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Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.


Introduction
Over the last few decades there has been increasing interest in how sexual selection and competition between males impact on evolution 1,2 . Experiments on mating behavior have played an important role in developing and testing theories of sexual selection 3,4 . In particular, research using species of the genus Drosophila, has contributed greatly to the understanding of sexual selection and behavior. However, it is important to investigate whether commonly used techniques might artificially bias the results of standard mating experiments 5,6 . Anaesthesia is often used for handling and identification in experiments 7 . For example, flies are commonly collected before mating, or sorted into genotypes or experimental treatments using carbon dioxide (CO 2 ) anaesthetic. In experiments where two or more individuals need to be distinguished, it is common practice to anaesthetise the flies and clip part of the wing off to identify each individual or treatment group 5,8 . It is vital, however, to understand how CO 2 treatment will affect behavior. The effect of CO 2 anaesthesia has been examined in Drosophilamelanogaster in which males exposed to CO 2 took significantly longer to mate and overall had lower mating success than nonanaesthetized males or males anaesthetized using exposure to cold 5 . This effect was observed both when anaesthesia was applied on the day of the experiment and when flies were given one day to recover. However, this study was limited in only examining trials where a single male was presented to each female 5 . A more realistic scenario is for a female to encounter multiple males 9,10 , allowing competition between males, which might allow the detection of more subtle losses of male fitness due to anaesthesia. The use of CO 2 anaesthesia has also been found to reduce fecundity and longevity of adult D. melanogaster when they are exposed shortly after eclosion, as is common when collecting virgin flies 11 . An alternative to CO 2 anaesthesia is to mark flies by feeding them food colored with dye 4,6,10,12,13 . This dye enters the intestines of the fly and is visible through the abdomen, allowing colored flies to be distinguished from uncolored flies, or from flies labelled with a different color. Methods differ in how this can be applied: being added directly to the food 12 , via dyed supplementary yeast paste 6 , or via exposure to a novel dyed food substrate 13 . These marking techniques appear to show no effect on mating performance 4,6 . However, a paper directly examining the effects of the same food coloring on adult D. melanogaster found a strong reduction in lifespan 14 . Previous work has also focused almost entirely on D. melanogaster, both with regard to the effects of CO 2 anaesthesia 5,11 and food coloring 14 methods. Currently, there is little information on how CO 2 anaesthesia or the use of intestinal coloring affects the mating behavior of other Drosophila.
The following study evaluates the effect of CO 2 anaesthesia on the mating behavior of three species of Drosophila (D. melanogaster, D. pseudoobscura, and D. subobscura). The effect of collecting flies on CO 2 was examined in both single and two male mating trials. The effect of CO 2 has also been found to vary in D. melanogaster 5 and so different latency periods between exposure to CO 2 and mating were tested. An alternative marking method to anaesthesia and wing clipping: the use of food dyes to stain the flies is also evaluated.
1. Take a standard Drosophila vial with approximately 20 ml of food in the bottom (Figure 1). Use the following recipe for food mix using 1 L of boiling water: 10 g of agar, 85 g of dextrose, 60 g of maize flour, 40 g of yeast, and stir for 5 min of simmering. Add 25 ml of 10% nipagen once the mixture has cooled to 75 °C. 2. After the food has cooled and solidified add two drops (approximately 0.5 -1 ml) of blue food coloring to the top of the food and spread over the whole surface of the vial (Figure 1). Use a different color dye if preferred. 3. Leave the food for two days in the fridge so that the dye is absorbed by the top layer of food; this avoids excessive moisture damaging the flies during the maturation period. Add a small piece of tissue paper if excessive moisture is still a problem to blot up extra moisture and then subsequently remove it. 4. Transfer flies onto the food either individually or in groups.
Note: Flies will gain intestinal staining within 1 day of being placed on the food. Alternatively, fully mature the flies on the dyed food prior to the experiment (increased mortality during the maturation period was not observed from exposure to food dye). 5. Check that the dyed flies can be easily distinguished from the non-dyed flies. If they cannot be distinguished, repeat steps 1.1 -1.4 using either a higher concentration of dye, or a different dye. 2. Expose half of the males to the desired treatment (CO 2 anaesthesia in this case). Use a CO 2 mat or tap to expose the flies for the required time. Store half of the males in each treatment on colored food until the mating takes place. This will make them visually distinguishable during the mating trials. 3. For transferring flies use an aspirator 17 . Label each vial to identify both the treatment and the color status of the male. Here, use four treatments (anaesthesia, non-colored = G-NC, anaesthesia, colored = G-C, no anaesthesia, non-colored, NG-NC, and no anaesthesia, colored, NG-C).

