Large-Scale Production of the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and Its Biopesticide: Nuclear Polyhedrosis Virus (HaNPV)

Abstract

A pilot-scale production system of NPV requires a continuous supply of large number of host insects that are disease-free, uniform in size, physiologically fit, and genetically efficient. Hence, the selection of healthy colony from the filed population is a critical process. Among the different populations collected from the fields, the population from Belgaum showed lesser level of microbial contaminations. Microscopic observations also revealed that the highest larval and pupal mortality was resulted due to protozoan pathogen, Vairimorpha sp. compared to other pathogens regardless of the populations collected from different geographical areas. The maximum fecundity and egg hatching and the time taken to stabilize the colony under laboratory conditions are considered as basic parameters to screen the most suitable breeding colony among the different populations for large-scale viral production. Although all the population collected from different geographical areas stabilized in the laboratory from fifth generation and onwards, Helicoverpa armigera population collected from Belgaum was found superior in terms of fecundity and egg hatching compared to other populations throughout the study period up to 25 generations. Helicoverpa armigera female moths emerged 1 day earlier than the male moths. The peak emergence period of female moths and male moths varied. More or less similar kind of peak emergence pattern was noticed irrespective of pupal age. After standardization of various components such as healthy population of H. armigera (Belgaum), adult nutrition (10% honey), sex ratio (1:2), and pigeon pea-based diet for mass production of H. armigera and its NPV, it was found that improved method increased the fecundity of H. armigera, and its HaNPV yield is significantly (P < 0.05) higher than that of standard rearing method. The difference of 368 extra number of HaNPV bottles (1 × 10¹¹ POBs/100 mL bottle) was produced from modified diet.

Appendices

Procedure for Mass Multiplication of Helicoverpa armigera

1.1 Safety

Follow appropriate personal protective equipment (PPE). Safety glasses, laboratory coats, toe-covered shoes, gloves, face mask, head cover, etc.

1.2 Materials Required

• 1. Black cotton cloth as ovipositional substrate.

• 2. Culture trays.

• 3. Semi-synthetic diet, honey, and vitamin syrup.

• 4. Plastic culture vials, plastic bowls with mesh lid.

• 5. Plastic jar for oviposition (3–5 L) and petriplates.

• 6. 0 or 1 numbered camel hair brush, blunt forceps.

• 7. Labels and markers.

• 8. Sodium hypo chlorite solution (0.25 and 1%). 0.25% Sodium hypochlorite for egg sterilization (4% available chlorine): Add 62.5 mL of concentrated sodium hypochlorite to 937.5 mL of reverse osmosis (RO)/distilled water, mix thoroughly, and fill in airtight bottle. 1% Sodium hypochlorite for pupal sterilization (4% available chlorine): Add 250 mL of concentrated sodium hypochlorite to 750 mL of RO water, mix thoroughly, and fill in airtight bottle. Label and store at room temperature for not more than 1 month.

• 9. Seventy percent alcohol for hand and surface sterilization (measure 300 mL of RO water into a 1000 mL beaker and add 700 mL of 99.9% ethanol and mix thoroughly and store in room temperature).

• 10. RO/distilled water.

• 11. Measuring cylinder (10–1000 mL) and beaker (50–500 mL).

• 12. Plastic trays, filter paper.

1.3 Methodology

Eggs

• 1. Select eggs/egg mass from screened/disease-free colony neonates (F3).

• 2. Soak the cloths/egg substratum containing 1- or 2-day-old eggs in a tray containing 1–2 L of 0.25% sodium hypochlorite solution for about 3 min for H. armigera. Remove the ovipositional substratum from sodium hypochlorite solution after gentle agitation.

• 3. Keep the egg cloth/egg substratum in a box with mesh lid and rinse under running tap water for 15–20 min.

• 4. Use filter lined with cloth to collect detached eggs.

• 5. Drain the water and place eggs over filter paper for drying (2–3 h).

• 6. After proper drying, put the egg cloths/papers in a plastic jar and place it in the incubator at 25 ± 2 °C until complete hatching.

• 7. Label the jars indicating date of egg collected.

Larval Rearing

• 1. Pour semi-synthetic diet into culture trays (3–5 mL/well) and culture tubes (3–5 mL/tubes) using 50 mL beaker/centrifuge tubes.

• 2. Allow the diet to cool for at least 30 min, before transferring the neonates.

• 3. Transfer active neonates into each well with the help of sterile camel hair brushes (No. 0 or 1) and cover properly.

• 4. Label the trays indicating date of transferring and expecting date of larvae reaching to later instars (fifth instar) and incubate in environmental chamber (25 ± 2 °C and RH 65 ± 5%).

• 5. Check trays periodically for growth and development of larvae, record any changes in diet, larval mortality, etc.

• 6. Handling later instars: After eighth day, transfer active grown up instar larvae into culture trays/tubes containing 4–5 mL of semi-synthetic diet and plug tightly to avoid larval escape.

• 7. Check culture tubes/trays every day for diet consumption by larvae and change to fresh diet if required until pupation.

