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Drosophila Egg-Laying Site Selection as a System to Study Simple Decision-Making Processes

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Science  21 Mar 2008:
Vol. 319, Issue 5870, pp. 1679-1683
DOI: 10.1126/science.1151842

Abstract

The ability to select a better option from multiple acceptable ones is important for animals to optimize their resources. The mechanisms that underlie such decision-making processes are not well understood. We found that selection of egg-laying site in Drosophila melanogaster is a suitable system to probe the neural circuit that governs simple decision-making processes. First, Drosophila females pursue active probing of the environment before depositing each egg, apparently to evaluate site quality for every egg. Second, Drosophila females can either accept or reject a sucrose-containing medium, depending on the context. Last, communication of the “acceptability” of the sucrose-containing medium as an egg-laying option to the reproductive system depends on the function of a group of insulin-like peptide 7 (ILP7)–producing neurons. These findings suggest that selection of egg-laying site involves a simple decision-making process and provide an entry point toward a systematic dissection of this process.

Decision-making, in one view, is the process by which animals deliberate whether to invest in one action or not by taking into account the values and costs associated with available options. Selection of egg-laying site by Drosophila provides a plausible system to investigate such decision-making processes. Egg production is costly; thus, the ability to weigh egg-laying options might have been selected to ensure better survival of offspring in an uncertain environment. Drosophila females are known to be selective toward egg-laying sites and will withhold eggs when there are no appropriate sites (16). It is less clear, however, whether Drosophila females value a given egg-laying site differently according to the availability of other laying options. We examined the selection of an egg-laying site by individual Drosophila females, in an attempt to find a genetically tractable system to study the molecular and cellular basis of simple decision-making processes.

The first indications that egg-laying site selection may employ a simple decision-based process emerged from our observations of females as they lay eggs. For consistent viewing of egg-laying events, we deprived females of egg-laying for 24 hours before placing them in a chamber filled with grape agar, an attractive egg-laying substrate (Fig. 1, A and B). By reviewing hundreds of egg-laying events, we found that immediately before each physical egg expulsion, females stereotypically bend the abdomen down-ward until it is nearly perpendicular to the substrate surface to extrude and insert the ovipositor into the substrate (Fig. 1C) before initiating a series of back-and-forth movements to expel and insert a single egg into the substrate (Movie S1). This behavioral component always accompanies the physical deposition of an egg and typically lasts about 6 s (Fig. 1I) on grape agar. We called this behavioral component the “ovipositor motor program” because it is reminiscent of the “oviposition motor program” of grasshopper egg-laying (7). We next found two more behavioral components that regularly follow the ovipositor program: An animal always grooms its ovipositor with its hind legs (Fig. 1D and Movie S1) for a few seconds and then stays immobile for a while as though it is resting (Fig. 1I). After the “clean and rest program” and before the initiation of the next ovipositor program, the animals presumably have the opportunity to locate an appropriate site for the next egg (Fig. 1H). Indeed, despite being placed in a relatively attractive grape-agar environment, the animals in virtually all cases display a “searchlike” behavior by walking around and probing the substrates with their proboscis and ovipositor (Fig. 1, E to G, and Movie S1). Because the proboscis, legs, and ovipositor all contain sensory receptors (8), this searchlike program should aid the animals in identifying appropriate egg-laying sites. In 199 out of the 200 cases that we observed (∼20 egg-layings each by 10 animals), we detected the searchlike behavior preceding the ovipositor program (Fig. 1J), although its duration varied. Thus, Drosophila females deposit their eggs one at a time and nearly always follow the cycle of search, oviposit, and clean and rest, with the searchlike program lasting from a few seconds to several minutes (Fig. 1J), whereas the other programs remain relatively constant in duration (Fig. 1I and fig. S1).

Fig. 1.

