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. 2014 Dec 9;3:e03785. doi: 10.7554/eLife.03785

Dopamine drives Drosophila sechellia adaptation to its toxic host

Sofía Lavista-Llanos 1,*, Aleš Svatoš 1, Marco Kai 1,†, Thomas Riemensperger 2,‡, Serge Birman 2, Marcus C Stensmyr 1,§, Bill S Hansson 1
Editor: Mani Ramaswami3
PMCID: PMC4270095  PMID: 25487989

Abstract

Many insect species are host-obligate specialists. The evolutionary mechanism driving the adaptation of a species to a toxic host is, however, intriguing. We analyzed the tight association of Drosophila sechellia to its sole host, the fruit of Morinda citrifolia, which is toxic to other members of the melanogaster species group. Molecular polymorphisms in the dopamine regulatory protein Catsup cause infertility in D. sechellia due to maternal arrest of oogenesis. In its natural host, the fruit compensates for the impaired maternal dopamine metabolism with the precursor l-DOPA, resuming oogenesis and stimulating egg production. l-DOPA present in morinda additionally increases the size of D. sechellia eggs, what in turn enhances early fitness. We argue that the need of l-DOPA for successful reproduction has driven D. sechellia to become an M. citrifolia obligate specialist. This study illustrates how an insect's dopaminergic system can sustain ecological adaptations by modulating ontogenesis and development.

DOI: http://dx.doi.org/10.7554/eLife.03785.001

Research organism: D. melanogaster, other

eLife digest

Many insect species rely on another animal or plant species for their own reproduction. For example, a fruit fly called Drosophila sechellia—which is found in the Seychelles—will only feed and lay its eggs on the fruit of a species of tree called Morinda citrifolia. This pairing is particularly unusual because these fruits, commonly called morinda, are toxic to all other Drosophila species.

Female Drosophila sechellia flies produce fewer eggs than other Drosophila species, which makes it difficult to raise this species in the laboratory. However providing these flies with morinda fruit, or chemicals from this fruit, was known to increase the expression of many genes involved in egg production and stimulate the flies to lay more eggs. Nevertheless, the reasons why this species of fruit fly depends on the toxic morinda fruit were unclear.

Now Lavista-Llanos et al. have confirmed that feeding Drosophila sechellia flies a diet of morinda fruit—instead of a typical laboratory diet—causes these flies to produce six-times as many eggs. Furthermore, this morinda diet had effects that went beyond the previously reported stimulatory effects of acidic chemicals in the fruits triggering the flies to lay more eggs.

Egg production in flies is controlled by dopamine, and a lack of this hormone is known to reduce the size of other fruit flies' ovaries and the number of eggs that they produce. Lavista-Llanos et al. went on to feed female Drosophila sechellia flies the chemical building blocks that make up the dopamine hormone, and one such chemical (called l-DOPA) caused the flies to produce more eggs. This did not occur when the flies were fed dopamine itself.

Lavista-Llanos et al. discovered that Drosophila sechellia flies have very high levels of dopamine but much lower levels of l-DOPA than other Drosophila fly species; and revealed that this was because a gene called Catsup is mutated in Drosophila sechellia. When Lavista-Llanos et al. mutated the same gene in another Drosophila species, the mutant flies produced fewer eggs and abnormally accumulated an enzyme (which makes l-DOPA) inside their developing eggs—just like Drosophila sechellia.

The presence of l-DOPA in morinda fruit partly compensates for the reduced fertility of Drosophila sechellia and the other flies with mutations in the Catsup gene. Lavista-Llanos et al. discovered that removing or replacing l-DOPA in the morinda fruit caused the flies to produce fewer eggs. Furthermore, the l-DOPA present in morinda increases the size of Drosophila sechellia eggs, which in turn helps them to survive their toxic environment.

Lavista-Llanos et al. also discovered that feeding dopamine to vulnerable Drosophila species helps them to cope with the toxic effects of a morinda diet. One of the next challenges will be to uncover how chemicals from the morinda fruit affect the dopamine system of the flies. It is also unknown if the dopamine hormone also influences the strong attraction that Drosophila sechellia feels towards its only host, the morinda fruit.

DOI: http://dx.doi.org/10.7554/eLife.03785.002

Introduction

Morinda citrifolia fruit (morinda) is the sole host of Drosophila sechellia (Tsacas and Baechli, 1981), a close relative of Drosophila melanogaster and endemic to the Seychelles archipelago (Louis and David, 1986). A peculiar aspect of the specialization is that morinda fruits are toxic to all other drosophilids (Legal et al., 1992). The toxicity stems from a high content of carboxylic acids (primarily octanoic and hexanoic acid) (Legal et al., 1994), to which D. sechellia appears to be resistant (Farine et al., 1996). The chemosensory system of D. sechellia is specialized in detecting and coding key volatiles produced by morinda (Dekker et al., 2006) while devoid of the repellence towards the acids (Matsuo et al., 2007). On the other hand, D. sechellia females exhibit a low reproductive potential, partly because of a low ovariole number and partly because of fairly low egg production (R'Kha et al., 1991; R'kha et al., 1997), making it difficult to raise D. sechellia under laboratory conditions. In turn, morinda stimulates egg production (R'kha et al., 1997), and D. sechellia clearly prefers to oviposit in medium containing morinda carboxylic acids (Amlou et al., 1998). On its host, D. sechellia increases expression of genes involved with oogenesis and fatty acid metabolism (Dworkin and Jones, 2009). Thus, we here examined the dependence of Drosophila sechellia on morinda, for optimal reproduction.

All animal embryos rely on maternally provided gene products for their initial development prior to zygotic genetic synthesis. Maternal effects can thus act as a form of cross-generational phenotypic plasticity, playing a role in an animal's adaptation to toxic environments. Embryonic survival in morinda is a maternally inherited trait and does not depend on the genotype of the embryo (R'Kha et al., 1991). We therefore considered if maternal effects sustained the evolutionary process that has driven the specialization of D. sechellia. Results enhance our understanding of the reproductive behaviour of Drosophila and suggest an ontogenetic mechanism of insect adaptation to a toxic host.

Results and discussion

We first examined the influence of chemicals found in morinda on the reproductive system of D. sechellia by testing the effect of diet on egg production. We raised two geographically different populations of D. sechellia on standard Drosophila cornmeal medium (standard diet), on morinda fruit (morinda diet), or on a non-host fruit (banana diet). For comparison, we also raised two strains of D. melanogaster (wild-type Berlin and Canton-S) on the same media (see ‘Materials and methods’). To score the rate of egg production, we transferred the flies to oviposition cages containing agar plates devoid of oviposition stimuli (i.e., yeast or morinda carboxylic acids). In agreement with previous reports, D. sechellia raised on standard diet produced few eggs compared to D. melanogaster wild-type Berlin and D. melanogaster Canton-S (Figure 1A and Figure 1—figure supplement 1), raised on the same media. The addition of morinda carboxylic acids to the standard diet did not increase egg production in D. sechellia (Figure 1B). D. sechellia raised on a non-host fruit (banana diet) showed a slight increase in the number of eggs laid (Figure 1B), but this number did not increase further with the addition of morinda carboxylic acids (Figure 1B). D. sechellia raised on morinda diet, however, showed a sixfold increase in egg production (Figure 1B and Figure 1—figure supplement 1). Morinda diet did not affect the number of eggs produced by D. melanogaster wild-type Berlin and slightly reduced those of D. melanogaster Canton-S (Figure 1C and Figure 1—figure supplement 1), as did the addition of morinda carboxylic acids to the standard diet (Figure 1C). Banana, however, tripled egg production in D. melanogaster wild-type Berlin (Figure 1C). This increase could be inhibited by the addition of morinda carboxylic acids to the banana (Figure 1C). These results demonstrate that the presence of a natural host modulates the reproductive capacity of Drosophila. In particular, morinda has a strong effect on D. sechellia egg production that goes beyond the reported stimulatory effect of the carboxylic acids on oviposition (Amlou et al., 1998).

Figure 1. Morinda increases egg production in D. sechellia.

(A–C) Egg production (egg/female/day) (N > 20) (A) and its relative change (N > 5) (B and C) in D. sechellia (14021–0248.25, sec) and D. melanogaster wild-type Berlin (wtB) fed a standard diet (sd), or morinda diet (md), or banana diet (bd), or diets supplemented with morinda carboxylic acids (+OA:HA). (D) Confocal images showing the surface (top) or the interior (middle) of ovarioles stained with nucleic acid specific dyes (sytox orange (SO) and TOTO) of D. sechellia (14021–0248.25) fed a standard diet (sec-sd) or a morinda diet (sec-md). The follicle cells (f) surrounding the oocyte (o) or stretched over the nurse cells (n) (arrow) are indicated for early (<S8) or vitellogenic cysts (>S8). Scale bar 100 μm. (E) Rate of vitellogenesis (>S8/<S8, top graph) and number of cysts (total cysts, lower graph) (N > 8) in D. sechellia (14021–0248.25, sec) and D. melanogaster wild-type Berlin (wtB) fed a standard diet (sd), or a morinda diet (md), or a diet supplemented with morinda carboxylic acids (+OA:HA). Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test (B–E); ****p < 0.00001 using Student's t test to compare species (A). Error bars represent s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03785.003

Figure 1.

