The overall goal of detecting UV reflectance on fish such as Poecilia latipinna is to understand how UV reflecting structures might impact the mating behavior of the species. This method can help us understand mating preferences such as whether UV cues are important, and if so, whether they are used by both sexes. The main advantage of this technique is that it allows us to detect cues that might not be visible for us, but might be very important to the fish.
For this protocol, first calibrate a spectrophotometer and a light source to a known white standard. The room lights should be low prior to all calibrations. Calibrate the instrument to all the wavelengths that will be measured which must include the UV wavelengths between 300 and 400 nanometers.
Now, put on gloves and anesthetize the fish in a 5%solution of ethyl 3-aminobenzoate methane sulfonic acid salt, known as MS222 buffered with an equal quantity of sodium bircarbonate. When the fish becomes unresponsive, immediately transfer it to a black non-reflective background. Before taking measurements, dim the room lights again.
To record the reflectance data, use analysis software. To match the setting here, set the integration time to 2000 seconds. Set the average number of scans to two, and set the pixel smoothing value to five.
Now, measure the reflectance of the fish using a fiber optic probe. Hold the probe to make a 45 degree angle between the fiber optics and the body. Take an average of several scans per body region.
Check for reflectance for several body areas and look for variation between individuals. When finished, optionally apply a commercial protectant to restore the mucus coating and then return the fish to a well oxygenated holding tank. Once recovered, the fish will show normal operculum beating and swimming.
This test makes uses of a choice compartment tank outfitted with UV and opaque filters. When the plexiglass partitions are removed, a fish in the neutral compartment is able to view full spectrum or non-UV light due to filters in different parts of the tank. For P.latipinna, use a 75.7 liter test aquarium.
Prior to each test, ensure that no water or olfactory cues are shared by the three tank sections. Use sexually receptive individuals for the test. For a female P.latipinna, choose fish within 48 hours postpartum and use males that have been isolated for at least 24 hours.
To conduct the test, first transfer the test animal to the neutral compartment. Next, choose two potential mating partners that resemble one another as closely as possible in size, coloration, and display characteristics. Color matching can be done precisely with instruments or roughly by eye.
Now, under full spectrum lighting, place one of the of the two potential mates in each of the the two choice compartments. The opaque and UV filters and the compartment dividers must all be in place. Let the animals acclimate to this condition for 15 minutes.
For each trial, randomly assign which compartment has the UV filtering. Then, remove the opaque covers from the dividers. After 15 minutes, start recording the time that the focal individual spends within specific preference zones using a stop watch.
The area within two body lengths of a choice compartment constitutes a zone of preference. Alternatively, use event recorder software. Record the activity of the fish for 15 minutes.
If the fish doesn't visit each preference zone at least once, treat it as being unresponsive and exclude it from the trial data. Measuring the UV reflectance of P.latipinna show that both sexes have UV characteristics which vary between individuals. The traits are most prominent along the sides of their bodies.
Once UV traits were found, the discrimination test revealed that females, but not males, used these characteristics in their mating decisions. Because both sexes possess UV traits, it is possible that they are involved in other social interactions such as shoaling or foraging. Once mastered, the UV measurement techniques can be done in less than two hours.
The mate preference trials can be completed in less than an hour for each fish. While attempting the procedure, it's important to ensure that the fish are reproductively receptive. Following this procedure, additional experiments to investigate the role of UV traits in individual or species recognition might be tested.
