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The overall aim of this procedure is to infect the insect larvae Galleria melanoma with pathogenic bacteria. To do this, insect larvae are rid to the appropriate growth stage and a bacterial solution is prepared. The larva then infected with the bacteria by either intra hemophilic injection or force Feeding larval mortality is then monitored over a 48 hour period.

Analysis of the resulting data describes bacterial virulence in an insect model of infection. Insects present the advantage to have an advanced system of antimicrobial defenses, and in particular, they possess a complex innate immune system, so avoid the cost and ethical issues that may be encountered when using mammalian models. Insects provide great alternative model for mammalian infection.

We work with the larvae of the greater wax moth G me, which are relatively large and can be used for infection studies conducted between 15 to above 37 degrees. To mimic a mammalian environment, Carina NI will demonstrate the rearing procedure and Elizabeth GME and Christophe Beon will demonstrate the infection protocols. The whole cycle from egg to last in star larvae lasts about five weeks.

At 25 degrees Celsius, one or two additional weeks are needed to obtain adult butterflies. Male butterflies measure 10 to 15 millimeters. The adult male moth is beige with faint light and dark markings.

Female butterflies measure around 20 millimeters. Females are darker than males with a brown to gray color place at least 100 puy or newly merged adult gene melanoma butterflies. In a five liter wire mesh cage suspend two packs of four layered paper in the cage for egg laying.

After two days, the adult female will lay eggs on the edge between the papers twice a week. Place the egg paper packs in new plastic rearing boxes with grids to let air circulate. The boxes should contain pollen and beeswax as a food source.

Eggs hatch in about three days, and small larvae start to develop by feeding on wax and pollen to provide appropriate ratio of food per lava. Regularly separate the larvae into new boxes and replenish the food every two days. Larvae ar reared during the six stages of larvae stadium.

Each stadium is characterized by the slippage of the head capsule of larvae. When larvae reached the last stage before pation, they stopped feeding and start building a light silk cocoon in their protective cocoons. The larvae will rest and transform into puby.Wax.

Worms remain in the pooper stage for one to two weeks to emerge into adult moths. The adult moths do not drink or eat. Mating occurs and the wax worms lifecycle begins again.

To standardize the pathology assay, take the larvae during the last larval stage. During this stage, they stop feeding, move to the cover of the rearing box and start to produce silk. This stage lasts around five days after which the larvae starts to shrink.

The cocoon becomes thicker and larvae build crystallises. Anytime during the larval stage, remove the nascent silk cocoon. Select last in star larvae, which are two to three centimeters long and 180 to 250 milligrams in weight, 24 hours before infection and put them into an empty box to starve them.

Priscilla, bacterial or spore suspensions are prepared as described in the accompanying document at final concentrations, ranging from 10 to the four colony forming units per milliliter to 10 to the eight colony forming units per milliliter. Each lava will receive 10 microliters of suspension at the desired concentration To prepare the cry one C toxin culture B, the CI strain 4 0 7 transformed with the plasmid PHTF 31 C, carrying the gene encoding cry one C in 100 milliliters, HCT medium with 10 milligrams per milliliter, erythromycin, and 30 degrees Celsius for 72 hours. This will allow full sporulation toxin, crystal production, and liberation in the culture sate.

To purify the crystals from the bacterial culture, prepare a sucrose gradient in a 40 milliliter tube. First, add 17 milliliters of 79%sucrose. Then carefully layer 17 milliliters of 72%sucrose on top of the 79%sucrose.

Finally, layer five to six milliliters of the 10 X concentrated sporulate culture and close the tube centrifuge the tube for 14 hours at 20, 000 times G at four degrees Celsius to separate the crystals from the spores. The toxin crystals will settle at the gradient interface while the dense spores will reach the bottom of the tube. Use a pipette to collect the crystals and then centrifuge at 20, 000 times.

G.After the spin reus suspend the crystal palette in 25 milliliters of cold, sterile water to wash it centrifuge at 8, 000 times G.This step is repeated three times to remove all sucrose sticking into the crystals. After crystal isolation, re suspend the crystals in five milliliters of sterile water and observe under the microscope. To evaluate purity, evaluate toxin protein concentration by classic Bradford staining on crystal solution pre solubilized in 50 millimolar sodium hydroxide.

