The behavior of adult Drosophila in the wild

Luis Soto-Yéber; José Soto-Ortiz; Pablo Godoy; Raúl Godoy-Herrera

doi:10.1371/journal.pone.0209917

. 2018 Dec 31;13(12):e0209917. doi: 10.1371/journal.pone.0209917

The behavior of adult Drosophila in the wild

Luis Soto-Yéber ^1,^#, José Soto-Ortiz ^1,^#, Pablo Godoy ^2,^¤,^#, Raúl Godoy-Herrera ^2,^*

Editor: Matthieu Louis³

PMCID: PMC6312304 PMID: 30596767

Abstract

Little is known about how Drosophila adults behave in the wild, including mating, allocation of food and space, and escape from predators. This lack of information has negative implications for our ability to understand the capabilities of the nervous system to integrate sensory cues necessary for the adaptation of organisms in natural conditions. We characterized a set of behavioral routines of D. melanogaster and D. simulans adults in three ecologically different orchards: grape, apple and prickly pear. We also investigated how the flies identify conspecifics and aliens in the wild to better understand relationships between group formation and adaptation of Drosophila to breeding sites. We characterized the locations by recording in each orchard humidity, temperature, illumination conditions, pH of fruits, the presence/absence of other Drosophila species and the predator ant Linepithema humile. Our findings suggest that the home range of these species of Drosophila includes decaying fruits and, principally, a variety of microhabitats that surround the fruits. The ecological heterogeneity of the orchards and odors emitted by adult D. melanogaster and D. simulans influence perch preferences, cluster formation, court and mating, egg-laying site selection, and use of space. This is one of the first large examinations of the association between changing, complex environments and a set of adult behaviors of Drosophila. Therefore, our results have implications for understanding the genetic differentiation and evolution of populations of species in the genus Drosophila.

Introduction

Interactions between parts of an organism and between complete organisms intersect in the concept of the biological organization, extending from the molecular level, such as assemblages of chemosensory proteins in Vibrio cholerae, to grouped distributions of animals in the wild [1, 2]. Such reciprocal influences ensure that biological organization is expressed in a coordinate manner in relation to environmental changes [3]. How animals act to affect survival, distribution and behavior of other animals is a central problem in ecology and evolution [4–7]. Association with conspecifics provides an individual animal a variety of benefits, such as opportunities to mate, the construction of habitats such as nests, and protection from predators. Conspecific aggregations may lead, however, to competition for food and space that affects individual fitness. In addition to a variety of signals emitted and processed by animals to attract and evade other animals, the heterogeneous distribution of resources may favor interactions between individuals [8]. The ability to move is another factor involved in interactions between animals [9–11]. Therefore, the capability to attract conspecifics and avoid or evade adults of other species has a principal role in the adaptation of animal species in natural conditions and has implications ranging from formation of social groups to the emergence of new species.

In this context, it is surprising that little research has been reported on how Drosophila adults behave in the wild. For example, associations with conspecifics and aliens have rarely been studied in wild populations of Drosophila by observation and experiment. Such an omission necessarily hinders and weakens comprehension of the role of behavior in the evolution of species in the genus Drosophila [12]. Larvae and imagoes have a remarkable variety of receptors and brain structures that process miscellaneous stimuli [13], suggesting an inherent flexibility to adapt to a wide range of complex environmental situations. In fact, responses to the odors emitted by Drosophila larvae are critical for the identification of conspecifics and aliens [14–16]. Species-specific odorants emitted by larvae of D. melanogaster, Drosophila simulans, Drosophila buzzatii, Drosophila hydei and Drosophila pavani participate in conspecific aggregation and provide cues regarding food availability and the physical features of pupation sites [17–19]. In larvae of D. melanogaster, Orco, Syn^97CS and rut loci are involved in cluster formation, orienting navigation toward conspecifics [15]. In this same species, a single larval chemosensory neuron is critical for detecting long-chain fatty acids that attract conspecifics [20]. Natural populations of D. melanogaster show genetic variation in their ability to identify conspecific larvae, suggesting that this behavior is under evolutionary selection [15,19]. Laboratory studies on the neurobiology of courtship and mating, egg-laying site selection and foraging confirm the importance of odor cues in the behavior of Drosophila [14–16, 21–23]. Adults of a number of Drosophila species are attracted by yeast odors, leading to aggregation around food in the wild [24]. These findings support the hypothesis that odor-based behavior of larvae and adults is important in the ecology and evolution of Drosophila and contribute to adaptation to the variable and changing environments in which these two life-stages live.

