Subjects

We tested wild type larvae of natural Chilean populations (Til-Til and Trana strains) and laboratory stocks (Oregon R-c and Canton – Special strains) of D. melanogaster. We also examined larvae of the vestigial (vg) strain. The Oregon R-c and vg strains differ in some larval behaviors. For example, Oregon R-c larvae dig deeper into the substratum than vg larvae [27]. We also tested three neurological mutants derived from the Canton-Special (CS) strain of D. melanogaster. Larval perception of odorants in the Orco mutant strain has been blocked because the dendritic localization of the receptors has been eliminated. Thus, the Orco mutation disrupts behavioral and electrophysiological responses to many odorants [29]. The Syn^97CS mutation affects presynaptic vesicle release in the entire larval brain, and olfactory associative learning is reduced in approximately 50% with respect to CS larvae; however, responsiveness to stimuli and motor performance in untrained animals are normal [28]. The rut locus participates in olfactory conditioning learning in D. melanogaster and it is expressed in neurons located in larval and adult mushroom bodies; rut does not affect larval locomotion and responsiveness to stimuli [30].

In 1952, Professor Danko Brncic brought the Oregon R-c and vg strains from Columbia University (USA) to the University of Chile. Dr Bertram Gerber, University of Würzburg in Germany sent the Canton-Special (CS), Orco, Syn^97CS and rut strains to our laboratory. The wild type Trana strain of D. melanogaster was established with 22 adults that emerged from 20 decaying grains of grape (Vitis vinifera, País variety); Trana is located 380 km southwest of Santiago. The wild type Til-Til strain of D. melanogaster was formed with 18 adults that emerged from three decaying prickly pear fruits collected in Til-Til, which is 50 km northwest of Santiago. The Til-Til and Trana larvae in the laboratory experiments were the fourth generation.

To investigate larval olfactory recognition in other Drosophila species, we examined the endemic South American sibling species D. pavani and D. gaucha (Subgenus Drosophila, mesophragmatica group) and the F₁ reciprocal hybrids [24]–[26]. The mesophragmatica group comprises many South American species and forms a phyletic unit [24]. D. pavani is predominantly Andean in distribution, whereas D. gaucha is distributed in Argentina, Uruguay and Southern Brazil. Both species share characteristics with domesticated species in that the adults can be collected from orchards, garden, and other plants associated with human activities [26]. Under laboratories conditions, the two species can produce viable but sterile hybrids [31]. D. pavani and D. gaucha have similar development durations for molting, wandering and pupating [25]. D. pavani and D. gaucha require approximately one and half year to assimilate to laboratory conditions. For this reason we tested the La Florida and Buenos Aires strains (see below).

In 1978, Professor Danko Brncic founded the La Florida strain of D. pavani with approximately 18 individuals collected from banana traps in La Florida, Santiago (Chile). Same prickly pear plants remain in this neighborhood today. In 1989, Dr Esteban Hasson sent the Buenos Aires strain of D. gaucha to us. This strain was created with 10 adults collected from banana traps in Buenos Aires (Argentina). The traps were established near prickly pear plants. The sex ratio was variable for the all strains.

The strains were all maintained by mass culture at 24±1°C, 70% humidity (D. melanogaster) and 18±1°C, 80% humidity (D. pavani and D. gaucha). D. pavani and D. gaucha grow better at this temperature and humidity than at 24°C. All stocks were maintained under constant light, because facilities to change the light/dark period were not available in the laboratory.

Crosses

Fifteen-day-old D. pavani (La Florida strain) and D. gaucha (Buenos Aires strain) males and females were reciprocally crossed. At this age, individuals are sexually competent [24]. Homogametic mating within strains also served as controls for the interspecific crosses. Crosses between the La Florida (D. pavani) and Buenos Aires (D. gaucha) strains provided abundant hybrid larvae of the two sexes [31].

Larva collection

Groups of 40–50 inseminated females of D. melanogaster, D. pavani, D. gaucha, and D. pavani females and D. gaucha females crossed with males of the other species were allowed to oviposit for 2–3 h on plastic spoons containing the culture medium. Thirty eggs of the species, strains and hybrids were randomly collected with a dissecting needle. Each batch of eggs was incubated on fresh spoons for 96–100 h at 24°C (D. melanogaster strains) and 168–172 h at 18°C (D. pavani, D. gaucha and the hybrids). D. melanogaster larvae emerged after 2–4 h. D. pavani, D. gaucha and hybrid larvae emerged 48 h after the eggs were laid. The culture medium was supplemented daily with 40% fresh baker’s yeast paste. One hour before an experiment, third-instar larvae were collected from the glass wall of rearing vials, washed twice with distilled water, and identified by the presence of protruded anterior spiracles [32]. All larvae were raised in half-pint bottles at 24°C (D. melanogaster) and 18°C (D. pavani and D. gaucha) on Burdick’s medium [33].

