Back to the light, coevolution between vision and olfaction in the "Dark-flies" (Drosophila melanogaster).

Trade-off between vision and olfaction, the fact that investment in one correlates with decreased investment in the other, has been demonstrated by a wealth of comparative studies. However, there is still no empirical evidence suggesting how these two sensory systems coevolve, i.e. simultaneously or alternatively. The "Dark-flies" (Drosophila melanogaster) constitute a unique model to investigate such relation since they have been reared in the dark since 1954, approximately 60 years (~1500 generations). To observe how vision and olfaction evolve, populations of Dark-flies were reared in normal lighting conditions for 1 (DF1G) and 65 (DF65G) generations. We measured the sizes of the visual (optic lobes, OLs) and olfactory (antennal lobes, ALs) primary centres, as well as the rest of the brain, and compared the results with the original and its genetically most similar strain (Oregon flies). We found that, whereas the ALs decreased in size, the OLs (together with the brain) increased in size in the Dark-flies returned back to the light, both in the DF1G and DF65G. These results experimentally show that trade-off between vision and olfaction occurs simultaneously, and suggests that there are possible genetic and epigenetic processes regulating the size of both optic and antennal lobes. Furthermore, although the Dark-flies were able to mate and survive in the dark with a reduced neural investment, individuals being returned to the light seem to have been selected with reinvestment in visual capabilities despite a potential higher energetic cost.

Introduction

The world is complex, and animals have evolved a large array of sensory systems to make sense of it. Getting information is so important for survival and reproduction that, at first sight, we may think that developing additional sensory capabilities in one or several modalities leads brain evolution. Obviously, those individuals that get more pertinent information have a clear advantage for finding food and sexual partners as well as for avoiding dangers. But, it also has an energetic cost for the brain to develop and maintain neural tissues [1-3]. Therefore, to invest in one or several sensory modalities makes sense only if it provides individuals with a reproductive and/or survival advantage; otherwise it is simply costly without any benefit, and not evolutionary stable.

Dim light/absence of light is a good example of an environment to observe evolution of sensory systems under environmental constraints. Dim light/absence of light exerts a selective pressure not only on vision, but also on other sensory systems. Until now, a wealth of comparative studies has shown that cave dwelling/nocturnal species have evolved either by developing specialised eyes, or by reducing the size of their eyes (for reviews: [1,4-7]). Not only dim light/absence of light is responsible for reduced eyes, but also captivity as observed in fruit flies (Drosophila melanogaster) [8]. Although effects of captivity on other sensory systems has not been documented, cave dwelling/nocturnal species with reduced investment in vision have also evolved with higher investment on alternative sensory systems, such as olfaction (e.g. [9-11]) or mechanoreception (e.g. [12-14]) to get information about their environment and to compensate the lack of visual information.

These observations initiated a common thinking that has relatively recently emerged about ‘trade-off’ between sensory systems, the fact that investment in one sensory modality correlates with decreased investment in another sensory modality. Trade-off between vision and olfaction has been described in several species such as in primates firstly [15], fish [7], butterflies/moths [10,16] and in ants [11,17]. However, whilst numerous comparative studies between several species have shown such trade-off between vision and olfaction (e.g. [7,10,11,15-19]), there is still no evidence that individuals are selected with inversed investments for vision and olfaction.

Empirical evidence is necessary not only to confirm this hypothesis of “trade-off”, but also to understand the mechanisms of change in the balance between sensory systems. Changes of sensory systems might occur simultaneously meaning that reduced investment in one sensory modality and increased investment in an alternative sensory system occur at the same time. However, they might also occur consecutively. In this case, reduced investment in one sensory modality appears before increased investment in an alternative sensory system. Order of changes is important since it may provide cues about the origin of these changes. For example, simultaneous changes may be the consequence of genetic/epigenetic factors that control investment in several sensory modalities concurrently whereas consecutive changes may reflect independent mechanisms. However, species lifespan makes difficult to observe such trade-off over generations.

The ‘Dark-flies‘, a strain of fruit flies (Drosophila melanogaster) reared in the dark for more than 60 years [20], constitute a unique model to investigate how individuals are selected based on sensory investment and to show a trade-off between vision and olfaction over generations. In the present study, we initially measured morphological differences (body and eyes sizes) between the Dark-flies and its parental and most genetically related strain (Oregon flies) [21]. To observe sensory investment in vision and olfaction over generations, a population of Dark-flies has been reared in normal lighting conditions for 65 generations (DF65G) at the time of this study. We then measured the sizes of visual (optic lobes) and olfactory (antennal lobes) primary centres, as well as the size of the brain in the Dark-flies, DF65G and Oregon flies. To ensure that the differences observed between the Dark-flies and DF65G were not due to rearing conditions, we also measured the same parameters in a population of Dark-flies that has been reared in normal lighting conditions for 1 generation. Overall, we expected to observe larger optic lobes (OLs) in the DF1G as a consequence of the presence of light on the development of visual system, and no change concerning the antennal lobes (ALs). Concerning the DF65G, we expected to observe that investment in vision increased beyond this developmental change (i.e. larger OLs compared to the DF1G), and to observe a reduced investment in olfaction in the DF65G (i.e. smaller ALs).

