REGULATION OF FEEDING BEHAVIOR IN ADULT DROSOPHILA MELANOGASTER VARIES WITH FEEDING REGIME AND NUTRITIONAL STATE

Transcription

1 J. exp. Biol. 97, 2 23 (99) Printed in Great Britain The Company of Biologists Limited 99 2 REGULATION OF FEEDING BEHAVIOR IN ADULT DROSOPHILA MELANOGASTER VARIES WITH FEEDING REGIME AND NUTRITIONAL STATE ROBERT S. EDGECOMB, CARA E. HARTH AND ANNE M. SCHNEIDERMAN Section of Neurobiology and Behavior, Division of Biological Sciences, Cornell University, Ithaca, NY , USA Accepted 2 July 99 Summary The regulation of feeding behavior in adult Drosophila melanogaster includes such elements as ingestion responsiveness, volume ingested in a single meal, food storage in the crop and rate of defecation. Our results suggest that feeding behavior varies in a manner dependent on feeding regime (food-deprived or ad-libitum-fed) and nutritional state. Fed flies that are subsequently food-deprived become increasingly more responsive to food stimuli over time and, when offered % agar diets containing different concentrations of sucrose, ingest greater amounts of diets that have higher sucrose concentrations. When fed ad libitum for 72 h on these same diets, D. melanogaster maintained much smaller crops on average than food-deprived flies fed a single meal. Additionally, ad-libitum-fed flies are grouped into two categories depending on the concentration of sucrose in the diet. Flies fed for 72 h on % agar diets having mmol l sucrose or more are not affected by the concentration of sucrose in the diet, while flies fed on diets of or 2 mmol l sucrose increase ingestion responsiveness, crop size and the rate of defecation with decreasing concentrations of sucrose in the diet. Flies fed on even lower sucrose concentrations ( or mmol l sucrose) for h exhibit both a shift over time to larger crop sizes and increased mortality over those of flies fed mmol l sucrose. These data suggest that flies fed ad libitum are capable of modulating their feeding behavior in response to their nutritional state. Introduction Feeding behavior in many insects involves the detection of food, the initiation of ingestion and the consumption of discrete meals (Bernays, 98). Many of the factors contributing to the regulation of feeding behavior have been elucidated for a muscoid fly, the black blowfly Phormia regina (for reviews, see Dethier, 976; Gelperin, 986; Stoffolano, 99). As the fly walks, taste hairs on the tarsi continually sample the environment. If a hungry fly steps in an adequate food source, it will extend its proboscis and begin to feed. During ingestion, liquid food passes through the anterior portion of the foregut and fills the crop, a collapsible food-storage sac. As the fly continues to feed, a Key words: proboscis extension, ingestion responsiveness, meal volume, food deprivation, ad-libitum feeding, nutrition.

2 26 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN number of factors contribute to terminating the current feeding bout and to increasing the threshold for subsequent ingestion. First, neurons in the taste hairs respond initially with high-frequency firing, then they rapidly habituate (Gelperin, 972). Second, two internal stretch receptor systems, one monitoring distension of the abdomen (Gelperin, 97) and the other monitoring foregut activity (Gelperin, 967), begin to provide negative feedback to the central nervous system (CNS) to terminate the meal and to decrease responsiveness to food stimuli, respectively (Dethier and Gelperin, 967; Bowdan and Dethier, 986; Edgecomb et al. 987). Thus, feeding behavior in P. regina is regulated primarily through interactions between peripheral taste neurons and internal stretch receptor neurons. Although these neural factors involved in feeding regulation have been well described in food-deprived blowflies, their role in ad-libitum-fed flies has received much less attention. P. regina deprived of food ingest large volumes of concentrated sugar solutions (Bowdan and Dethier, 986; Edgecomb et al. 987). However, when fed ad libitum on sugar solutions of similar strength, the blowfly Lucilia cuprina ingests frequent, small meals and maintains relatively small crop volumes (Simpson et al. 989). The mechanisms that govern these differences in meal frequency and crop volume between food-deprived and ad-libitum-fed flies are not clear. Our goal was to understand the differences between regulation in food-deprived and ad-libitum-fed flies and to establish a basis for future neurogenetic studies of feeding regulation. Therefore, we assayed several variables of feeding regulation in the adult fruitfly Drosophila melanogaster. We have found that feeding regulation variables, such as ingestion responsiveness, meal volume, meal storage and rate of defecation, are affected by changes in feeding regime or nutritional state. We maintained D. melanogaster in either a food-deprived or an ad-libitum-fed condition and describe how these feeding regulation variables vary according to the fly s feeding regime. Also, by feeding D. melanogaster on different concentrations of sucrose ad libitum, we show that the nutritional state affects ingestion responsiveness, meal storage and defecation rate. Our results from both ad-libitum-feeding and food-deprivation experiments with D. melanogaster are interpreted in the light of the known mechanisms for feeding regulation in muscoid flies. Materials and methods Fly rearing Wild-type (Canton-S) Drosophila melanogaster were raised in half-pint bottles on a standard yeast/glucose diet ( g of yeast, g of glucose, 2 g of agar, ml of acid mix, ml of H2O) in an incubator under a 6 h:8 h L:D cycle (dawn at 7: h EST). Temperature was maintained at 2± C:22± C day:night at 7 % relative humidity. Flies were tested between 2: h and 7: h in ambient laboratory conditions, with overhead illumination provided by fluorescent lighting. Since the state of hydration can affect feeding behavior in P. regina (Edgecomb et al. 989), D. melanogaster were removed from the humidity-controlled incubator less than 3 min before the start of each trial in the laboratory. For all experiments, newly eclosed male and female

