processing or formulation later on.
However, where a centrifuge is not available prolonged settling followed by the pouring off of the
liquid may be effective but it must be done under cooled conditions, (<10C), in a refrigerator if
decay and subsequent bad smells are to be avoided in the final product.
DIFFERENTIAL CENTRIFUGATION
The basic procedure is to use a short, low speed spin to separate off any remaining hard parts;
followed by a longer, high speed spin to sediment the NPV and separate it from the water, fats and
liquid insect wastes.
The initial centrifugation is best done using a large volume, low speed centrifuge. An appropriate
machine would be one capable of 3000-6000 RPM (capable of producing forces of 5000g)4 with 4
x 750ml buckets capable of processing 3 litres of homogenate at a time (equivalent to 1000-2000
infected insects). Such a machine costs 10,000 for a refrigerated model, which is best as it
guarantees that the samples can be kept cool for reasons discussed above. A non-refrigerated
centrifuge with similar specifications costs half as much, but more care and effort is required, (e.g.
pre cooling-samples, buckets etc.), to ensure the samples do not become too warm for too long.
4
The speed setting needed to get this gravitational force depends upon the design of machine used and its
instruction manual should be consulted. There is also a standard equation that can be used to determine the
necessary speed, based upon the dimensions of the rotor.
RCF (g) = 11.18 x r x (N/1000)2
where
r = radius in cm from centre of rotor to point at which RCF value is required

N = speed of rotation in revolutions per minute.
For a required g force, the speed in RPM can be calculated by re-arranging thus:
N=
(RCF/11.18 x r ) x 1000
50
For pelleting virus, angle rotor are fastest but swing-out buckets are adequate, with most large
buckets of >100ml being of the swing out type. Therefore using large swing-out buckets of 500750ml capacities, may be much quicker than having to make a number of runs with small angled
rotors.
A short, low speed spin is used initially to pellet any hard parts or skin that have got through the
filtering, but this should last no more than 1-2 minutes at 100g. After this, the liquid which is still
turbid and full of NPV is poured off and retained, while the pellet of hard parts left in the bottom of
the bucket is washed out and discarded.
The NPV OB themselves are then pelleted using 2500g for 20-30 minutes. At the end of this run,
the liquid supernatant is poured off and discarded. This discarded supernatant contains soluble
material, some bacteria, lipids (especially if done cold), and other insect wastes. The pellet of NPV
is re-suspended in a small quantity of water (preferably sterile, distilled water). This pellet should
contain concentrated semi-pure NPV at a concentration of 109-1010 OB/ml The following
procedure can be used.
1. Weigh and homogenise infected insect cadavers in an equal volume (weight) of 0.1% sodium
dodecyl sulphate (SDS) in sterile, iced, distilled water.
2. Filter through cheese cloth or muslin to remove gross debris.
3. Wash debris through with additional 0.1% SDS and squeeze.
4. Spin the crude virus extract at 100g for 30 seconds to pellet gross contaminants. Decant and
retain the supernatant. Re-suspend the loose pellet in 0.1% SDS and sediment again at 100g
for 30 seconds. Combine the supernatants and discard the final loose pellet. NB: g force is
determined by the angular velocity of the rotor and the distance of the sample from the axis. See
Footnote 4 for the formula to calculate g.
5. Spin the supernatants at 2500g for 10 minutes to pellet viruses and other similar sized particles.
Discard the supernatant which contains very small particles and a floating lipid layer.
6. Re-suspend the pellet in a small volume of distilled water. This procedure will often give virus of
adequate purity.
Greater purity can subsequently be obtained by sedimenting the product through a 40% sucrose
solution in a long, high speed spin. Low density material will remain above the sucrose solution
while heavier particles, including the virus, will sediment.
One centrifuge run is usually sufficient to give NPV of adequate purity for pesticide formulation.
Clean-up is easiest with larvae harvested and frozen before death. In these samples bacterial levels
(< 106 bacteria ml-1) and bacterial spore levels (<102 m1-1) are low. In some samples which are
very dirty, e.g. where a high proportion of dead larvae have been included, it may be necessary to
repeat this stage twice to remove the gross contamination. In samples containing many dead
51
harvested larvae, bacterial levels are high (> 109 ml-1), and spore forming bacteria will have invaded
the cadavers and multiplied (> 106 ml-1). As a result fermentation of insect body contents has often
started and the clean-up becomes more difficult.
If bacterial spores are present it is impossible to separate these from the NPV without expensive
gradient centrifugation, as their size and pelleting characteristics are similar to OB. It is also difficult
to inactivate the spores because they are highly resistant and you cannot destroy them without
inactivating NPV.
In well-infected samples of insects, NPV infection degrades fat body cells and the lipid content of
the homogenate is very low. If much lipid is seen to be present at the centrifugation stage it usually
means the insects in the sample are either poorly infected with NPV or the infection has not been
allowed to develop properly.
Thus, getting the infection and harvesting stages right reduces processing problems enormously. This
simple primary centrifuged material is ideal for use as pesticide and there is no need to process the
sample further. In fact, further processing can reduce the effectiveness of NPV as a pesticide. It
has been found in studies of UV protectants that this primary purified NPV has a better UV stability
than pure NPV with the addition of UV protectants.
4.12 STORAGE OF INSECT VIRUSES