Methods

Subjects and housing

In 1954, the Dark Flies Project was started by putting and maintaining in the dark populations of fruit fly (Drosophila melanogaster) taken from an original Oregon-R-S strain [20]. During the Dark-flies Project, the Dark-flies were maintained in sterilized milk-bottles plugged with a cotton-ball or silicon plug, and provided with a low-nutrient food source, Pearl’s synthetic medium [22], to accentuate selection of individuals. The flies were kept in the dark by placing the bottles in a light-proofed can that was painted black inside and had a blackout curtain to cover the lid. In parallel, the Oregon-R-S strain was maintained under 12:12 LD lighting conditions, and fed within a standard cornmeal medium. All flies were kept at 25°C in a temperature-controlled room. In 2002, the original Oregon-R-S strain from which the Dark-flies had been taken was lost, and a new population of Oregon-R-S was re-obtained and established from the original source. This new population of Oregon-R-S flies is the most genetically similar strain to the Dark-flies of those that have been analysed [23], and were used for comparisons in our study.

In November 2014, a population of Dark-flies was placed in a 12:12 L:D condition, similarly to the Oregon flies (Fig 1). This new strain, the original Dark-flies and Oregon-R-S flies were obtained in February 2017 from the Dark Flies Project based at Tohoku University, Japan [20]. Upon arrival, we kept all flies under the same standardised conditions for 6 months (about 12 generations) before making any measurements and starting experiments. This was to help reduce any effects of their past rearing environment or nutritional status on our results. All flies were reared in vials (28.5 x 95mm) of K-resin (VWR International) containing a fresh mixed plain white drosophila medium (Blades Biological Ltd). They were kept in a room at 25±3°C with a 12:12h L:D photoperiod (light phase: 0700–1900). The tubes containing the Dark-flies were maintained in a metal container surrounded by black tissue paper to keep out the light.

Fig 1

Representation of the experimental design and timeline.

Body measurements

At 4 days old, Oregon flies and Dark-flies that had been kept under their standard rearing conditions were collected to measure their mass, length (Oregon: 49 males, 67 females; Dark-flies: 24 males, 41 females; Fig 2). The flies were anesthetised by putting their tubes into ice. Photographs of the flies were taken against a piece of graph paper (for scale) using a camera fixed on a microscope (Brunel eyecam plus fixed to a BMDZ Brunel Microscopes Ltd). The length of each fly was measured using GIMP 2 (version 2.8.22), and the body mass was measured using a balance with a range of 0.01mg (Mettler AT261 Professional Analytical Balance, © Mettler-Toledo).

Fig 2

Images of male (A) and female (B) flies from each strain. In each image, Dark-flies are on the left, and Oregon flies on the right. The mean (+SEM) length (C) and mass (D) of male and female Oregon flies and Dark-flies.

Brain extraction and histology

After sacrificing the flies by immerging them in ethanol (100%), we extracted their brains in a Ringer solution (NaCl: 180mM; KCl: 6mM; CaCl2: 3mM; NaHCO3: 3mM; Ph: 7.3). We then followed the method used in our previous study, which is a variant of Stölck and Heinze’s method [24]. On the day of extraction, we fixed the brains in a zinc-formaldehyde fixative solution (ZnCl2: 18.4mM; NaCl: 135mM, sucrose: 35mM; 1% paraformaldehyde; pH 7.3) at room temperature overnight. On the second day, we rinsed the brains 8 x 20min in PBS (NaCl: 140mM; KCl: 2mM; Na2HPO4: 10mM; KH2PO4: 2mM; pH 7.3) and bleached them in a fresh solution of 10% hydrogen peroxide in 0.05M Tris-buffered saline solution (Tris-HCl: 0.05mM) for 6 hours. After bleaching, we again rinsed the brains with a Tris-HCl solution (3 x 10min), put them in a fresh mixture (20:80) of dimethyl sulfoxide (DMSO):Methanol for 85min, and rinsed a final time with a Tris-HCl solution (3 x 10min). After the last wash, we pre-incubated the brains overnight at 4°C in a solution of PBT (PBS with 0.3% of Triton X-100) containing 5% goat serum. On day 3, we incubated the brains at 4°C with 1:25 anti-synapsin antibodies (3C11 anti SYNORF1; Developmental Studies Hybridoma Bank; dshb.biology.uiowa.edu) added to PBT containing 1% goat serum for 5 days. On day 6, we washed the brains with a solution of PBT (8 x 20min), and then incubated them at 4°C with 1:200 of the secondary antibody (goat anti-mouse, IgG (H+L) conjugated to rhodamine; JacksonImmunoResearch, WestGrove, PA, USA) for 5 days. On day 13, we first again washed the brains with a solution of PBT (2 x 30min), and then with a solution of PBS (6 x 30min). After these washes, we dehydrated the brains in an ascendant series of ethanol solutions (70%: 2 x 10min; 80%: 10min; 90%: 10min; 100%: 2 x 30min) before stocking them in methyl salicylate at -20°C.

