The overall goal of the following experiment is to use a developmentally timed manipulation of food availability to create a large range of body sizes, which can then be used to estimate the scaling relationship among morphological traits. First, eggs are collected from the same population of flies over three days, creating three age cohorts. When the oldest cohort of larvae begins to pate, larvae of all cohorts are removed from the food.
The result of this dietary modification is an enhanced range of size variation of the pupi. Next pupil and adult wing images are taken of all individuals to allow for the estimation of the morphological scaling relationship between body and wing size. Ultimately, results are obtained that show how wing size scales with the full range of possible body size by fitting a type two linear regression to the data.
In this study, we use a developmentally time manipulation of access to nutrition to experimentally increase body size variation among adult flies. This enables us to estimate morphological scaling relationships across the full range of body sizes. This method can help answer key issues regarding the expression and evolution of morphological scaling, such as identifying the proximate basis of variation in the parameters of scaling relationships among biological groups.
Move a population of flies into a mating chamber and then collect eggs every 24 hours for three days. Ultimately creating three age cohorts with the aid of a dissecting microscope. Move batches of 50 eggs from each cohort into vials containing 10 milliliters of standard fly food.
Incubate the vials at the desired temperature for larva development. When the oldest cohort reaches the wandering stage, it is time to apply the diet manipulation by removing the larvae from the food. At this point, larvae in the oldest cohort are well beyond the minimal viable weight for occlusion and will be near the natural maximal body size.
Larvae in the intermediate cohort have exceeded the minimal viable weight, but will produce smaller adults than larvae from the oldest cohort. Larvae and the youngest cohort are very close to the minimal viable weight and thus will produce very small adults. To remove the larvae from the food.
Add approximately five milliliters of 40%sucrose solution to the vials, and then agitate for 15 to 20 minutes on a shaker table on a mid-range setting. After agitation, remove the floating larvae from the sucrose solution with a fine paintbrush and place them by cohort into vials containing a wet cotton plug for moisture. Incubate at the desired temperature until pupation occurs.
Arrange the pupi on a microscope slide ventral side up. Once the pupi are in place, capture images using a digital camera attached to the microscope. Be sure to configure the settings so each pupa appears as a dark silhouette on a white background.
After imaging, transfer each pupa to an individual two milliliter epit tube containing one milliliter of fly food. Estimate the pupil size as the number of pixels of the pupil silhouette. Other size metrics such as length or width could also be used under a dissecting scope.
Lightly anesthetize the fly using carbon dioxide and position it using a wing grabber, a suction device that pulls the wing between two pieces of glass and holds it perpendicular to the fly's body For imaging, although a wing grabber is used to image wings of live flies in this study, wings and other traits can be dissected and imaged with normal tools. Image the wing using the microscope camera attached to the computer and note the sex of the fly After the wing is imaged and saved, estimate the wing size by either using the minimum convex polygon that circumscribes marginal fixed landmarks as linear measurements or as the pixel count of the wing silhouette. Here imaged pupi showed the extremes of size distribution due to diet manipulation.
After the pupa has achieved the minimal viable weight for occlusion. Also shown are the corresponding silhouettes for the measurement of pupil size. The corresponding wing images for those same individuals at the extreme ends of the size distribution are shown here alongside the silhouette.
Used to estimate wing size extraction of the scaling relationship. Parameters that describe how wing size scales with body size allows for comparison of how overall shape covas with size across groups. Here, data are shown for individuals of each sex that were fully fed starved one day or two days as larvae.
Although the individual components of this procedure are quick, overall, it can be laborious. Generally, it takes about 350 person hours to process approximately 3, 500 flies from egg to adult. With practice, individuals can greatly improve their flight handling times, And this procedure can be used to measure other morphological traits, which allows the estimation of additional morphological scaling relationships.