We detected groups of D. melanogaster adults away from clusters composed of D. simulans flies in a grape orchard, suggesting that imagoes of the two species can identify conspecifics and aliens at distance. Recognition could be based on species-specific odorants [14, 25, 26]. For adult D. melanogaster and D. simulans that coexist in the same orchard, the referenced clusters could favor conspecific mating and prevent hybridization between species [27]. This notion requires that, for example, species-specific odorants attract conspecifics, repelling flies of alien species. We investigated this proposition by observing and comparing routines of D. melanogaster and D. simulans adults in three ecologically different orchards. We also taxonomically classified groups of flies collected in the orchards. We substantiated our observations by testing the capability of virgin and non-virgin D. melanogaster and D. simulans adults to recognize at distance other individuals. We discuss some consequences of such behavior for the ecology and evolution of species in the genus Drosophila.

Materials and methods

Orchards

We observed routines of Drosophila adults in the wild in Til-Til, 33° 05’ 00” S 50 km Northwest Santiago at the end of the Chilean summer and in autumn (March to May). In these months, Drosophila populations in Chile reach their peak abundance [28]. Observations were made between 9:00 am and 6:00 pm in three types of orchards: (i) grape (Vitis vinifera, Sultanina, Red Globe and Crimson Seedless varieties; Figs 1–3), (ii) apple (Malus domestica, Red Delicious variety) and (iii) prickly pear (Opuntia ficus-indica, Fig 4). We are grateful to Don Juan Ignacio Herrera and Don Gonzalo Herrera, owners of the Fundo La Capilla in Til-Til, who gave permission to collect flies and fruits, for their tolerance while we conducted our study on their land.

Fig 1 — The plants are Sultanina variety.

Fig 4 — The orchard is located in Til-Til, 2 km away from the grape orchard shown in Fig 1.

We recorded humidity and temperature at different sites in each of the orchards (N = 16 measures per orchard and parameter). The pH of mature grape, apple, prickly pear fruits hanging from the plants and fresh pieces of cladodes 5 cm in diameter cut from the plant (N = 50 units per type of fruit and tissue) was also recorded. We followed the same procedure for decaying fruits and tissues.

Fly clusters in the wild

Drosophila adults were grouped in the grape orchard. Lying on the ground, we crawled toward clusters of flies perched on decaying grape bunches and the dry leaves of grape plants (Figs 2 and 3) to prevent the flies from detecting our presence and fleeing. By using a 30 x 0.5 cm (length x width) glass aspirator, we carefully collected each group of flies [29]. This procedure was also employed to collect flies in the apple orchard. Some flies escaped after detecting our presence and so were not collected from those groups; escaped adults may have belonged to other Drosophila species. In the prickly pear orchard, an abundance of cladode thorns were a formidable obstacle that impeded use of the aspirator to collect adults. However, with great patience, we managed to collect some flies. Each cluster was deposited into a 10 x 2.5 cm (length x width) labeled vial. Each vial contained 2.5 cm³ of Drosophila Burdick’s medium [30].

Fig 2 — The orchard is in Fig 1. Adult flies of D. *melanogaster* are enclosed by a red oval; those of D. *simulans* are encircled by a yellow oval.

By examining external genitalia and morphological traits, we taxonomically classified the flies deposited in each vial [31]. The food was also scanned to search for eggs and larvae. A Leica M7 stereomicroscope was used for these purposes. Vials with pre-adults were maintained at 22 °C until emergence, and the imagoes were taxonomically identified. The possibility that some emerged adults could be interspecific hybrids was also scrutinized [32, 33]. The flies were individually transferred to a vial filled with Drosophila medium. When a new generation did not emerge, the parents were classified as interspecific hybrids. We did not determine whether the collected flies were virgins.

