Supporting Online Material for

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1 Supporting Online Material for Drosophila Egg-Laying Site Selection as a System to Study Simple Decision-Making Processes Chung-hui Yang, Priyanka Belawat, Ernst Hafen, Lily Y. Jan, Yuh-Nung Jan* *To whom correspondence should be addressed. yuhnung.jan@ucsf.edu This PDF file includes: Materials and Methods Figs. S1 to S7 Published 21 March 2008, Science 319, 1679 (2008) DOI: /science Other Supporting Online Material for this manuscript includes the following: (available at Movies S1 to S3

2 Supporting online material Supplementary Figures fig. S1. Time spent on each of the three egg-laying behavioral components by w1118 females. 32 egg-laying events on grape-agar substrate by two w1118 females were quantified and compared to CantonS animals. The average times for ovipositor program, clean and rest program, and search program are: 6.63, 93.35, and No significant statistical differences were found when durations for each behavioral component were compared between w1118 and CantonS females, p>0.5, Mann-Whitney test for three comparisons. fig. S2. Tendency to avoid laying eggs on sucrose-containing medium (in a standard sucrose-lobeline two-choice chamber) was observed in animals of different genetic background. Preference index for CantonS: (N=29, mean= -0.82), OregonR (N=9, mean= -0.69), w1118: (N=28, mean= 0.695). All are significantly deviated from 0. p<0001 for CantonS and W1118, p=0.001 for OregonR. Wilcoxon Signed Rank Test. fig. S3. Hatching rates of embryos deposited on the lobeline-containing medium and the sucrose-containing medium are comparable. Hatching rate for embryos deposited on lobeline medium: (85%, N=18 assays), on sucrose-containing medium: (87%, N=18 assays). p>0.5, Mann-Whitney test. fig. S4. Specificity of ILP7-GAL4 1

3 (A) ILP7 antibodies staining in the adult brain. (B) mcd8-gfp staining of ILP7-GAL4 labeled neuronal processes in the same brain as in (A). (C) Merged picture of (A) and (B). (D) ILP7 antibodies staining in the whole adult VNC. (E) Nuclear RFP (UAS-redstinger) signal of ILP-GAL4 labeled neurons in the same VNC as in (D). Note that the extreme low background in this picture is because no staining was involved. (F) Merged picture of (C) and (D). fig. S5. Egg-jamming phenotype ILP7-neurons silenced animals (A) Internal reproductive tract of a wild-type female. (B) Internal reproductive tract of ILP7-neurons silenced animals. Note the jamming of the eggs within the thin tube. fig. S6. Validation of transgenic constructs that over-express ILP7 (A) ILP7 antibodies staining of in the nervous system of wild-type larval animals. (B) ILP7 antibody staining of animals that carry an HS-ILP7 construct. (C) ILP7 antibodies staining of a WT animal. (D) Staining of the V5 tag in animals that over-express an UAS-ILP7-V5 by a 477-GAL4. Staining of larval VNC was used because of the ease of dissection. We note here that function of larval ILP7 neurons is not yet known; however, 8 of out 16 of the total larval ILP7 neurons (see green arrow) project axons onto the larval gut (data not shown), suggesting a potential involvement of these neurons in feeding-related behaviors. fig. S7 Many of the ILP7-neurons are also positive for fruitless expression 2

4 (A-C) Three confocal images showing that many ILP7-neurons (in green, labeled by ILP7 antibodies staining) are also positive for fruitless expression (in magenta, labeled by using a fruitless-gal4 to express a UAS-nuclear-RFP). Supporting online material Supplementary Movies Movie S1. Drosophila egg-laying behavioral sequence This video shows a female displaying the stereotypical behavioral programs that accompany each physical act of egg-laying: probing the substrate with its proboscis and ovipositor, inserting the ovipositor into the substrate, moving back and forth to push an egg out, cleaning the ovipositor with its hind legs. Movie S2. Drosophila female makes contacts with the sucrose-containing medium prior to depositing an egg on the lobeline-containing medium This video shows a female exploring and probing the sucrose-containing medium prior to moving towards the lobeline side of the chamber to deposit an egg in a single-female assay. Movie S3. Drosophila females prefer to forage on the sucrose-containing medium in the two-choice chamber This video shows two females foraging on the sucrose side (in red color) of the twochoice chamber. The consumption of sucrose medium can be inferred from the animals persistent proboscis extension onto the sucrose-containing medium. 3