Confocal laser scanning microscopy (LSM)

We observed the stained brains using a confocal microscope (Nikon A1; Nikon corporation) with a X10 0.45-NA plan Apo λ objective. We used a helium-neon laser with a long-pass emission filter (561nm) to visualise the antibodies (anti-synapsin), and an argon laser with a band-pass emission filter (488nm) for background autofluorescence. We made optical sections at a resolution of 1,024 x 1,024 pixels with 2μm intervals through the entire depth of the brains following a ventral-dorsal neural axis [25]. In total, we obtained brains of 14 (7♂ and 7♀) Dark flies, 14 (7♂ and 7♀) DF1G, 10 (5♂ and 5♀) DF65G and 10 (2♂ and 8♀) Oregon flies.

Analyses and statistics

Body, eyes and brain measurements were made blind to fly strain: the optical image files were renamed with random numbers generated by Excel (Microsoft® Excel® for MAC 2011, version 14; © 2010 Microsoft Corporation) and were given to an observer who was unaware which strain each image was from. The volumes of the whole brain, the optic lobes (OLs), the antennal lobes (ALs) and the hemisphere (i.e. the whole brain without the OLs and ALs) were measured from the optical image files using FIJI software [26] and a plugin (“measure stack”: Bob Dougherty, Copyright (c) 2002, 2005, OptiNav, Inc.). In the case of OLs, we measured the whole volume of optic lobes (i.e. medulla, lobula and lobula plate including neuropils and cell bodies) whereas the whole volume of glomeruli was considered for the antennal lobes. The relative volumes of OLs and ALs were calculated by dividing their volumes by the volume of hemisphere.

Statistical analyses were performed using SPSS v22 (IBM Corporation) using Generalized Linear Models (GLMs). For body and eye sizes, we performed GLMs to test the effects of fly strain (Dark-flies or Oregon flies) and sex (male or female) on the measurements. For brain measurements, we initially tested the effects of fly strain (Dark flies, DF1G, DF65G or Oregon flies) and sex (male or female) on the data. Because we did not find any main effect of sex or interaction of sex with any other factor (see result section), we removed sex from the models, and performed GLMs with strain as a factor. Finally, we performed post-hoc tests with pairwise comparisons using Fisher's Least Significant Difference (LSD).

Results

Body and whole brain size

To explore whether there were any morphological and anatomical differences between the Dark-flies and the Oregon flies, we first compared the body and brain sizes between the two strains reared under their standard rearing conditions, i.e. Dark-flies reared in 24D and Oregon flies reared under a 12L:12D condition. There was no effect of strain on either body mass (χ21 = 0.517, P>0.05; Fig 2D) or length (χ21 = 0.001, P>0.05; Fig 2C), although females of both strains were consistently heavier (GLM; χ21 = 45.53, P<0.001; Fig 2D) and larger than males (χ21 = 97.59, P<0.001; Fig 2C), with no interaction between sex and strain for either measure (mass: χ21 = 0.687, P>0.05; length: χ21 = 0.012, P>0.05).

Although there were no significant differences between the strains in their body size, Dark-flies had smaller brains than Oregon flies (GLM: χ21 = 64.48, p<0.001; Fig 3D). In contrast to body size and mass, we did not find any effect of sex in the measurements (χ21 = 0.99, p>0.05). We also did not find any interaction between strain and sex (χ21 = 2.54, p>0.05) on whole brain measurements. Therefore, both male and female Dark-flies had consistently relatively smaller brains to body size than the Oregon flies.

Fig 3

Flies’ brains.

Brain images from a brain of a male Dark-fly showing a series of optical sections from ventral (top left) to dorsal (bottom right) side following a neural axis (A). A 3D representation of a brain of a male Dark-fly (B) and a male Oregon fly (C). The scales and depth location are presented in the figure. The mean (+SEM) size of whole brain (including OLs and ALs) (D) in males and females Oregon and Dark-flies.

Absolute brain measurements

There was a main effect of the strain on the size of the hemisphere (GLM: χ23 = 19.81, p<0.001; Fig 4): the Dark-flies and DF1G had smaller hemispheres compared to the DF65G (p<0.05) and Oregon flies (p<0.001). Interestingly, there were no differences between the DF1G and the original Dark-flies (p>0.05), or between the DF65G and the Oregon flies (p>0.05).

Fig 4

Hemisphere, OLs and ALs absolute sizes.

Absolute volumes ± SEMs of hemisphere (black), OLs (grey) and ALs (white) in Dark-flies, DF1G, DF65G and Oregon flies. Statistics are represented with letters associated to dots, and significant differences (p<0.05) are represented with different letters.

The volume of the OLs (GLM: χ23 = 13.94, p<0.01) and ALs (GLM: χ23 = 8.13, p<0.05) also changed according to the strains (Table 1). The Dark-flies had smaller optic lobes compared to the DF65G (p = 0.011) and Oregon flies (p = 0.001) and, conversely, the Dark-flies possessed a larger absolute size of the ALs compared to the DF65G (p = 0.044) and Oregon flies (p = 0.008). Although there was no significant difference between the DF1G and DF65G flies (p = 0.061), the size of the optic lobes in the DF1G was significantly smaller than that of the Oregon flies (p = 0.011). For the ALs, there were no significant differences between the DF1G, DF65G and Oregon flies (pairwise comparisons, for all values: p>0.322).