For ease of storage and subsequent formulation, harvested infected larvae are homogenised and
then dried. Drying can be achieved by either freeze -drying or spray-drying.
4.12.1 Freeze-drying
Freeze drying is one of the standard methods of drying biological material while retaining full
biological activity. Freeze drying is used to dry samples of bacteria and viruses where it is required
to reduce their bulk or to produce a dry, stable active powder for long term storage. It is also
commonly used to help purify or concentrate unstable biological materials such as enzymes or
antibiotics.
Thus in freeze drying, the sample dries while remaining at sub-zero temperatures so that no heat
degradation of biologically active molecules occurs. At the very low vacuums used, even with
samples frozen to -40C, samples of several litres can be dried out completely within 24 hours
without ever raising their temperature above freezing. Typically the water vapour coming from the
sample is drawn from the sample chamber by the vacuum pump into a frozen condenser chamber at
-60C, where the water crystallises out on the walls as ice and can be removed separately when the
run is over.
When freeze drying NPV from insects, the infected insects are first macerated and filtered to remove
gross insect debris (Refer to Section 4.11.1). It has been found that trying to freeze insects that are
intact and whole is slower than with macerated insects, presumably because the intact skin acts as a
barrier and slows down the escape of the water vapour from the insect bodies.
52
53
Freeze drying can be applied to samples of any size, however the process gets increasingly slow as
the thickness of the sample increases. Thus samples of depth no greater than 1-1.5cm are easily
dried out in 24 hours but samples deeper than that take very much longer, (the corpse of a baby
elephant has successfully been freeze dried at London Zoo but that took 18 months to complete the
drying!)
Insect material, when freeze dried, is often very light and flaky and can be difficult to handle. The
process is made easier by using a starting paste containing as little liquid as possible (no more than
85-90% total water). Another way to make samples easier to handle is to freeze dry them with an
inert carrier such as china clay or lactose to help give the final product bulk.
For freeze drying to be successful the sample must be completely frozen. If any part remains
unfrozen it will not dry out completely and the whole batch will warm up and melt during the drying
run. Usually if a homogenate of infected insects is cooled to below -20C it will completely freeze.
However if the sample contains many insects harvested after death in which there has been microbial
degradation of insect tissues, some of the bacterial fermentation products may act as an antifreeze
and a lower temperature is required. If freezing thoroughly becomes a problem it can often be
overcome by pelleting the NPV and discarding the liquid phase, which will contain the freezing point
depressant fermentation products, and re-suspending the pelleted NPV in clean water.
Once a freeze drying run starts it must be continued until the process is complete. If stopped
halfway through, e.g. by a power cut, the sample can rapidly warm up and melt, making it
impossible to continue until the sample is re-frozen. It is important to note that leakage of virus into
the vacuum pump occurs if the ice melts and the vacuum is reinstated. This will damage the pump
and will require expensive repair or replacement. It is recommended that a trip switch is
incorporated into the system that will cut power in the event of a power cut during out of office
hours.
Plate 17
Trays of NPV homogenate (
freeze-dryer, Edwards SuperModulyo.
54
) in a
FREEZE DRYING PROTOCOL FOR VIRUS -INFECTED INSECTS