3 Regulation of feeding behavior in Drosophila 27 D. melanogaster were transferred together to fresh yeast/glucose diet for 3 day before being transferred to glass vials (2 mm 9 mm) containing ml of % agar or sucrose/ % agar diet. Proboscis prints The proboscis print assay is an indicator of a fly s readiness to ingest. It measures the number of proboscis extensions made as the fly ingests a thin sucrose/gelatin coating. When the fly feeds, it leaves a print of the oral disc in the coating (Fig. ). After ingesting a spot of coating, the proboscis retracts at least partially before extending towards another portion of the coating. These proboscis prints are visualized by darkfield illumination under a dissecting microscope. Since each proboscis print represents an extension of the proboscis, readiness to feed (ingestion responsiveness) can be expressed as a specific number of prints in the coating. The uniform coating is prepared by dipping acid-cleaned microscope slides into a mmol l sucrose/ % gelatin solution (w/v) and air-drying the slides for 3 h. Because the amount ingested with each print is slight and the duration of the assay is relatively brief, ingestion responsiveness is unlikely to be affected during the assay period (see Discussion). To determine the number of proboscis extensions made, adult D. melanogaster were anesthetized briefly on ice (2 min), and flies were placed individually in adjacent wells of a flat-bottomed 96-well plate (two columns of five flies). Each group of wells was covered by a strip of waxed paper and a sucrose/gelatin-coated slide. After the flies had recovered for 3 min, the waxed paper was removed and the flies were allowed free access to the coating on the slide for 2 min. At the end of the test period, the slides were removed, and the proboscis prints made in the coating were counted for each well and averaged for the number of flies that had access to each slide. Fig.. Proboscis prints are readily apparent under darkfield illumination. The fly ingests only the coating that is below the oral disk. When a spot of food is consumed, the fly retracts its proboscis either partially or fully. The proboscis must extend each time to produce additional proboscis prints. Scale bar, m.

4 28 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN To prepare flies for screening, newly eclosed flies were fed for 3 days on a yeast/glucose diet in bottles and then subjected to two different feeding regimes. Some flies were food-deprived by transferring them to glass vials containing ml of % agar, and then were tested, 6, 2, 8, 2, 36 or 8 h later. In the second regime, flies were transferred to vials containing % agar with a particular concentration of sucrose (,, 2, 2, 3, or mmol l ). Sucrose-fed flies were allowed to feed ad libitum for 3 days on the selected pre-trial diets and then assayed for the number of proboscis prints made on the sucrose/gelatin-coated slides. As a control for the effect of ingestion of the gelatin substratum on the number of proboscis prints made, flies were fed a pre-trial diet of mmol l sucrose for 3 days and tested on % gelatin alone. Although flies fed on such a pre-trial diet are very responsive to the sucrose/gelatin coating, no prints were observed on the % gelatin coating. This suggests that the gelatin substratum itself has little effect on the number of proboscis prints produced. Mean values are reported ± S.E.M. With the exception of the crop score assay (see below), comparisons among means for all experiments were tested for significance (P<.) using analysis of variance (ANOVA) and comparisons between means were tested using either a sequential Bonferroni test (Rice, 989) or Duncan s multiple range test (Statistical Analysis System, SAS Institute, Cary, NC). Meal volume The exact volume of a single meal can be determined by feeding D. melanogaster on a diet that contains the food dye FD & C Blue No. (Tanimura et al. 982; Shimada et al. 987). Fed flies were homogenized and the amount of dye ingested was assayed spectrophotometrically. Blue No. absorbs light strongly in a narrow band around a wavelength of 62 nm. This dye remains within the digestive tract and passes out of the fly unaffected by gut ph and enzymes. For our experiments, groups of either 2 male or 2 female flies, previously food-deprived for 2 h on % agar, were fed for min on ml of sucrose/agar diets containing. % Blue No.. No adverse effects on feeding behavior were observed in flies fed on either. % or. % Blue No.. Five minutes was chosen as optimal because, by that time, most flies had completed the initial meal, but had not yet begun defecating food dye. After feeding for min, the flies were anesthetized with CO 2 and homogenized in ml of. mol l of phosphate buffer (ph 7.2) and microcentrifuged (2 g) for 2 min. A.9 ml sample was taken immediately from the supernatant and brought to a final volume of. ml with phosphate buffer. The solution was centrifuged again for 2 min and the supernatant was transferred to cuvettes. The homogenization and centrifugation steps were performed as rapidly as possible to prevent eye pigments from leaching into the solution and interfering with the absorbance spectrum of the dye. To correct for background absorbance of the fly, the absorbance of the supernatant from unfed 2 h food-deprived flies was measured at 62 nm and was subtracted from the absorbance of the supernatant from flies of the same sex fed a single meal. The net absorbance was used to calculate the amount of food ingested. The relative amounts of food in the crop and more posterior portions of the digestive

