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First published online October 5, 2007
Journal of Experimental Biology 210, 3547-3558 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006924

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Natural variation in food acquisition mediated via a Drosophila cGMP-dependent protein kinase

Karla R. Kaun¹, Craig A. L. Riedl¹, Munmun Chakaborty-Chatterjee¹, Amsale T. Belay¹, Scott J. Douglas¹, Allen G. Gibbs² and Marla B. Sokolowski^1,*

¹ Department of Biology, University of Toronto, 3359 Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada
² School of Life Sciences, University of Nevada, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA

View larger version (39K): [in this window] [in a new window]   Fig. 1. for affects food intake. (A) Mid-third-instar rover (forR), sitter (fors) and sitter mutant (fors2) larvae with ingested dye visible through the ventral cuticle. (B) Well-fed rovers (forR) had significantly lower food intake than well-fed sitters after 10 min (ANOVA, F(2,84)=12.03, P<0.0001), 15 min (F(2,85)=11.46, P<0.0001) and 20 min (F(2,85)=7.09, P<0.001) on dyed yeast paste. A two-way ANOVA on strain (forR, fors, fors2) and time (10, 15, 20 min) showed a significant effect of strain (F(2,254)=12.12, P<0.001), no effect of time (F(2,254)=0.13, P=0.9) and no strain-by-time interaction (F(2,254)=0.23, P=0.9). (C) Rovers had significantly less blue area than sitters at early third (F(2,89)=4.24, P=0.02) and mid-third-instar (F(2,87)=10.52, P<0.0001) stages. (D) Spectrophotometric quantification of homogenates from larvae fed a Erioglaucine (FD and C Blue No. 1) dyed yeast paste for 15 min showed that forR larvae ingest significantly less dye than fors or fors2 larvae (F(2,26)=7.88, P<0.01).

View larger version (22K): [in this window] [in a new window]   Fig. 2. for affects feeding rate and food acquisition. (A) Gut dissections from larvae fed blue yeast paste for 15 min also indicated that forR larvae ingested significantly less food than fors or fors2 larvae (F(2,26)=5.32, P<0.05). (B) The rate that the gut filled with food was measured by feeding larvae on a yeast paste with Brilliant Blue dye, extracting the gut, and comparing the length of the gut with food to the length of the entire gut. Gut size did not differ significantly between strains (F(2,67)=0.86, P=0.43). Sitter (fors and fors2) larvae filled their gut more quickly than rover (forR) larvae (10–20 min feeding, two-way-ANOVA F(8,36)=70.53 P=0.0008; effect of strain, F(2,36)=5.82, P=0.007; effect of time, F(2,36)=5.52, P=0.008; one-way ANOVAs: 5 min, P=0.82; 10 min, P=0.10; 15 min, P=0.05; 20 min, P=0.04). This difference stabilized after about 25–30 min of feeding when the midguts of all larvae become saturated with the blue yeast paste, resulting in no measurable difference in food intake (one-way ANOVAs: 25 min, P=0.85; 30 min, P=0.68). (C) Rovers showed significantly less food intake on fructose–agarose (F(2,87)=12.20, P<0.0001; forR vs fors, P<0.0001; forR vs fors2, P<0.0002; fors vs fors2, P=0.92) and glucose–agarose (F(2,87)=3.98, P=0.02; forR vs fors, P=0.0078; forR vs fors2, P=0.048; fors vs fors2, P=0.47) when food intake was measured using Carmine dye. (D) Drosophila larvae use their mouthhooks for both food ingestion and locomotion. We found no significant correlation between mouthhook movements and amount of dye ingested when larvae fed on a glucose–agarose substrate (R2=0.18, P=0.11).