Two Male Mating Trials Using Food Coloring
3. Transfer newly emerged females into fresh food vials to mature as groups of 10. 4. Allow flies to mature to the mating age (D. melanogaster -3 days, D. pseudoobscura -5 days, D. subobscura -7 days 18 . Store flies at a suitable temperature for the species being studied (e.g., 22 °C for D. pseudoobscura and D. subobscura and 25 °C for D. melanogaster). 5. Move females to individual vials (containing ~ 20 ml of food) 1 day before the mating trial for acclimatization to the mating vial. Label these vials so that vials can be differentiated. Be careful to blind the experiment by using neutral labelling (i.e., 1 -150) so it is not possible to guess the identity of the flies in any vial. Note: The person who places the flies into each vial will have to know the identity of the flies placed in each vial as they will note which treatment was stained. However, the observer who watches the mating should not know their identity. To do this at least two experimenters will be needed, one to set up and one to observe. 6. Begin the mating trials between 10 -12 A.M., or at a time that coincides with the light coming on in the light/dark cycle the flies are exposed to ("dawn" for the flies). Add two male flies to each mating vial (containing a single female fly) using an aspirator. Ensure that the two males are from different treatments (anaesthesia or control) and that one has intestinal staining to make it possible to differentiate them from each other, and note which male is stained. 7. If copulation occurs, record the status of the male that mates (either colored or non -colored). If trials last for 2 hr, assume the female will not mate. Note: 2 hr is suitable for these species, but other Drosophila may need more or less time. 2. Check the mating latency and mating duration data for normality, by plotting frequency histograms of latency and duration for each treatment 20 , and using a test for normality such as Shapiro-Wilk. If it is not normal, transform it, or use non-parametric equivalent statistics 20 . Note: For the example data from the single male experiments log transformation met the requirements of normality and equal variances. 3. If the data can be normalized, use t-tests to examine differences between mating latency and duration in the single male mating trials when using two treatments 21 . If multiple treatments are used, try an Analysis Of Variance (ANOVA) 20 . If the data cannot be normalized, try equivalent non-parametric tests 21 . 4. Use binomial tests to test for an effect of either food coloring or CO 2 anaesthesia on the mating success of competing males 20 . If multiple treatments are used, as is the case with the example data, use a GLM with binomial error structure 21 . 5. For the two male trials in the example data, use GLMs with binomial error structures. One GLM examined color as a response variable (colored = 0 and non-colored = 1) with species, gas status, and gas treatment fitted as explanatory variables.One GLM examined CO 2 as a response variable (gassed = 0 and not-gassed = 1) with species, color status, and gas treatment fitted. In each case, produce the maximal model, and perform model simplification based upon AIC 20 .

Representative Results
Two male mating trials -The effect of CO 2 anaesthesia on mating behavior The best model found to explain the variation in the effect of CO 2 anaesthesia contained species as a factor (with D. pseudoobscura and D. subobscura fused as they showed no differences between each other). In D. pseudoobscura and D. subobscura there was no significant effect of CO 2 anaesthesia on mating success in two male trials (Z 1,589 = 0.087, P = 0.931). For D. melanogaster, males exposed to CO 2 anaesthesia had significantly lower mating success (Z1,589 = 2.467, P = 0.014). There was also a significant interaction between species and treatment (χ 2 1,589 = 6.83, P = 0.009) with a greater effect being seen when D. melanogaster were exposed to gas at collection or 1 day before trials ( Table 1). However, D. melanogaster males exposed to CO 2 two days before the experimental trial did not show an effect of CO 2 .

Two male mating trials -The effect of intestinal coloring on mating behavior
Model simplification showed no significant effect of food coloring being found for any of the three species (P > 0.1). When treatment or gas status were included in the analysis these were also not significant (P > 0.1). The proportions of successful matings for colored flies across treatments are shown in Table 2. The difference in coloration between flies kept on colored and uncolored food can be seen in Figure 1. The intensity of the intestinal food coloring was greater in D. pseudoobscura and D. subobscura than in D. melanogaster.
Single mating trials -The effect of using CO 2 anaesthesia on mating behavior There was no difference in mating latency for any of the three species when CO 2 anaesthesia was used to collect recently emerged adults. An effect was found for mating duration in D. subobscura when it was exposed to CO 2 two days before mating trials (Figures 2 and 3; Table 2).