5 Regulation of feeding behavior in Drosophila 29 tract were determined by feeding D. melanogaster on food colored with. % Blue No. in % agar. The dye can be seen through the cuticle in portions of the gut located in the abdomen, but not in the thorax. Thus, dye is visible in the crop, most of the midgut, the hindgut and the rectum, but not in the remainder of the foregut or anterior midgut. We created a scoring system for the size of the dye-filled crop: () no dye is visible in the crop, but it may be present in the midgut, anterior hindgut or rectum; (2) a small spot is visible in the crop; (3) the crop extends towards the lateral aspects of the abdomen and is wider than it is long; () the crop extends towards the lateral aspects of the abdomen and is at least as long as it is wide; the abdomen is slightly swollen; and () the crop is maximally distended and the abdomen is extremely swollen. If the abdomen of a gravid female was extremely swollen, but the crop was not maximally distended, the fly was given a crop score of. This crop score assay is applicable to both single-meal and longterm (ad libitum) feeding studies. In single-meal-fed flies, the crop score reflects the amount of that meal stored in the crop. In ad-libitum-fed flies, the crop score reflects nearequilibrium conditions in which crop size is presumed to depend on long-term interactions of multiple sensory inputs. To determine whether sucrose concentration in the diet affects the distribution of crop scores, single-meal and ad-libitum feeding regimes were employed. Flies fed previously for 3 days on yeast/glucose diet were either food-deprived for 2 h and fed a single meal on a sucrose/ % agar/. % Blue No. diet for min or fed ad libitum on a sucrose/ % agar/. % Blue No. diet for up to 72 h. A minimum of 2 flies was used for each sample. Individual samples were pooled and the data were tested for significance (P<.) using a contingency table (G-test). To prevent division by, cells with a frequency of were assigned a value of.. Although frequencies were used to calculate significant differences, data were plotted as relative frequencies. For comparisons between crop score distributions from only two feeding categories, the value of was adjusted according to the number of individual comparisons made. Defecation The rate of defecation in adult D. melanogaster fed on Blue No. was also measured. Because the dye passed out in the excreta, defecation rate was quantified as the number of fecal spots deposited on a glass surface over time. For each trial, approximately male or female flies previously fed for 3 days on a yeast/glucose diet were fed ad libitum for 3 days on diets ranging from to mmol l sucrose containing % agar and. % Blue No.. The flies were then transferred to empty glass vials for 2 min. The vials were cleared and the number of flies and blue fecal spots on the vial counted. The rate of defecation was calculated as the number of spots per fly per minute. Fecal spot size was not noticeably affected by sucrose concentration in the diet. Results Food deprivation and single-meal feeding The number of proboscis prints made by male D. melanogaster fed previously on a yeast/glucose diet increases with the duration of food deprivation (ANOVA, P<.)

6 22 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN (Fig. 2). Male flies taken directly from a yeast/glucose diet and tested for 2 min produce, on average,.6±. prints per fly. No significant difference is observed in the average number of prints between these replete flies and flies deprived of food for 6 or 2 h. After 8 h of food deprivation, however, the average number of prints per fly is significantly elevated (6.±.7 prints per fly) over that of replete flies. The number of prints continues to rise with increasing length of food deprivation and, after 8 h of food deprivation, flies produce an average of.6±2. prints (Fig. 2). Beyond 8 h of food deprivation, the mortality of male D. melanogaster approaches %, and the few survivors were not tested. When male or female flies food-deprived for 2 h are fed a single meal ( min) on a diet containing,,,, or mmol l sucrose, the volume ingested in min varies with the sucrose concentration (ANOVA, male, P<.; female, P<.) (Fig. 3). There is no difference in volume ingested for flies of either sex when fed, or mmol l sucrose. However, at sucrose concentrations of mmol l or higher, both male and female flies ingest significantly more than flies of the same sex fed on sucrose concentrations below mmol l. In male flies, the volume ingested increases significantly with elevation of sucrose concentration above mmol l. This relationship is similar in female flies except that the volumes ingested of and mmol l sucrose in % agar do not differ significantly. When presented for min with the highest sucrose concentration tested ( mmol l ), male and female flies Number of prints per fly Time of food deprivation (h) Fig. 2. Effect of food deprivation on proboscis prints. The number of proboscis prints made by male D. melanogaster increases with the length of food deprivation. Flies were fed for 3 days on a yeast/glucose diet, food-deprived on % agar for 8 h and then assayed for 2 min for the number of proboscis prints made on mmol l sucrose/ % gelatin-coated slides. No significant increase in the number of prints made by food-deprived flies is observed until 8 h after food deprivation. Each point represents the mean ± S.E.M. from eight trials of 8 flies each.

7 Regulation of feeding behavior in Drosophila 22 ingested 22± nl and 98± nl, respectively (Fig. 3), and the abdomens of both sexes became expanded. There is no difference between the sexes in the volume of diet consumed in min over the sucrose concentration range tested (ANOVA, P=.6). In addition to measuring volume ingested, the percentage of flies that ingested visible amounts of dye was determined. More than 8 % of flies of either sex ingested some food during the min assay period. The relative amount of food stored in the crop can also be determined in flies fooddeprived for 2 h and then fed for min on dye-colored diets with varying sucrose content (Fig. ). In these single-meal-fed flies, the distribution of crop scores is dependent on the concentration of sucrose in the diet (G-test, male, P<.; female, P<.). Larger percentages of flies have bigger crops when fed on higher concentrations of sucrose. However, as with meal volume, no difference is observed in the relative frequencies of crop scores in male flies fed or mmol l sucrose (P=.39) (Fig. A). Over 8 % of the flies fed these sucrose concentrations have crop scores of or 2, indicating that most flies had little or no food in their crops at the end of the single meal. The relative frequency of crop scores in male flies fed a single meal of mmol l sucrose differs from those fed mmol l sucrose (P=.7), although the majority of flies (72 %) still have small crops. Low percentages of male flies fed on sucrose concentrations above mmol l have small crops (Fig. A). The distributions of crop scores in food-deprived female and male flies fed for min are similar (Fig. B). As with males, females fed on or mmol l sucrose 2 2 Volume ingested (nl) Male Female [Sucrose] in % agar (mmol l ) Fig. 3. Effect of concentration of sucrose in diet on volume consumed in food-deprived flies. The volume ingested by food-deprived male or female D. melanogaster is positively correlated with the concentration of sucrose in the diet. Flies were food-deprived for 2 h and then fed for min on ml of,,,, or mmol l sucrose in % agar and. % Blue No.. Each column represents the mean + S.E.M. from four trials.