Figure 1—figure supplement 1. Morinda stimulates egg production in D. sechellia.

Figure 1—figure supplement 1.

Egg production (egg/female/day) in D. sechellia 14021–0248.25 (sec.25, N = 3), D. sechellia 14021–0248.31 (sec.31, N = 3), D. melanogaster wild-type Berlin (wtB, N = 3) and D. melanogaster Canton-S (CS, N = 3), fed a standard diet (sd) or morinda diet (md). ns = non-significant, *p < 0.05 using Student's t test to compare diets in each group. Error bars represent s.e.m.
DOI: http://dx.doi.org/10.7554/eLife.03785.004

Figure 1—figure supplement 2. Apoptosis in D. sechellia ovaries.

Figure 1—figure supplement 2.

Confocal fluorescent images (right) and light transmission images (left) of D. sechellia (14021–0248.25, sec) ovaries of flies fed a standard diet (sd) (top) or morinda diet (md) (bottom), stained with the vital die acridine orange to label apoptosis (Arama and Steller, 2006). The number of apoptotic cysts per ovary (average ± s.e.m.) is indicated for a standard diet (N = 11) and morinda diet (N = 10). Insets show in detail apoptosis occurring at the germanium (top) and an example of the occasional apoptotic cysts in ovaries of flies fed morinda (bottom). Scale bar 20 μm.
DOI: http://dx.doi.org/10.7554/eLife.03785.005

Figure 1—figure supplement 3. Feeding behavior in D. sechellia.

Figure 1—figure supplement 3.

(A) Individual weight (mg/fly) (N > 20) of D. sechellia (14021–0248.25) mated females and males grown on standard diet (sd) or morinda diet (md). (B) Food intake (N = 3) measured as light absorbance (a. u., arbitrary units) of ingested sulforhodamine B added to a standard diet (sd) or morinda diet (md). *p < 0.01 using Student's t test to compare diets in each group. Error bars represent s.e.m.
DOI: http://dx.doi.org/10.7554/eLife.03785.006
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To study the mechanism behind the dietary modulation of egg production, we next examined oocyte development. The germarium in each ovariole continuously produces oocyte-cysts, each composed of 15 nurse cells and one oocyte, surrounded by a layer of follicular cells (Figure 1D). Each oocyte-cyst follows 14 stereotypical consecutive stages of pre-vitellogenic (S1–S7) and vitellogenic (S8–S14) growth, each easily distinguishable by morphological criteria (Spradling et al., 1997). Mated D. melanogaster females typically exhibit multiple ovarioles carrying vitellogenic cysts (Spradling et al., 1997). Females of D. sechellia had cysts normally composed of 15 nurse cells and one oocyte, surrounded by follicular cells (Figure 1D). Kept on standard diet, mated D. sechellia held oocytes in early developmental stages (<S8) (Figure 1D) with few ovarioles, if any, carrying vitellogenic cyst (>S8), what resulted in a significantly lowered rate of vitellogenesis (calculated as >S8/<S8) in D. sechellia compared to D. melanogaster wild-type Berlin (Figure 1E). The low number of ovarioles in D. sechellia (R'kha et al., 1997) was reflected in the low number of total cysts compared to in D. melanogaster wild-type Berlin (Figure 1E). The halt in oocyte development explains the low production of eggs in D. sechellia raised on standard diet. D. sechellia fed on morinda diet showed a significantly increased vitellogenesis rate (Figure 1D,E). Adding the morinda carboxylic acids, however, had no effect on vitellogenesis in D. sechellia (Figure 1E). We thus conclude that D. sechellia requires components of the morinda fruit other than just the carboxylic acids to induce vitellogenesis and increase egg production.

Environmental and physiological stressors can cause egg chambers in the ovaries of Drosophila to be eliminated by apoptosis at oogenesis checkpoints in region-2/3 of the germarium or cyst stage S7/S8 (the mid-oogenesis checkpoint) (Drummond-Barbosa and Spradling, 2001; McCall, 2004). To analyze if apoptosis occurs in D. sechellia, we stained live ovaries with the vital dye acridine orange (Arama and Steller, 2006). Flies fed a standard diet showed massive apoptosis occurring at S7/S8 cysts, and to a lesser extent in region-2/3 of the germarium (Figure 1—figure supplement 2); suggesting that apoptosis could be the reason for the depressed oviposition on standard media. On the other hand, D. sechellia flies fed a morinda diet showed strikingly few apoptotic cysts (Figure 1—figure supplement 2), in line with the stimulatory effect of morinda in egg-production. Low food availability can trigger the mid-oogenesis checkpoint (Terashima and Bownes, 2004). Thus, we asked if the observed apoptosis in D. sechellia raised on standard diet was a consequence of starvation or aberrant feeding behaviour. In fact, D. sechellia adults raised on the standard diet showed slightly higher weights (Figure 1—figure supplement 3), and also consumed more food than did flies raised on morinda diet (Figure 1—figure supplement 3). Hence, we conclude that the halt in oogenesis observed in D. sechellia is not a consequence of starvation or aberrant feeding behaviour.

Oocyte-cyst progression is under tight maternal hormonal regulation: juvenile- and steroid hormones are mutually controlled by the biogenic amine dopamine (DA), ensuring normal oogenesis (Gruntenko and Rauschenbach, 2008). Interruption of DA during development in D. melanogaster results in small ovaries and poor egg production (Neckameyer, 1996), both of which are features that characterise adult D. sechellia (R'kha et al., 1997). Moreover, genes involved in DA differentiation were shown differentially expressed in D. sechellia compared to the generalists species D. melanogaster and the sister Drosophila simulans (Dworkin and Jones, 2009; Wurmser et al., 2011). Accordingly, we wondered if D. sechellia suffers from a DA deficiency. Therefore, we first tested if the addition of monoamines was sufficient to rescue egg production by feeding D. sechellia standard diet supplemented with amines and amine precursors. Indeed, supplementing the standard diet with the DA precursor 3,4-dihydroxyphenylalanine (l-DOPA) significantly increased the number of eggs produced by D. sechellia (Figure 2A), whereas the addition of DA itself had no effect (Figure 2A). Likewise, adding the monoamine octopamine (OA), or its precursor tyramine (TA), did not affect egg production (Figure 2A). l-DOPA supplementation did not change the total number of D. sechellia oocyte-cysts (p = 0.28103, one-way ANOVA). Instead, it drastically reduced apoptosis in the ovary (Figure 2B) increasing vitellogenesis significantly (Figure 2C), whereas DA, TA and OA had no such effect. These results show that the administration of the DA precursor l-DOPA is sufficient to stimulate oocyte progression and egg production in D. sechellia.

Figure 2. Morinda l-DOPA is required to stimulate egg production.

(A) Egg production (egg/female/day) (N > 6) (B) quantification of apoptosis (apoptotic cysts/ovary) (N > 6) and (C) rate of vitellogenesis (>S8/<S8) (N > 12) in D. sechellia (14021–0248.25) flies fed a non-supplemented (−) standard diet (sd) or a standard diet supplemented with L-3,4-dihydroxyphenylalanine (1 mg/ml, l-DOPA); dopamine (1 mg/ml, DA); tyramine (2 mg/ml, TA) or octopamine (2 mg/ml, OA); or a non- pretreated morinda diet (md) or a morinda diet pre-treated with catechol-O-methyltransferase (2.5 U per gram of fruit, md + COMT). Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test. Error bars represent s.e.m. (D) The total ion chromatogram (top trace) shows all compounds present in morinda extract, and the extracted ion chromatogram (lower trace) corresponds to the exact mass of sum formula of L-3,4-dihydroxyphenylalanine (l-DOPA) present in the fruit; as analysed by UHPLC-MS. (E) Egg production (eggs/female/day) (N > 3) in D. sechellia (14021–0248.25) flies fed a morinda diet (md) non-pre-treated (−), pre-treated with catechol-O-methyltransferase (2.5 U per gram of fruit, COMT) or α-methyl-DOPA (0.4 mM, mDp). Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test. Error bars represent s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03785.007

Figure 2.

Figure 2—figure supplement 1. D. sechellia female fertility is modulated by morinda.

Figure 2—figure supplement 1.

