The overall goal of this procedure is to rapidly screen a large number of host plant volatiles for potential biological activity. This is accomplished by first collecting and analyzing by GCMS, the host plant volatiles of interest. Then the chemical components are identified and verified.
The third step is to screen the individual components in sufficient replicates for bioactivity via electrographic analysis. The final step is to formulate and evaluate blends of active components. Ultimately, results can provide a truncated series of components or blend of components ready for further investigation in behavioral studies with the insect of interest.
The main advantage of this technique or existing methods, such as time intensive behavioral studies, is that rapid screening by electrographic analysis significantly decreases the number of compounds needed to be taken on for other studies demonstrating a procedure. Is MR Wage a technician for my laboratory? Once the relevant volatiles have been identified by gas chromatography and mass spectrometry, it is possible to perform electro iconography analysis or EAG of each available volatile first.
However, the available volatiles must be prepared. Prepare a solution of each volatile in pentane at a concentration of five milligrams per milliliter in a container that can be tightly sealed and stored at four degrees Celsius. Next, prepare the stimulus pipettes.
Label 10 past star pipettes per volatile and wrap their tips with a small piece of param. 10 pipettes is sufficient to deliver the volatile to each sex in a low replicate number preliminary screen. Now line up the remaining set of five pipettes in a rack holder and place a bioassay disc into the open end of each pipette.
Gently fold a disc with tweezers and partially place it into the large end of the pipette so that two to three millimeters of the disc is exposed. Using a pipetter or syringe load 10 microliters of volatile solution onto each disc. It is extremely important to correctly calculate, formulate, and prepare the solutions to ensure consistent replicates.
Allow two minutes to pass and then fully push the discs into their pipettes. Once loaded, immediately seal each pipette with param. Repeat this pipette preparation for the desired number of replicates.
Using the same protocol. Prepare a negative control of just pentanes and positive controls, acetophenone and sex hormone in this instance. Now proceed with preparing the insect antennae.
In this experiment, a pre amplifier delivers the signal from a fork electrode to the EAG acquisition instrument. The software provided with the EAG instrument must be configured appropriately in the software. Configure the settings by first setting the sampling rate to 106.7.
Set the rectify toggle to know and set the filter to zero to 42 hertz Under the filter tab. Set the EAG filter to on and set the low cutoff to 0.1 hertz. Then save the settings to a configuration file for future use.
Operating the program is then as simple as opening a new project file, saving it under a specific name and proceeding with a recording. The red triangle icon starts the recording process and remains on for a predetermined amount of time for each individual puff. When all of the puffs have been performed for the antennae, the black X icon stops the recording to make the EAG responses easier to read accurately.
A small ruler can be taped onto the computer screen at the baseline of the signal. The response is recorded in millimeters and then later converted to the amplitude in microvolts. If needed, the recordings can be played back for further evaluation.
In this example, three to four day old naval orange were male and mated female moths are used. However, this protocol can be tailored to other insects. Transfer each insect to a small litted plastic container.
On the day of analysis, just before the bioassay, insert the insect into the large opening of a pipette tip, which has a smaller opening, just large enough to fit its head. Then using a smaller pipette tip, carefully push the insect toward the smaller opening until its head is exposed under a stereoscope. Ensure that the antenna are secured within the holding tube and the base of the antenna are exposed for excision.
Next position the fork holder with a small film of electrode gel in close proximity to the antenna using micro scissors tease out, then excise both antennae and start a timer to record the time between this event and that of the ensuing stimuli. Using a wire tipped tool with a dab of gel on the end, pick up one antenna and place it on the fork. Ensure that the base of the antenna is placed on the non red indifferent ground electrode of the fork.
Do the same for the second antenna. Placing the base of the antenna on the same side of the fork, two to three millimeters apart with the antenna positioned. Trim the tips of the antenna as needed.
Apply electrode to gel with a brush and ensure that their base and tip are immersed in electrode gel so that there are no bubbles in the gel. Avoid covering the central portion of the antenna with gel to maximize the antenna surface area exposed to the puff of volatiles. Once the preparation is properly jelled, connect the fork to the probe pre amplifier.
Now apply a flow of humidified air over the preparation to maintain the viability of the an antenna prep. After 10 minutes from the time of excision, proceed with applying the first stimuli. The order of operation for stimulating a preparation begins and ends with a positive control in between stimulate with a randomized sequence of compounds with no identical consecutive compounds, and with at least one negative control, the number of compounds delivered to an antenna varies by species.
A naval orange worm moth preparation can stay active for more than 30 minutes, allowing for up to 10 volatiles to be readily analyzed in a low replicate number. The same principle of sample delivery applies to subsequent screens of blended compounds that were found to elicit responses in the preliminary screen. The first stimuli pipette must be prepared 30 seconds before applying the puff unseal the pipette and position it in the holder so it will direct airflow two to three millimeters from the antennae at 10 minutes from the antenna excision.
Deliver the first stimulus puff over one to two seconds at 300 milliliters per minute. Now, quickly replace the pipette so that one minute after the first stimulus, the second is delivered. Having consistency among samples will minimize the variability of volatile amounts that are actually puffed.
After the final positive control puff is administered, remove the antenna and clean the fork. With an ethanol saturated wipe allow it to dry off completely before subsequent use. To increase the throughput of assays, an assistant should prepare the next antenna with clean forks 10 minutes before the end of the previous preparations.
Testing sequence, more than one fork is necessary for this method. After each experiment, euthanize the test moths in a dry ice environment and dissect the female moths to check their mating status. Assume the cohabitating males must have mated naval orange worm antenna Preparations were stimulated with two second puffs and ten second recordings were made with a ten second window on a five millivolt scale.
A negative deflection was the typical response. Yet the absolute values were recorded. All female preparations with a weak response to the positive control were discarded.
The average response of the remaining preparations was 2, 600 microvolts. Similarly, the average male response to the sex pH control was typically 3000 microvolts. Thus anani with a substandard response were discarded data from an antennae that showed degraded response over the trial were also discarded.
Degradation occurred if the Anton response to the puff of the final positive control was either less than 75%of the first puff of the pre-con control, or less than the second puff of the pre-con control for the remaining experimental runs. The response values were analyzed by taking the raw measurements from each run and subtracting the negative control response of that run from every value in that run. Next, all the positive controls in each run were averaged and corrected to 1000 Microvolts noting the ratio for correction to 1000 microvolts for each run.
The values for tested compounds were then multiplied by the positive control correction ratio. Lastly, the average corrected response of each tested compound over all the runs was compared to evaluate each compound's candidacy for further investigation. Don't forget that working with chemicals can be extremely hazardous and proper precautions should always be taken.