8 222 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN diets show no difference in the distribution of crop scores (P=.78). With increasing sucrose concentrations above mmol l, the relative frequencies of flies having higher crop scores increases. One chief difference is that only 3 % of females (versus 7 % of males) fed mmol l sucrose have maximal crop scores of (Fig. ). Ad libitum feeding on mmol l sucrose diets When flies are allowed unlimited access to the food source, we found that the number of proboscis prints made, the crop score distribution and the rate of defecation are all affected by dietary sucrose concentration. Male flies fed ad libitum on pre-trial diets having sucrose concentrations ranging from to 3 mmol l produce fewer proboscis prints as sucrose concentration increases (67.7±3. and.±.2 prints per fly for and 3 mmol l sucrose respectively) (Fig. ). No difference in the number of proboscis prints occurs among flies fed 3, and mmol l sucrose. Females made significantly more prints than males when fed ad libitum for 72 h on diets containing mmol l sucrose (sequential Bonferroni test, P=.) or mmol l (P=.6) sucrose, but not on 2 mmol l (P=.6) or mmol l (P=.6) sucrose (Table ). In flies fed ad libitum on dye-colored diets, the amount of food maintained in the crop increases with decreasing concentrations of sucrose in the diet (G-test, male, P<.; female, P<.) (Fig. 6). Regardless of sex, all D. melanogaster had blue dye visible in some portion of the gut. In contrast to food-deprived flies fed a single meal, however, the majority of male flies fed ad libitum for 72 h on diets containing, or mmol l sucrose have little or no food stored in the crop (Fig. 6A, c.f. Fig. 3). The crop score A Male (mmol l ) B Female (mmol l ) Fig.. s of food-deprived male (A) and female (B) D. melanogaster fed a single meal. Flies were food deprived for 2 h and then fed for min on,,,, or mmol l sucrose and. % Blue No. in % agar. Each point represents the percentage of flies in the sample exhibiting a particular crop score. s ranged from (no blue dye is visible in the crop, but dye is present in the midgut, hindgut or rectum) to (crop is fully distended). 6 male and female flies were assayed. The relative distributions of crop scores are similar in male (A) and female (B) flies, although in flies fed, or mmol l sucrose, greater percentages of males had high crop scores than females.

9 Regulation of feeding behavior in Drosophila 223 distributions are not different among flies fed for 72 h on these sucrose concentrations. At concentrations of sucrose below mmol l, however, the percentage of flies having larger crops increases. The distribution of crop scores of males fed 2 mmol l sucrose for 72 h is intermediate to, and significantly different from, that of males fed or mmol l sucrose (P=. and P=., respectively). Flies fed ad libitum for 72 h on mmol l sucrose show the greatest percentage of larger crops (Fig. 6A). Although capable under other feeding conditions of consuming large meals, no flies fed ad libitum on, 2,, or mmol l sucrose for 72 h were observed to have crop scores of. As in male flies, the distributions of crop scores in female flies fed ad libitum shifts towards larger crops when they are fed on weaker sucrose concentrations (Fig. 6B). Again, the distribution of crop scores of females fed 2 mmol l sucrose is significantly different from that of females fed mmol l sucrose (P<.). The shift in the relative frequency of higher crop scores in females fed mmol l sucrose, however, is not as great as the shift observed in males fed on the same sucrose concentration (Fig. 6). Only 3 % of females (versus 6 % of males) have crop scores ranging from 3 to. In addition to proboscis prints and crop score distribution, the rate of defecation also is affected by the concentration of sucrose in the diet of flies fed ad libitum for 72 h (ANOVA, male, P<.; female, P<.) (Fig. 7). For flies fed in this manner, there is no overall difference between the sexes in the number of fecal spots produced (ANOVA, P=.3). Flies fed on concentrations ranging from to mmol l sucrose 8 6 Number of prints per fly [Sucrose] in pre-trial diet (mmol l ) Fig.. Effects of sucrose concentration in the pre-trial diet on the number of proboscis prints made in a 2 min period on mmol l sucrose/ % gelatin-coated slides. Male D. melanogaster were fed for 3 days on % agar with concentrations of sucrose ranging from to mmol l and then assayed for the number of proboscis prints made. Each column represents the mean + S.E.M. from trials of 8 flies each.

10 22 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN Table. Sexual dimorphism in mean number of proboscis prints in Drosophila fed ad libitum on diets containing different concentrations of sucrose Numbers of Sucrose prints made per fly concentration (mmol l ) N Female Male 3 79.±2.7 a 6.±. b ±.8 a 3.3±.7 a 3.8±2.9 a.2±.3 b 8.6±3. a.±. a Flies were fed for 3 days on % agar having either, 2, or mmol l sucrose. The flies were then assayed for the number of proboscis prints made in 2 min. Each sample represents a trial of flies of a single sex. Means within rows followed by the same letter are not significantly different (sequential Bonferroni test, P<.). Values are means ± S.E.M. did not differ from each other in production of fecal spots and produced fewer than.3 fecal spots min on average. The rate of defecation in flies fed ad libitum on 2 mmol l sucrose is significantly higher than the rates for flies fed on diets containing higher sucrose concentrations. Furthermore, in flies fed mmol l sucrose, the rates of defecation,. and.2 fecal spots min for males and females, respectively, are significantly higher than those for flies fed 2 mmol l sucrose. Thus, the rate of A Male B (mmol l ) Female (mmol l ) Fig. 6. s of male (A) and female (B) D. melanogaster fed ad libitum for 3 days on, 2,, or mmol l sucrose in % agar and. % Blue No.. Each point represents the percentage of flies in the sample exhibiting a particular crop score. 236 male and 87 female flies were assayed. The relative distributions are similar between male (A) and female (B) flies. Greater percentages of males fed and 2 mmol l sucrose and of females fed mmol l sucrose have high crop scores than of males and females fed on diets having higher sucrose concentrations.

