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Methods of collecting vectors and their maintenence

Vectors and their rearing techniques

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Methods of collecting vectors and their maintenence

  2. 2. Three orders in class Insecta contain important vectors Homoptera : Aphids Leafhoppers Whiteflies Mealybugs Thysanoptera : Thrips Coleoptera : Beetles
  3. 3. APHIDS Most important group of virus vectors as they can transmit a large number of viruses by more than one mechanism.
  4. 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. 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. 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
  8. 8. LEAF CAGE
  9. 9. CLIP CAGE
  10. 10. 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.
  11. 11. • 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. 13. 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
  14. 14. 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
  15. 15. 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.
  16. 16. 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.
  17. 17. Diet sachets
  18. 18. LEAFHOPPERS They cause huge losses in major crops like rice
  19. 19. 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.
  20. 20. LIGHT TRAPS Are trapped using light traps and collected with an aspirator
  21. 21. 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
  22. 22. • 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. 24. 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.
  25. 25. • 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
  26. 26. 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.
  27. 27. COLLECTION Collected from underside of leaves of host plants using an aspirator
  28. 28. 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
  29. 29. • 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
  31. 31. Gauge Perspex Jar Shoot of source plant Cotton plug Lid with hole Flask with water Gauge CAGE FOR TRANSMISSION STUDIES
  32. 32. TUBE CAGE
  33. 33. • 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
  34. 34. • 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.
  35. 35. MEALYBUGS Planococcus sp. is reported to spread Grape leaf roll virus
  36. 36. COLLECTION
  38. 38. THRIPS • Thrips of genera Thrips and Frankliniella are important polyphagous pests and transmit viruses like Tomato spotted wilt virus.
  39. 39. COLLECTION
  40. 40. 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
  41. 41. 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.
  42. 42. DIABROTICA BEETLES These are known to spread cucumber mosaic virus in cucmber and bacterial wilt of maize in maize.
  43. 43. 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.
  44. 44. 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.