Larval odor recognition and pupation site selection

We employed three treatments to test the response of D. melanogaster larvae to conspecific and D. pavani larval odors. In the first treatment, one 2.0×2.0 cm piece of Whatman cellulose filter paper was moistened for 1 h in Burdick’s medium used for 4–5 days by D. melanogaster (or D. pavani) larvae, whereas another identically sized filter paper was moistened in sterile Burdick’s medium for a similar period of time. Before transferring the two filter paper types to Petri dishes, each piece of paper was carefully examined under stereomicroscope to verify that no food was adhered to the surface. In the second treatment, one piece of paper was impregnated for 1 h with food worked by larvae of the strain (Canton-Special, Til-Til, Trana, vestigial, Orco, Syn^97CS and rut strains of D. melanogaster). The other filter paper was moistened for a similar time with food processed by the Oregon R-c larvae. The test for larvae of this strain was an Oregon R-c filter paper, and a Canton – Special filter paper. In the third treatment, the larvae were placed together with a filter paper that had been moistened for 1 h in food occupied by larvae of D. melanogaster strains, and another moistened for a similar time with the food used by D. pavani larvae. These treatments were also applied to D. pavani, D. gaucha and the interspecific reciprocal hybrids.

For each treatment, 10-cm Petri dishes were filled with 10 ml of 3% agar gel. Each filter paper type was deposited onto agar in Petri dishes on opposite sites at 6 cm one from the other. Batches of 20 third-instar larvae of each species and strain were introduced into corresponding Petri dish and gently deposited onto the middle of the agar 3 cm from each piece of paper. Once the larvae actively moved, we transferred the Petri dishes to the culture room maintained at 24±1°C (D. melanogaster), and 18±1°C (D. pavani, D. gaucha and the hybrids). To decrease possibilities that temperature differences and illumination conditions would interfere with pupation site preferences, we always deposited the Petri dishes in the identical location in the culture room. The number of pupae on/under each paper was recorded two (D. melanogaster), four–five days later (D. pavani, D. gaucha and the hybrids). Some pupae were observed on the agar surface near the paper. Those individuals detected within 10 mm of the border were counted as belonging to that paper. Replicate measurements (10 measurements, 200 larvae) were performed for each strain by dividing the larvae into groups of 5 replicates and conducting the measurements in parallel. Thus, we were able to estimate magnitude of environmental odor variation on larval response to chemical cues. We did not know whether the substances present in the paper could diffuse through the agar, which might have introduced an additional source of experimental error to our measurements because of possible larval gustatory responses. To address this variable, we tested Orco mutant larvae. There were no odor-evoked responses for the specialized olfactory sensory neurons of Orco larvae. The Orco mutation does not affect gustatory neurons [29].

We also monitored the pupa distributions on the agar in the virgin food/D. pavani food and virgin food/Oregon R-c food treatments. In the first treatment, wild type and vg pupae of D. melanogaster were clumped across the agar, whereas Orco, Syn^97CS and rut pupae were randomly distributed. In the virgin food/Oregon R-c food treatment, wild type, vg, Orco, Syn^97CS and rut pupae were scattered across the agar. We calculated the Clark and Evans aggregation index [34] to compare the pupa distributions in the two treatments. We estimated the average distance to the nearest neighboring (rA) pupa using a 0.5-cm Cartesian grid and then compared this average value with the expected value (rE) for the identical number of individuals randomly distributed on an area of equal size (rE = ½√ρ), where ρ is the pupa density. The ratio R = rA/rE, reflects the spatial distribution of the individuals (aggregated, random, over-dispersed) with values ranging between R = 0.0 (maximum aggregation) and R = 2.15 (uniform). When individuals are randomly distributed, R = 1.0 [34]. We followed a similar procedure for the pupa distributions of D. pavani (La Florida strain), D. gaucha (Buenos Aires strain) and the reciprocal interspecific hybrids in the virgin food/D. pavani (D. gaucha, hybrid) food, and virgin food/Oregon R-c food treatments.