11 Regulation of feeding behavior in Drosophila 22 defecation is inversely related to sucrose concentration in D. melanogaster fed ad libitum on diets ranging from to mmol l sucrose and is independent of sucrose concentration for flies fed on diets ranging from to mmol l sucrose. Ad libitum feeding on mmol l sucrose diets Mortality To investigate further how sucrose concentrations in the diet affect feeding regulation during ad libitum feeding conditions, adult D. melanogaster were fed for 3 days on a yeast/glucose diet and then transferred to % agar diets containing,,, or mmol l sucrose. Percentage mortality was recorded for male and female flies fed for 2, 36, 8 and 6, and 72 h on these diets. In male flies, the percentage mortality rises most rapidly over time in flies fed or mmol l sucrose (Fig. 8A). As the concentration of sucrose in the diet increases, percentage mortality rises more slowly over time. Flies fed on mmol l sucrose have percentage mortalities of less than 3 % throughout the 72 h test period. As with male flies, female flies exhibit the most rapid rise in percentage mortality over time when fed on or mmol l sucrose and exhibit a more gradual rise in percentage mortality over time with increasing concentration of sucrose in the diet (Fig. 8B). The percentage mortality in female flies, however, is not as high as that found.6 Number of fecal spots per fly per minute.2.8. Male Female 2 [Sucrose] in % agar (mmol l ) Fig. 7. Effect of sucrose concentration in diet on the number of fecal spots produced by ad-libitum-fed flies. Male or female D. melanogaster were fed ad libitum for 3 days on, 2,, or mmol l sucrose and. % Blue No. in % agar and assayed for the number of fecal spots produced in 2 min. No difference is observed in the rate of defecation between male and female flies fed on the same diet. In both sexes, the rate of defecation is low, ranging from.2 to.3 fecal spots fly min when they are fed ad libitum on, and mmol l sucrose diets. The rate of defecation increases significantly, however, when flies are fed on diets of and 2 mmol l sucrose. Each column represents the mean + S.E.M. from five trials of flies each.

12 226 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN in male flies fed for the same length of time on the same concentration of sucrose. By 72 h of ad libitum feeding, the percentage mortality has reached only 6, 7 and 6 % in females fed, and mmol l sucrose, respectively, whereas males fed on the same diets have greater than 98 % mortality. As for male flies, less than 3 % mortality is observed at any given time for female flies fed mmol l sucrose. distribution In addition to calculating percentage mortality, we determined the size of the crop in adult D. melanogaster fed ad libitum for 2 72 h on dye-colored diets having,,, or mmol l sucrose. Since these flies have unrestricted access to food, some will have just consumed a meal, while others may not have fed for some time. Thus, flies in a range of feeding states should be represented in each sample. Flies were fed the same diets and 8 A Male Percentage mortality B Female 8 Percentage mortality Time fed ad libitum (h) Fig. 8. Percentage mortality over time of male (A) and female (B) D. melanogaster fed ad libitum for up to 3 days on ( ), ( ), ( ), ( ) or ( ) mmol l sucrose in % agar and. % Blue No.. Each point represents the mean ± S.E.M. from five trials of flies each. 7

13 Regulation of feeding behavior in Drosophila 227 assayed at the same times as in the mortality study. The crop score was determined only for groups whose percentage mortality was below %. When male flies are fed ad libitum, their crop score distributions are variable over time in a manner dependent on the sucrose concentration in the diet (Fig. 9). In male flies fed for 2 h on diets ranging from to mmol l sucrose, the crop score distributions do not differ significantly (G-test, P=.9) (Fig. 9A). The majority of these 2 h-fed flies (88 9 %) have crop scores of or 2. These flies also have crop score relative frequencies similar to flies fed ad libitum for 72 h on, and mmol l sucrose diets (cf. Fig. 6A). At 36 h post-feeding, the crop score distributions for flies fed, or mmol l sucrose were similar to each other and to the distributions for flies fed on the A 2 h fed B 36 h fed D 8 h fed 2 3 (mmol l ) 6 2 C 6 h fed 2 3 (mmol l ) (mmol l ) (mmol l ) Fig. 9. s of male D. melanogaster fed ad libitum for 2 h (A), 36 h (B), 8 h (C) and 6 h (D) on,,, or mmol l sucrose in % agar and. % Blue No.. Each point represents the percentage of flies in the sample exhibiting a particular crop score. s were recorded only for groups of flies in which the percentage mortality for that time point was below %. The numbers of flies tested for each time point were 86, 877, 9 and 37 for 2 h (A), 36 h (B), 8 h (C) and 6 h (D), respectively.

14 228 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN A B 2 h fed 36 h fed C D 8 h fed 6 h fed E 72 h fed 2 3 (mmol l ) (mmol l ) (mmol l ) (mmol l ) (mmol l ) Fig.

