1. Insect Pathology Prof. Abdelwahab A. Ibrahim Entomology Department, Faculty of Science, Benha University

2. CULTURE OF INSECT PATHOGENS VIRUSES  Viruses cannot be cultured on artificial media. They only grow in a living insect host. Use an extract of the internal contents of an infected insect and introduce the virus particles into the mouth of the host by:  1 using a hypodermic syringe or,  2 contaminating the food supply. BACTERIA  Same procedure with viruses, or culture the bacteria on nutrient agar. FUNGI  Deuteromycotina and Ascomycotina fungi are easily cultured.  Use agars and/or boiled grains (rice, wheat, barley) as culture media.  Other fungi may need special media, such as meal worm agar. Entomophthorales are difficult to grow in culture. DO NOT sub-culture fungi continuously.  Infect some healthy insects after 3 or 4 tranfers and reisolate. Prof. Abdelwahab A. Ibrahim

3. STORAGE OF INSECT PATHOGENS VIRUSES Store tissues containing the virus in the fridge or freezer. All occluded insect viruses can survive freeze drying. BACTERIA Store in a cool, dry place. FUNGI 1 Freeze-dry or store in liquid nitrogen. 2 Grow fungi in glass tubes on weak media agar slants (potato carrot agar, fifth strength potato dextrose agar; Seal with wax or screw caps. Add sterile araffin oil to prolong survival. Label and date the flasks clearly. Make sub-cultures every three to five (3-5) years. 3 Spores growing on cadavers will survive at 4°C in the refrigerator as long as they are kept dry. They can also be frozen. PROTOZOA Store resistant stages in the refrigerator. NEMATODES Adult or egg stage of mermithids and third stage juvenile of rhabditids can be stored in a moist environment at about 5-15°C. Prof. Abdelwahab A. Ibrahim

4. how to rear Entomopathogenic nematodes  Place 2 pieces of filter papers in a petri dish.  Transfer 2 or 3 ml of water containing about 1000-5,000 nematodes into the dish, on the filter paper (be sure the papers become wet but NO EXTRA WATER CAN BE SEEN).  Depending on the size of the petridish, a number of wax worms (or other insects) will be released into the dish. With the petri dish 100 x 15 mm, about 10- 25 wax worms can be used for 1000-5,000 nematodes (you should know that to use too many nematodes, they will die in the body of wax worms and you will not have many nematode offspring). Larger petri dish can be used for a larger number of insects.  After 2 days, all wax worms will be killed by nematode. The wax worms will be washed in a cup of water and transferred to a White trap as shown in the pictures. Prof. Abdelwahab A. Ibrahim

5. How to make a White trap for Nematodes Place a top of 100 x15 mm petri dish in a larger petridish (150 x25 mm), cover the former with filter paper as in Fig. A, arrange the dead wax worms on the filter paper as shown above. After the arrangement is finished, add water into large petridish so that the water reaches the edge of the filter paper which will absorbed water and become wet. Be sure the paper underneath of wax worms to be wet, add some water on the paper. Keep the prepared petri dish in the dark. After about 5-10 days, the infective stage of nematode will emerge and move into water. Nematode can be collected, washed several time using a sieve. Now the nematodes can be used to infect insects or stored in a refrigerator for future use. Prof. Abdelwahab A. Ibrahim

6. How to arrange the dead wax worms in White traps to collect nematodes Prof. Abdelwahab A. Ibrahim. A: for Steinernema. B-D: for Heterorhabditis.

7. Prof. Abdelwahab A. Ibrahim