All behavioral tests were performed between 10∶00 and 14∶00 hours under controlled temperature, 22.00±1.0°C, light, 6.2±0.2×10⁻⁵ lx, and humidity, 70±3%.

Statistical analysis

We tested for homogeneity between replicates within a treatment and strain. We applied a G-test of Independence to compare the pupa percentages: N = 10 replicates per strain, N = 8 strains of D. melanogaster, N = 3 treatments. The test was also applied to the pupa percentages obtained for replicated experiments: N = 10 replicates within a treatment, N = 2 treatments, with D. pavani, D. gaucha, and hybrid larvae, N = 4 genotypic groups. None of the individual chi-square tests detected a significant deviation from the hypothesis of homogeneity between the replicates within a strain or treatment (the Chi-square values were all lower than the critical value χ² = 23.59, df = 9; P>0.05; Table S1). Thus, we pooled the replicate data within treatments, strain, and species.

We also applied a binomial test to the data. The null hypothesis stated that there was no difference between the probability of selecting one filter paper type and the probability of selecting another paper within a treatment or strain (p = q = ½). The binomial test was selected because the data were in two discrete categories and the experimental design utilized one sample (see above homogeneity between replicates within a treatment and strain and Table S1). The rejection region of the null hypothesis was equal or less than α = 0.01.

An analysis of variance was applied to compare pupa aggregation indexes (R) of D. melanogaster in the presence and absence of D. pavani larval volatiles. The analysis was also applied to pupa aggregations of D. pavani, D. gaucha and the hybrids stimulated by D. melanogaster larval volatiles.

Pupation site selection: D. melanogaster

More than 60% of wild type and vg pupae of D. melanogaster were on the conspecific paper, thus indicating that they were stimulated by odors emanating from the two types of paper one moistened with sterile food and the other with food processed by larvae of the own strain (Fig. 1 A–E). Differences in pupa percentages on the two paper types are statistically important (Table S2). By contrast, confronted with paper moistened with virgin food and food worked by D. pavani larvae more than 60% of the wild type and vg D. melanogaster pupae were located on the agar, and approximately 17% were located on the two paper types (Fig. 1 I–M, and Table S2). Clearly, the wild type and vg D. melanogaster larvae recognized and responded to conspecific and D. pavani chemical cues. In the two treatments, more than 60% of the Orco pupae were scattered across agar (Fig. 1 F–N, and Table S2), thus suggesting that the Orco larvae did not detect the odors emitted from the two paper types. In a similar way, stimulated by conspecific and D. pavani larval odors, Syn^97CS and rut larvae also were randomly distributed across agar (Fig. 1 N–P, and Table S2). Thus, the Orco, Syn^97CS and rut loci are necessary for pupation site selection in D. melanogaster.

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Figure 1. Pupation behavior of third-instar larvae D. melanogaster stimulated by the odors emanating from filter paper moistened with virgin food, food processed by conspecific larvae, and food worked by D. pavani larvae.

Preferences are shown as percentage of pupae ± SE on the papers, N = 10 replicates, 200 larvae per strain. White column (A–P), filter paper moistened with virgin food. Black column, filter paper moistened with food worked by larvae of the strain (A–H) and D. pavani larvae (I–P). The strains tested were: Oregon R-c (Or) and Canton-Special (CS), wild type, laboratory stocks, and Til-Til (TT) and Trana (Tr), wild type, natural populations. Mutant strains were also tested: vestigial (vg); Orco, dendritic localization of odorant receptors is abolished; Syn^97CS learning mutant, deletion for phosphoproteins in presynaptic terminals; rut learning mutant, encodes a calmodulin dependent adenylate cyclase that converts ATP to cyclic AMP. Statistical significance is in Table S2.

https://doi.org/10.1371/journal.pone.0102159.g001

The pupa aggregation (R) indexes were calculated for the wild type and mutant strains of D. melanogaster. In the virgin food/D. pavani food treatment (Fig. 2 A–H), the aggregation R-values for the wild type and vg strains of D. melanogaster were approximately R = 0.0, which indicated grouped pupae (Fig. 2 A–E, black circles). In the presence of chemicals emanating from the two types of papers moistened with virgin food and Oregon R-c food, the calculated R-values were approximately R = 1.0, which indicated that the pupae were randomly distributed across the substrate (Fig. 2 A–E, white triangles). These differences in the pupa aggregation indexes were statistically important (ANOVA, Table S3). In the presence of conspecific and alien larval odors, the pupa aggregation (R) indexes of the Orco, Syn^97CS and rut strains were approximately R = 1.0, which confirmed that the larvae pupated randomly across the agar (Fig. 2 F–H, and Table S3). The results again indicated that these three loci are necessary for D. melanogaster pupation behavior. For all strains tested, the aggregation indexes were independent of the number of pupae on the agar (Fig. 2 A–H).