This protocol is based upon a freeze dryer with integral freezing water trap and separate freezer
trays, e.g. Modulyo 12K with tray freezer attachment (Plate 17).
1.
Macerate the infected insect sample in water, (approximately two parts water to one part
insects).
2.
Filter through muslin to remove gross debris, skin and hard parts such as jaws.
3.
Pellet the NPV by centrifugation at 3000g for 20 minutes.
4.
Discard the supernatant and re-suspend the pellet in one part water. At this point add the
inert carrier if desired.
5.
Mix thoroughly and pour into the freeze dryer trays. These should be filled no deeper than
10-15mm. If the freeze dryer has temperature probes, place them so they are frozen into
the sample but not in direct contact with the metal of the freezer trays (taping them in place
before you add the sample to the tray is usually necessary).
6.
Freeze at -40C until fully frozen, usually 8-15 hours, e.g. overnight. (If the freeze dryer
has a drying chamber with a built in high capacity freezer unit, this can take only a couple
of hours).
7.
Check that the oil in the freeze-drier's vacuum pump is at the right level and that it is clean.
8.
Start the freezing unit freeze dryer water trap and allow it to run for 30 minutes to cool the
water condenser chamber to working temperature (-50C). Be aware that the
temperature gauge on most freeze dryers shows the temperature of the coolant in the
system not that of the chamber. If you run the freeze dryer before the condenser chamber
is cold enough to trap all the water vapour the water will get into the oil of the vacuum
pump and this will ultimately cause the pump to seize.
9.
When ready load the frozen trays into the dryer racks, close the chamber and start the
vacuum pump immediately. Watch the pressure gauge reading and this should start to fall
rapidly within a minute or two. If not check for leaks or a valve left open.
10. The sample temperature should start to fall as sublimation starts and the sample cools even
further. If there is any part of the sample not frozen it may cause a meltdown and
liquefaction of the sample at this point. However it may alternatively completely freeze
during this initial fall in temperature and the run will be OK. Keep the sample under
observation for the first 30 minutes as this is when meltdowns occur.
11. If the freezer trays have thermostatically controlled heaters set these at -20 initially. This
will feed heat into the sample to drive the sublimation of the water without heating it up
enough to melt the sample.
12. Once the drying process is successfully started after 2-3 hours the heater setting can be
raised to 10C to speed things up. Some freeze dryers have programmable heater trays
and a progressive rise in the temperature profile back to room temperature can be set to
ensure rapid drying.
55
13. On successful completion the sample will return to room temperature or the heater tray set
temperature, and this indicates all the frozen water has sublimed and the sample has
completely dried.
14. To stop the run switch off the vacuum pump and open the valve to allow air to enter the
drying chamber. Do this slowly or some of the dry sample may blow out of the tray all
over the drying chamber and into the condenser chamber.
15. Open the drying chamber and remove the dried sample. You should quickly transfer it to
a sealed container as crude insect virus samples are very hygroscopic and will quickly reabsorb water from the air which will de-stabilise them and causes the powder to clump
and clog.
56
4.12.2 Spray drying