The egg-laying behavioral sequence of Drosophila females. (A) The grape-agar chamber used for observing egg-laying behavior. (B) Top view of the chamber through the camcorder. (C) Animal performing the ovipositor motor program. Note the downward-bending of the abdomen. Red arrow, ovipositor. (D) Animal cleaning the ovipositor (red arrow) with its hind legs. (E) Animal probing the substrate with its proboscis (red arrow). (F and G) Animal probing the substrate with its ovipositor (red arrow). (H) Egg-laying temporal sequence. The physical deposition of an egg starts from “A” and continues to “B.” Between the end of “B” and the beginning of the next “A” is period “C,” in which animals are consistently seen displaying the probing actions described in [(E), (F), and (G)]. Ovi, ovipositor motor program. (I) Time spent on each of the programs described above. “A”: 6.7 ± 0.25 s; “B”:101±4.7 s; “C”: 113.2 ±18.9 s. For programs “A” and “B,” N = 50 egg-layings displayed by four animals from four chambers. For program “C,” N = 77 egg-layings displayed by four animals from four chambers. Error bars indicate SEM. A comparison of behaviors between CantonS and W1118 is shown in fig. S1. (J) Distribution of time spent on the searchlike program. N =77 individual egg-layings displayed by four animals from four chambers. Unless otherwise mentioned, all quantifications in this work were conducted by using CantonS animals or transgenic animals in CantonS background.

We next examined what the females seek by presenting them with different egg-laying options in the behavioral chamber. We placed in the chamber a sweet (sucrose-containing) and a bitter (lobeline-containing) (9) soft agarose medium separated by a region of hard agarose that deters egg-laying and prevents simultaneous detection of the two soft media. Unexpectedly, animals consistently laid more eggs on the lobeline side of the chamber (Fig. 2, A to C and G, and fig. S2). This bias against sucrose medium was not caused by intrinsic attraction to the lobeline medium for egg-laying, however, because animals tended to avoid lobeline when the other option was a plain medium (Fig. 2, E and G), consistent with previous findings on quinine avoidance in egg-laying (10). Furthermore, females avoided laying eggs on the sucrose-containing medium in our chambers no matter whether the alternative was lobeline-containing, plain, or a substrate containing sodium chloride at the same concentration as the sucrose (Fig. 2, A to D and G).

Fig. 2.

Drosophila avoid laying eggs on media containing high levels of sucrose. (A to E) Representative outcomes of various two-choice tests. Single-female assay was used in (C), the rest were three-female assays. S, sucrose; L, lobeline; P, plain. The same designations will be used throughout the figure legend. (F) Animals extending their proboscis onto the sucrose medium (arrows) for foraging. (G) Preference index (PI) of various two-choice conditions (see supporting online material for derivation). N, NaCl; l, agar with 15% grape juice; h, agar with 45% grape juice. “Single” indicates that single animals were used in this assay; no difference was found between PI from single-versus three-female assay (P > 0.5, Mann-Whitney Test). Significant deviations from 0 were found for all conditions tested (P = 0.01 for L/P, P < 0.0001 for the rest, Wilcoxon Signed Rank Test). All box-whisker plots in this work display median, 25/75%, and 10/90% quartiles of each data set. The last entry shows that females prefer agar with less grape juice for egg-laying. The number of assays used and the mean PI are (29, –0.82), (26, –0.79), (10, –0.89), (34, –0.27), (50, –0.83), and (34, –0.62). (H) Females avoid sucrose medium less as its concentration drops in the sucrose versus plain two-choice assays and even showed a slight preference at the 0.5 mM setting (P = 0.0116, Wilcoxon Signed Rank Test). No significant avoidance can be found at 1 mM (P = 0.095, Wilcoxon Signed Rank Test). The rest of the entries all show significant avoidance of sucrose medium (P < 0.0001, Wilcoxon Signed Rank Test). The number of assays used and the mean PI are (49, 0.18), (34, –0.102), (36, –0.33), (34, –0.56), (34, –0.83), and (35, –0.90). (I) For each egg deposited on the lobeline medium in two-choice chambers, animals pay 1.51 ± 0.17 and 1.61 ± 0.15 respective previous visits to each substrate. N = 185 egg-depositions by eight females. (J and K) Hyperpolarizing Gr5a neurons decreases egg-laying bias against the sucrose-containing medium. The difference between animals carrying Gr5a-GAL4 with or without UAS-Kir2.1 is significant (Mann-Whitney Test). Number of assays used and mean PI are (38, –0.52), (40, –0.79), (41, –0.09). ****P <0.001. The exact cause(s) for the reduction of PI of Gr5a-GAL4/+ animals is unclear; however, these animals contain two insertions of Gr5a-GAL4 trans genes, so an excess of GAL4 proteins in these neurons alone could potentially contribute somewhat to reduction of neuronal function.