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Figure 2. Pupa aggregations indexes (R) and number of pupae on agar of the Oregon R-c (A), Canton-Special (B), TIl-Til (C), Trana (D), vestigial (vg) (E), Orco (F), Syn^97CS (G), and rut (H) strains of D. melanogaster.

White triangle indicate aggregation in the presence of conspecific larval odors; black circle, aggregation in the presence of D. pavani larval odors. R = 1.0 indicates that individuals are randomly distributed over substrates. R = 0.0, maximum aggregation. For statistical significance, see Table S3.

https://doi.org/10.1371/journal.pone.0102159.g002

Fig. 3. A–E shows that D. melanogaster larvae that were stimulated by conspecific odors used the two types of paper to pupate. However, in the presence of conspecific and D. pavani larval cues, the wild type and vg D. melanogaster pupae were preferentially observed on the conspecific paper (Fig. 3 I–M; Table S2). In the two treatments, similar percentages of Orco, Syn^97CS and rut pupae were found on the two paper types (Fig. 3 F–H and N–P; Table S2). These results and those in Figs. 1 and 2 again indicate that in D. melanogaster olfaction is required for pupation site selection and that genes expressed in the neurons of the peripheral olfactory circuits [29], synapses [11], and mushroom bodies [2] participate in D. melanogaster pupation behavior.

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Figure 3. Pupation site selection in the presence of conspecific and D. pavani larval cues in D. melanogaster larvae.

A–H; white column, filter paper moistened with food used by larvae of the strain; black column, paper moistened with Oregon R-c food. I–P, white column, filter paper moistened with food of the strain; black column, paper moistened with food processed by D. pavani larvae. For details, see Figure 1; statistical analysis in Table S3.

https://doi.org/10.1371/journal.pone.0102159.g003

Pupae were also detected outside the two paper types on the Petri dish agar. In the treatment with two types of conspecific food (Fig. 3 A–H), a few pupae were present on agar. The calculated (R) aggregation indexes (N = 10 replicates) were (i) Oregon R-c, 0.88±0.02; (ii) Canton-Special, 0.83±0.09; (iii) Til-Til, 0.70±0.04; (iv) Trana, 0.91±0.05; (v) vg, 0.79±0.06; (vi) Orco, 0.81±0.08; (vii) Syn^97CS, 0.95±0.10; and (viii) rut, 0.97±0.05. In the conspecific food/D. pavani food treatment (Fig. 3 I–P), 79±2.31% of the wild type and vg D. melanogaster pupae were adhered one another, as observed in nature (Figs. S1 and S2). In these two identical treatments, the Orco, Syn^97CS and rut pupae were scattered across the substrate. These results are consistent with those in Figs. 1–3.

Pupation site selection: the sibling species D. pavani and D.gaucha

In the virgin food/conspecific food treatment, approximately 68% of D. pavani and the pavani female x gaucha male hybrid larvae pupated on the conspecific paper (Fig. 4 A, C, Table S2). In this identical situation, D. gaucha and the gaucha female x pavani male hybrid larvae used both paper types to pupate (Fig. 4 B, D, Table S2). Notably, more than 70% of the pupae of the four groups of genotypes were located on the agar, and less than 14% were located on the paper types moistened with virgin food and food processed by D. melanogaster larvae (Fig. 4 E–H, Table S2).

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Figure 4. D. pavani, D. gaucha and the hybrids pupation site preferences.

The larvae were stimulated by signals emitted from a paper moistened with virgin food and a paper moistened with food worked by conspecific larvae (A–D), and from a paper moistened with virgin food and a paper moistened with the Oregon R-c strain (E–H). For details, see Figure 1; statistical analysis in Table S2.

https://doi.org/10.1371/journal.pone.0102159.g004

Figure 5. A–D shows that D. pavani and the pavani female x gaucha male hybrid larvae that were stimulated by D. melanogaster larval volatiles pupated near congeners (Fig. 5 A, B, black circles). Aggregation decreased in the presence of conspecific larval chemicals (Fig. 5 A, B, white triangles). These differences between the two treatments were statistically important (Table S3). The results confirmed that olfaction is needed for pupation site selection in D. pavani and pavani female x gaucha male hybrid larvae. By contrast, in the presence of conspecific and D. melanogaster larval cues, D. gaucha and gaucha female x pavani male hybrids randomly pupated across the agar (Fig. 5 C, D, white triangles and black circles), which suggested that these larvae did not use olfaction to select their pupation sites.