Observations in the wild

We directed our attention to the behavior of adults in grape, apple and prickly pear orchards between 9:00 am and 6:00 pm (Figs 1–4). In the grape and apple orchards, most flies exhibited very little movement. By contrast, Drosophila adults in prickly pear orchards were mobile, moving from place to place. In the grape and apple orchards, we observed (i) courtship and mating behavior and (ii) a careful scan of the fruits (a description of the behavior is provided in the Results section). Fermented areas of the fruit were explored approximately twice as long as non-fermented areas. We examined such fruits under a stereomicroscope to locate eggs and larvae (see Results). In the prickly pear orchard, we observed courtship and feeding. We also observed that males and females met on grape, apple, and prickly pear fruits in which the decay process had started. We presumed that the flies were feeding. We carefully collected the groups and identified sex and species. We also looked for aggressive encounters between flies [34]. The Drosophila adults that we observed seemed vigorous, agile and attentive to the ecological quality of breeding sites.

Stocks

We formed strains of D. melanogaster and D. simulans (subgenus Sophophora, melanogaster Group) with flies collected in the grape orchard (see above). Each stock was formed with 3 males and 3 females of D. melanogaster and D. simulans aspirated at random from each of two vials containing flies collected from grapes of the Sultanina variety (N = 12 adults; Figs 2 and 3). A similar number of males and females were taken from vials containing flies collected from dry grape leaves. Thus, each stock included 24 individuals. Before being introduced into culture bottles, adults were again taxonomically checked. The amount of genetic variability in the stocks was not estimated. The flies used in the experiments reported below were the third or fourth generation of the cultures established in the laboratory.

Experiments

To determine whether D. melanogaster and D. simulans associated in the laboratory similar to those detected in the wild, we introduced 8, 10, 25 and 50 non-virgin males and females of 4–5 day old individuals of each species into 10-cm-diameter glass Petri dishes. Adults of both sexes of both species formed distinct clusters distributed within the Petri dishes (Fig 5). Individual perch preferences in the Petri dishes of virgin and non-virgin 4–5 day old flies of these stocks were determined (N = 50 individuals per sex and species). For dispersal, groups of 10 individuals were used in 10 independent replicates (details below). Virgin flies were collected without anesthesia 3–4 h after emergence from pupae. They were aged, the two sexes separately, until they were 4–5 days of age in vials filled with synthetic Drosophila food [30]. Non-virgin males and females were also separated without anesthesia 2 days after emergence and aged in vials with food until 4–5 days old. Experiments on perch preferences and dispersal were carried out at 20 °C under homogenous lighting conditions and 80% humidity. Illumination conditions were monitored with a Leitz-Westlar Microsix-L 2875 photometer.

Fig 5 — The adults (N = 50 males) shown belong a strain formed with individuals collected in the grape orchard (Fig 1).

Perch preferences

Each Petri dish offered the flies three different places to perch: (i) the lid of the plate; (ii) a vertical surface, namely, the wall of the Petri dish; and (iii) the bottom of the plate. Each fly tested was aspirated into the Petri dish through a 0.5 cm diameter hole made in the wall of the Petri dish. The hole was stoppered with a cotton wool plug to prevent flies escaping from the dish. Ten minutes later, once flies had recovered from handling and had acclimated to the Petri dish, we recorded the location (ceiling, wall, floor) where each individual perched. For each fly tested, a new Petri dish was used.

The controls for perch preferences of virgin and non-virgin D. melanogaster and D. simulans adults consisted of Petri dishes without congeners and alien fly odors (treatment 1). In treatments 2 and 3, Petri dishes contained, respectively, 8 non-virgin or 8 virgin D. melanogaster males for 30 min. After this time, the flies were liberated; eight was the mean number of adults composing clusters observed in the wild (see Results). In treatments 4 and 5, the Petri dishes contained, respectively, non-virgin and virgin conspecific female odors. Treatments 2–5 were repeated with 8 non-virgin and virgin individuals each of D. simulans introduced into the Petri dishes for 30 min (treatments 6–9). The experiment was repeated with D. melanogaster females. The whole experiment was also run for both sexes of D. simulans.