NPV is commonly produced in vivo through a process of larval inoculation and subsequent harvest.
For ease of storage, harvested infected larvae are homogenised and then dried. Drying can be
achieved by either freeze drying (see Section 4.12.1) or spray drying.
Spray drying involves atomisation of feed into a spray, and contact between the spray and a drying
medium results in moisture evaporation. With baculoviruses, a suspension of homogenised infected
larvae (the feed) is combined with a high pressure air flow and atomised through a hydraulic nozzle.
The spray generated combines with a hot air stream and moisture evaporation takes place from
droplet surfaces. Evaporation is rapid due to the vast surface area of droplets in a spray. Dried
particles suspended in hot air are then removed in a cyclone.
To achieve rapid evaporation the hot air stream can exceed input temperatures of 200C. Like
almost all biological material, baculoviruses such as NPV are heat sensitive. However, because
these viruses are encapsulated in a protective protein crystal they are much less sensitive than most
viruses or vegetative bacteria. However, there is a risk of denaturing virus if exposure to heat is
excessive. Rapid evaporation, on the other hand, produces cooling, which mitigates the heating
effect. Once dry however, particles will be heated to the output temperature, and rapid separation
of virus from the hot air stream is essential.
Careful control of input temperature and feed flow rates is essential. Output temperature is critical
to the spray drying of heat-sensitive material. Despite the above comments about virus heat
sensitivity we have found that NPV is surprisingly heat tolerant. Output temperatures in the range 70
- 100C do not appear to destroy activity although exposure to these temperatures is extremely
short.
Spray drying is an efficient alternative to freeze drying and grinding. Careful control of temperatures
is required and bench top models such as the Buchi, (see below), should not be left entirely
unattended and constant attention should be paid to the spraying conditions such as inlet and outlet
temperature to prevent overheating or clogging. Failure to watch this could lead to the material
being lost or inactivated.
Spray drying eliminates the need for the separate grinding stage required for freeze drying, provided
the homogenates are first filtered to remove large particles. The powder produced is sufficiently fine
to be passed through knapsack sprayer filters without causing blockage. Moisture content of dried
powders is acceptable at between 1 - 4% and does not seem to be related to output temperature.
SPRAY DRYING WITH BUCHI 190 MINISPRAY DRYER
The following protocols are for a Buchi 190 mini spray dryer, the commonest research scale
machine in development laboratories (Plate 18). This is used for preparing small batches of material
for testing purposes. For mass production much larger machines, often used in the food industry,
with capacities of at least 15 -100 litres per hour can be used for preparing bulk batches. When
using these machines exact procedures may differ and the instruction manuals should be consulted
57
but the principles and general technique are the same. A high-pressure air supply is required for this
process.
Spray drying carried out in a Buchi 190 mini spray drier is in an open cycle. Air drawn into the
spray drier under control of an aspirator, is heated to the required temperature and then passed into
a drying chamber. The suspension to be dried is delivered by a peristaltic pump through silicone
tubing to a hydraulic nozzle (0.5mm diameter orifice), where it is atomised by high pressure air (6-7
bar). Evaporation takes place in the drying chamber where the atomised sample and heated air
combine. Suspended particles in the drying air are drawn out of the drying chamber and into a
cyclone under the action of the aspirator. Here, product particles are spun out of the air stream and
passed on to a product collector. Humid air passes upwards and out through a filter before being
exhausted.
Suspension flow rates of between 0.3 and 1.5 litres per hour can be achieved in the Buchi 190.
This should be seen as a guide only. Differences in suspension viscosities and pump roller
adjustments may alter the flow rates seen with distilled water alone. At high feed flow rates, even at
the highest input temperatures, the process is inefficient as wet feed accumulates on the drying
chamber walls.
Output temperature is principally controlled by feed flow rate and feed water content. Higher feed
flow rates lead to lower output temperatures because of increased evaporative cooling. For any one
feed flow rate, higher input temperatures give rise to higher output temperatures. Minimum product
residence time in the drying chamber can be assumed to be the average residence time of the air
(Masters, 1985). Throughput of air, assuming linear response of the aspirator control setting, is
approximately 33.75m3 hr-1. Residence time is calculated by dividing the chamber volume (3.29x103 3
m ) by throughput, giving 0.35 seconds. Most of the product however has a much higher residence
time because of particle re-circulation and retention in areas of lower air velocities.
PROTOCOL TO SPRAY DRY UNPURIFIED SUSPENSIONS OF INSECT VIRUSES:
Plate 18
Spray drying NPV using
a mini-spray dryer,
Buchi-190.
58
1.
Prepare a homogenous suspension of infected larvae in distilled water.
2.
Using a double layer of muslin, filter out all particles greater than 0.5mm diameter which
could otherwise accumulate and block the hydraulic nozzle.
3.
