This document provides details on methods for collecting and maintaining important insect vectors of plant viruses. It discusses the rearing of aphids, leafhoppers, whiteflies, mealybugs, thrips, and beetles. For each insect group, it describes collection techniques from host plants and rearing procedures to maintain virus-free colonies, including necessary equipment, host plants, and environmental conditions. The goal is to obtain virus-free insects for transmission studies of various plant viruses.

1. METHODS FOR COLLECTING VECTORS
AND THEIR MAINTENANCE
G.Anitha
RAD/13-45
Dept of Entomology

2. Three orders in class Insecta contain important
vectors
Homoptera : Aphids
Leafhoppers
Whiteflies
Mealybugs
Thysanoptera : Thrips
Coleoptera : Beetles

3. APHIDS
Most important group of virus vectors as they can
transmit a large number of viruses by more than
one mechanism.

4. COLLECTION
• 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

5. Maintenance on host plants
Materials required
• 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
too dense.
• Large aphid cage (40X40X55cm) for virus-free stock
colony
• 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.

6. • Leaf cages
• Small artist’s paint brush
• Labels, Small stickers
• Magnifying glass/stereomicroscope
• Vials
• Pieces of Parafilm membrane or aluminium foil
• Infected host plants
• Virus-free host plants (usually tobacco) (4-5 leaf
stage)
• Apterous aphids

7. CAGE REARING

8. LEAF CAGE

9. CLIP CAGE

11. 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
day cycle.
• Removal of aphids from a leaf should be done very
carefully to prevent breaking their stylets
(mouthparts) when feeding.

12. • 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
transmission studies.

13. MAINTENANCE ON ARTIFICIAL DIET

14. S.No. Item Qty (mg/100ml)
1. Amides
Asparagine 500
Glutamine 200
2. Microelements
Copper chloride 0.2
Iron chloride 11
Manganese chloride 0.4
Zinc sulphate 0.8
3. Amino acids`
Alanine 100
Arginine 200
Aspartic acid 200
Cysteine HCl 100
Glutamic acid 200
Glycine 50
Histidine 80

15. S.No. Item Qty (mg/100ml)
Isoleucine 100
Leucine 100
Lysine 100
Methionine 100
Phenylalanine 40
Proline 40
Serine 50
Threonine 150
Tryptophan 50
Tyrosine 40
Valine 100
Ascorbic acid 10
Biotine 0.1
Choline chloride 50
Folic acid 0.5
Meso inositol 50

16. Preparation of diet (Harrewijn, 1983)
• Tyrosine is dissolved in 75 ml of double distilled water
(DDW)
• 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.

17. Preparation of virus suspension and
diet sachet
• 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
transmission studies.

18. Diet sachets

19. LEAFHOPPERS
They cause huge losses in major crops like rice

20. COLLECTION
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
material.

21. LIGHT TRAPS
Are trapped using light traps and collected with an
aspirator

22. MAINTENANCE ON HOST PLANTS
Materials required
• 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

23. • 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
plants)
• Nymphs and newly emerged leafhopper adults

24. REARING EQUIPMENT

26. REARING VIRUS-FREE LEAFHOPPERS
• Collected insects are released into the transfer
chamber and the hoppers are collected with an
aspirator.
• 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.

27. • 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
200C).
• 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

28. WHITEFLIES
• Whiteflies transmit viruses of Geminivirus group
cause serious diseases in many crops such as bean,
cassava, cotton, tobacco and tomato transmitted by
Bemisia tabaci.

29. COLLECTION
Collected from underside of leaves of host
plants using an aspirator

30. MAINTENANCE
Materials required
• Glasshouse compartment (28-320C)
• Large cages (40X40X40cm)depending on the plants
used
• 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.
• Aspirator
• Black cloth

31. • Pots (10cm diameter)
• Labels
• Insecticide
• 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

32. REARING VIRUS-FREE WHITEFLIES

33. Gauge
Perspex Jar
Shoot of source plant
Cotton plug
Lid with hole
Flask with water
Gauge
CAGE FOR TRANSMISSION STUDIES

34. TUBE CAGE

36. • 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

37. • Plants with eggs and larvae are moved to a larger
cage for continuous production of whitefly stock
culture
• 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
nature.

38. MEALYBUGS
Planococcus sp. is reported to spread Grape leaf roll
virus

39. COLLECTION

40. MAINTENANCE ON VIRUS-FREE HOST

43. THRIPS
• Thrips of genera Thrips and Frankliniella are
important polyphagous pests and transmit
viruses like Tomato spotted wilt virus.

44. COLLECTION

45. MAINTENANCE
Materials
• Glasshouse compartment at a 16:8 h photoperiod (light:dark) at
25+30C.
• Cages to host the potted Impatiens plant-host plant
• Small artist’s paint brush
• Microfuge tube
• 24-well plates
• Corkborer
• Pots
• Parafilm
• Diseased plants of Impatiens inoculated by infected
F.occidentalis
• Virus-free plants of Impatiens
• Virus-free pollen as feed for thrips

48. REARING 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
parafilm membrane.
• 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.

49. DIABROTICA BEETLES
These are known to spread cucumber mosaic
virus in cucmber and bacterial wilt of maize in
maize.

50. COLLECTION AND REARING
PROCEDURE
• 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
high temperatures.
• 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.

51. REFERENCES
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.