Dispersal orientation

We also studied the orientation of the movements of the sexes of D. melanogaster and D. simulans stimulated at distance by odors of conspecifics and aliens. The apparatus used is depicted in Fig 6. It consisted of a 1-liter conical flask fitted at the base with 8 regularly spaced tubes. An 18-cm-Y-shaped long tube was connected to each tube. The distal end of each Y-tube was fitted with a cell. In treatment 1, 10 non-virgin (or virgin) D. melanogaster males (or females) were deposited into the flask when the cells were empty and had no odors (first control treatment). The number of flies in each of the Y-tubes was recorded 10 min later. In treatment 2, each empty cell had previously contained 8 virgin (or non-virgin) males (or females) of the same species on one side for 30 min, and the corresponding cells on the opposite side had contained 8 virgin (or non-virgin) D. simulans males (or females) for a similar time (second control treatment). In treatment 3, each empty cell on one side had 8 virgin (or non-virgin) conspecific males (or females), and the corresponding cells on the opposite side were empty and had no fly odors. Treatment 4 was similar to treatment 3 except that the cells on one side had contained non-virgin (or virgin) D. simulans males (or females). S1 Fig shows a diagram depicting the essays described (see also Fig 6). Each treatment was replicated 10 times. The whole experiment was repeated by testing virgin and non-virgin D. simulans males (or females). The apparatus was carefully washed and dried before each treatment and replication. The hole of the flask was plugged with a cotton wool plug to prevent flies from escaping the apparatus.

Fig 6 — The flies were confronted with odors of the two species. The distal end of each Y-tube was connected to an empty cell impregnated with odors of the species.

Statistical analysis

Perch selection

We performed a multinomial logistic regression for the perch selection data; the dependent variable was nominal with three levels (wall, ceiling, floor). The link function was the logit function (Ln). We assumed that the log-odds followed a linear model [35]. As the base comparator, the percentage of flies detected on the floor of the Petri dishes was used. This place was the least preferred section of the dishes (see Results).

The equation was:

\begin{array}{l} Ln (percentage of flies detected on the wall of the Petri dishes) = odor + sex + sexual \\ experience + species + odor x sex + odor x sexual experience + odor x species \\ + \dots \dots \dots \dots ‥ + odor x sex x sexual experience x species \end{array}

Box plots

We first examined the data on perch preferences, noting that we were dealing with skewed data. We changed the scale (log scale), but the bias was not rectified. Therefore, we used the median to express the central tendency together with the lower and upper quartiles and minimum least and maximum greatest values to describe the distribution of the dataset in the applied treatments. Outlier values were not found. We built box-and-whisker plots to compare those distributions. For example, we added percentages of non-virgin males of D. melanogaster calculated from the number of flies recorded on the wall of the Petri dishes in the four treatments with conspecific odors; such a box plot was compared with the distribution of non-virgin males on the wall of the Petri dishes with D. simulans odors (Figs 7–10).

Perch preferences were examined in two types of Petri dishes: (i) impregnated with conspecific adult odorants (white columns), and (ii) saturated with adult odorants of D. *simulans* (grey columns). Fig 7 A–C and Fig 7 D–G show, respectively, percentages of non-virgin and virgin males of D. *melanogaster* perched on the wall, ceiling and floor of the Petri dishes. The horizontal black lines inside the boxes indicate median values. Bars outside the boxes show, respectively, maximum greatest values and minimum least values of the distributions. Conspecific is an abbreviation of Petri dishes with conspecific odors; *simul* stands for Petri dishes with odors of D. *simulans*.

Statistical analysis of dispersal

Dispersal data were analyzed by applying a generalized linear model based on a binomial distribution because the dependent variables are binary: (i) number of flies in the flask and (ii) number of flies in the Y-tubes. For each variable, we conducted an analysis based on the linear independent explanatory variables of odor, sex, sexual experience, species, and the corresponding interactions. The link function was the logit function for binary data [35]. The equation was:

\begin{array}{l} Ln (number of flies within the flask) = odor +sex + sexual experience + species + odor x \\ sex + odor x sexual experience + odor x species + \dots \dots \dots \dots ‥ + odor x sex x sexual \\ experience x species . \end{array}

For statistical analysis we used STATA MP 14.0 for Windows 32 bits, 2015, Serial 10699393.