To determine whether females actually encountered both options before laying an egg on the lobeline medium, we next tracked individual animals for 2 hours and found that for each egg deposited on the lobeline side, the animals had paid an average of 1.5 previous visits to the sucrose option (Fig. 2I). Animals often probed the sucrose substrate actively but rarely activated the ovipositor program while they were still in contact with this medium (Movie S2); in contrast, activation of the ovipositor program was frequently seen when the animals were probing the lobeline medium (Movie S2). Thus, lack of substantial egg-laying on the sucrose medium is not due to animals' not making regular contacts with this medium. General repulsion to the sucrose-containing medium in our chamber is not the cause either—animals were often seen actively feeding on the sucrose medium by extending their proboscis onto it (Fig. 2F and Movie S3). Moreover, the avoidance of egg-laying on the sucrose medium (when it is paired with a plain one) is concentration dependent: It attenuates and can even turn into mild attraction as the concentration of sucrose decreases (Fig. 2H).

The reliance on neuronal activities of the sweet taste receptor (Gr5a) neurons (11) is evident in the experiments employing the GAL4-UAS method to express in these Gr5a-expressing neurons a hyperpolarizing Kir2.1 potassium channel (12). This manipulation significantly increased egg-laying on the sucrose-containing medium (Fig. 2, J and K), which suggests that sucrose detection through the Gr5a neurons is important for the low egg-laying on this medium in the two-choice chamber.

To determine whether the desirability, or “value,” of the sucrose-containing medium for egg-laying might change according to context, we examined egg-laying in single-choice chambers that contain either only the sucrose-containing medium or only the lobeline-containing medium. Interestingly, females lay comparable numbers of eggs in the two single-choice chambers (Fig. 3, A to C); thus, in the absence of other options, females readily accept the sucrose-containing medium for egg-laying. Furthermore, the same sucrose-containing medium can even become a preferred option for egg-laying if it is paired with a more repulsive medium that contains twice the amount of sucrose (Fig. 3, D and E).

Fig. 3.

Avoidance of sucrose-containing medium is context-dependent. (A to C) Comparable numbers of eggs were laid in the two single-choice (sucrose versus lobeline) chambers, P > 0.5, Mann-Whitney Test. Number of assays used and sample means are (39, 67.6) and (38, 67.6). (D and E) Pairing the same sucrose medium (100 mM, S) with different options alters the number of eggs deposited on (D) and the PI against this medium (E). 2S, 200 mM sucrose. Assay number and sample mean for (D) are (29, 7.3), (35, 4.9), (39, 33.26), and (36, 40.89). For (E), (29, –0.82), (35, –0.82), (39, –0.01), (36, 0.37). ****P < 0.0001, **P < 0.005, Mann-Whitney Test. (F) Comparison of the PI shows that animals avoided the sucrose-containing medium less when distance between the sucrose and lobeline media increased. Single females were used. White bars, without food deprivation; gray bars, 24-hour food deprivation before experiments. In both treatments, there were significant differences between 1X and 3X and between 3X and 10X. Food deprivation did not significantly impact PI for all three distances tested. Assay number and mean PI are: (52, –0.84), (23, –0.84), (47, –0.5), (55, –0.46), (31, –0.002), and (35, –0.08). **P < 0.005, ***P < 0.001, Mann-Whitney Test. (G) Exposure assay set-up. Animals were placed in two-choice “exposure” chambers for 1 hour before being transferred into sucrose-only or lobeline-only single-choice chambers. (H) Despite robust preference for lobeline medium in the exposure chambers (S1 versus L1), the difference in egg numbers between the two single-choice chambers [S2(a+b) versus L2(a+b)] is less, although still significant. Number of assays used and the sample means are (69, 2.15), (69, 25.54), (34, 30.09), and (35, 36.16). *P = 0.05, ****P < 0.0001, Mann-Whitney Test.