Table 1

Absolute and relative sizes of brain, OLs and ALs.

	Hemisphere (μm3)		OLs size (μm3)		ALs size (μm3)		OLs relative size		Als relative size
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Dark-flies	3,16E+06	7,96E+04	1,42E+06	4,33E+04	1,91E+05	1,16E+04	0,45	0,01	0,060	0,002
DF1G	3,11E+06	6,55E+04	1,51E+06	3,33E+04	1,67E+05	6,38E+03	0,49	0,01	0,054	0,002
DF65G	3,54E+06	2,05E+05	1,76E+06	1,97E+05	1,62E+05	1,38E+04	0,50	0,05	0,046	0,003
Oregon flies	3,81E+06	1,97E+05	1,85E+06	1,07E+05	1,53E+05	9,00E+03	0,49	0,02	0,041	0,003

Relative brain sizes

Concerning the relative sizes, there was an effect of the strains on the relative sizes of the ALs (GLM: χ23 = 34.14, p<0.001; Fig 5), but not on that of the OLs (GLM: χ23 = 3.1, p = 0.376). The Dark-flies and DF1G possessed larger relative ALs compared to the DF65G (p<0.05) and Oregon flies (p<0.001); however, no differences were observed between the Dark-flies and the DF1G (p = 0.062) or between the DF65G (p = 0.28) and the Oregon flies.

Fig 5

OLs and ALs relative sizes.

Mean values of the relative sizes ± SEMs of OLs (grey) and ALs (white) in Dark-flies, DF1G, DF65G and Oregon flies. The number of individuals used is shown in Fig 4. Statistics are represented with letters associated to dots, and significant differences (p<0.05) are represented with different letters.

To confirm a tendency of a trade-off between vision and olfaction, we performed GLMs and tested whether the relative size of the OLs co-varied with the relative size of the ALs. By performing such statistics, we found a major effect of the strains on the ALs (GLM: χ23 = 46.00, p<0.001), on the OLs (GLM: χ23 = 10.49, p<0.05), and a significant effect of the covariate (GLM: χ21 = 8.39, p<0.01).

Discussion

Returned to the light, the Dark-flies, whose sensory investment seems in favour of olfaction, have clearly been selected with a decreased investment in the antennal lobes and increased investment in the optic lobes and whole brain in general, beyond developmental processes. This constitutes the first experimental evidence of a trade-off between vision and olfaction. This trade-off seems simultaneous at first sight, since we observed changes in both sensory systems in the DF1G and DF65G, and might be associated to possible genetic or/and epigenetic processes regulating the size of both optic and antennal lobes. Furthermore, our results highlight the fact that darkness has a negative influence on brain size, which might be the consequence of a decreased investment in the visual system. In addition, after having adopted less energetically costly strategies for survival and reproduction in the dark, individuals have been selected with higher investment in the visual system after being returned to the light despite its potential energetic cost. Since there were no predatory or other environmental pressures (other than light condition) in our flies’ raising conditions, selection of the flies might have been the result of sexual selection and less probably of foraging. In other words, it is unlikely that individuals possessed an advantage in terms of survival since food was really easy to find in the tubes. However, it is likely that individuals possessing better visual capabilities had reproductive skills (e.g. better access to females during male competition) and inherit this trait on the following generations.

Our results experimentally show for the first time that the trade-off between vision and olfaction occurs simultaneously. The Dark-flies returned to the light for 1 and 65 generations possess bigger OLs associated with smaller ALs compared to the original Dark-flies (although the results were not significant in the case of the DF1G). We still do not know if changes in a single sensory modality (i.e. only reduced investment in obsolete sensory systems or only increased investment in more reliable sensory systems) may evolve or not. But, although we do not exclude the possibility that sensory systems may evolve independently, our results show mechanisms regulating both vision and olfaction at the same time in accordance to a new environment, in terms of developmental (i.e. rearing environment of individuals) and evolutionary processes (increased changes through generations).

Gradual changes were observed between the Dark-flies, DF1G and DF65G tend to show that both developmental and evolutionary processes affect the size of hemisphere and sensory systems and play a role in the trade-off between vision and olfaction. Although differences measured between the dark-flies and DF1G were not significant, we do not exclude the hypothesis that light had a positive effect on the size of hemisphere and OLs, and a negative effect on the size of ALs in the DF1G. Indeed, it is already known that rearing flies in the dark for one generation has a negative impact on the size of mushroom bodies (neuropils present in the hemisphere) and OLs [27,28]. In our experiment, the DF1G were made to simply observe the reversed mechanism and to distinguish between changes due to developmental processes and changes due to due to evolutionary processes. By doing it, we have clearly observed that the size of hemisphere and the relative size of ALs significantly differed between the DF65G and DF1G. More generally, the differences observed between the Dark-flies and DF1G were accentuated between the dark-flies and the DF65G. Indeed, although these differences were not significant between the DF1G and the Dark-flies, they were significant between the DF65G and the Dark-flies for all the parameters measured (except for the relative size of OLs), reflecting a process in two steps.