15 Regulation of feeding behavior in Drosophila 229 same concentrations at 2 h post-feeding (Fig. 9B). Larger percentages of males fed for 36 h ad libitum on and mmol l sucrose, however, have bigger crops than either flies fed for 36 h on, or mmol l sucrose or flies fed for 2 h on the same sucrose concentration. The greatest increase in flies with larger crops occurs in the group fed mmol l sucrose. This shift towards larger crops persisted in flies fed for 8 h on mmol l sucrose (Fig. 9C). Flies fed for 8 h on either or mmol l sucrose also exhibit an increased percentage of larger crops. By 6 h post-feeding, 83 % and 7 % of the male flies fed on either and mmol l sucrose, respectively, have crops scores of 3 or higher (Fig. 9D). The crop score distributions also change over time for female flies fed ad libitum on diets ranging from to mmol l sucrose (Fig. ). Unlike males, however, no significant difference is observed in the crop score distribution among females fed on these diets for the same length of time up to 72 h post-feeding. During this time, the majority of females have crop scores of or 2. When crop score relative frequencies are compared among flies fed the same concentration of sucrose over time, a change in the distribution of crop scores is observed among flies fed ad libitum on, and mmol l sucrose. A significantly greater percentage of flies fed mmol l sucrose for 8 h (G-test, P=.3) and fed or mmol l sucrose for 6 h (P<.) have large crops than flies fed on the same concentration for 2 h (Fig. ). No significant difference is observed in the distribution of crop scores between 6 and 72 h for flies fed on the same concentration of sucrose. Discussion Proboscis print behavior Proboscis print behavior has been described previously both for the housefly (Hewitt, 9) and for blowflies (Graham-Smith, 93; Dethier, 976). When these muscoid flies are allowed to feed on a thin film of sugar, they consume with each proboscis extension only the sugar directly below the oral disk. Thus, each proboscis print marks the full extension of the proboscis and the initiation of ingestion. We have confirmed that D. melanogaster exhibit proboscis print behavior by using slides coated with mmol l sucrose and % gelatin. Proboscis prints are made in response to the sucrose and not to the gelatin, since well-hydrated flies that are responsive to mmol l sucrose/ % gelatin do not respond to % gelatin alone. Additionally, we have found that, in D. melanogaster, the number of proboscis prints serves as an indicator of a fly s ingestion responsiveness. Each proboscis extension signifies the fly s readiness to ingest and the proboscis print itself provides a record of the event. The total amount of food ingested from proboscis prints over the 2 min assay period is Fig.. s of female D. melanogaster fed ad libitum for 2 h (A), 36 h (B), 8 h (C), 6 h (D) and 72 h (E) on,,, or mmol l sucrose in % agar and. % Blue No.. Each point represents the percentage of flies in the sample exhibiting a particular crop score. s were recorded only for groups of flies in which the percentage mortality for that time point was below %. The numbers of flies tested for each time point were 397, 38, 338, 8 and 276 for 2 h (A), 36 h (B), 8 h (C) 6 h (D) and 72 h (E), respectively.

16 23 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN slight, corresponding to a small meal. Male flies food-deprived for 2 h and provided access to dye-colored mmol l sucrose/gelatin slides for 2 min have empty or slightly filled crops (crop scores of or 2) (R. S. Edgecomb, unpublished observations). Minimizing the volume of food ingested is important in limiting post-ingestional effects on subsequent feeding behavior (Edgecomb et al. 987; Sudlow et al. 987). The small meals ingested by D. melanogaster during the proboscis print assay appear to have little effect on ingestion responsiveness over the course of the proboscis print test. Fooddeprived flies or flies fed ad libitum on a pre-trial diet of 2 mmol l sucrose/ % agar were often observed still making prints in the sucrose/gelatin coating at the end of the 2 min test period. Because of the brief assay time and small volume ingested, the proboscis print assay provides a useful measure of the fly s readiness to feed and, hence, its ingestion responsiveness. Although the rate of ingestion is slow for D. melanogaster fed on thin coatings, flies fed on thicker semi-solid foods ingest more rapidly. For example, 2 h food-deprived flies fed on a thin mmol l sucrose/gelatin coating consume small volumes in 2 min. In contrast, flies similarly food-deprived, but fed on ml of mmol l sucrose/ % agar diet in glass vials, ingest large volumes in only min. Most of the latter have large crops (crop scores of or ) and distended abdomens. Since flies fed on large volumes of diet cannot exhaust the supply of food beneath the oral disk, they are able to ingest more food with each proboscis extension than flies fed on a thin coating. In fact, flies fed large volumes of mmol l sucrose/ % agar often ingest most of their meal in one location (R. S. Edgecomb, unpublished observations). These results suggest that the nature of the food source (thin film or large reservoir) can affect feeding patterns. That flies are capable of detecting and ingesting minute food sources on plant surfaces is supported by studies on foraging behavior in the apple maggot fly Rhagoletis pomonella (Tephritidae) (Hendrichs and Prokopy, 99; Hendrichs et al. 993a,b). When food is presented in discrete quantities, the form of diet presentation affects food handling and processing time (Hendrichs et al. 993b). Owing to the time required to liquefy solid food, feeding on dry sucrose costs R. pomonella more time than feeding on aqueous sucrose having the same weight of solute. Flies fed on dry sucrose may increase the risk of predation due to prolonged exposure on the leaf surface. Regulation of feeding behavior in food-deprived flies Our results demonstrate that food-deprived adult D. melanogaster are capable of regulating their ingestion responsiveness and single-meal volume. Male D. melanogaster fed ad libitum on a yeast/glucose diet have a low responsiveness for ingestion (.6±. prints per fly) at the onset of food deprivation. As the period of food deprivation lengthens, ingestion responsiveness gradually increases until male D. melanogaster produce an average of.6±2. prints per fly at 8 h of food deprivation (Fig. 2). This value is probably nearly maximal, since by 8 h of food deprivation the percentage mortality of male flies has already reached 7 %. In addition to ingestion responsiveness, food-deprived D. melanogaster regulate meal size. When male or female D. melanogaster deprived of food for 2 h are fed a single