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Figure 5. Pupa aggregation indexes (R) and number of pupae on agar of D. pavani (A), D. gaucha (C), and the interspecific hybrids (B, D).

White triangle aggregation in the absence of D.melanogaster larval cues; black circle aggregation in the presence of D. melanogaster larval signals. For details, see Figures 1 and 2; statistical analysis in Table S3.

https://doi.org/10.1371/journal.pone.0102159.g005

Stimulated at the same time by conspecific and D. melanogaster larval cues, D. pavani and pavani female x gaucha male hybrid larvae pupated more on conspecific target paper than D. melanogaster paper (Fig. 6 A, B, Table S2). However, a similar percentage of D. gaucha and gaucha female x pavani male hybrid pupae were detected on the two paper types (Fig. 6 C, D, Table S2). Clearly, in the two sibling species divergent evolutionary changes in the organization and function of the larval nervous system involved in pupation behavior have occurred.

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Figure 6. D. pavani (A), D. gaucha (C) and the interspecific hybrids (B, D) pupation site preferences.

Preferences are expressed as percentages ± SE of pupae on a paper moistened with food worked by conspecific larvae, white column, and on a paper moistened with food processed by D. melanogaster larvae (the Oregon R-c (Or) strain) paper, black column. For details, see Figure 1; statistical analysis in Table S2.

https://doi.org/10.1371/journal.pone.0102159.g006

D. melanogaster

D. melanogaster has a shorter larval period than D. pavani - 4 to 6 days versus 8 to 12 days, at 24°C - [32]. This difference may mean that D. melanogaster larvae searching for pupation sites do not encounter D. pavani larvae and/or pupae. However, this cannot account for why the third-instar larvae of the two species modified their pupation behavior when stimulated by the odors of the other species. Perhaps conspecific and alien larvae identification occurs when first and second instar larvae are feeding within the breeding sites. Therefore, it is important to investigate whether first and second instar larvae of Drosophila associate larval odors with the presence of conspecific and alien larvae. Further studies are planned to investigate this possibility.

The chemicals present in the paper used could diffuse through the agar. Thus, larval gustatory responses could be also involved in our measurements. Nevertheless, in the presence of conspecific and D. pavani larval odors, the Orco larvae pupated randomly across the agar, and distance between nearest neighboring Orco pupae did not change. These mutant larvae were without the Orco odor co-receptor but had gustatory ones [29]. The Orco mutation interferes with larval odor recognition, thus affecting pupation behavior. These results confirm that olfaction, not gustation, is essential for pupation site selection in D. melanogaster.

On the other hand, stimulated by conspecific odors and D. pavani odor cues, Syn^97CS and rut larvae also pupate randomly across the Petri dish agar. Thus, these two mutations cause notable changes in the pupation behavior of D. melanogaster. These two loci affect olfactory associative learning in D. melanogaster [28], [30]. We suggest that larval odor-based learning could be involved in the pupation behavior of D. melanogaster. D. buzzatii and D. simulans larvae reared in isolation from conspecifics pupate randomly across the substrate, but form pupa aggregations when reared with congeners [14]. Notably, larvae of Drosophila hydei, subgenus Drosophila, repleta group, hydei subgroup and Drosophila busckii, subgenus Dorsilopha also form pupa aggregations away from D. melanogaster and D. pavani pupae [15]. These results suggest that social larval odor-based learning could be an integral component of pupation site selection and it could be widely extended throughout the genus Drosophila.