We could not determine the origin of mechanisms responsible for changes in the size of OLs and ALs, but they might correspond to a competition between axonal terminations coming from different sensory systems and/or to genetic/epigenetic processes. Indeed, one explanation may lie in the neurons at higher levels that might have limitations in the number of dendritic connections that they can form. In such case, these connections would be favoured for one sensory modality to the detriment of another sensory modality in accordance with the environment (see [28]). It is likely that this process is involved in developmental differences. In particular, the Hox genes or the allele Lobe (L1) could be at the origin of such explanation. It is well known that the Hox genes are involved in establishing regionalized identity of neurons in the embryonic brain and control the termination of neuronal proliferation by posteriorly inducing apoptotic cell death during postembryonic brain development [29]. More recently, the allele L1 has been showed to have an antagonistic action in the number of trichoid sensilla (on antennae) and the number of ommatidia (on eyes) in fruit flies [19].

However, another explanation might be the control of the size of both visual and olfactory systems by genetic and epigenetic processes that act anteriorly to develop one sensory system to the detriment to the other and might correspond to evolutionary changes. In particular, both visual and olfactory systems are developed from the differentiation of cells from the eye-antennal imaginal disc in fruit flies [30]. It has recently been shown that this differentiation is directly controlled by the gene Paired box 6 (Pax6) [31], and that this gene (Eyeless/Pax6) is involved in the trade-off between vision and olfaction [32]. In the present case, we still do not know if this gene is responsible for the differences between the different strains, however, we may wonder if this would be equally applied to species where the olfactory and visual neuroepithelia develop from other and less competitive processes.

Our data tend to show that sensory systems are not equally weighted in terms of magnitudes concerning changes in neural substrates. We do not know if, other than vision and olfaction, any change took place in other sensory modalities such as the mechanosensory system. However, to compensate a decrease of ~0.3x105 μm3 in terms of neural substrate in the antennal lobes, the DF65G have developed ~0.34x106 μm3 of neural substrate in the optic lobes in association with ~0.4x106 μm3 in the hemisphere (Fig 3, Table 1). These changes in neural substrates seem directly associated with higher food consumption [33]. These results might indicate that the energetic cost of information is relative to sensory systems. For example, visual information seems more energetically costly than olfactory information in terms of neural substrates since the development of OLs is ten times higher than the reduction of ALs. However, the development of visual system seems to be relatively less costly in terms of evolutionary benefits that it provides for survival and reproduction.

This leads to consider that the balance between the visual and olfactory systems reaches an equilibrium where information is optimised to benefit individuals according to environment. It is interesting to notice that the brain areas measured in the DF65G tend to reach those of the Oregon flies. But, would these values reach those of the Dark-flies if these DF65G or other flies are put in the dark? This raises other questions such as to know if the reduction in the visual system in the dark would have been so drastic if the flies had abundance of food, or if the presence or absence of scents in the environment plays a role in the evolution of olfaction in the dark, and consequently on the evolution of vision. Here, there is a large horizon for questions in order to understand the co-evolution of sensory systems, questions we do not have the answers.

In the present study, we did not measure the eyes shape, the size of ommatidia and did not count their number to determine intra- and inter-specific differences (see [34]). However, since we did not find any difference in the surface of the eyes between the Dark-flies and Oregon flies (see S1 Fig), our results are towards a contradictory standpoint from those made in crickets Gryllus bimaculatus showing that individuals reared in the dark present an increase of the surface of their eyes in parallel to an increase in the number of ommatidia [35]. Since the crickets were reared for a single generation in the dark, we do not know if this difference between crickets and fruit flies is attributed to differences between developmental adaptations and long term rearing in the dark. Another explanation may lie to different developmental processes between crickets and fruit flies, i.e. hemimetabolous vs holometabolous developmental processes, which may raise the same question as previously on differences between species having different developmental processes.

Whilst many comparative studies have put the spotlight on cognitive abilities being central to brain size evolution (e.g. [36-38] but [39,40]), our empirical approach demonstrates that changes in sensory input, specifically vision, can play a key role in determining absolute hemisphere size. This is perhaps not surprising, given that processing visual information takes up so much of the brain in the Drosophila: the optic lobes represent around 30% of the whole brain, and are an order of magnitude larger than the antennal lobes. Additionally, other species also show such correlation between brain size and the size of the visual system such as in guppies [41], birds [42] and primates [18]. Our data complete these studies and support the idea of concerted brain evolution, the fact that there are constraints that cause correlated size changes across different component areas [43]. In the present case, darkness rather than captivity (since both Dark-flies and Oregon flies were reared in the same conditions of captivity) had a negative effect on brain size that may be explained by the reduced investment in the visual system, data that join those of Barth et al. on neural development in the dark in fruit flies (Drosophila melanogaster) [27,28].