View larger version (33K): [in this window] [in a new window]   Fig. 3. for affects glucose absorption third-instar larvae. (A) No differences in path length between rover (forR), sitter (fors) and sitter mutant (fors2) larvae were found during a 15 min test on a non-nutritive substrate (agar; F(2,56)=0.34, P=0.71). However, as expected, rovers showed significantly longer foraging trails than sitter and sitter mutants during a 15 min test on a yeast substrate (F(2,53)=35.77, P<0.0001). (B) After 15 min of feeding on a yeast–water paste containing 14C-labeled glucose, rover larvae ingested significantly less 14C than sitters (F(2,17)=52.66, P<0.001). When subsequently exposed to unlabeled medium for 3 h, rovers retained significantly more 14C label, indicating a higher level of glucose absorption compared to sitters (F(2,17)=7.36, P<0.006). (C) After 15 min of feeding on a yeast–water paste containing 14C-labeled L-U-leucine, well-fed rover larvae ingested significantly less 14C than sitters (F(2,15)=11.03, P=0.001; forR vs fors, P=0.0003; forR vs fors2, P=0.03; fors vs fors2, P=0.04). When subsequently exposed to unlabeled medium for 3 h, rovers did not differ significantly from sitters in amount of 14C label (F(2,15)=1.92, P=0.18). (D) Rovers (forR) and sitters (fors and fors2) do not differ significantly in the number of contractions of the anterior midgut (F(2,27)=0.17, P=0.85), acidic region (F(2,27)=1.83, P=0.18) or posterior midgut (F(2,27)=1.37, P=0.27). (E) No significant differences were found in the amount excreted by forR, fors or fors2 larvae (F(2,57)=0.049, P=0.95). In addition, no significant interaction between the number of fecal spots and strain was found with a logistic regression analysis on excreted food concentration (F(2,5)=2.40, P=0.100).

