Long term research and use of a bio-control agent depend on the availability of effective and economical rearing techniques, and this protocol has proven to be efficient for rearing this parasitoid. There is currently enormous interest in rearing this parasitoid for research or field release, and the protocol will benefit future long-term research and use of this promising bio-control agent. First users may need to understand some basic biology of the involved insects through reading the relevant references provided in the article and some practices of the methods.
For making the host diet, first, prepare the liquid portion of the diet as described in the manuscript. Next, add 25 to 30 grams of mashed blueberries to an empty 250-milliliter flask. Then, pour the liquid into the flask with the liquid just covering the top of the mashed blueberries.
Next, for rearing the host Drosophila suzuki, first cut a piece of absorbent paper towel and twist it at the center. Then, place the twisted middle section of the paper towel in the diet flask. For retaining moisture, use distilled water to wet the paper towel and the diet.
Then, to transfer sexually mature adult flies, carefully remove the stopper of a fly colony flask and invert it into the diet flask. After a week of this exposure, hold the flies in an environmental chamber for three weeks for fly emergence. For exposing host larva to parasitoids, first remove the adult flies and the paper towel from the flask, leaving the fly eggs and larvae.
To serve as a pupation substrate for the larvae, fold a new absorbent paper towel and insert it into the flask. Then aspirate six female and male pairs of the parasitoid G3 Ganaspis brasiliensis into the flask and streak a thin layer of honey on the bottom of the foam stopper. For collecting and storing the parasitoids, remove the adults after five days.
Then store the flask in the environmental chamber for 35 days under the conditions mentioned earlier. With the emergence of adult wasps, aspirate them three times per week. For holding the wasps, first place a paper towel moistened but not saturated with distilled water at the bottom of a 2.5-by-9.5 square centimeter Drosophila vial.
Then, transfer about 60 wasps to the vial and mark the emergence date. Store the vial in the environmental chamber. Twice a week, streak a thin layer of honey on the bottom of the foam stopper of the vials.
First, arrange large scale rearing of Drosophila suzuki by placing 1, 500 to 2, 000 sexually mature adult flies at a 50:50 sex ratio in a large, knitted, mesh-covered cage. Then, place a plate with water-soaked cotton and four to six Petri dishes with standard Drosophila medium in the cage. Twice every week, replace the exposed Petri dishes with fresh ones.
Place each infested Petri dish without the lid in an 800-milliliter plastic cup and cover the cup with a fine mesh. Then, incubate the cups at 23 degrees Celsius and 75%relative humidity for 12 to 15 days. After incubation, transfer newly hatched adult Drosophila suzuki to a rearing cage.
Next, begin preparing the host larvae by rinsing blueberries in cold water for one minute and soak the fruits in a basin filled with a bleach solution, diluted to 5%for three minutes. Then, fill nine-centimeter Petri dishes with the washed blueberries. In the late afternoon hours, put the Petri dishes in the rearing cage and leave them overnight.
The next morning, gently tap on the Petri dishes to dislodge flies from the fruits and remove the fruits from the rearing cage. Meanwhile, prepare a parasitism cage and a wasp emergence cage for the parasitoid G1 Ganaspis brasiliensis. Both the cages should have a thin string below the ceiling for suspending one or more feeders.
In each cage, suspend a Drosophila vial containing water and seal with a cellulose acetate plug upside down from the ceiling every five to seven days, depending on the relative humidity. Immediately after taking out infested blueberries from the Drosophila suzuki cage, put them in the parasitism cage with 1, 500 to 2, 000 Ganaspis brasiliensis wasps for two to three days. Next, collect the fruits in an 800-milliliter plastic cup with absorbent paper at the bottom.
Incubate the open cup in the emergence or eclosion cage at 21 degrees Celsius and 65%relative humidity for at least 28 days. During the second and third weeks of incubation, check the cage weekly for early host eclosion. To facilitate successive parasitoid collection, remove the adult flies.
At the end of the fourth week of incubation, provide a feeder and a water source to the cage. Collect about 10 to 15%of the adult wasps and transfer them to the parasitism cage to replace old and unproductive wasps. From the remaining wasps of the eclosion cage, store up to 700 adults at a 50:50 sex ratio with an 800-milliliter plastic cup containing a two-milliliter tube filled with water and sealed with a dental cotton roll at the bottom of the cup.
Store the cup with a marked emergence state in an environmental chamber for up to one month. For shipping parasitoids, place about 200 adults in a 50 milliliter conical tube. Then, put all the tubes in an insulating shipping container along with ice packs.
This protocol resulted in successful small-scale rearing of G3 Ganaspis brasiliensis, a parasitoid agent, against the agricultural pest Drosophila suzuki in the laboratory. Offspring production remained unaffected by differences in parasitoid density and exposure time. Large-scale generation of G1 Ganaspis brasiliensis could also be achieved, with the production beginning five weeks after the initial exposure to the host Drosophila suzuki.
From week 8 to week 22, the parasitoid production became proportional to the amount of fruit exposed to them. It is critically important to maintain a suitable rearing temperature, control the humidity, and always provide food for adult insects. The methods could be further improved by optimizing the rearing densities of host and parasitoid to maximize the reproduction of wasps and by exploring the possibility of rearing this parasitoid on alternative hosts.