• 8. Examine dead larvae for pathogen infection whenever the proportion of larval mortality exceeds 5%.

• 9. Collect 1-day-old healthy and fully formed pupae from the culture tubes/trays and keep them separately in plastic bowls and cover with mesh lid, keep in isolate chamber until sterilization.

Pupae Handling

• 1. Select the healthy and fully formed 4 or 5 days old pupae.

• 2. Soak in a bowl containing 0.5 or 1 L of 1% sodium hypochlorite solution for 3–5 min.

• 3. Collect floating pupae and discard.

• 4. Keep selected pupae in a plastic bowl and cover with mesh lid. Rinse under running water for 15–20 min to remove the hypochlorite residue.

• 5. Drain the water and place pupae over filter paper and dry.

• 6. After complete drying, place in sterilized and labeled pupal bowls for moth emergence (8–10 days).

• 7. If required, sex the pupae according to Standard protocol and keep in separate jars for moth emergence.

Moth Handling

• 1. Black cotton cloth as egg substratum and earthen pot as an oviposition jar for H. armigera.

• 2. Place moth food plastic lids inside the oviposition jar.

• 3. Preparation of moth food—Soak the absorbent cotton wad in the 10% honey solution (Add 100 mL of Dabur/any pure brand honey and 20 mL of vitamin syrup into 880 mL of RO/distilled water mix properly and pour into airtight bottle and after use store in a refrigerator. Prepare always fresh honey solution as and when required amount only) similarly, dip absorbent cotton wad in RO/distilled water and place it on another plastic lid. Keep one each of the plastic lid in the oviposition jars and replace every day till moths exhaust.

• 4. Select female and male moths from pupal bowls (vigorous adults that emerge early are to be used for continuation of culture).

• 5. Release 10–15 pairs of moths/ovipositional jar. The males are plain greenish and the females(chocolate brown).

• 6. Cover the mouth of the oviposition jar with black cotton cloth and secured with rubber bands.

• 7. Collect eggs from second day and end on fifth day from each jar and estimate number of eggs.

• 8. Keep selected egg sheets in plastic containers with date of collection. Sterilize 1-day-old eggs and keep again in the plastic containers till they hatch.

• 9. Examine malformed adults for pathogen infection whenever the proportion of malformation exceeds 5%.

1.4 Labeling of Insect Culture

• 1. Insect Name: Helicoverpa armigera

• 2. Date:

• 3. Number of larvae/pupa/moths:

• 4. Location:

• 5. Name of contact person:

Procedure for Mass Multiplication of H. armigera Nuclear Polyhedrosis Virus (HaNPV)

1.1 Safety

Follow appropriate personal protective equipment (PPE). Safety glasses, laboratory coats, toe-covered shoes, gloves, face mask, head cover, etc.

1.2 Materials Required

• 1. Culture trays

• 2. Semi-synthetic diet

• 3. Early fifth instar larvae of H. armigera

• 4. Stock culture of virulent strain NPV

• 5. 1000 μL to 10 μL pipet

• 6. Blunt-end polished glass rod and blunt forceps

• 7. Refrigerator

• 8. Refrigerated centrifuge

• 9. Plastic storage container (2 L)

1.3 Methodology

• 1. Maintain sufficient stock culture of NPV in the laboratory.

• 2. Select active early fifth instar larvae of H. armigera for the NPV propagation.

• 3. Pour semi-synthetic diet into multi-cavity trays (3–5 mL/well) and allow the diet to cool for at least 30 min.

• 4. Apply the dose of 1 × 107 POBs/mL in 10 μL per cavity on the semi-synthetic diet in multi-cavity trays.

• 5. Spread the virus suspension uniformly over the diet surface by using blunt-end polished glass rod. Inoculated trays are dried for at least 15 min prior to larval transfer.

• 6. Release the larvae individually into each cavity of multi-cavity trays and incubate trays at 25 ± 1 °C in a laboratory incubator.

• 7. Provide freshly prepared untreated diet when the food exhausted in the cavities.

• 8. On the fifth day, observe the larvae for the development of virosis and collect the cadavers carefully from the individual cavities with the help of sterile blunt forceps.

• 9. Store the collected cadavers in a refrigerator up to processed.

• 10. Remove these cadavers from the refrigerator and thaw very rapidly by agitation in distilled water.

• 11. Homogenize the cadavers using electric mixer and dilute the concentrate with distilled water.

• 12. Filter the homogenate through a sterile, double-layered muslin cloth. Use distilled water repeatedly to wash the homogenate for maximum extraction of the polyhedra.

• 13. Spun the filtrate for 3 min at 500 rpm in the refrigerated centrifuge to remove debris and larger particles.

• 14. Re-spun the supernatant for 20 min at 5000 rpm in the refrigerator to collect the pellet-containing polyhedra.

• 15. Resuspend the pellet in distilled water and wash thrice for obtaining semi-purified HaNPV. Label the HaNPV concentrate bottle and store in a cold chamber.