Centrifuge the suspension and discard the supernatant with any fat layer.
4.
Re-suspend the pellet with distilled water, the suspension should not be more than 25%
suspended solids. Above this the viscosity will be too great for efficient flow and
atomisation.
5.
Stabilise the input and output temperatures using distilled water before starting to dry the
sample using the procedure below.
6.
Turn on the power supply and switch on the spray dryer; turn on the high pressure air
supply; attach the cooling water supply to hydraulic nozzle assembly; open the air supply
to the nozzle and set at approximately 700 NI h-1.
7.
Switch on the aspirator and heater and allow the input temperature to stabilise at
approximately 150C by adjusting the heater temperature control little by little as
necessary. Ensure that any residual moisture from cleaning has evaporated and that there
is no contamination from previous use.
8.
Place a flask containing approximately 500ml distilled water on the shelf below the
peristaltic pump and place the feed tube in the water.
9.
Switch on the pump and, with water flowing, adjust the gap between the rollers and the
tube seating such that there is no reverse flow. The gap should be approximately 0.2mm
less than twice the tube wall thickness. A gap greater than this will lead to inefficient
delivery and reverse flow; a lesser gap than this will risk straining the pump and rupturing
the delivery tube.
10. With distilled water being pumped through the nozzle adjust the pump speed a little at a
time such that the output temperature is between 70 and 75C.
11. The input temperature should remain at approximately 150C. If not, adjust the heater
temperature accordingly. Note that this will effect the output temperature also, so
additional adjustments to the flow rate will be needed.
12. When the input and output temperatures have stabilised at 150 and 70C, replace the
distilled water with the suspension of crude virus.
13. Activate the automatic nozzle cleaning. Because the suspension contains a lower water
content, there is likely to be a rise in output temperature. This should be compensated for
by slightly increasing the pump speed.
14. Note that some suspensions are prone to sedimentation, in this case the suspension should
be mounted on a magnetic stirrer.
15. When the suspension has finished drying, replace the empty flask with distilled water but
do not allow the spray dryer to run dry at this stage as this will raise the output
temperature, putting the dried product at risk. The change from virus suspension to water
must be immediate.
59
16. Allow the water to flow until the feed supply tube and nozzle are clear of suspension.
17. Turn off the pump. At this point, output temperatures will rise quickly and it is important
that all dried product has collected in the product collector below the cyclone where it is
isolated from increases in output temperature.
18. Once all traces of atomised water have disappeared, (within a few seconds), turn off the
heater but do not turn off the aspirator. The aspirator must be left running until the input
temperature has fallen to below 70C.
19. Once this point is achieved the aspirator is turned off.
20. When air flow has stopped, the product can be removed in its collector and should be
stored in a sealed sachet or container immediately, preferably with silica gel, as spray dried
crude virus will rapidly absorb ambient humidity which leads to loss of activity during
storage.
21. A small sample should be retained separately for moisture analysis.
22. Disassemble the spray dryer and clean all glassware and metal components with a
proprietary disinfectant, rinse thoroughly with distilled water, dry and re-assemble.
60
4.13 SUMMARY OF MAIN POINTS
Production of NPV can only be carried out in live insect cells, either in whole insects or
in cell culture.
In whole insects virus production can be highly productive and all commercial
production is done this way, as cell culture is still under development as a practical
technique.
Key to production in insects is good inocula, healthy insects, correct choice of NPV
dose, harvesting time and stable rearing conditions.
While harvesting live insects from production takes more care it results in a cleaner
more consistent product.
All processing involves loss of NPV do what you need, no more. Gradient purified
NPV is needed for production inocula but not for field use.
All processing must be carried out at 4-10C to avoid the development of bad smells
and the build up of bacterial contamination.
Properly processed centrifuged NPV is better for formulation as it has high activity and
low smell.
It is easier to clean up well-infected, live harvested insects into a low smelling product.
Drying of virus suspension is the most convenient way of storage.
4.14 REFERENCES
Dobrata, M & Hinton, R (1992) Conditions for density gradient separations. In: Preparative
centrifugation a practical approach. Rickwood D. (ed.) IRL Press, pp. 77-142.
Hunter, F R; Crook, N E and Entwistle, P F (1984) Viruses as pathogens for the control of
insects. In Microbiological methods for environmental biotechnology.
(ISBN 0-12-295040-2).
Masters, K (1991) Spray drying handbook. (5th Edition), Longman Scientific, Harlow England.
Rickwood, D (ed.) (1987) Centrifugation - a practical approach. (2nd edition) IRL Press, 354 pp.
Roth, H & Rickwood, D (1992) Centrifuges and rotors. In: Preparative centrifugation - a
practical approach. Rickwood, D (ed.) IRL Press, pp 42-76.
Rowe, T W G & Snowman, J W (1978) Edwards freeze drying handbook. Edwards High
Vacuum, Crawley, England.
61