Results

Physical and chemical features of Drosophila breeding sites

In the grape orchard, the canopy decreases the overall impact of sunlight, but foliage density is heterogeneous, leaving sites with more or less sunlight (Fig 1). Exposure to sunlight affects the duration of fruits as breeding sites due to loss of water (Figs 2 and 3). At 2:30 pm, the mean environmental temperature in the grape orchard was 24.60 ± 2.80 °C (N = 37 measurements), environmental humidity reached 79.80 ± 9.52% (N = 48 measurements), and pH of mature, unfermented grape grains hanging from the plants was 7.0 ± 0.4 (N = 47 measurements). In totally fermented grape grains of the same variety, the pH was 2.7 ± 0.8 (52 measurements); a strong acetic acid aroma was also detected over decaying fruits. We also occasionally found adults of the predator Argentinian ant Linepithema humile.

In the prickly pear orchard, a canopy is absent (Fig 4). Unfermented and decaying fruit units and pieces of cladodes used as breeding sites by Drosophila species are all exposed to extreme solar radiation. At 2:30 pm, the mean environmental temperature was 48.20 ± 2.56 °C (34 measurements), environmental humidity reached 42.34 ± 2.07% (34 measurements), and the pH of unfermented prickly pear fruits was 6.8 ± 0.5 (52 measurements); in decaying fruits fallen on the ground, the pH was 3.4 ± 0.5 (58 measurements). The pH of unfermented cladode tissue was 7.4 ± 2.3 (48 measurements); the pH of decaying cladode tissue was 4.4 ± 1.2 (59 measurements); an acetic acid aroma was not detected. The prickly pear orchard had abundant L. humile.

In the apple orchard, the canopy casts a shadow covering an area of 2–3 m around each tree, leaving sunny places 3–5 m in diameter between neighboring trees. We found decaying apple units within and outside the shadowed area. At 2:30 pm, the mean environmental temperature in a shady habitat was 28.75 ± 2.79 °C (35 measurements), and the temperature in sunlit microhabitats was 42.56 ± 1.93 °C (31 measurements). Mean humidity reached 63.67 ± 5.87% (34 measurements), and the pH of fruit fluctuated between 6.2 ± 0.4 in non-decaying apples hanging from the trees (Red Delicious variety; 46 measurements) and 3.4 ± 1.5 in decaying fruits of the same variety (59 measurements); acetic acid aroma was not detected. No L. humile were found.

Fly routines in the grape orchard

In the grape orchard, D. melanogaster (54.78 ± 2.87%), D. simulans (45.03 ± 6.71%) and Drosophila busckii (0.19 ± 0.04%) were perched on dry leaves scattered on the ground and on unfermented grains of grape forming clusters (Figs 2 and 3). The remarkable absence of movement was broken by very few attempts at courtship and copulation. A male courted a female within a cluster (Figs 2 and 3); the female left the group, walking quickly followed closely by the courtier male vibrating a wing. Other males of the same and neighboring groups joined in, aligning behind. Headed by the female, the train snaked among fly clusters. Abruptly, the courting male stopped following the female and after a few seconds, the escort train dispersed and the males returned to the groups. In other instances, the female stopped walking while the leader male increased the frequency of semicircles around the female. Males of the escort train remained near the partners, vibrating their wings. After copulation, the escort males returned to the groups. These observations suggest cooperative male courtship. Courtship and copulation were also observed on unfermented grains of grape fallen on the ground (Figs 2 and 3). However, participation of other males was not observed. We did not detect aggressive encounters between flies perched on dry leaves and on grains of grape.

On the surface of unfermented grape grains, some female D. melanogaster and D. simulans were observed scanning the fruit. The process took approximately 10.74 ± 3.51 min (45 observations). We observed that 2–3 females landed at unfermented grape grains or grains where fermentation was just starting; after 8–11 sec, one of the flies walked two or three steps and stopped, spreading the trunk and leaving the end of it in contact with the surface of the fruit. After 15–20 sec, the fly again walked two to three steps, extending the trunk until the whole of fruit surface was explored; the other flies remained motionless. We collected the scanned grains (241 units); we did not detect eggs or larvae on 206 grains (85.48%), whereas 35 grains (14.52%) had eggs or larvae. Adult D. melanogaster or D. simulans emerged from 34 of these grains (99.95%); adults of both species emerged from 1 grain. Abundant colonies of microorganisms consumed by the larvae of Drosophila were present on the decaying grape grains. A few large larvae were also observed moving on decaying grape grains and on the ground; presumably, they were searching for pupation sites. We also detected clusters of Drosophila pupae adhered to the stalks of bunches of grapes; the pupae were principally located between the grapes and the ground.