The toxin can be stored at minus 20 degrees celsius before use. Next, the insects will be injected with the bacterial solution. For each condition, fill a one milliliter hypodermic syringe per condition with 300 microliters of water or the bacterial solution for the infection of 20 to 25 larvae.

Remove the bubbles by carefully tapping the syringe. Then attach a 0.45 by 12 millimeter needle to the syringe. Place the syringe in an automated syringe pump with set off speed to one microliter per second, and set up volume of 10 microliters.

Perform a blank injection of 10 microliters in an empty tube to control the injected volume. Place the insect manually between the thumb and forefinger. Then insert the needle fixed in the injector in the lava skin or cuticle at the base of the last prole, and inject 10 microliters of the bacterial solution.

Carefully remove the insect from the needle. Place the infected insect in a five centimeter Petri dish. Five larvae per dish, infect at least 20 larvae for each experimental condition, place the dishes at 37 degrees Celsius in an incubator.

Insects can also be infected orally as demonstrated in this section of the video in a two milliliter fendor tube. Mix 0.2 micrograms per microliter of cryo one C toxin with either spore or vegetative cell suspension to obtain final concentrations ranging from three times 10, the three to one times 10, the six per 10 microliters filler, one liter syringe fitted with a 30 gauge 25 millimeter hypodermic needle with 300 microliters of the bacterial cry. One C toxin solution for the infection of 20 to 25 larvae.

Remove the bubbles from the syringe manually place the insect so that its mouth enters the needle fixed in the injector and force feed it with 10 microliters of the bacterial solution. Using the automated syringe pump following oral injection, the solution will spread in the insect gut. In this example, 10 microliters of blue dye solution is introduced.

Notice that the dye quickly fills in the intestinal lumen of the insect larvae After the insect has ingested the solution, place it in a small five centimeter Petri dish, infect at least 20 larvae for each experimental condition, place five larvae in each dish After forced feeding or injection experiments. Keep the larvae in the Petri dish without food at 25 degrees Celsius or 37 degrees Celsius, check larval mortality regularly. Over a 48 hour period, dead larvae are brown to black and unknown motile.

At the end of the experiments, free all of the larvae debt and alive at minus 20 degrees Celsius for 24 hours in an autoclavable bag, and then autoclave them to kill all the bacteria. Analyze the mortality data using log prob bit software or similar software. This program tests the linearity of dose mortality curves, and provides lethal doses.

LD 50 intra hemophilic injection of bacteria into g Melan has proved very useful for the identification of many virulence factors dealing with tissue damage and resistance to innate immune factors of several human pathogens to determine the role of the potential bacterial cell wall Peptidase C-W-P-F-M in virulence insect mortality, after various doses of wild type and mutant strains of BOUs bacteria was assessed as shown here. Insect mortality was dose dependent, but significantly decreased when larvae received the bacteria with mutant CWP fm. Thus, this cell wall peptidase plays a crucial role during priscilla's serious virulence towards insect larvae to demonstrate the synergistic effect of the spores of bether on the insecticide activity of cryo one C toxin cryo one c crystals were added to spores of the wild type strain of bether and fed to Galleria.

Melanoma beys without crystals were used as a control. After 48 hours at 25 degrees Celsius larvae mortality was evaluated as shown in this graph, the mortality rate following the ingestion of spore toxin mixture is significantly increased over the mortality rate following either cryo one C toxin or spore ingestion alone. This demonstrates a synergistic effect, the spores or vegetative bacteria on the insecticidal activity of the cryo one C toxin.

The role of cryo one C in Galleria melanoma is to help the bacteria passing through the para atrophic matrix, a chitin and mucus rich structure protecting the intestine of insects from direct contact with food and pathogens. The use of insects, especially in the level stage as infection models for several pathogens is becoming increasingly common. After watching this video, you should have a good understanding of how to perform hemel and oral infections, and we call insect mortality in gallium melan.

These methods can be easily applied to other model systems such as bombex, ma, and spotter species since these genomes have been sequenced, so hosts can be shut down by RNA methods or other gene manipulations.