(A) Rate of vitellogenesis (>S8/<S8) (N > 6) in D. sechellia (14021–0248.25) flies fed a morinda diet (md) non-pre-treated (−) or pre-treated with catechol-O-methyltransferase (2.5 U per gram of fruit, COMT). **p < 0.002 using Student's t test. Error bars represent s.e.m. (B) Confocal image shows D. sechellia (14021–0248.25, sec) egg retention in the ovary of a fly fed a morinda diet (md) supplemented with α-methyl-DOPA (0.4 mM, mDp). Dissected ovaries were stained with nucleic-acid-specific dyes (sytox orange (SO) and TOTO). The oviduct (ov) is indicated. Scale bar, 100 μm. (C) Bar graph shows ovipositon (egg/female/day) (N = 3) stimulated in D. sechellia (14021–0248.25) flies offered fresh morinda (md) as oviposition substrate compared to flies offered a standard oviposition substrate (sd). *p < 0.02 using Student's t test. Error bars represent s.e.m.
DOI: http://dx.doi.org/10.7554/eLife.03785.008
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The findings so far thus led us to speculate that morinda contains monoamines, which stimulate oogenesis in D. sechellia. Indeed, we detected 180.4 ± 3.5 ng l-DOPA (mean ± s.e.m; N = 3) per gram of fruit pulp (Figure 2D) in morinda extracts analysed by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Notably, we did not detect DA, OA or TA in morinda. Unripe (150.99 ± 8 ng l-DOPA [mean ± s.e.m; N = 3] per gram of fruit) and overripe (147.47 ± 8 ng l-DOPA [mean ± s.e.m; N = 3] per gram of fruit) morinda fruits contained l-DOPA in equal amounts, what suggests that oxidisation, known to occur commonly in monoamines through atmospherical exposure, is prevented in morinda. The preservation of l-DOPA is likely due to the high carboxylic acid content—octanoic and hexanoic acids have been shown to inhibit diphenol oxidase activity (Guo et al., 2010)—and the ensuing low pH of the fruit (4.1 ± 0.1 [N = 7] and 3.6 ± 0.1 [N = 5] [mean ± s.e.m.], ripe and overripe morinda, respectively; for comparison, 5.7 ± 0.2 [mean ± s.e.m.], [N = 4], ripe banana; p = 0.0001; one-way ANOVA), known for its antioxidant properties. Banana is reported to contain up to 1 mg DA per gram of fresh weight (Kanazawa and Sakakibara, 2000). Notably, DA in banana is synthesized directly from TA (Deacon and Marsh, 1971) and not via synthesis of l-DOPA. Presumably, the absence of l-DOPA in banana renders this non-host fruit unable to stimulate egg production in D. sechellia (see Figure 1B).

Is the presence of l-DOPA in morinda then necessary for the stimulatory effect on egg production? To address this question, we depleted levels of l-DOPA in morinda by pre-incubating fruit pulp with catechol-O-methyltransferase (COMT, 2.5 U/g morinda), an enzyme that catabolises l-DOPA into an unusable product (Gordonsmith et al., 1982). Treatment with COMT suppressed the anti-apoptotic effects of morinda (Figure 2B) and hindered the fruit from stimulating vitellogenesis (Figure 2—figure supplement 1) and egg production in D. sechellia (Figure 2E), reducing the number of eggs to levels in a standard diet (compare Figure 2A and Figure 2E). Additionally, we added α-methyl-DOPA (mDp, 0.4 mM), a non-hydrolysable l-DOPA analogue and a competitive inhibitor of the enzyme dopa decarboxylase that converts l-DOPA into DA, separately to the fruit. The presence of mDp in morinda strongly reduced egg production (Figure 2E); feeding a corresponding mDp-supplemented diet to D. melanogaster wild-type Berlin did not have such an effect (not shown). Notably, mDp in the fruit did not hinder oogenesis; instead, eggs were retained in the ovaries (Figure 2—figure supplement 1). This halt in ovulation was completely reversed one day after flies were moved to fresh medium (Figure 2—figure supplement 1), and, remarkably, oviposition was significantly enhanced when morinda was offered as an oviposition substrate (Figure 2—figure supplement 1). We conclude that morinda contains l-DOPA and that its presence in the fruit pulp is both sufficient and necessary to stimulate egg production in D. sechellia.

Morinda, accordingly, provides D. sechellia with the DA precursor necessary for the progression of oogenesis. Although providing a critical chemical, the acidity of the fruit, which helps preserve l-DOPA by preventing oxidisation, creates a hostile environment for the eggs to develop in. How do flies ensure that the eggs survive in this toxic environment? An interesting observation provides one clue. Eggs of D. sechellia are characteristically large compared to those of sibling species D. melanogaster, D. simulans, Drosophila ananassae, Drosophila erecta, Drosophila mojavensis, Drosophila persimilis, Drosophila pseudoobscura, Drosophila virilis, Drosophila willistoni and Drosophila yakuba (Markow et al., 2009). In accordance, we observed D. sechellia eggs to be 45% larger in size compared to eggs of D. melanogaster wild-type Berlin (Figure 3A), in a standard diet condition. Upon being fed a morinda diet, eggs of D. sechellia increased in volume twofold compared to eggs of conspecifics fed standard medium (Figure 3A and Figure 3—figure supplement 1); the resulting eggs had an almost threefold larger volume than did D. melanogaster wild-type Berlin eggs in standard conditions (Figure 3A). This effect could be replicated by supplementing standard diet with l-DOPA or, notably, DA (Figure 3B). On the other hand, adding COMT or mDp to a morinda diet prevented the fruit from having any effect on the volume of eggs (Figure 3C). These results indicate that egg size is also under a dopaminergic control regime during egg development. The increased size may provide buffering capacity to the eggs, helping them to cope with the toxic environment of the host. Indeed, the hatching rate of eggs transferred onto a morinda-containing medium was significantly higher in the enlarged eggs of flies fed morinda than in the smaller eggs of individuals fed only standard medium (Figure 3D). The low number of ovarioles of D. sechellia (R'kha et al., 1997) thus seems to be a result of a trade-off between number and size (Figure 3—figure supplement 2), favouring the enlarged eggs of females fed morinda.

Figure 3. Morinda enhances early fitness.

(A–C) Volume (mm3(10−3)) of D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) eggs produced by flies fed a standard diet (sd) or morinda diet (md) (N > 15) (A); D. sechellia (14021–0248.25) flies fed a non-supplemented (−) standard diet (sd), or supplemented with L-3,4-dihydroxyphenylalanine (1 mg/ml, l-DOPA) or dopamine (1 mg/ml, DA) (N > 23) (B); D. sechellia (14021–0248.25) flies fed a non-pre-treated (−) morinda diet (md), or pre-treated with catechol-O-methyltransferase (2.5 U per gram of fruit, COMT) or α-methyl-DOPA (0.4 mM, mDp) (N > 10) (C). (D) Egg hatching rate (N > 5) in D. sechellia (14021–0248.25) fed (feed) a standard diet (sd) or morinda diet (md), ovipositing (ovip.) in either media. Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test. Error bars represent s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03785.009

Figure 3.

Figure 3—figure supplement 1. Volume of D. sechellia eggs is modulated by morinda diet.

Figure 3—figure supplement 1.

Volume (mm3 (10−3)) of D. sechellia 14021–0248.25 (sec.25) and D. sechellia 14021–0248.31 (sec.31) eggs produced by flies fed a standard diet (sd) or morinda diet (md) (N > 7). Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test. Error bars represent s.e.m.
DOI: http://dx.doi.org/10.7554/eLife.03785.010

Figure 3—figure supplement 2. Female resource investment on fertility is conserved in D. sechellia.

Figure 3—figure supplement 2.

Histograms of egg volume (mm3 (10−3)) (top), ovariole number (middle), and egg mass production (egg mass, calculated as egg volume × ovariole number) (bottom) in D. sechellia 14021–0248.25 (red bar) and D. sechellia 14021–0248.31 (light red bar) fed a morinda diet, compared to 12 Drosophila siblings (D. ananassae (14024–0371.13), D. erecta (14021–0224.01), D. melanogaster (wild type Berlin and 14021–0231.36), D. mojavensis (15081–1352.22), D. persimilis (14011–0111.49), D. pseudoobscura (14011–0121.94), D. sechellia (14021–0248.25, white bar), D. simulans (14021–0251.195), D. virilis (15010–1051.87), D. willistoni (14030–0811.24), D. yakuba (14021–0261.01), (data from Markow et al., 2009), (grey bar)) fed a standard diet.
DOI: http://dx.doi.org/10.7554/eLife.03785.011
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Genetic changes in a single gene of the steroid hormone biosynthetic pathway made Drosophila pachea dependent on the uncommon sterols of its host plant, the toxic senita cactus (Lang et al., 2012). Likewise, the requirement of dietary l-DOPA suggests that its metabolism is impaired in D. sechellia. We next quantified l-DOPA in fly-tissue extracts by UHPLC-MS. D. sechellia—kept on standard diet—showed significantly lower levels of l-DOPA in whole flies, bodies and ovaries than did D. melanogaster wild-type Berlin (Figure 4A). However, in D. sechellia fed a morinda diet, l-DOPA levels were significantly increased compared to levels in conspecifics fed a standard diet, and surpassing those of D. melanogaster wild-type Berlin in standard diet (Figure 4B). In short, these results demonstrate that under laboratory conditions, l-DOPA is greatly reduced in D. sechellia, and that this l-DOPA deficiency can be remedied by a diet supplemented with morinda fruit.