To explore further how context affects the valuation of sucrose medium, we varied the physical separation between the sucrose- and lobeline-containing media in the two-choice chamber. We found that when the distance between the two choices was increased by a factor of 3 to 10, egg-laying on the sucrose medium progressively increased (Fig. 3F), and some of the individual animals tested actually laid more eggs on the sucrose-containing medium than on the lobeline medium (Fig. 3F). This result suggests that the preference of sucrose medium over lobeline is not absolute and that animals can weigh the “desirability” of egg-laying sites versus the “effort it takes to locate them” in making their decisions. Interestingly, this distance-dependent response to sucrose-containing medium was not altered by food deprivation before assays (Fig. 3F), suggesting that egg-laying and foraging are distinctive tasks with distinctive substrate preferences.

Sequential placement of the same animals first in a two-choice chamber and then in a sucrose medium–only single-choice chamber (Fig. 3G) revealed speedy recovery of egg-laying interests on the sucrose-containing medium (Fig. 3H), suggesting that the substrate evaluation process is efficient and performed predominantly on an egg by egg (search by search) basis. However, compared with the number of eggs deposited in a lobeline medium–only chamber, there were fewer eggs in the sucrose medium–only chamber (S2 versus L2, Fig. 3H), whereas no difference was observed when naïve flies were introduced to the single-choice chambers (Fig. 3C); it thus appears that previous experience may exert some influence in egg-laying decisions. Taken together, our results suggest that Drosophila females possess some neural process that assigns “acceptability” or “value” to a given egg-laying substrate by taking readily into account the availability of other options. Such “value” can then be used by the motor output systems to decode whether a particular option is appropriate to trigger the physical egg-laying action in the given context.

To begin discerning the neural circuitry that underlies egg-laying site selection, we first sought to identify specific neurons that regulate the egg-laying rate as they might be engaged by the “value system” to control egg-laying on a given substrate. Neurons that express an insulin-like neuropeptide, ILP7, are of interest because ILP7 shares some homology with Relaxin, an important reproductive hormone in mammals (13, 14). Our antibodies revealed that ILP7 is present in only very few cells in the larval and adult central nervous system (Fig. 4, A and B, and fig. S6), with some cells sending projections to the subesophageal ganglion (SOG) and distinct positions in the ventral nerve cord (Fig. 4A, B), which are all sites where gustatory information relay might occur. Moreover, projection of ILP7 to the female internal reproductive tract can also be found (Fig. 4C), in support of the notion that ILP7 may be the Drosophila Relaxin. Interestingly, many of the ILP7-neurons are also positive for fruitless expression in adult males (fig. S7). This result suggests a potential role of these neurons in male reproductive behavior, because fruitless has been shown to be a master regulator of male courtship behavior in Drosophila (15).

Fig. 4.