Discussion
This data shows that the impact of CO 2 anaesthesia is inconsistent between species, with two of three species showing little impact. Our results suggest labelling with food dye had a lower impact on male mating success than CO 2 anaesthesia for D. melanogaster. These experiments demonstrate that food dyes can easily and cheaply be used to label flies for mating assays involving multiple males.
Of the three model Drosophila species examined, only D. melanogaster showed an effect of CO 2 anaesthesia on mating performance in a competitive situation. In contrast, none of the species showed an effect of collection on gas in single mating trials in terms of mating latency, contrary to previous results for D. melanogaster 5 . The effect of competition could therefore be highlighting more subtle fitness effects of CO 2 anaesthesia, which are only detectable under situations where there is male-male competition. Exposure at early collection and one day prior to the trial have a negative effect on the ability of males of D. melanogaster to gain a mating. Exposure two days prior to the trial however did not show any effect. Both D. pseudoobscura and D. subobscura did not show any effect of exposure to gas in any of the trials. One explanation is that D. melanogaster was vulnerable to early exposure to CO 2 because it must be collected earlier in life (0 -6 hr old) than the other species to ensure males are virgin. Hence male D. melanogaster of this age may be more sensitive as the cuticle of the fly is still hardening, compared to the other species which have had longer for their cuticle to harden. In general, this supports the idea that the effects of CO 2 anaesthesia are species specific and investigators should appropriately test the effect in their target species. Currently, the majority of work on the effect of CO 2 anaesthesia has been carried out on Drosophila melanogaster 5,11,22 and therefore may not be appropriate to apply to other related species.
The alternative non-invasive method presented to differentiate flies is food dye. Results suggest this treatment had no effect in across all the species examined. However, while its use was successful in providing a cheap and easily visible marker for distinguishing between individuals it should be noted that the dye was easier to distinguish in D. pseudoobscura and D. subobscura than in D. melanogaster. Previous authors have used several colors (red, green and blue) 4,6 . We found blue coloring to be the easiest to distinguish in all species, particularly D. pseudoobscura and D. subobscura. Using several colors would potentially allow more complex experiments with many individually marked flies. However, preliminary tests of different dyes are essential, as some food dyes fail to color the flies, possibly being digested when consumed. Other dyes can have toxic effects and reduce survival of the flies, and should be avoided 14 ). Alternative food coloring methods using more expensive stains have also been used for examining intestinal integrity for D. melanogaster 23 . These may provide an alternative, although more expensive, dyeing method 23 .
The dye method is as quick as CO 2 wing clipping as flies can be stored on dyed food from collection. Uptake of the food was rapid (~ 3 hr), so storage O/N on colored food would also be sufficient to mark flies, as used in other studies 6 . However, the duration of the coloring is relatively short (~ 4 -5 hr) compared to wing clipping (permanent) or fluorescent dust marking (10 -12 days) 24 . As Drosophila species vary in appearance, different dyes will be more or less effective for different species, and as some strains (e.g., knock-out mutants) can be vulnerable to changes in diet, any use of dye requires a preliminary test of its effectiveness particularly if longer term exposure to dyes can be toxic 14 . In contrast to the study by Kalaw et al. 14 , we found no significant mortality after storage for multiple days on colored foods for D. melanogaster (3 days), D. pseudoobscura (5 days), or D. subobscura (7 days), probably due to the difference in dye used.
The critical step for successful use of the dye technique is step 1.5, validating that the chosen dye works well with the species and strain being used. An alternative technique involves applying colored dust to the outside of the fly prior to use in field experiments 24 . This method has been used for tracking individuals in the field due to the duration of marking and the ease of mass marking flies 24 .
Although we have not explicitly tested this method in mating trials, it would be important to examine any effects that dust could have on the senses important in mating, particularly in Drosophila 25,26 . In species, however, where intestinal dyeing is not possible, these methods could be suitable.
In conclusion we found that in two of the three species tested (D. pseudoobscura and D. subobscura) there was no effect found of either CO 2 anaesthesia or food coloring on mating ability of males. For D. melanogaster a negative effect of CO 2 anaesthesia was detected, but food coloring did not affect mating success in this species. Overall, the dye method provides a simple and cheap non-invasive method for identifying individual Drosophila that is equivalent or better than methods that require CO 2 anaesthesia. It is likely this method would work across a range of species.

Disclosures
The authors declare that they have no competing financial interests.