Larvae of many Drosophila species develop within unpredictable, heterogeneous and rapidly changing breeding environments [8], [16]–[18], [35]. Fermentation increases the acidity of fruits, and microbial action also produces alcohols, esters and other odoriferous, toxic substances that cumulate within the breeding sites surrounding the larvae [14], [17], [18]. On the other hand, decaying fruits are exposed to desiccation reducing their availability as breeding sites. These are all heterogeneous processes because some sections of a fruit may be in an advanced state of dryness and decay, whereas desiccation and fermentation are only beginning in other sections of the fruit. The presence of other species of larvae within the identical decaying fruit unit is another difficult variable to predict. In Chilean orchards three to four Drosophila species may emerge from a fruit, whereas only one species may emerge from the identical fruit type located 15–18 cm away (unpublished data). Thus, many biotic and abiotic variables cause important ecological changes to which Drosophila larvae must respond. In these changing environments, larval odor-based learning could be important to pupation behavior [21]. The spatial positions on the breeding sites where conspecifics emit odor signals could be associated with favorable locations to pupate. Alien larval odors could be indicative of an unfit area colonized by the larvae of other species. Orco, Syn^97CS and rut mutations interfere with the association process by causing disturbances and blocking the recognition of odors of congeners and alien larvae. Thus, Orco, Syn^97CS and rut loci are crucial for D. melanogaster pupation behavior because they influence (i) the spatial orientation of larval movements, (ii) the identification of conspecifics and (iii) the ability to form groups away from alien larvae.

D. pavani and D. gaucha

Notably, when stimulated by D. melanogaster larval cues, D. pavani pupae were grouped, but D. gaucha pupae were observed scattered across the substrate. Thus, D. gaucha larvae show a markedly different behavior to that of D. pavani larvae. Namely, D. gaucha larvae did not appear to use the olfactory system to select their pupation sites. However, these conjectures could be not completely correct. Fig. 4 B shows that approximately 35% of D. gaucha pupae were present on the conspecific and sterile food impregnated paper, but in the sterile food/Oregon R-c food treatment, 10% of the pupae were located on the paper (Fig. 4 F). These results may be indications that D. gaucha larvae detected D. melanogaster larval volatiles. However, as in nature, D. gaucha was ecologically isolated from D. melanogaster, this result could have occurred because of a delay in processing and transmitting information to central structures. In D. melanogaster, larval olfactory responses may change according to the post-eclosion odor experience [37]. Further experiments will be required to clarify these results.

D.pavani x D.gaucha hybrids

The pupation behavior of the D. pavani x D. gaucha hybrids confirms that the larvae of the two species differed remarkably in their olfactory sensitivity. The pavani female x gaucha male hybrid larvae used olfaction to select their pupation sites, but the gaucha female x pavani male hybrid larvae did not do. However, in the presence of D. melanogaster larval cues, the pavani female x gaucha male hybrid pupae were aggregated, but the gaucha female x pavani male hybrids pupated randomly across the substrate. These results indicate that in D. pavani and D. gaucha genes on the X chromosome may have important behavioral effects.

Alternative alleles appear to be localized on the X chromosome of each species. Reciprocal differences in behavior in crosses between the species could also reflex cytoplasmic heredity. Relatively little attention has been paid to olfactory responses in intra-and interspecific hybrid Drosophila larvae. These investigations may provide key information about the neurogenetics and evolution of behavior in sibling species of Drosophila. Although our experiments demonstrate significant differences in odor responsiveness between D. pavani and D. gaucha, definitive evidence will require an analysis of chemosensory receptors in larvae of the two species.

Our study suggests that in species of Drosophila that coexist in the wild and belong to phylogenetically distant clades, such as D. melanogaster, subgenus Sophophora, and D. pavani, subgenus Drosophila, olfaction is used for selection of pupation sites. In D. gaucha which is phylogenetically close to D. pavani, the larvae do not coexist with D. melanogaster or D. pavani and do not require olfactory signals to select pupation sites. Thus, our results emphasize that the presence of larvae from two or more Drosophila species at breeding sites is significant to understand the role of olfactory system and most likely larval odor-based learning in pupation site selection. Our investigation also emphasizes the significance of genotype, thus providing some information about the genetics of neural circuits underlying to pupation behavior.

In summary, Drosophila larva pupation behavior contributes to the partition of space between species with similar ecologies. This behavior has implications for a stable coexistence of many Drosophila species in nature. Pupation behavior also helps to organize Drosophila communities so that they persist on ephemeral and fragmented breeding sites. The larvae appear to produce some pheromones similar to those observed in adults [16]. In Drosophila, imagoes produce cuticular pheromones that chemically correspond to unsatured long-chain hydrocarbons, and the position of double bonds is key for recognize partners. In D. melanogaster, males produce abundant monoenes, such as 7- tricosene and 7-pentoacosene; whereas females produce 7, 11-heptacosadiene and 7, 11-nonacosadiene [38]. Some components of salivary gland secretions might function as pheromones in the larvae. Further studies will be necessary to support this hypothesis.