No data to date have referred to any correlation or absence of correlation between brain size and the size of the olfactory system until now. The optic lobes being ten times larger than the antennal lobes may explain why we found no correlation between the sizes of ALs and brain, in the sense that size variations of OLs will involve the largest variations of brain size. These data support the idea that different areas of the brain can change size relative to one another (mozaic brain evolution: [44,45]), and that environmental constraints and changes in the investment in sensory systems are important in determining brain size, in agreement with recent studies [46-48].

Trade-off between sensory systems is not limited to vision and olfaction only, and investment in sensory systems depends not only on species’ environment, but also on lifestyle. For example, Barton et al. [15,18] found that primates have evolved visual capabilities to the detriment of olfaction. However, although they found a negative correlation between these two sensory systems in primates, they also found a non-significant negative relationship in insectivores and no correlation in bats [15]. Obviously, species did not evolve in the same manner, and vary in their reliance on sensory systems. For example, it is well known that cavefish or fish living in the deep sea have not only developed capabilities in olfaction, but also in mechanoreception [7,14]. The naked mole-rat and star-nosed mole are other examples of species possessing tiny eyes that mostly rely on tactile cues [12,13]. Therefore, trade-offs/coevolution between sensory systems might concern the senses in general. In the case of the fruit flies, although this might not have affected the balance between vision and olfaction, mechanoreception might also play a role, notably because it was observed that the Dark-flies possess longer bristles to detect tactile information and vibrations [49]. One of the challenges for the future will be to integrate other senses in comparative studies, and to understand the coevolution between sensory systems by taking into account 1) the sensory specificities of each species; 2) the fact that the mechanisms of neural integration differ between sensory systems. This might make measures of brain areas not sufficient, and other alternatives might have to be found in the future for measuring sensory capabilities.

To conclude, our observations are based on artificial selection in fruit flies in accordance with their lifestyle, but other observations might be made on different species/environments. Here, we have brought new highlights about the coevolution between vision and olfaction in the light of darkness, but also many new questions. These questions are not only essential to understanding how species have evolved in the past, but are oriented to anticipate the future of species. For example, it has been shown recently that human activity pushes animals to adopt a more nocturnal lifestyle [50]. Therefore, it becomes important to understand how individuals are selected and will perceive the world in the dark, and whether our impact on species’ shift to nocturnal life is reversible, since we are at the origin of such evolutionary change. However, human activities are not only pushing species to more nocturnal environments, but are, in general, changing the lifestyle and evolutionary pressures on species, which may open the way to new studies.

Ethics

The work was with insects, which are not subject to the same legal and regulatory rules as vertebrates, and do not fall under EU Directive 2010/63/EU on the protection of animals used for scientific purposes. However, a general ethical approval for the project was granted by Newcastle University for this project.

Images of the eyes of Dark-flies (A) and Oregon flies (B) in females (top) and males (bottom). The mean (+SEM) surface of a single eye (c) of male and female Dark-flies and Oregon flies.

(TIF)

Click here for additional data file.

Eyes measurement’s method and results.

(PDF)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

4 Nov 2019

PONE-D-19-27069

Back to the light, coevolution between vision and olfaction in the “Dark-flies” (Drosophila melanogaster)

PLOS ONE

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Impacting environmental changes provide a core driving force for evolutionary adaptations. How the nervous system is remodelled to cope with such changes remains largely unknown. The rapid generation time of fruit flies allows to investigate the genetic basis of adaptations.

In the work described in the current manuscript aims to address changes in brain architecture in flies kept in constant darkness for generations. The authors provide evidence that there are adaptations in the size of the antennal lobe, optic ganglia and overall brain size.

While the overall motivation for the study is indeed of interest there are several points that remain unclear.

- It is entirely unclear what flies were used for which type of analysis. In the method section the authors try to elaborate on the history of the “dark flies”, however it remains unclear if the original dark-fly stock was lost or if only the control stock was lost. Also, how this relates to the flies from 2014 is unclear.

It is critical that the authors clearly describe how the current experiment was done.

The authors may choose to add a graphical representation of the actual experiments performed.

- In the first result section “Body, eye and whole brain size” the eye-size analysis seems to have been forgotten!

While I assume that there was no difference (based on the supplemental data), there are a couple points taken into account.

1- Since the eye is an uneven surface the measurement may not be accurate. This may be improved by taking a high-resolution image of the eye in the focal plane (or scanning electron microscopy).

2- While not absolutely critical, this notion raises the question if the number of ommatidia is decreased.

- The volumetric analysis, a main point in the current manuscript remains poorly explained. The authors used the “measure stack” plugin in FIJI, however it is not described how different brain areas were distinguished and if this was based on the entire optic lobe ganglia (including cell bodies/cortex) or only neuropil (which I would assume was done for the antennal lobe neuropil).

- I feel it is critical to note that simply by exposing the animals to light it is unclear if and what type of selection occurred. Since flies are raised in rather artificial laboratory conditions and no controlled selective pressure was applied it is unclear what the relevant changes reflect. This should be clearly defined and discussed.