Table 1 shows the distribution of adult flies on the dry leaves of grape plants and unfermented grape grains on the ground (Figs 1–3).

Table 1. Number of male and female D. melanogaster and D. simulans collected from dry leaves and grains of grapes on the ground.

Each collected group of flies was deposited into a vial. The sexes of individuals in each sample vial were taxonomically identified.

Drosophila species and type of substratum where collected
N° Vial	dry leaves of grape plants				N° Vial	grains of grape				Total
	melanogaster		simulans			melanogaster		simulans
	male	female	male	female		male	female	male	female
1	4	5	–	–	22	2	1	–	–	12
2	5	3	–	–	23	1	2	–	–	11
3	3	2	–	–	24	3	1	_	_	9
4	4	3	–	–	25	–	–	2	1	10
5	3	4	–	–	26	–	–	2	2	11
6	3	5	–	–	27	2	2	–	–	12
7	–	–	3	4	28	–	–	3	1	11
8	–	–	4	4	29	–	–	4	–	12
9	–	–	4	3	30	–	–	1	3	11
10	–	–	5	4	31	–	–	–	4	13
11	–	–	3	4	32	3	–	–	–	10
12	–	–	4	4	33	2	2	–	–	12
13	2	4	–	–	34	–	3	–	–	9
14	–	–	5	3	35	–	–	–	2	10
15	4	1	–	–	36	3	1	–	–	9
16	6	3	–	–	37	–	–	1	2	12
17	–	–	1	6	38	3	3	–	–	13
18	–	–	4	4	39	2	2	–	–	12
19	5	1	–	–	40	1	4	–	–	11
20	3	3	–	–	41	2	2	–	–	10
21	–	7	–	–	42	1	3	–	–	11
Total	42	41	33	36		25	26	13	15	231

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Each vial contained adult D. melanogaster or D. simulans. Hybrids between the species were not detected. These findings confirm that D. melanogaster adults distributed differentially from D. simulans in this grape orchard.

Fly routines in the prickly pear orchard

In the prickly pear orchard (Fig 4), we found adults of D. melanogaster (13.82%), D. simulans (37.08%), Drosophila buzzatii (48.67%), Drosophila nigricruria (0.28%) and Drosophila pavani (0.15%). Compared to observations in the grape orchard, adults were actively moving from place to place on prickly pear fruits and cladodes. We observed courtship and mating, and feeding; fly clusters were not observed. We also detected groups of 5–6 flies of D. buzzatii on the cladodes of the plants, arranged in circles with heads directed toward the center of the circle. The functional significance of such behaviors remains to be investigated. Nearly 100% of the fallen prickly pear fruits had an orifice of 2.5 ± 0.8 cm in diameter and 1.5 ± 0.5 cm deep made by wild Chilean birds, the Austral blackbird (Curaeus curaeus), the Austral thrush (Turdus falcklandii) and the Chilean mockingbird (Mimus thenca). Adult and larval Drosophila were detected inside the holes, on the edges of the holes and on peels of the fruits.

Courtship and mating of D. melanogaster adults was observed on the peel and inside the holes of prickly pear fruits. Individuals of D. simulans were detected principally on cladodes fallen on the ground on which the decay process was starting. Cooperative male courtship as that observed in the grape orchard was not observed. We found D. buzzatii adults mating on cladodes on the plant. These results suggest that the three more abundant Drosophila species mate on different substrates in the prickly pear orchard.

The fruits and cladodes in the prickly pear orchard were awash with Argentinian ants (L. humile). The ants captured larval and occasionally adult Drosophila. We noted that, upon arrival of the ants at prickly pear fruits, Drosophila adults flew away. Two or three ants cooperatively captured larvae. We observed that larvae quickly detected the arrival of ants, as suggested by an increase in locomotion and a decrease of turns, followed by a rapid navigation toward the liquefied part of the fruit, where they sank and disappeared. The behavioral changes of the larvae seem to alert the adults of the ant’s arrival.