5 Supporting online material Material and Methods Fly Stocks. The following stocks were used for this study: Canton-S, w1118, ILP7- GAL4 (this work), UAS-Kir2.1-GFP (a gift from Dr. Graeme Davis), UAS-red-stinger (Bloomington Stock Center), UAS-mCD8-GFP (Bloomington Stock Center), and Gr5a- GAL4 (a gift from Dr. Kristin Scott and Dr. John Carlson). ILP7-GAL4 was created by cloning the 1.6 Kb upstream genomic sequence of ILP7 into a pcasper-aug-gal4 vector (a gift from Dr. Bruce Baker). HS-ILP7 was constructed by cloning the 660bp genomic region containing the ILP7 coding sequence into a pcasper-hs vector. UAS- ILP-V5 was constructed by cloning the same region plus the V5 tag into puast vector. All GAL4/UAS lines were backcrossed to Canton-S twice prior to behavioral assays. For ILP7 over-expression studies, the animals were raised at 29 degree to maintain higher level of gene expression. Immunohistochemistry. ILP7 polyclonal antibodies were generated in rabbits. The epitope peptide NH2-RSQSDWENVWHQETHS-CONH2 was chemically synthesized, purified by RF HPLC (30mg, minimum 85% purity), and conjugated with KLH. Tissues dissected for antibody staining were fixed in 4% formaldehyde (EM grade) for 40 minutes at room temperature. Fixed tissues were then washed in wash buffer (PBS with 0.3% Triton) for 1 hour before incubation with primary antibodies (in PBS-Triton plus 5% donkey serum) overnight at 4 degree. Antibodies against ILP7 were diluted at 1/1000. Cy2- or Cy3- conjugated Donkey anti-rabbit secondary antibodies were used at 1:500. 4

6 Behavioral analysis. Handling of animals: animals were handled in the following manner: for each test chamber, 5 virgin females of a specific genotype and 16 virgin males (Canton S) were placed in a vial and allowed to mate for 2 hours at the 4 th day after collection. After mating, only the females were transferred into a fresh empty vial that contained wet yeast paste but no other substrate appropriate for egg-laying. Mated females can initiate egg-laying soon after mating but do not like to do so on yeast paste, therefore this empty-vial treatment can deprive the females of egg-laying without starving them. For food deprivation experiment, however, females were transferred into vials that contained only a piece of kimwipe soaked with 2.5 ml of distilled water. For 3- female assay, 3 females (out of 5 from a vial) were selected after hours of deprivation and placed in a behavior chamber for 2 hours. For single-female assay, only one female out of 5 was selected (out of 5 from a vial) to be put into a behavior chamber for 2 hours. Behavior chambers: behavior chambers were made of small plastic containers purchased from TAP plastics (style FT-2: 0.76"L x 0.62"W x 1.69"H) with ventilation holes custom-drilled on the sides. For two-choice assays, 7 ml of 3% agarose (with 1% v/v of acetic acid and ethanol) was placed into the chamber first. 100 µl of sucrose medium (100 mm sucrose in 1% agarose that contains 1% v/v ethanol and red food color) and 100 µl of lobeline medium (500 µm of lobeline in 1% agarose that contains 1% v/v ethanol and green food color) were added on opposite sides of a chamber after the 3% hard agarose solidified. 3X and 10X chambers were custom made by Tap plastic with the chamber length specified to 5 cm and 18 cm respectively. Video acquisition: videos were recorded by a SONY DCR-H1000 camcorder and subsequently processed using the Ulead Video10 software. Egg-laying behavioral sequence: the 5

7 ovipositor motor program was the first one to be spotted because of the unique body posture. The rest of the programs were determined by observing behavioral events that occurred before and after the display of the ovipositor program. Calculation of preference index: the preference index (PI) is calculated by the following formula: (# of eggs on choice A - # of eggs on choice B)/( # of eggs on choice A + # of eggs on choice B). A/B for each two-choice experiment is always labeled above each bar presented in a graph. Scoring ovipositor motor program: the number of times an animal inserted it ovipositor into the substrate to expel the egg was counted. 6

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