- The data displayed in Figure 3 are surprising. While it seems that the overall brain volume and optic lobe volume increases from Dark-flies to DF1G to DF65G and OR flies, the main change in antennal lobe size appears to occur from Dark-flies to DF1G flies. How can this be explained? Such large adaptation occurring in one generation seems rather strange.

Wording:

130 “After killing the flies “ should be “Flies were scarified” or similar.

Reviewer #2: 1. Due to lack of clarity in several parts of the manuscript it is hard to be fully confident that all conclusions are sound (see the review document).

4. As mentioned above, the manuscript needs to be revised for clarity.

**********

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Reviewer #1: No

Reviewer #2: Yes: Primoz Ravbar

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Submitted filename: Review_BackToLight.doc

Click here for additional data file.

13 Dec 2019

Reviewer #1

- It is entirely unclear what flies were used for which type of analysis. In the method section the authors try to elaborate on the history of the “dark flies”, however it remains unclear if the original dark-fly stock was lost or if only the control stock was lost. Also, how this relates to the flies from 2014 is unclear. It is critical that the authors clearly describe how the current experiment was done. The authors may choose to add a graphical representation of the actual experiments performed.

We thank the reviewer for this remark. We agree that a graphical representation is better to understand the evolution of flies, and have included a new figure (Figure 1) in order to explain it.

- In the first result section “Body, eye and whole brain size” the eye-size analysis seems to have been forgotten!

While I assume that there was no difference (based on the supplemental data), there are a couple points taken into account.

1- Since the eye is an uneven surface the measurement may not be accurate. This may be improved by taking a high-resolution image of the eye in the focal plane (or scanning electron microscopy).

2- 2- While not absolutely critical, this notion raises the question if the number of ommatidia is decreased.

Since our analysis for the eye-size is far for being complete, we decided to keep this analysis as supplemental data in order not to weaken this article. The questions of the reviewer are very interesting. However, we are not able to make images with a better resolution since we are not located in Newcastle anymore. This also justified our choice to keep these data as supplemental data.

- The volumetric analysis, a main point in the current manuscript remains poorly explained. The authors used the “measure stack” plugin in FIJI, however it is not described how different brain areas were distinguished and if this was based on the entire optic lobe ganglia (including cell bodies/cortex) or only neuropil (which I would assume was done for the antennal lobe neuropil).

We added details within the text.

- I feel it is critical to note that simply by exposing the animals to light it is unclear if and what type of selection occurred. Since flies are raised in rather artificial laboratory conditions and no controlled selective pressure was applied it is unclear what the relevant changes reflect. This should be clearly defined and discussed.

We appreciate this comment and we added a part at the beginning of the discussion about it (Lines 371-377).

- The data displayed in Figure 3 are surprising. While it seems that the overall brain volume and optic lobe volume increases from Dark-flies to DF1G to DF65G and OR flies, the main change in antennal lobe size appears to occur from Dark-flies to DF1G flies. How can this be explained? Such large adaptation occurring in one generation seems rather strange.

We thank to the reviewer for this substantial insight. We do not have any explanation about the fact that the ALs decreased in size mostly between the Dark-flies and the DF1G. Since, we do not have any explanation about it, we were unable to discuss about it. If the reviewer would have some idea about such phenomenon, we are ready to make changes accordingly.

Wording:

130 “After killing the flies “ should be “Flies were scarified” or similar.

The line has been changed accordingly.

-----------------------------------------------------------------------------------------------------------------------------------

Reviewer #2

- Overall the paper is interesting, the methods, results and the conclusions seem generally credible. However, the manuscript needs major revisions in terms of clarity. In the review below only several specific cases where the revisions is necessary are stated, however, the manuscript probably needs major revisions way beyond these examples. In particular the hypothesis and predictions should be clearly stated. The Figures and the Results should address the predictions clearly and any discrepancies between the predictions and the results should be pointed out and thoroughly discussed. The justifications for the control groups used should also be made more clear.

- This reviewer recommends the manuscript for publication based on intriguing, indeed fascinating, question of how development and evolution of sensory systems can be affected by depriving flies of one sensory modality (vision) across many generations, in a controlled environment. The reviewer is confident that the authors will be able to present their current results and conclusions in a clear form in the revised versions.

Line 14: “the Dark-flies returned back to the light” Does this refer to DF65G flies?

The line refers both to DF1G and DF65G. Appropriate clarifications are made.

Line 36: “environment to see evolution” Maybe: “… to observe evolution...”

The line has been changed accordingly.

Line 54: “between extent species” Maybe: “Between several species”?

We kept the term extent in the sense that we would like to refer to species that are still present nowadays on the earth, in opposition to fossils and other species that disappeared. Since analyses may be made on fossils, we would like to keep this distinction and hope that this is suitable for the reviewer.

Line 64: “...or alternative...” Do the authors mean “consecutive”? This paragraph should be re-written for clarity. In particular lines 66-68 are hard to comprehend by this reviewer.

We thank the reviewer for this suggestion. The whole paragraph has been adjusted in order to be more unambiguous. We also kept the work « consecutive » as suggested.