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THE

HELICOVERPA ARMIGERA NPV

Using the manual

Insect Viruses - an Introduction

Cytoplasmic Polyhedrosis Viruses

The Life Cycle of Baculoviruses

Introduction to H. armigera NPV

Insect Culture for Virus Production and Testing

Importance of Clean Culture, Sanitation and Hygiene

Entry preparation room

Adult moth room

Larval holding room

Egg handling room

Diet preparation room

Insect culture in the laboratory

Establishment of a culture from the wild

Quarantine and colony clean up

Procedure for larval diet

Insect Rearing Protocol

Rearing of late stage larvae

Selection for vigour

Supply of Insects for Virus Production

Problems in Rearing Larvae

Mould on diet caused by Aspergillus niger

The Production of Insect Viruses

Tissue Culture Production

Production in Whole Insects

Protocol for purifying inocula

4.6.1.1 Primary processing

4.6.1.2 Gradient centrifugation

Incubation and rearing

Sanitation of production facilities

Model protocol for the production of Helicoverpa armigera NPV

Preparation of production trays

Inoculation of production trays and loading larvae

Harvesting infected larvae

Processing of infected larvae

Processing insect viruses for use as biopesticides

Homogenising and filtering

Storage of insect viruses

Summary of Main Points

Quality Control in NPV Production

The Importance of Quality Control

The importance of routine protocols

Quality Control Techniques

Microscopic examination of NPV & GV

Microscopes and choice of illumination

Counting baculoviruses and the use of standards

Microscopic staining techniques

Sikorowski quick stain for CPV polyhedra (& NPV)

Method for differential Giemsa staining of occluded insect

Giemsa stain with acid hydrolysis for nucleopolyhedrovirus

Counting NPV using the improved Neubauer haemocytometer or

Identifying contaminants under the microscope

Bioassay Techniques and Microbial Pesticides

Lethal dose50 and lethal concentration50

Summary of main points

Surface Dose Bioassay to Determine LC50 in 1 Instar Larvae

Artificial diet for polypots

Diet Plug Bioassay to Determine LD 50

Leaf Dip Bioassay

Analysis of bioassay data

Graphical method for estimation of the median lethal