Figure 4. Dopamine metabolism is impaired in D. sechellia.

(A) L-3,4-dihydroxyphenylalanine quantification (l-DOPA pg/fly) (N = 3) in whole fly, bodies and ovaries of female D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd). *p < 0.05 and **p < 0.002 using Student's t test. (B) Relative L-3,4-dihydroxyphenylalanine (% pg l-DOPA per mg body, % l-DOPA) (N = 3) in female D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd) or morinda diet (md). p = 0.0062 and p = 0.018 using Student's t test D. melanogaster vs D. sechellia fed, respectively, a standard diet or morinda diet. (C) Western blots of total protein whole-fly extracts for TH-PLE, CATSUP, and α-TUBULIN as a loading control, in D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd) or morinda diet (md). The numbers under TH-PLE and CATSUP protein lanes indicate the relative protein levels (normalised to α-TUBULIN). (D) Ratios of L-3,4-dihydroxyphenylalanine (l-DOPA/tyr) (N = 3) and dopamine (DA/tyr) (N = 3) to tyrosine substrate in female D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd). **p < 0.007 and ***p < 0.000007 using Student's t test. (E) Drosophila CATSUP protein structure scheme showing a signal peptide (grey box) and six trans-membrane domains (black boxes). Deletions (dash) and exchanges (grey or white) of amino acids in D. sechellia (14021–0248.25, sec) compared to in D. melanogaster wild-type Berlin (wtB) and in D. melanogaster DGRP-357 (DGRP-357) CATSUP are indicated. (F–H) Dopamine (DA ng/fly) (N = 3) (F), relative rate of vitellogenesis (% >S8/<S8) (N > 10) (G), and egg-volume (% mm3(10−3)) (n > 10) (H) in D. sechellia (14021–0248.25, sec), D. melanogaster wild-type Berlin (wtB) and D. melanogaster DGRP-357 (DGRP-357) fed a standard diet (sd). (I) Egg production (egg/female/day) (N > 3) in D. melanogaster wild-type Berlin (wtB), heterozygote flies (Catsup1/CyO), D. melanogaster DGRP-357 (CatsupIn270Del/CatsupIn270Del), trans heterozygote flies (CatsupIn270Del/Catsup1) and D. sechellia (14021–0248.25, sec), fed a standard diet (sd). Different letters denote significant differences (p < 0.05) using ANOVA followed by Tukey's test (F–I). Error bars represent s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03785.012

Figure 4.

Figure 4—figure supplement 1. TH-PLE and CATSUP expression in Drosophila.

Figure 4—figure supplement 1.

Western blots of total protein whole-fly extracts for TH-PLE, CATSUP, and α-TUBULIN as a loading control in D. melanogaster Canton-S (CS), D. sechellia (14021–0248.31, sec), D. simulans (14021–0251.004, sim), D. mauritiana (14021–0241.01, mau) and D. melanogaster DGRP-357 (DGRP-357) fed a standard diet. The numbers under TH-PLE and CATSUP protein lanes indicate the relative protein levels (normalised to α-TUBULIN).
DOI: http://dx.doi.org/10.7554/eLife.03785.013

Figure 4—figure supplement 2. Tyrosine quantification in Drosophila.

Figure 4—figure supplement 2.

D. sechellia (14021–0248.25) females (sec) show higher tyrosine content (expressed as picogram per fly [pg/fly]), compared to D. melanogaster wild-type Berlin females (wtB) and D. melanogaster DGRP-357 (DGRP-357) females, fed a standard diet. N = 3. Different letters denote significant differences (p < 0.05) using ANOVA followed by Tukey's test. Error bars represent s.e.m.
DOI: http://dx.doi.org/10.7554/eLife.03785.014

Figure 4—figure supplement 3. Sensory bristles.

Figure 4—figure supplement 3.

(A) Histograms of total (macro and micro quetas) number of bristles per sternopleural plaque and (B) graph of mode (N is indicated for each species) of female D. melanogaster wild-type Berlin (wtB), D. melanogaster Canton-S (CS), D. melanogaster DGRP-357 (DGRP-357), D. melanogaster DGRP-437 (DGRP437), D. melanogaster DGRP-304 (DGRP-304), and D. sechellia (sec) original from Praslin (14021–0248.31 (sec.31) and 14021–0248.08 (sec.08), Seychelles 14021–0248.27 (sec.27)) and Cousin (14021–0248.25 (sec.25) and 14021–0248.28 (sec.28)) islands.
DOI: http://dx.doi.org/10.7554/eLife.03785.015

Figure 4—figure supplement 4. L-DOPA rescues diminished CatsupIn270Del/Catsup1 egg production.

Figure 4—figure supplement 4.

Relative egg production (% egg/female/day) (N = 3) in D. melanogaster wild type Berlin (wtB) and D. melanogaster trans heterozygote flies (CatsupIn270Del/Catsup1) fed a non-supplemented (−) standard diet or a diet supplemented with L-3,4-dihydroxyphenylalanine (1 mg/ml, l-DOPA). ns = non-significant; *p < 0.015 using Student's t test to compare diets in each group.
DOI: http://dx.doi.org/10.7554/eLife.03785.016

Figure 4—figure supplement 5. Conserved Catsup sequence in D. sechellia.

Figure 4—figure supplement 5.

Drosophila CATSUP amino acids sequence showing deletions (dash) and exchanges (grey or white) of amino acids in D. sechellia (14021–0248.03 (sec_03), 14021–0248.07 (sec_07), 14021–0248.08 (sec_08), 14021–0248.25 (sec_25), 14021–0248.28 (sec_28)) compared to in D. melanogaster wild-type Berlin (wtB).
DOI: http://dx.doi.org/10.7554/eLife.03785.017
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The oxidation of the precursor amino acid tyrosine into l-DOPA is the first and rate-limiting step in the DA biosynthetic pathway (Levitt et al., 1965). This oxidation is catalysed by the enzyme tyrosine hydroxylase (TH), which is encoded by the pale (ple) locus in Drosophila (Neckameyer and White, 1993). The whole-fly expression of TH-PLE was increased in D. sechellia relative to in D. melanogaster wild-type Berlin (Figure 4C) and Canton-S (Figure 4—figure supplement 1), as revealed by Western blots. Thus, l-DOPA impairment in D. sechellia seems not to result from a low expression of its synthesizing enzyme. To assess if the activity of TH-PLE was altered in D. sechellia, we compared the ratios of product (l-DOPA and DA) to substrate (tyrosine) in whole-fly extracts. D. sechellia showed a 3.3-fold decrease in the l-DOPA/tyrosine ratio as compared to in D. melanogaster wild-type Berlin (Figure 4D) with a 1.4-fold increase in tyrosine content (Figure 4—figure supplement 2). However, in D. sechellia the DA/tyrosine ratio was increased 2.4 times with respect to in D. melanogaster wild-type Berlin (Figure 4D). DA itself was increased 4.8 times in D. sechellia (Figure 4F). In sum, we conclude that TH-PLE is active; in fact, DA levels are drastically enhanced in D. sechellia.

TH-PLE activity is negatively regulated via direct physical interactions with the protein Catecholamines up (Catsup) (Stathakis et al., 1999). Interestingly, Catsup loss-of-function mutations cause hyperactivation of TH-PLE and abnormally high levels of catecholamines (Stathakis et al., 1999), as well as infertility due to maternal arrest of oogenesis (Schupbach and Wieschaus, 1991; Stathakis et al., 1999). These phenotypes prompted us to investigate whether Catsup is impaired in D. sechellia. We cloned the ortholog of Catsup from D. sechellia, which revealed a 45 bp in-frame deletion of 15 amino acids in a predicted zinc-binding region of the protein (O'Donnell et al., 2002) and seven single amino acid exchanges (Figure 4E). Additionally, Western blots of whole-fly extracts revealed 2- to 3.7-fold decrease in CATSUP expression in D. sechellia compared to CATSUP expression in D. melanogaster wild-type Berlin (Figure 4C) and Canton-S (Figure 4—figure supplement 1). The reduced expression of D. sechellia CATSUP would explain the high DA levels in D. sechellia (see Figure 4F).