Fly routines in the apple orchard

The dominant species in the apple orchard was D. simulans (96.10%). Adults of D. melanogaster (2.13%), Drosophila repleta (repleta species Group, 1.02%), D. hydei (repleta species Group, 0.56%) and a few adults of Drosophila subobscura (obscura species Group, 0.19%) were also observed. Flies were perched on decaying apples that had fallen on the ground and on herbs and dry branches distributed around such fruits. As in the grape orchard, an absence of movement was the most remarkable behavior of the adults. Clusters of flies were not detected.

Perch site selection

D. melanogaster males

Fig 7A–7G shows the perch preferences of non-virgin and virgin D. melanogaster males in Petri dishes with conspecific odorants (white columns) and odorants of D. simulans (gray columns). Places available to perch include the wall, ceiling and floor of the Petri dishes as noted above. Most non-virgin and virgin D. melanogaster males perched on the wall of the Petri dishes. However, the distribution of male D. melanogaster on the wall of Petri dishes with conspecific odorants was clearly different than in the Petri dishes with D. simulans odorants, as shown by median values, lower and upper quartiles, and minimum least values and maximum greatest values of the distributions (Fig 7A and 7D. See below Statistical analysis of perch site selection and S1 Table).

In line with the above results, the distribution of non-virgin and virgin males perched on the ceiling (Fig 7B and 7F) and floor (Fig 7C and 7G) of Petri dishes with conspecific odorants (white columns) differed from the distribution in the same sites in Petri dishes impregnated with D. simulans odorants (gray columns; see below: Statistical analysis of perch site selection and S1 Table).

D.melanogaster females

Perch preferences of non-virgin and virgin females of D. melanogaster were also affected by odorants left by conspecific adults of the two sexes, and adult D. simulans (Fig 8A–8G). As in the case of the males, distribution of D. melanogaster females perched on the walls of the Petri dishes with conspecific odorants were significantly different from those obtained in Petri dishes with D. simulans odorants (Fig 8A and 8D; see below: Statistical analysis of resting place selection and S1 Table). Similar results were obtained for the distribution of females perched on the ceiling (Fig 8B and 8F) and floor (Fig 8C and 8G) of Petri dishes with conspecific odorants and Petri dishes with D. simulans odorants (see below: Statistical analysis of resting place selection and S1 Table).

Fig 8 — White columns show distribution in Petri dishes with conspecific odors. Grey columns stand for distribution patterns in Petri dishes with odorants of adult D. *simulans*. See Fig 7A–7G for further details.

D. simulans males

Fig 9A–9G shows perch preferences of non-virgin and virgin D. simulans males in Petri dishes with conspecific and D. melanogaster odorants. Most D. simulans males perched on the walls of Petri dishes (Fig 9A and 9D). Distribution patterns of non-virgin and virgin males of D. simulans detected on the wall, ceiling and floor of Petri dishes with conspecific odorants were significantly different than in Petri dishes with D. melanogaster odorants (Fig 9A–9G; see below: Statistical analysis of perch site selection and S1 Table).

D. simulans females

In agreement with the above results, perch preferences of non-virgin and virgin D. simulans females on the wall, ceiling and floor in Petri dishes with conspecific odorants differed from those in Petri dishes with D. melanogaster odorants (Fig 10A–10G; see below: Statistical analysis of perch site selection and S1 Table). Taken together, the results of perch preferences of non-virgin and virgin adult D. melanogaster and D. simulans indicate that odor cues emanating from the sexes of the two species play an important role in the selection of places to perch and rest.

Statistical analysis of perch site selection

Perch selection data were analyzed with a multinomial logistic regression (see Materials and methods and S1 Table). Because the floor of Petri dishes was scarcely used by the flies to perch, we used it as a Base comparator [35]. The statistical analysis indicates that odor x sex x sexual experience x species interaction explains intra-and inter-specific differences in perching preferences in Petri dishes: i) preference for wall of Petri dishes yielded an odds ration value = 0.04, confidence interval [0.01–0.19], P-value > |z|< 0.05 = 0.001; ii) ceiling preference produced an odds ratio value = 0.16, confidence interval [0.03–0.92], P-value > |z|< 0.05 = 0.04. We conclude that adult odors of the same and the sibling are important in perch site selection of D. melanogaster and D. simulans adult flies.