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Methods of collecting vectors and their maintenence
METHODS FOR COLLECTING VECTORS
AND THEIR MAINTENANCE
Dept of Entomology
Three orders in class Insecta contain important
Homoptera : Aphids
Thysanoptera : Thrips
Coleoptera : Beetles
Most important group of virus vectors as they can
transmit a large number of viruses by more than
• As they are sedenatry, collection for initial
stock culture is easy and is done by collecting
the plant part where they are present and
transferring using a small brush
Maintenance on host plants
• Glasshouse compartment at a 16:8 h photoperiod
(light:dark) at 22+30C. This light:dark hours prevents
development of alate forms, if the population is not
• Large aphid cage (40X40X55cm) for virus-free stock
• Cylindrical Perspex cages of different sizes (from 5-
8cm diameter and 10-14cm height) with top and
side holes covered with a 400µm mesh to confine
the aphids after their transfer to individual plants.
Rearing Virus-free aphids
• As most aphids do not transmit virus transovarially,
virus-free aphids can be reared from new born ones.
• 10-15 apterous adults (8-9 day old) from a stock
culture are placed in a leaf cage on a host plant and
transferred daily to another plant leaving behind the
nymphs which are less than 1 day old.
• After 3 days, the old adults are killed and new adults
from a previous series are used to start the next 3
• Removal of aphids from a leaf should be done very
carefully to prevent breaking their stylets
(mouthparts) when feeding.
• The abdomen is gently touched with the brush until
the insect withdraws the stylets and starts walking
• 3-4 day old apterous aphids which are efficient
transmitters of virus are transferred from the virus-
free stock with a paint brush to a vial for starvation.
• Then the insect is transferred to another vial for
Preparation of diet (Harrewijn, 1983)
• Tyrosine is dissolved in 75 ml of double distilled water
• Amino acids, amides,sucrose and magnesium sulphate
is added (solution A). All other vitamins are added.
• Ascorbic acid and citric acid are dissolved in 15ml DDW
and microelements are added to it (solution B)
• Both solutions are mixed
• pH is adjusted with potassium hypo sulphate buffer
• Diet made upto 100ml and filtered with a sterile
bacterial filter and kept under sterile conditions.
• Just before use, 1% Bovine serum albumin is added to
prevent it from sticking to the parafilm.
Preparation of virus suspension and
• Pieces of Parafilm are exposed to UV radiation for
a period of 5 min.
• Their sides are stretched and the diet(100µl) is
poured between them to form a ‘Diet sandwich’.
• This is placed inside a leaf cage or a petridish for
They cause huge losses in major crops like rice
Sweep nets are made of a heavy material (such as
canvas) that can be dragged through dense
vegetation without being damaged or thin net-like
Are trapped using light traps and collected with an
MAINTENANCE ON HOST PLANTS
• Glass house/screehouse
• Insect sweepnet
• Aspirator to collect insects
• Metal cages (55X55X75cm) with aluminium mesh
screen for rearing host plants
• Insect transfer chamber (50x50X40cm) for
convenient collection and introduction of insects
onto test plants, made either of hard plastic or
glass in a wooden frame with an opening
• Black cloth
• Seedling boxes
• Water trays
• Pots and labels
• Pair of forceps for transplanting the seedlings
• Test tube rack for 4X10 tubes
• Glass tubes (18X150mm)
• Test tube caps (18mm)
• 40-45 day-old virus-free rice plants (food plants)
• 35-40 day-old diseased rice plants – TN1 variety
• 7-day old virus-free TN1 ssedlings (1-2 leaves) (test
• Nymphs and newly emerged leafhopper adults
REARING VIRUS-FREE LEAFHOPPERS
• Collected insects are released into the transfer
chamber and the hoppers are collected with an
• Transferred onto 45-day old TN1 plants in a metal
insect cage designated as “egg cage” for egg laying
• They are retained there for two days to obtain eggs
of uniform size.
• Egg laden plants are then transferred into a rearing
cage leaving the adults in the egg cage for
successive egg laying processes.
• After emergence of the nymphs, fresh food plants
are regularly supplied.
• During 2-3 weeks, a leafhopper passes through five
nymphal instars before becoming an adult, with the
duration of development depending on the
temperature (14days approx. at 380C and 37 days at
• A new instar develops in approx. 2-4 days.
• First instar nymphs are more numerous on the
lower surface of older leaves while second instar
nymphs are distributed evenly on all leaves
• Whiteflies transmit viruses of Geminivirus group
cause serious diseases in many crops such as bean,
cassava, cotton, tobacco and tomato transmitted by
Collected from underside of leaves of host
plants using an aspirator
• Glasshouse compartment (28-320C)
• Large cages (40X40X40cm)depending on the plants
• Cages made from Perspex household jars.
• A small hole is cut out for insertion of the plant;
bottom of the jar is cut and replaced with muslin
cloth and nylon gauze.
• Ten conical flasks are to be collected to small cages.
• Black cloth
• Pots (10cm diameter)
• Magnifying lens
• Tomato plants infested with virus
• Young virus-free tomato plants (test plants)
• Young plants of species immune to the virus that
attacked the tomato plants
• Whiteflies collected in field
• Virus-free whiteflies
• To establish whether the insects are free from
the virus, they are allowed to feed on tomato
seedlings for 48hrs and they are observed for
development of symptoms
• Thereafter the whiteflies are transferred to a
cage with young virus-free seedlings of tomato
• Ten days later all the surviving adults are
removed and killed
REARING VIRUS-FREE WHITEFLIES
• Plants with eggs and larvae are moved to a larger
cage for continuous production of whitefly stock
• To keep the culture in good condition, the insects
are to be transferred to virus-free plants at
regular intervals and this frequency depends on
plant species used
• Since, the insects are very “lively”, they are to be
checked from time to time for their virus-free
Planococcus sp. is reported to spread Grape leaf roll
• Glasshouse compartment at a 16:8 h photoperiod (light:dark) at
• Cages to host the potted Impatiens plant-host plant
• Small artist’s paint brush
• Microfuge tube
• 24-well plates
• Diseased plants of Impatiens inoculated by infected
• Virus-free plants of Impatiens
• Virus-free pollen as feed for thrips
• Adults are transferred with a small paintbrush to
pods of French bean (Phaseolis vulgaris)
• For oviposition in a growth chamber at 270C, these
pods are placed in a preserver pots along with a
pierced microfuge tube containing pollen and a
small container with sugared water touching the
• Central part of the metal lid of Preserver pot is cut
and replaced by a metal mesh
• After 24h, pods are taken out and cleaned by
removing the thrips
• They are placed separately in preserver pots at 250C
• Thus nymphs of same age can be obtained.
These are known to spread cucumber mosaic
virus in cucmber and bacterial wilt of maize in
COLLECTION AND REARING
• Initial cultures are obtained by sweep net studies
from which they are aspirated by mouth and
emptied into cardboard cartons. Adequate moisture
is to be provided and they are to be shielded from
• They are transferred to a cage of 50X50X50cm
dimensions and feed with a diet given by Guss and
Krysan (1973) along with slices of sweet potato
roots,sweet potato foliage which increase fecundity
and longevity of adult.
Dijkstra J and Jager C P d.1998. Practical Plant
Virology. Springer Lab Manual. 460pp.
Schalk J M 1986. Rearing and Handling of
Diabrotica balteata. In Methods for the study
of Pest Diabrotica ed: Krysan J L and Miller T A
Springer-Verlag, New York. 86 pp.