Is Catsup then responsible for the reproductive phenotypes in D. sechellia? An allele (In270Del) similar to D. sechellia Catsup has been described for a natural D. melanogaster population (DGRP-357) (Carbone et al., 2006) (Figure 4E). CatsupIn270Del has been associated with polymorphisms in the number of sensory bristles, starvation resistance and locomotor behavior (Carbone et al., 2006). As for D. sechellia, we found increased DA levels in adult D. melanogaster DGRP-357 compared to in D. melanogaster wild-type Berlin (Figure 4F). Reciprocally, D. sechellia showed an increased number of sensory bristles that were present in flies of three geographically different populations (Figure 4—figure supplement 3). To test if CatsupIn270Del was sufficient to generate egg phenotypes on par with those of D. sechellia, we next examined egg growth in D. melanogaster DGRP-357 females. Both D. sechellia traits—low levels of vitellogenesis (Figure 4G) and enlarged eggs (Figure 4H)—were present in D. melanogaster DGRP-357. Furthermore, D. melanogaster DGRP-357 produced fewer eggs than did D. melanogaster wild-type Berlin (Figure 4I), and the ovariole rate of egg production in D. melanogaster DGRP-357 females did not differ significantly from that of D. sechellia (p = 0.2104; Student's t test). To control for non-identified gene mutations present in D. melanogaster DGRP-357 that could contribute to lowering egg production, we generated heteroallelic flies carrying CatsupIn270Del in trans-heterozygosis with the null Catsup1 allele (Stathakis et al., 1999). CatsupIn270Del/Catsup1 females displayed significantly decreased egg production, below that of D. melanogaster wild-type Berlin and parental lines (Figure 4I), that could be rescued by feeding the flies a standard diet supplemented with l-DOPA (Figure 4—figure supplement 4). In sum, these results show that D. melanogaster flies carrying a D. sechellia Catsup-like allele mirror D. sechellia's low production of eggs.

The formation of TH-PLE functional protein begins during the maternal phase of egg development (Pendleton et al., 2007). Pharmacologically inhibiting TH-PLE arrests oocytes at S8 stage of development, which is prevented by the co-administration of l-DOPA (Pendleton et al., 1996). We thus asked if the diet requirement of morinda for D. sechellia oocyte progression results from a maternal impairment of the enzyme synthesizing l-DOPA. Ple mRNA has been shown expressed by in situ hybridization in nurse- and follicle cells (Neckameyer, 1996). We detected TH-PLE in the ooplasm of D. melanogaster wild-type Berlin developing oocytes (Figure 5A). Remarkably, D. sechellia oocytes showed big masses of TH-PLE abnormally accumulated in their ooplasm (Figure 5B), suggesting that the nurse-to-oocyte traffic of maternal Ple is hindered in this fly.

Figure 5. Expression of TH-PLE and CATSUP in Drosophila female reproductive system.

(A–C) Confocal images showing striking accumulation (arrowhead) of TH-PLE (green, anti-TH) in D. sechellia (14021–0248.25) (B) and D. melanogaster DGRP-357 (C), compared to in D. melanogaster wild-type Berlin (wtB) (A) oocytes. (D–F) Confocal images showing CATSUP (magenta, anti-SLC39A7) expressed in the nurse cells (arrow) of D. melanogaster wild-type Berlin (wtB) oocytes (D) and absent in the nurse cells of D. sechellia (14021–0248.25, sec) (E) and D. melanogaster DGRP-357 (DGRP-357) (F) oocytes. (G–I) Confocal image showing CATSUP expressed in the nuclei (arrow) and the membrane (arrowhead) of D. melanogaster wild-type Berlin (wtB) spermatheca secretory cells (G), and absent from D. sechellia (14021–0248.25) (H) and D. melanogaster DGRP-357 (DGRP-357) (I). Scale bar 20 μm.

DOI: http://dx.doi.org/10.7554/eLife.03785.018

Figure 5.

Figure 5—figure supplement 1. Egg hatching in morinda.

Figure 5—figure supplement 1.

(A) D. melanogaster DGRP-357 first-instar larvae (L1) hatching inside the female reproductive system. U: uterus; S: spermatheca; SR: seminal receptacle. Scale bar 20 μm. (B) Proportion of eggs hatched or moulted to larva 1 (L1) larva 2 (L2) or larva 3 (L3), in D. melanogaster wild-type Berlin (wtB), D. melanogaster pferta (pferta), D. melanogaster Oregon-R (OR), D. melanogaster Canton-S (CS), D. melanogaster DGRP-357 (DGRE-357) D. melanogaster DGRP-437 (DGRP-437), D. simulans (14021–0251.004, sim) and D. mauritiana (14021–0241.01, mau).
DOI: http://dx.doi.org/10.7554/eLife.03785.019
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Catsup encodes the Drosophila ortholog of the mammalian ZIP7/SLC39A7 zinc transporter and has recently been associated with trafficking of proteins during development (Groth et al., 2013). Flies carrying the I288T amino acid exchange shared in D. sechellia CATSUP and D. melanogaster DGRP-357 CATSUP (see Figure 4E) accumulate abnormal amounts of proteins in developing tissue (Groth et al., 2013). Notably, as for D. sechellia, we found big masses of TH-PLE abnormally accumulated in the ooplasm of D. melanogaster DGRP-357 oocytes (Figure 5C) strongly suggesting that CATSUP could regulate the nurse-to-oocyte traffic of maternal Ple in Drosophila. CATSUP was expressed in the perinuclear endoplasmic reticulum- and golgi-like cisternae of the nurse cells of S8 oocyte cysts (Figure 5D), what results a suitable intracellular distribution for a protein with traffic nurse-to-oocyte function. We found, additionally, CATSUP expressed in the spermathecal secretory cells (SSC) of the female reproductive tract (Figure 5G). As for whole flies extracts, D. sechellia and DGRP-357 showed diminished expression of CATSUP in the nurse cells (Figure 4E,F) and SSC (Figure 4H,I). A recent study showed that the SSC require a not-yet identified secretory pathway to control the ovulation and downstream movement of the eggs (Sun and Spradling, 2013). The ablation of the SSC causes ovoviviparity in D. melanogaster (Schnakenberg et al., 2011), a condition naturally occurring in D. sechellia (Markow et al., 2009). Remarkably, 81.8% of D. melanogaster DGRP-357 mated females had an egg in their uterus (against 22.7% of D. melanogaster wild-type Berlin; p < 0.0001, chi-squared test); many eggs showed an advanced embryonic development and even hatched inside the female reproductive tract (Figure 5—figure supplement 1).

Null ple alleles (Neckameyer and White, 1993) and conditional ple mutants at its restrictive temperature (Pendleton et al., 2007) are lethal at late embryonic stage. Zygotic ple expression starts at later stages of embryonic development (Lundell and Hirsh, 1994; Pendleton et al., 2007), with early stages depending on maternal TH-PLE. Maximal survival of D. sechellia egg (R'Kha et al., 1991) matches the proportion of eggs hatching in the term of 2.5 hr after oviposition (Markow et al., 2009)—that is: laid at ∼22 hr of embryonic development—what coincides with the peak of DA synthesis during embryogenesis (Wright, 1987). Thus, being ovoviviparous (Markow et al., 2009) may benefit D. sechellia embryos with dietary maternal l-DOPA and, additionally, provide the eggs with a protective cuticle (secreted at a late stage of embryonic development) highly resistant to environmental hazards. In fact, susceptible siblings embryos resist contact with morinda at late developmental stages (R'Kha et al., 1991). While no D. melanogaster (N = 345) eggs or D. sechellia sister species D. simulans (N = 58) and D. mauritiana (N = 226) eggs completely hatched in morinda pulp, 33% of D. melanogaster eggs produced by flies carrying CatsupIn270Del allele DGRP- (N = 271) hatched to larva 1 (Figure 5—figure supplement 1), some even reaching larva 2 and larva3. These results illustrate how a mutation that reduces female fertility can concomitantly contribute to increase fecundity in a novel niche.