Adequate level of ILP7 is important for normal egg-laying and proper bias in egg-laying site selection. (A to C) ILP7 antibody staining in adults. (A) ILP7-neurons send projection into SOG (red arrow) in the brain. (B) ILP7 is present in very few cells (blue bracket), with distinctive projection in the thoracic segments in ventral nerve cord (red arrow). (C) ILP7 is also present on the female internal reproductive tract. (D and E) Specificity of ILP7 antibodies was confirmed by the reduced signal in animals that overexpress an ILP7-RNAi construct (E). (D) Staining control. (F to H) ILP7-GAL4 specificity was confirmed by the colocalization of signals from ILP7 staining (F) and UAS-nuclear-RFP expressed by ILP7-GAL4 (G) in the same cells (H). (I) Reducing neuronal function of ILP7-neurons through Kir2.1 overexpression causes a loss of ovipositor program. For the two control groups, number of eggs laid was used to substitute for the number of ovipositor program. The number of assays and the sample means are (20, 59.7), (20, 67.2), and (10, 0). ****P < 0.0001, Mann-Whitney Test. (J) Animals carrying an HS-ILP7 construct or overexpressing UAS-ILP7 by ILP7-GAL4 avoided sucrose medium significantly less than control animals in sucrose versus lobeline two-choice assays. Number of assays used and sample means are (21, –0.87), (23, –0.51), (15, –0.73), (20, –0.86), and (20, –0.11). ****P < 0.0001, Mann-Whitney Test. HS-ILP7 animals that were raised at 18°C show comparable sucrose avoidance to control animals in sucrose versus plain two-choice assays (P > 0.5, Mann-Whitney Test). Number of assays used and sample means are (14, –0.88) versus (34, –0.9).

We next created an ILP7-GAL4 to specifically alter ILP7-neurons' function (Fig. 4, F to H, and fig. S4). Using the GAL4-UAS approach, we found that females with hyperpolarized ILP7-neurons showed no discernable developmental defects but displayed virtually no ovipositor motor programs and are thus sterile (Fig. 4I). To distinguish between the possibility that ILP7-neurons are required merely for housekeeping processes to push the egg through the internal reproductive tract [which could account for the fully penetrant egg-jamming phenotype due to silencing ILP7-neurons (fig. S5)] and the possibility that ILP7-neurons also participate in conveying the “acceptability” of potential laying options to the reproductive system (e.g., the reproductive tract and the ovipositor motor program), we examined the effect of elevated ILP7 level—in ILP7-neurons or ubiquitously—on egg-laying choice (Fig. 4J and fig. S6). In both cases, the elevation of ILP7 level caused the animals to be more receptive to laying eggs on the sucrose medium in the “regular” two-choice chambers (Fig. 4J). These results are consistent with the idea that ILP7 might participate in the relay of the appropriateness of a given option to the reproductive systems to execute egg-laying on that option.

Drosophila females can accept or avoid a given sucrose-containing medium for egg-laying depending on context. Whereas such context-dependent avoidance of high-sucrose medium shows little discernable advantage in the laboratory setting [embryo hatching rate on sucrose-containing and lobeline-containing media are comparable (fig. S3)], it could have been selected by virtue of predation avoidance and larval dietary balance (protein/carbohydrate ratio). Our finding that Drosophila employs a simple decision-making process in selecting egg-laying sites raises the possibility that the fruit fly has the capacity to compare and assess available options by performing integrations and amplifications in its nervous system. Dopamine and octopamine are both candidates for mediating such amplification/integration processes: The former is important for decision-making in primates and flies (1618) and is used to signal the unconditional stimulus of “punishment” during learning tasks in Drosophila (19, 20); the latter is a reinforcing signal for appetitive conditioning in both Drosophila and honeybees (1921) and an important regulator of egg-laying in Drosophila (22). In addition, our work suggests that the ILP7-expressing neurons are important for proper execution of egg-laying decisions, thus providing an additional anatomical and molecular entry point into dissecting the decision-making processes during egg-laying site selection.

Supporting Online Material

www.sciencemag.org/cgi/content/full/319/5870/1679/DC1

Materials and Methods

Figs. S1 to S7

Movies S1 to S3

References and Notes

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