17 Regulation of feeding behavior in Drosophila 23 meal of mmol l sucrose or higher, they ingest meal volumes proportional to the concentration of sucrose in the diet. Significant differences in meal sizes also occur between food-deprived flies fed either or mmol l sucrose (Shimada et al. 987). Under the feeding conditions we employed, there is no difference in volume consumed in males and females. However, only 3 % of females (versus 7 % of males) fed mmol l sucrose have a maximum crop score of. This indicates that females have the potential for ingesting more food. Females are generally larger than males (Ashburner, 989) and, if the crop is also proportionally larger, then females should be able to store more food in their crops than males. Because we fed flies for several days on a yeast/glucose diet before food-depriving them for 2 h, females were gravid. Hence, much of the abdomen was taken up by the enlarged ovaries, reducing the space available for crop distension. From these observations, we expect that female flies with lessdeveloped ovaries may be able to ingest greater volumes of food than male flies treated similarly. The positive relationship in adult D. melanogaster between sucrose concentration and volume consumed does not apply to sucrose concentrations of mmol l or less. Similarity in volume ingested is not due to changes in the frequency of ingestion across this sucrose concentration range, since more than 8 % of the flies have some blue dye visible in their gut, regardless of sucrose concentration. Further, previous investigations on chemosensory discrimination indicate that D. melanogaster can respond with proboscis extension to aqueous sucrose solutions as low as.2 mmol l sucrose (Rodrigues and Siddiqi, 978). In a two-choice test between % agar and mmol l sucrose/dye-colored % agar, 8 9 % of wild-type (Canton-S) flies over a h period preferentially ingested the sucrose-containing diet (Balakrishnan and Rodrigues, 99). Despite this preference, we observe no difference in the volumes of or mmol l sucrose consumed in a no-choice situation. This uniformity we observe in the volume of diet ingested and the percentage of flies having ingested suggests that, when flies are fed on mmol l sucrose, their low levels of ingestion may depend more on maintaining an adequate water balance than on replenishing energy stores. This is supported by the observation that flies fed mmol l sucrose ad libitum have the same percentage mortality as flies deprived of food. Thus, although mmol l sucrose/ % agar may be preferred over % agar alone in a two-choice assay, the nutritional benefit of mmol l sucrose is negligible and the total volume of diets ingested does not differ. In D. melanogaster, the relationships between ingestion responsiveness and duration of food deprivation and between meal size and sucrose concentration are similar to those observed in other insects (Barton Browne, 993). Of particular interest are the similarities between D. melanogaster and Phormia regina. As in D. melanogaster, P. regina become more responsive to sugar as the length of food deprivation increases (Evans and Dethier, 97; Bowdan and Dethier, 986; Edgecomb et al. 987). Likewise, in P. regina the volume of food ingested increases with the strength of the stimulus (Bowdan and Dethier, 986). The similarities in proboscis extension responsiveness and meal size determination during food deprivation between these two muscoid flies suggest that these behaviors are regulated similarly in the two species.

18 232 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN Regulation of feeding behavior in ad-libitum-fed flies Flies fed ad libitum can respond to the presence of food primarily in two ways. First, they can ingest large single meals with long periods of non-feeding, during which the crop slowly empties. Alternatively, they can ingest small, frequent meals and maintain their crop size at a determined volume. If long intermeal intervals are characteristic for D. melanogaster in ad libitum feeding conditions, then the flies would represent a wide spectrum of feeding states from having fed recently to having fed much earlier. In this situation, we would expect flies that had fed recently to have large crops, while those that had fed earlier to have smaller crops. In fact, we observed no ad-libitum-fed flies having a maximal crop score of and a maximum of 6 % having a crop score of. The majority of flies had either no food in the crop (crop score of ) or a small amount of food present in the crop (crop score of 2). Our results indicate that flies fed ad libitum do not regulate their feeding behavior in the same way as food-deprived flies, which ingest large meals. Rather, ad-libitum-fed flies maintain relatively small crop sizes regardless of the sucrose concentration fed. This shift in behavior between food-deprived and ad-libitum-fed D. melanogaster could conceivably benefit flies in their natural environment. If food is scarce, flies may become food-deprived. When these flies encounter food, they will probably ingest large meals. Ingestion of large meals when food is plentiful, however, may be disadvantageous, since the extra weight from crop reserves will probably impede the fly during take-off and flight and lead to increased susceptibility to predation (Berrigan, 99; Marden and Chai, 99). In our ad libitum feeding regime, adult D. melanogaster had only to travel the length of a vial to encounter a food source. This regime represents very high food density conditions, whereas food deprivation simulates very low density conditions. The natural situation is probably a continuum between the two laboratory-generated extremes in feeding conditions. If this is the case, then the likelihood of encountering food in the environment becomes an important variable for feeding regulation in natural populations of D. melanogaster. Estimates of the number of feeding sites in the flies natural environment range from a few of small volume for Hawaiian D. melanogaster (Spieth, 986) to superabundant, but diffuse for Drosophila obscura and Drosophila subobscura in a British mixed deciduous woodland (Begon and Shorrocks, 978). Although ad-libitum-fed D. melanogaster maintain relatively small crops, they do not all regulate their feeding behavior in the same way. The concentration of sugar in the diet during ad libitum feeding affects ingestion responsiveness, crop size and rate of defecation (Figs 7). To determine whether D. melanogaster can compensate for the changes in sucrose concentration to maintain approximately the same total sugar intake, we measured rates of defecation in ad-libitum-fed flies. Since fecal spot sizes appear similar for D. melanogaster fed on different sucrose concentrations, changes in spot number can be used to calculate the extent of compensation of ingestion. Our results show that D. melanogaster do not compensate for changes in sucrose concentration when fed on diets of mmol l sucrose and above. Below mmol l sucrose, however, they can compensate for up to % of the sugar lost between and mmol l sucrose by more than tripling the rate of defecation (Fig. 7). Thus, the degree of compensation in