How do D. sechellia adults cope with the toxic environment of morinda fruits to lay their eggs in the first place? In non-sechellia Drosophilids, morinda acids toxicity is mostly manifested as locomotion disorders followed by slowness in movement until complete immobilization and death (Legal et al., 1992). These behavioral phenotypes resemble the neurotoxic effects described in Drosophila upon exposure to fungal-derived organic volatiles of similar chemical structure to morinda carboxylic acids (Inamdar and Bennett, 2014), which have been portrayed as reducing DA in the flies (Inamdar et al., 2013). Thus, we asked if incrementing DA could aid the adult flies in overcoming the behavioral effects of morinda carboxylic acids. To test this, we fed adult D. melanogaster wild-type Berlin a synthetic diet supplemented with increasingly amounts of DA and tested their survival upon exposure to morinda carboxylic acids. After a few minutes of experiencing a natural concentration mix of octanoic and hexanoic acids, D. melanogaster wild-type Berlin flies fed a standard diet showed general restlessness and altered equilibrium with incorrect positioning of tarsi, followed by slowness in movement until complete immobilization. A 66% of females (Figure 6A) and 53% of males (Figure 6B) were completely immobilized after 120 min of exposure to the mix of acids. Dietary administration of DA declined the locomotion behavioral effects of morinda carboxylic acids in a dose-dependent manner (Figure 6A,B). D. melanogaster wild-type Berlin flies (females and males) treated with 100 mg/ml DA reached a survival degree not significantly different from that of D. sechellia (Figure 6C,D). These results suggest that the endogenously increased DA levels in D. sechellia (see Figure 4F) contribute to override the behavioral effects of morinda carboxylic acids. On the other hand, DGRP-357 flies—of intermediate DA levels (see Figure 4F)—showed D. melanogaster levels of resistance to morinda (41.2 ± 4.7 and 52.5 ± 2.8% survival in morinda, respectively in DGRP-357 [N = 6] and D. melanogaster wild-type Berlin [N = 4]; p = 0.09 using Student's t test), suggesting that further genes might contribute to elevate the levels of DA in D. sechellia. D. sechellia shows protein levels of TH-PLE, the rate limiting enzyme in DA production, higher than in the sister species D. simulans and D. mauritiana, and in wild type D. melanogaster (wild-type Berlin, CS) and DGRP-357 (see Figure 4E and Figure 4—figure supplement 1). Additionally, in a screen for genes that differ in expression between D. sechellia and four geographically distinct populations of its generalist sister species D. simulans, Wurmser et al. (2011) showed lower expression of the DA catabolic enzyme (Dopamine-N-acetyltransferase), what probably contributes to almost quintuplicate DA in D. sechellia (see Figure 4F). Thus, the enhancement of DA could have served as a founder event in the history of morinda becoming an obligate host for the specialist D. sechellia.

Figure 6. DA contributes to the behavioral resistance to morinda carboxylic acids.

Figure 6.

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(A–B) Survival kinetic curves for D. melanogaster wild-type Berlin (wtB) (A) females (f, N > 3) and (B) males (m, N > 3) exposed to morinda carboxylic acids (OA:HA) and fed a standard diet supplemented with either no (−), or increasing doses of DA (5 mg/ml, 10 mg/ml and 100 mg/ml). *p < 0.02 and **p < 0.002 using Student's t test. (C–D) Survival (%) (N > 3) of D. sechellia (14021–0248.25, sec) and D. melanogaster wild-type Berlin (wtB) (C) females (f) and (D) males (m), fed a non-supplemented (−) or DA (+DA, 100 mg/ml) supplemented standard diet upon 2 hr exposure to morinda carboxylic acids (OA:HA). Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test. Error bars represent s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03785.020

Our results are compatible with an evolutionary scenario in which an original Catsup allele carrying a 45-bp deletion and six non-synonymous mutations was present in the ancestor of present-day D. sechellia (Figure 4—figure supplement 5), probably showing differentially low expression of CATSUP compared to D. melanogaster and the sister species D. simulans and D. mauritiana (see Figure 4E and Figure 4—figure supplement 1); what caused diminished egg production and concomitantly enhanced early survival in the fruit. A second event, further elevating DA (see Figure 4F), would have redeemed adult D. sechellia from the toxic effects of morinda acids allowing them to oviposit in ripe fruits, benefiting from morinda stimulating effect on egg production and from the lack of siblings competition, rendering D. sechellia dependent on morinda. This dependency would have, in turn, shaped the super specialization of the chemosensory system (Dekker et al., 2006; Matsuo et al., 2007; Dworkin and Jones, 2009) present in today's D. sechellia morinda obligate specialist.

Conclusions

The molecular traits underlying adaptations and endurance to new toxic-hosts remain unknown. We present a novel role for the Drosophila catecholamine regulatory protein Catsup in maternal secretory functions and suggest that the malfunction of CATSUP contributed to D. sechellia becoming an obligate specialist on its toxic host. We propose an evolutionary scenario in which an initial mutation in D. sechellia DA metabolism caused impaired female fertility and fecundity, but concomitantly provided eggs and adults with resistance to morinda toxic acids. Together with an early loss of repellence (Matsuo et al., 2007), this initial maternally inherited tolerance allowed individuals to develop in morinda and feed the DA precursor, particularly enriched in this fruit, which strongly increased adult female fertility. In turn, the lack of repellence (Matsuo et al., 2007), and instead the strong attraction to morinda volatiles (Dekker et al., 2006), combined with the beneficial morinda effect on ovulation and egg laying (Figure 2—figure supplement 1), shaped D. sechellia's preference to oviposit in its natural host (Amlou et al., 1998).

Materials and methods

Chemicals

All used chemicals were of commercial origin and used without further purification. UHPLC-MS- grade water and methanol were used for chromatography.

Fly stocks and rearing

D. sechellia (14021–0248.03, 14021–0248.07, 14021–0248.08, 14021–0248.25, 14021–0248.27, 14021–0248.28, 14021–0248.31) were obtained from the Drosophila Species Stock Center (DSSC, https://stockcenter.ucsd.edu). D. sechellia (14021–0248.25), D. simulans (14021–0251.004) and D. mauritiana (14021–0241.01) were kindly provided by the Division of Chemical Ecology, Swedish University of Agricultural Sciences. D. melanogaster line wild-type Berlin was kindly provided by Silke Sachse. D. melanogaster line Oregon-R was kindly provided by Rafael Cantera. D. melanogaster pforta was captured in the Weingut Kloster Pforta (Naumburg, Germany). D. melanogaster lines Canton-S (BL1), DGRP-357 (BL25184), DGRP-437 (25194), DGRP-304 (BL25177) and Catsup1/CyO (BL5138) were obtained at the Bloomington Stock Center at Indiana University (http://flystocks.bio.indiana.edu/). The CatsupIn270De/Catsup1 flies were selected as not carrying CyO from the F1 of a corresponding crossing of the parental lines DGRP-357 and Catsup1/CyO. Flies were reared in vials (50 mm diameter × 95 mm high) at 25°C, 70% RH in L:D 12:12 on standard cornmeal–yeast–agar medium (standard diet), supplemented as specified in the text, or on fresh morinda pulp (morinda diet) collected from fruits of M. citrifolia plants kept in our greenhouse, or on banana. We observed no differences in survival to morinda fruit in our D. sechellia experimental stock along the successive generations (5 days survival in ripe morinda: 70.7 ± 5.6% and 83.2 ± 3.6% for new and old females [N = 3], p = 0.147 using Student's t test to compare stocks, respectively; 83.3 ± 16.7 and 64.7 ± 18.2 for new and old males, [N = 3], p = 0.493 using Student's t test to compare stocks, receptively), discarding an artificial adaptation to morinda acids. On the other hand, morinda toxicity (Legal and Plawecki, 1995) hindered a permanent experimental stock of D. melanogaster in the fruit, for what flies were fed morinda for as long as the experiment lasted. Morinda carboxylic acids were added to the standard diet or agar plates as indicated in the text, using a range of natural concentrations (0.07% vol/V of 3:1 octanoic:hexanoic mix) (Legal et al., 1994).

Adult survival

Triplicates of 10–20 flies were placed in vials (25 mm diameter × 95 mm high) containing ripe morinda and maintained at 25°C, 70% RH in L:D 12:12. Live adults were flipped daily to new vials, recording female and male survival. Data is expressed as percentage of females and males alive.

Egg production and oviposition

Egg production was scored in groups of 10–20 females and males kept during 4 days in cages (37.5 mm diameter and 58 mm high) holding agar plates containing 5% sucrose and devoid of yeast at 25°C, 70% RH in L:D 12:12. Agar plates were changed daily and the total number of eggs was summed. Egg production is expressed as number of eggs per female per day, averaged over >5 independent experiments. Oviposition of the eggs retained by mated females fed a morinda diet supplemented with α-methyl-DOPA (0.4 mM, mDp) was scored in groups of 20 females transferred to non-supplemented standard diet or morinda diet as oviposition substrates. The number of laid eggs was averaged over three independent experiments and expressed as number of eggs per female per day.

