The overall goal of this protocol is to study the effect of a Densovirus, HaDV2, on its host, Cotton Bollworm. This method can help answer key questions in the virus host interaction field such as the effect a now path-standing viruses on their hosts. The main advantage of this technique that larvae use to construct virus positive and negative strains are derived from the same par-ne-co-hosts, ensuring the same genetic background.
To begin the experiment, eight single-pair mating of newly enclosed moths by using a plastic cage per pair covered with cotton gauze to ensure good ventilation and to serve as an ovum position substrate. To increase the likelihood of obtaining a virus-free colony, utilize at least 30 pairs of single-pair matings. Feed adult moths with a 10%sugar and 2%vitamin solution-soaked cotton ball, and replenish the solution daily.
Today's post-mating, as female adults lay their eggs on the cotton gauze, begin to change the gauze daily. Collect and maintain gauze-containing eggs in the same growth room as the adult moths until the eggs hatch. Immediately transfer newly hatched larvae to new 24-well plates with one individual per well.
Five days after the first egg-laying date, collect the parent moths and store them in liquid nitrogen. Check for HaDV2 of the parents using PCR with specific primers, and keep the HaDV2-free individuals as the HaDV2-free noninfected strain. Collect adult moths individually and immediately store them in liquid nitrogen.
Completely grind collected individual moths in liquid nitrogen using a sterilized mortar and pestle. Transfer around 10 micrograms of debris into a clean 1.5 milliliter tube. Then transfer the remaining debris into a new tube then immediately store them at 80 degrees Celsius.
Extract DNA from the 10 micrograms of debris following the protocols provided by the manufacturer. Once the moth DNA has been isolated, check for HaDV2 using the protocol. According to the results, divide the remaining debris into two groups:HaDV2 positive and HaDV2 negative.
Add one milliliter of phosphate-buffered saline, or PBS, to each individual to completely dissolve the remaining larval debris. Pre-cool the centrifuge four degrees Celsius, then spin the homogenate. On ice, filter the liquid supernatant with the 0.22 micron membrane filter.
Collect 200 microliters per tube of the filtered liquids, and immediately store them at 80 degrees Celsius. To determine the oral infection efficiency with the HaDV2 infected filtered liquids of different concentration by diluting the fluid containing HaDV2. In a fume hood evenly spread 200 microliters of HaDV2 containing filtered liquids for each concentration on the surface of the solid artifical diet.
Next, begin to transfer 100 freshly hatched larvae to the HaDV2 containing artificial diet in a petri dish. Gently knock the cotton gauze including freshly hatched larvae to transfer the larvae from the gauze to a sheet of white paper. Knock the paper gently so that the larvae spin silk and balloon off the paper.
Use sterilized forceps to touch the silk and move the larvae to a petri dish. After 48 hours, transfer one individual larvae per well on the artificial diet to 24-well plates. Re-ar the larvae inoculated with HaDV2, using the same concentrations to the adult stage.
Check for HaDV2 infection status of the adults using the protocol. Adults inoculated with 10 to the eighth copy number per microliter of HaDV2 yielded 100%infection and are kept as HaDV2 positive strain. To prevent contamination, sterilize the forceps and scissors in 75%ethanol three times and then heat them in the flame of an alcohol lamp.
Using an analytical balance weigh the empty tubes that are to be used to store the tissues. Next place the larvae and adult moths from the infected strain on ice for approximately five minutes. Fix the frozen larvae on cystosepiment using two insect pins inserted in the intersegmental membranes near the head and the last abdomen segment.
Then in the posterior abdomen, cut the proleg of the larvae so that the hemolymph flows out. Use a pipette to pipette about 10 microliters of larval hemolymph within about 10 seconds to prevent browning and coagulating of the hemolymph. With a pair of scissors make an incision in the cuticle ranging from the last intersegmental membrane to the head.
Using two forceps, dissect foregut, midgut, hindgut, fat body, and malpighian tubules. To prevent the contamination of the remaining tissues with the hemolymph during the dissection, wash the dissected tissues in PBS buffer. Mix tissue samples from three individuals together as one replicant.
Immediately transfer each tissue to the tubes in liquid nitrogen. In a clean petri dish containing PBS buffer, dissect the tissues of female and male adults and immediately transfer each tissue to the tubes in liquid nitrogen. Weigh the tubes and the above tissues together separately and calculate the mass of tissues.
Extract DNA from these tissues and perform kPCR to quantify the viral titer of each tissue. Place at least 100 pupae or newly emerged adult moths derived from the noninfected strain in a cotton gauze-covered 5-liter wire mesh cage. After two days, mated adult females will start to lay eggs on the cotton gauze.
Collect and maintain the cotton gauze containing the eggs. Proceed with neonate larvae inoculation by transferring the neonate larvae of the noninfected strain to the artificial diet containing HaDV2 to establish the infected strain. Similarly, transfer neonate larvae to the artificial diet covered with HaDV2-free filtered liquid to establish the control groups, the noninfected strain.
Include at least 240 larvae in each treatment. After 48 hours transfer one individual larva per well to 24-well plates. Sequentially number each larva in each well, and record the instar stage and survival status daily.
About five days later, when the artificial diet deteriorates, simultaneously transfer these individuals to a new 24-well plate containing fresh artificial diet. Weigh the larvae from day seven to 11 using an analytical balance. After weighing, individually return the larvae to the 24-well plate.
Upon molting to the fifth instar, around ten day after hatching, transfer the larvae to glass tubes. Transfer the individuals to the glass tube at the same time each day. Fifth instar larvae can also be identified by the width of the head capsule.
Next, code each larva with its original reference number on the glass tube using paint or markers. Record the larval status daily. Record the pupae and eclosion date for each individual in the glass tube, and record the mass of three-day old pupae.
After eclosion, place one female and one male into a single cage with 10%sucrose and 2%vitamin solution. Replenish the sucrose solution every day. Record the date of death for each moth.
When eggs are laid, change the cotton gauze daily. Count the number of eggs immediately. Finally, count newly hatched offspring while they hatch.
Tri-als were run to ensure the quality of the specimens used in the experiment. Progenies from the male and female parents that we HaDV2-free were reared as the noninfected strain. We successfully amplified the actin gene in parents and progenies using the same DNA template suggesting that the DNA templates were of good quality.
The randomly selected eight progenies were also free of HaDV2, HaNPV, and Wolbachia. A standard curve defining the relationship between the log of the quality of starting material and the gained CT values shows a strong linear correlation between the CT values and the log of the initial gene copy number of HaDV2. The HaDV2 copy number was calculated per microgram of each tissue, and the percentage of the total HaDV2 titer for the specific tissue revealed that HaDV2 was mainly distributed in the fat body.
While attempting this procedure, it is important to remember to handle the delicate larvae during the first five days after they hatch during the en-tire procedure.