View larger version (63K): [in this window] [in a new window]   Fig. 4. Food level affects survivorship to eclosion, development rate and food intake in rovers and sitters. (A) Larvae were reared from egg-hatch on media containing 100%, 75%, 50%, 25% or 15% of the yeast and sucrose content of standard medium (100%; see Materials and methods). Values are means ± s.e.m. Between-strain survivorship analysis revealed that differences were not significant at 100% (F(2,21)=0.91, P=0.4), 75% (F(2,20)=1.70, P=0.2), 50% (F(2,21)=1.17, P=0.3) or 25% (F(2,21)=0.73, P=0.5) food levels, but at 15% rovers differed significantly from fors (F(1,14)=6.00, P<0.03) and fors2 (F(1,14)=13.29, P<0.003). Between-strain comparisons of developmental delay revealed that rovers differed from both fors and fors2 at 15% (F(2,21)=4.33, P<0.03) and rovers differed from fors2 at 100% (F(2,21)=4.42, P<0.03), 50% (F(2,21)=7.06, P<0.005) and 25% (F(2,21)=3.80, P<0.04) food levels but not at 75% (F(2,20)=2.58, P=0.1). (B) Rovers (forR) ingested less yeast paste after 15 min feeding than sitters (fors and fors2) when raised on high levels of food (100%, 75%, 50%), but not when reared on low levels of food (25%, 15%) [two-way ANOVA for strain (F(2,435)=5.13, P<0.006) and strain-by-food level (F(8,435)=2.28, P<0.02); one-way ANOVA on 100% (F(2,87)=6.86, P<0.002), 75% (F(2,87)=14.33, P<0.0001), 50% (F(2,87)=6.34, P<0.003), 25% (F(2,87)=0.14, P=0.9), 15% (F(2,87)=0.11, P=0.9)]. (C) Rovers and sitters differ in food intake measured after 5 min feeding on yeast paste when reared at 100%, food quality but not when food-deprived (reared on 25% and 15% food) [ANOVA, at 100%, F(2,87)=5.53, P<0.0006, 25% F(2,87)=0.69, P=0.50), 15% F(2,87)=0.33, P=0.7]. (D) All larvae reared under low food levels were staged to mid-third instar prior to measuring food intake. Rover and sitter larvae reared in dilute (15% and 25%) food were smaller in size at mid-third instar compared to larvae reared in normal (100%) food. However, no within-strain differences were found at any food level [two-way ANOVA, F(8,260)=56.50, P<0.0001; strain, F(2,260)=0.19, P=0.82; food quality, F(2,260)=73.25, P<0.0001, with significantly smaller larvae at 25% (P<0.0001) and 15% (P<0.0001) compared to 100%; strainxfood quality F(2,4)=0.23, P=0.92]. (E) At 15% food, rovers, sitters and sitter mutants ingested similar amounts of 14C-labeled media in15 min (F(2,15)=0.67, P=0.3), but rovers absorbed more than sitter and sitter mutants (F(2,17)=45.51, P<0.0001). forR larvae had a twofold increase in absorption compared to fors and fors2 larvae at 15% food levels (F(2,12)=41.96, P<0.0001; forR vs fors, P<0.0001; forR vs fors2, P<0.0001; fors vs fors2, P=0.12). (F) After 15 min of feeding on a yeast–water paste containing 14C-labeled L-U-leucine, rover larvae reared on 15% food did not differ significantly from sitters in amount of 14C ingested (F(2,15)=0.37, P=0.70). When subsequently exposed to unlabeled medium for 3 h, rovers absorbed significantly more 14C label compared to sitter mutants (F(2,15)=4.55, P=0.03; forR vs fors, P=0.15; forR vs fors2, P=0.009; fors vs fors2, P=0.53).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Variation in for mediates nutrient acquisition strategies. (A) fors;hs-GAL4/UASforT2 flies reared at 23°C show an increase in T2 protein level (lane 1) compared to control flies reared under the same conditions (lanes 2 and 3). The actin bands show that equal amounts of protein were loaded in each lane. (B) Expression of a forT2 transgene in sitters resulted in a rover-like decrease in food intake (ANOVA: at 5 min, F(1,57)=3.03, P=0.09; 10 min, F(1,58)=7.60, P=0.008; 15 min, F(1,58)=11.92, P=0.001; 20 min, F(1,57)=5.46, P=0.02). (C) Ubiquitous expression of forT2 in sitters using a leaky hs-GAL4 transgene significantly decreased food intake (F(2,89)=19.56, P<0.0001) and (D) increased glucose absorption rate (F(2,17)=47.51, P<0.0001).

View larger version (36K): [in this window] [in a new window]   Fig. 6. Variation in for affects response to food quality. (A) Expression of forT2 was sufficient to increase developmental rate and survivorship in food-deprived sitter larvae raised on low nutrient media (25% or 15%) [development time: at 25% (F(2,27)=17.78, P<0.0001) and at 100% (F(2,26)=0.95, P=0.4); survivorship: at 15% (F(2,29)=4.07, P<0.03) and at 100% (F(2,28)=0.66, P=0.5)]. (B) PKG enzyme activity is inversely related to food intake. Rovers (forR) had significantly higher PKG activity than sitters (fors and fors2) when reared on high food levels, but this difference diminished when larvae were food-deprived [two-way ANOVA: for strain (F(2,63)=57.36, P<0.0001), food level (F(2,63)=95.26, P<0.0001), strain-by-food level (F(4,63)=11.67, P<0.0001); ANOVA: for 100% (F(2,21)=87.9, P<0.0001), 25% (F(2,21)=14.39, P<0.0001), 15% (F(2,21)=0.70, P=0.5) food level]. (C) Sitter larvae expressing a forT2 transgene expressed a rover-like pattern of food [two-way ANOVA: for strain (F(2,261)=3.78, P=0.02), food level (F(2,261)=2.85, P=0.06), strain-by-food level (F(4,261)=1.60, P=0.2); one-way ANOVA by strain: for 100% (F(2,87)=6.87, P<0.002), 25% (F(2,87)=0.32, P=0.7), 15% (F(2,87)=0.42, P=0.7) food level].