Lines 82-88: Another control for DF65G flies should be Dark-flies treated exactly the same as the experimental group but without light. If this is what was done, authors should clarify. It would be nice to observe any dosage effect between flies raised in light for various numbers of generations.

Although the suggestion makes sense and we agree that the DF65G without light would be interesting to observe, unfortunately we do not have the appropriate data and cannot perform this experiment anymore. We really thank the reviewer for this suggestion and this would be an interesting point to investigate in the future, although Barth et al. investigated brain and OLs sizes in fuit flies raised in the dark for one generation.

There are two possible mechanisms for the sensory trade-off that the authors observe. One is a per-existing stimulus-dependent plasticity, whereby brain development of these sensory areas is a function of sensory input (activity-dependent plasticity). The other mechanism for the trade-off would be “hard-wired” changes of the sensory areas resulting from evolutionary selection. If the former mechanism is the case, we should expect to observe the differences in sensory areas between Dark-flies and DF1G, while in the case of the latter mechanism, the evolutionary selection, the differences should only be observed between Dark-flies (or DF1G flies) and the DF65G flies. The authors should clarify their motivation for setting the control groups.

We thank to the reviewer for this substantial insight. We included a new paragraph in the discussion to make things less confusing (lines 413-430). We hope that this paragraph may satisfy the reviewer and reply to questions that were unclear until now.

Line 98: the line should read: “...12:12 LD lighting conditions...”

Appropriate changes have been made.

Lines 194-198: authors should state the N (number of flies) for each group across the comparisons.

The number of flies that were used for each experiment was already included in the material and method section. In order to make things clearer, we added these numbers on the figures. We hope that this change is adequate for the reviewers.

Line 202: the text is probably referring to Fig. 2D, not Fig 3D .

Appropriate changes have been made. More generally, our references to figures were erroned and we apologize for such mistake.

Lines 208-214: Should the text be referring to Fig. 3 rather than Fig. 4? If so, then it makes sense. Otherwise it is difficult to see how these results are reflected in the figure.

Appropriate changes have been made.

Lines 224-232: the text is probably referring to Fig. 4, not Fig 5. The authors state that there was no effect on OL size in Fig. 4 yet there seems to be a significant difference between the Dark-flies and the other three categories in terms of OL size.

Appropriate changes have been made.

Line 235 (and elsewhere): should “main effect” be stated as “major effect”?

Appropriate changes have been made.

Lines 258-260: If the difference in OL and AL sizes were significant between Dark-flies and DF1G flies, wouldn’t this suggest a developmental rather than evolutionary cause? Are the authors suggesting that a combination of developmental and evolutionary mechanisms is implicated in the observed differences? Please clarify.

We appreciate the comments. Indeed, this is the case. We added a paragraph in order to make the discussion clearer (lines 413-430) and included this way of thinking by talking about a process in two steps.

Line 295: “… i.e. mechanosensory system ...” “e.g.”?

Appropriate changes have been made.

Lines 292-304: this paragraph is difficult understand and the references to the figure and the table are unclear. Should be re-written.

We modified this paragraph in order to make it more comprehensible. We hope that the changes that were made are adequate and made it understandable.

Line 380: “the light of darkness” should read “the light or darkness”

We kept the terms « light of darkness » since our wish was to make a word play here, in order words, we would like to express the fact that darkness (the fact that we used darkness) enabled us to bring new insights.

Figure 1: The number of flies in each group should be stated in the figure.

For the figures, please note that the number of the figures changed since we added one more figure. For the previous figure 1, newly figure 2, we added the number of flies as requested.

Figure 2: Label the fly groups in the figure.

We have labeled the brain with the group. Concerning the bar plot, the groups are indicated. If there is something else that we missed for changing the figure and in accordance with the reviewer, we are open for making changes.

Figure 3: Add the number of flies in each category. Add p-values or other indicators of significance to the figure. Confidence intervals are hard to read for DF65G group for (OL and AL).

The number of flies is already present on the figure (on the bottom). If the reviewer could be more explicit, we can make changes according to his request. We changed the confidence interval to make it clearer and added letters to represent significant changes. We hope that these changes are adequate for the reviewer.

Figure 4: Add the legend. Units on y-axis are not defined. Confidence intervals are hard to read for DF651 group for (OL and AL).

We added the legend and changed the confidence intervals in order to make it more clear. Concerning the X-axis, we added some marks to make it clearer.

28 Jan 2020

Back to the light, coevolution between vision and olfaction in the “Dark-flies” (Drosophila melanogaster)

PONE-D-19-27069R1

Dear Dr. Carle,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Matthieu Louis

Academic Editor

PLOS ONE

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the revised manuscript the authors have removed the incomplete eye analysis, which makes the manuscript more coherent (Except in method section line 170 the eye is still included). Similarly most of the methodology is more appropriately represented and can be easily followed.

My only minor remaining concern lies in the surprising finding of the antennal lobe size of DF1G.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Primoz Ravbar

4 Feb 2020

PONE-D-19-27069R1

Back to the light, coevolution between vision and olfaction in the “Dark-flies” (Drosophila melanogaster)

Dear Dr. Carle:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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