19 Regulation of feeding behavior in Drosophila 233 feeding behavior to changes in dietary sucrose concentration is highly dependent on the concentration range selected. Blowflies also change their feeding behavior to compensate for changes in sugar concentrations in the diet. Adult P. regina fed. or. mmol l sucrose alternating in a 3 day cycle ingest an average of 3 l day of. mmol l sucrose and 3 l day of. mmol l sucrose with an average compensation of about % of the total sucrose consumed (Gelperin and Dethier, 967). Male Lucilia cuprina can also compensate partially for changes in glucose concentration in the diet by regulating meal size, meal frequency and crop volume (Simpson et al. 989). L. cuprina fed ad libitum on mmol l glucose (which is equivalent to mmol l sucrose) ingest more frequently and a larger volume per meal than those fed mmol l glucose. Despite these changes, L. cuprina fed on mmol l glucose ingest only 3 % of the total glucose ingested by those fed on mmol l glucose (Simpson et al. 989). The maximum degree of compensation for L. cuprina fed on other concentrations of glucose may be higher. Although there is no difference between the sexes in defecation rates of male and female D. melanogaster fed ad libitum, a sexual dimorphism occurs in the number of proboscis prints and in crop score distributions. Adult female D. melanogaster fed ad libitum for 72 h on or mmol l sucrose produce more proboscis prints than males fed on the same pre-trial diet (Table ). Both males and females were fed protein for 3 days prior to being fed for 72 h on a sucrose/agar diet. This sexual dimorphism may result from sex-specific effects of protein ingestion (e.g. oogenesis in females) or may persist in the absence of protein from the adult diet. Male and female D. melanogaster also differ in percentage mortality and crop score distributions when fed on diets ranging from to mmol l sucrose (Figs 8 ). The rise in percentage mortality in females is delayed and occurs more slowly than in male flies fed on the same diet. Also, crop score distributions take longer to shift to higher crop scores in females than in males. Again, because all flies were first fed a yeast/glucose diet for 3 days, it is possible that females have accrued more reserves on which to draw and are thus able to delay the changes in feeding behavior observed in male flies. That female tephritid flies may use developing ova as a possible nutritional source is suggested by the observation that 2 h food-deprived flies show no tendency to dump eggs (Prokopy et al. 993). Removal of all protein in the adult diet from the onset of eclosion may eliminate the observed sexual dimorphism in feeding regulation. Role of nutrition in ad libitum feeding Adult D. melanogaster fed ad libitum for 72 h on sucrose/agar diets ranging from to mmol l regulate ingestion responsiveness, the amount of food stored in the crop and the rate of defecation (Figs 7). Under these feeding conditions, the three variables share similar trends. Flies fed on diets ranging in sucrose concentration from to mmol l have indistinguishable behaviors. They all make few proboscis prints, maintain small crops and have low defecation rates. At sucrose concentrations of 2 mmol l or below, the number of proboscis prints, crop size and the rate of defecation all increase in response to decreasing concentrations of sucrose in the diet. Thus,

20 23 R. S. EDGECOMB, C. E. HARTH AND A. M. SCHNEIDERMAN D. melanogaster fed ad libitum can be separated into two groups: those fed on mmol l sucrose or higher, whose behavior is independent of sucrose concentration, and those fed on sucrose concentrations of 2 mmol l or less, whose behavior is affected by the sucrose concentration in the diet. In a previous study on the survival of adult D. melanogaster on aqueous sucrose solutions, survival increased with increasing sucrose concentration up to mmol l (Hassett, 98). Beyond that concentration range, no increase in survival was observed, suggesting that flies raised on sucrose concentrations below mmol l are ingesting a nutritionally deficient diet. The shift in feeding regulation behavior that we observe in response to diets containing less than mmol l sucrose is probably an attempt by the fly to compensate for this deficiency. The mechanism by which D. melanogaster shift over time to maintaining larger crops when fed ad libitum on 2 mmol l sucrose or less is not known. This behavior does not fit well with the known excitatory and inhibitory inputs that participate in feeding regulation. The combined effect of increased inhibitory input due to large crops and decreased excitatory input due to lower sucrose concentrations argues against these known neural mechanisms for regulating the shift in crop size. The strong relationships between concentration of sucrose in the diet and both the shift towards larger crops and increased mortality suggest that the change in crop size and rate of defecation are related to the nutritional state of the fly. Adult D. melanogaster may measure such factors as lipid or glycogen reserves, rate of glycogen to sugar conversion or blood sugar levels to assess their nutritional state and to modulate how they respond to gustatory or stretch receptor input. That this shift in crop size does not occur in D. melanogaster fed or mmol l sucrose (Figs 9, ) indicates that, although flies may need to compensate for a nutritional deficit, they also need a minimum food stimulus to alter their feeding behavior. We would like to thank Dr D. Frick of Warner Jenkinson Company, St Louis, MO, for technical assistance in dye selection and for generously providing the dye for these experiments. We would also like to thank Dr T. Eisner for use of an incubator, Dr R. Booker for use of a microcentrifuge, Dr S. Smedley and J. Dale for statistical assistance, and Dr S. Smedley, Dr S. Adamo, J. Trimarchi and two anonymous reviewers for comments on earlier drafts of this manuscript. This work was supported in part by NSF IBN (A.M.S.). References ASHBURNER, M. (989). Drosophila: A Laboratory Handbook, p. 33. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. BALAKRISHNAN, R. AND RODRIGUES, V. (99). The Shaker and ShakingB genes specify elements in the processing of gustatory information in Drosophila melanogaster. J. exp. Biol. 7, 6 8. BARTON BROWNE, L. (993). Physiologically induced changes in resource-oriented behavior. A. Rev. Ent. 38, 2. BEGON, M. AND SHORROCKS, B. (978). The feeding- and breeding-sites of Drosophila obscura Fallen and D. subobscura Collin. J. nat. Hist. 2, 37. BERNAYS, E. A. (98). Regulation of feeding behavior. In Comparative Insect Physiology, Biochemistry and Pharmacology, vol., Regulation: Digestion, Nutrition, Excretion (ed. G. A. Kerkut and L. I. Gilbert), pp. 32. New York: Pergamon Press.