Ovariole staining and immunofluorescence

Ovaries (N > 8) were dissected and fixed in 500 μl of 4% paraformaldehyde in PT (phosphate buffer saline [PBS], 0.1% Triton X-100 [Sigma, St. Louis, MO]) during 30 min at room temperature. After being rinsed three times during 10 min in 1 ml PT, ovaries were incubated in 500 μl PT + 0.5 μl TOTO-3 (1 mM, Invitrogen, Life Technologies GmbH, Germany) + 0.5 μl SYTOX Orange (5 mM, Invitrogen, Life Technologies GmbH, Germany) and protected from light, overnight (ON) at 4°C. Ovaries were rinsed three times during 10 min in 1 ml PT and mounted in Vectashield (Sigma, St. Louis, MO). Oocyte-cyst stages were visually determined by inspection of stained ovaries under fluorescent microscope. Alternatively, fixed ovaries were blocked in PBST (PT, 5% normal goat serum [Sigma, St. Louis, MO]), incubated 24 hr at 4°C with primary antibody anti-TH (1:100, mouse, ImmunoStar, Acris Antibodies, GmbH, Germany) or anti-SLC39A7 (1:1000, rabbit, Sigma, St. Louis, MO), diluted in PBST, further rinsed three times in 1 ml PT during 10 min at room temperature (RT) and incubated respectively with fluorescently conjugated secondary antibodies anti-mouse (AlexaFluor488, Invitrogen, Life Technologies GmbH, Germany) and anti-rabbit (AlexaFluor546, Invitrogen, Life Technologies GmbH, Germany), diluted 1:250 in PBST at RT, protected from light. Ovaries were rinsed three times in 1 ml PT during 10 min and mounted in Vectashield (Sigma, St. Louis, MO). Confocal images were obtained at 1-μm intervals over 20 μm Z-stack using a LSM510 Meta confocal microscope (Zeiss, Jena, Germany).

Apoptosis

Apoptosis in D. sechellia ovaries (N > 6) was visualized following the protocol by Arama and Steller (McCall, 2004). Briefly, live ovaries were dissected in PBS and incubated in a freshly prepared solution of 0.6 μg/ml acridine orange (Sigma, St. Louis, MO) for 5 min, at RT. Ovaries were rinsed briefly in PBS and mounted in a drop of Halocarbon 700 oil (Sigma, St. Louis, MO) and observed immediately. Confocal images were obtained at 1-μm intervals over 20 μm Z-stack using a LSM510 Meta confocal microscope (Zeiss, Jena, Germany).

Feeding assay

Feeding assay was performed as described previously (Riemensperger et al., 2011). 10 D. sechellia 5-day-old flies (five females and five males) were starved for 2 hr at 25°C and transferred to vials with cornmeal-yeast food (standard diet) or morinda fresh pulp (morinda diet), containing 10 mM sulforhodamine B (Sigma, St. Louis, MO). Flies were allowed to feed for 1 hr and were immediately frozen at −20°C for 2 hr. Females and males were processed separately. Heads were removed to prevent contamination with eye pigments, and the bodies were homogenized in 250 μl of PBS. Samples were micro-centrifuged at 17,000×g (Eppendorf Centrifuge 5415 R) for 7 min at 4°C, after which the supernatant was collected, mixed with 60 μl of chloroform and micro-centrifuged for 6 min. The optical density of the supernatant was determined at 570 nm (BioSpectrophotometer, Eppendorf). Results are the mean of three independent determinations in each food condition.

Amine and precursors quantification

Fresh morinda fruits (10 g) were homogenized in 15 ml 0.1 M perchloric acid, incubated during 5 min at RT and micro-centrifuged at 17,000×g (Eppendorf Centrifuge 5415 R) for 5 min at 4°C. The resulting supernatant was measured in a RS-3000-LTQ-Orbitrap XL instrument (Dionex and Thermo Fischer) (Docimo et al., 2012). l-3,4-dihydroxyphenylalanine (l-DOPA) values were estimated by calibration curve regression. Arithmetic mean and standard deviation were calculated over three independent samples and values expressed as nanogram of l-DOPA per gram of fruit. Other amines, DA, TA and OA, were not detected in morinda fruit under these conditions. Fly samples (5 whole flies, or 6 bodies or 10 ovaries) were processed in 50 μl of 0.1 M perchloric acid with 0.3 mM mDp as an internal standard, using ceramic beads (peqlab, Biotechnology GmbH) in TissueLyser LT (QIAGEN). Samples were micro-centrifuged twice at 17,000×g (Eppendorf Centrifuge 5415 R) for 5 min at 4°C and the supernatant measured as above. Tyrosine, l-DOPA and DA absolute values were calculated by calibration curve regression using XCMS/MZMatchR program (Tautenhahn et al., 2008). Arithmetic mean and standard deviation were calculated over three independent samples. For flies fed a standard diet, values were expressed as picogram of l-DOPA or DA per fly (fly weights showed no statistically significant difference [p = 0.85564545, D. melanogaster vs D. sechellia, Student's t test]). To compare flies fed different diets, values were expressed as picogram of l-DOPA per milligram of body tissue.

Fruit pH measurements

The fruit pulp of ripe or overripe morinda and banana was mashed lightly and the pH was measured with a glass electrode. Results are the mean of three independent determinations for each fruit.

Egg-hatching rate and size

D. sechellia 10–20 females and males fed a standard diet were placed in oviposition cages (37.5 mm diameter and 58 mm high) holding plates containing either a standard diet or fresh morinda pulp, at 25°C, 70% RH in L:D 12:12. In parallel, D. sechellia 10–20 females and males fed a diet of morinda were placed in oviposition cages (37.5 mm diameter and 58 mm high) holding plates containing fresh morinda pulp, at 25°C, 70% RH in L:D 12:12. Plates were changed every 0.5 hr to get pools of synchronized eggs and further incubated at 25°C. Hatching rate was expressed as the relative number of hatched eggs (larvae 1) to the total number of eggs produced. Egg size was measured within 1 hr of oviposition or on pre-fertilized eggs inside the ovary (there was not a statistically significant difference between these groups). For each species, the lengths (L) and widths (W) of 15–30 eggs were measured and their volumes were determined according to the formula (1/6)πW2L (Markow et al., 2009). Values were expressed as mm3 (10−3).

Western blots

For each species, 20 adult flies were homogenized in 200 μl lysis buffer (50 mM Tris–HCl pH 7.5, 0.1% [vol/V] Triton X-100, 100 mM NaCl, 1 mM DTT, 10% glycerol, 15 mM EDTA) freshly prepared with protease inhibitor cocktail (Roche) added immediately before use, using ceramic beads (peqlab, Biotechnology GmbH, Germany) in TissueLyser LT (QIAGEN GmbH, Germany). Whole protein extracts of one same experiment were separated in parallel by 10% SDS-PAGE plus electronic transfer to PVDF membranes (BioRad, Germany). After being blocked in 5% non-fat milk in TBS-tween (TBS, 0.05% Tween-20 [Sigma, St. Louis, MO]) for 2 hr, at RT, membranes were incubated with 1:1000 dilutions of primary antibodies (anti-TH [mouse, ImmunoStar, Acris Antibodies, GmbH, Germany], or anti-SLC39A7 [rabbit, Sigma, St. Louis, MO] or anti-α-tubulin [mouse, Sigma, St. Louis, MO]) diluted in 2.5% non-fat milk in TBS tween ON at 4°C. Membranes were further washed in TBS-tween at RT and re-blocked in 10% non-fat milk TBS-tween for 10 min at RT before being incubated with 1:10000 dilution of corresponding secondary HRP-conjugated anti-mouse or anti-rabbit (BioRad, Germany). Proteins were detected using an enhanced chemiluminescence detection kit (Thermo scientific pierce, Germany). The densitometry of bands was performed using ImageJ package (http://imagej.nih.gov/ij/). Relative densities of TH-PLE and CATSUP were scaled using the relative densities of the loading-controls (α-Tubulin).

Gene cloning and sequencing

For each species, we prepared total RNA (Trizol, Invitrogen, Life Technologies GmbH, Germany) and synthesized cDNA (SuperScript III First-Strand Synthesis System, Invitrogen, Life Technologies GmbH, Germany) that was used to amplify Catsup transcript by PCR (Advantage HD Polymerase, Clontech) with specific primers (forward 5′-ATGGCCAAACAAGTGGCTGA-3′ and reverse 5′-TTACTCGAACTTGGCGATAAC-3′). Each PCR product was cloned in pCRII vector (Invitrogen, Life Technologies GmbH, Germany) and at least 10 colonies were picked for plasmid DNA purification and sequencing. These sequences were aligned using MegAlign (DNASTAR) and a consensus sequence was obtained for each species. The sequence of CatsupIn270Del was obtained from the Drosophila Polymorphism Database (DPDB) (Casillas et al., 2005).

Sternopleural bristle quantification

Total (macro and micro) bristles of each sternopleural plaque were counted in females of D. melanogaster wild-type Berlin (N = 34), D. melanogaster Canton-S (N = 54), D. melanogaster DGRP-357 (N = 61), DGRP-437 (N = 18), DGRP-304 (N = 16)—three independent lines carrying CatsupIn270Del allele—and D. sechellia lines 14021–0248.08 (N = 98), 14021–0248.25 (N = 74), 14021–0248.27 (N = 25), 14021–0248.28 (N = 56) and 14021–0248.31 (N = 50). Data is expressed as frequency histograms and a bar-graph showing the mode of each population.

Behavioural assay

5-day-old flies were fed a standard diet either not- or supplemented with increasingly amounts of DA (5 mg/ml, 10 mg/ml and 100 mg/ml) during 16 hr prior to their behavioural assessment, at 25°C, 70% RH in L:D 12:12. The locomotion behaviour was scored in groups of 30 females and 30 males kept in cages (37.5 mm diameter and 58 mm high) holding agar plates containing 5% sucrose and a mix of 0.07% vol/V 3:1 octanoic:hexanoic acids. M. citrifolia fruits contain highly volatile compounds that confer toxicity (Legal et al., 1994), with the two most abundant being octanoic acid (58%) and hexanoic acid (19.24%) (Farine et al., 1996). Octanoic acid is the most toxic compound in the ripe fruit (Legal et al., 1994), which D. sechellia has been able to overcome by the development of both larval and adult tolerance mechanisms (R'Kha et al., 1991; Legal et al., 1992; Amlou et al., 1997, 1998; Jones, 1998, 2001, 2005). Given these observations, the ratio provided on par. A drop of fresh yeast was added to the plates forcing the attracted flies to enter in contact to the acids. Flies were carefully observed under a stereoscope and the number of immobilized flies was recorded every 5 min independently for females and males. Results are the mean of three to five independent determinations in each food condition.

Acknowledgements

We wish to thank Regina Stieber, Sandra Scholz and Céline Callens for expert technical support; and Emily Wheeler, Boston, for editorial assistance. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work was supported by the Max Planck Society (SL-LL, AS, MK, MCS and BSH) and by grants from the ESPCI ParisTech and the Fondation de France to SB (TR and SB).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

  • Max-Planck-Gesellschaft to Sofía Lavista-Llanos, Aleš Svatoš, Marco Kai, Marcus C Stensmyr, Bill S Hansson.

  • Centre National de la Recherche Scientifique ESPCI ParisTech to Thomas Riemensperger, Serge Birman.

  • Fondation de France to Thomas Riemensperger, Serge Birman.

Additional information

Competing interests

BSH: Vice president of the Max Planck Society, one of the three founding funders of eLife, and a member of eLife's Board of Directors.

The other authors declare that no competing interests exist.

Author contributions

SL-L, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

AS, Acquisition of data, Analysis and interpretation of data.

MK, Acquisition of data, Analysis and interpretation of data.

TR, Acquisition of data, Analysis and interpretation of data.

SB, Conception and design, Drafting or revising the article.

MCS, Conception and design, Drafting or revising the article.

BSH, Conception and design, Drafting or revising the article.

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eLife. 2014 Dec 9;3:e03785. doi: 10.7554/eLife.03785.021

Decision letter

Editor: Mani Ramaswami1

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Dopamine drives Drosophila sechellia adaptation to its toxic host” for consideration at eLife. Your article has been favorably evaluated by K VijayRaghavan (Senior editor) and 3 reviewers, of whom Mani Ramaswami is a member of our Board of Reviewing Editors.

The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Giovanni Bosco and John Jaenike.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The paper identifies the genetic basis for the evolution of a feeding behavior in Drosophila species. It maps the mutation conferring preference for morinda fruit to mutations in catsup, a Drosophila catcholamine regulatory protein encoding gene, which affects dopamine synthesis as well as the presence of a dopamine precursor l-Dopa in morinda fruit. It further shows a role for Catsup in maternal secretory function.

The paper is on balance a very nice piece of work that is potentially suitable for publication in eLife. However, there are some experimental gaps in the paper, as well as additional experimental and/or informational details the need to be included to tighten and complete the study.

The essential revisions that we request are:

1) Please provide data to show that a morinda diet does not affect egg-laying in Drosophila melanogaster. Particularly as previous work shows that contact with the ripe fruit kills any member of the D. melanogaster complex with the exception of D. sechellia, which is attracted by and prefers it (Legal and Plawecki 1995).

2) Please provide DAPI visualization of the ovaries to either confirm or deny apoptosis as a possible culprit for the depressed oviposition in D. sechellia when on standard media. Alternatively, some other method of detecting apoptosis, like TUNEL or activated caspase staining could be used to determine whether an oogenesis checkpoint has been triggered. Subsequently, demonstrating that apoptosis is no longer present when the flies are raised on Morinda fruit is also necessary. This is required because stressors can cause egg chambers in the ovaries to be eliminated by apoptosis at oogenesis checkpoints in region-2/3 of the germarium or stage 7/8 egg chambers (the mid-oogenesis checkpoint) (Drummond-Barbosa 2001; McCall 2004), which could be a possible explanation for the deceased egg numbers.

3) Please test whether heteroallelic D. melanogaster fly lines carrying Catsupln270del and the null Catsup1 allele, show egg depression that can be rescued by feeding the flies l-DOPA (as with the D. sechellia experiments). This is a very important experiment that would complete the story.

4) The paper states that no D. melanogaster wild-type eggs hatched in Morinda pulp but 13.5% of DGRP-357 eggs do. Could this be a strain difference in D. melanogaster lines? This issue could be addressed by testing a few other D. melanogaster lines or by genetic rescue.

5) At the end of the Results and Discussion please present a clear and complete description of the evolutionary scenario by which D. sechellia first became tolerant to and then dependent on morinda fruit. Even if the scenario includes some speculations, it is important to see the whole thing put together.

Minor comments to which we request a constructive response:

1) The authors state that some D. sechellia in Figure 1 were raised on a Morinda diet (pulp from the fruit collected from the greenhouse). On its native islands, D. sechellia specializes on the ripe fruit of Morinda citrifolia (Louis and David 1986, Lachaise et al. 1998), which is native from the region of the Indian Ocean and is not present in Africa (Lecomte 1924, Jones 2005). How many generations was the stock raised on this media? If more than one, is it possible that you have forced selection pressures on the fly lines to be octanoic acid resistant? Additionally, are they allowed to only feed on ripe fruit, or rotten as well?

2) The Methods state that 3 different lines of D. sechellia were acquired from the species stock center. However, I cannot see which lines were used for what figures. Could you please clarify how many lines were tested, and what lines were used for the experiments? Was only one line tested, or was an outbred population bred? Having multiple lines to make the result generalizable is important. At least one additional D. melanogaster line would be preferred as well in order to make the overall claims of the paper more generalizable.

3) The authors state several times that genes involved in biogenic amine dopamine (DA) is “differentially expressed when compared to generalist Drosophila siblings” followed by two citations. I found the need to look at the citations, as the language of the sentence confused me. It could be referring to D. sechellia's relationship to D. melanogaster, D. simulans, or D. mauritiana (or some combination thereof). Please make this clear.

4) “We screened for genes that differ in expression between D. sechellia and its generalist sister species, D. simulans. We also screened for genes that are differentially expressed in D. sechellia when these flies chose their preferred host vs. when they were forced onto other food. D. sechellia increases expression of genes involved with oogenesis and fatty acid metabolism when on its host.”

Two peers are cited. Please provide values and the comparisons (species wise) from the papers cited here.

5) The authors state that fresh and overripe Morinda contains l-DOPA in equal amounts. Please show this data in supplement to confirm to readers that the state of the fruit being used (ripe/overripe) is not skewing the results.

6) Figure 2D demonstrates that depleting levels of l-DOPA in Morinda by using COMT hinders egg production stimulation. To further convey this synthetic alteration of the Morinda being the same as standardized food, please provide >S8/<S8 ratios as in Figure 2B.

7) Figure 3A demonstrates that eggs of D. sechellia are larger in size when compared to those of sibling species. Please state what siblings are being compared to. Additionally, the eggs of D. sechellia fed Morinda are 3-fold larger than wild-type D. melanogaster. It is unclear to me what media the D. melanogaster lines are being fed in this experiment; banana food or standardized media? Given that the food substrate (and many other factors) is so important for these experiments, please explicitly write exactly what media each species is on, temperature, humidity, number of flies per vial/bottle, etc.

8) Figure 3C shows a Western blot of total protein whole-fly extracts for TH-PLE, Catsup, and ∝-tubulin. To make the claims more generalizable, please provide one additional strain for both D. melanogaster and D. sechellia. While it may be beyond the scope of the paper, it would be interesting to provide the other sister species on the Western blot as well (D. simulans, D. mauritiana).

9) Consider performing an LD50 assay with this DGRP line as this could be very useful to show that Catsup deficiencies could confer resistance to an otherwise toxic fruit, demonstrating multiple roles for this gene.

10) Figure 5 demonstrates the accumulation of TH-PLE in D. sechellia and D. melanogaster oocytes using antibody staining. D-E shows Catsup expression in nurse cells of D. melanogaster. If possible, accompanying images of D. sechellia and DGRP-357 ovaries should accompany the panels in this figure to further confirm Catsup expression changes.

11) Figure 6 shows that DA contributes to the behavioral resistance to Morinda, specifically to octanoic and hexanoic acid. Increased DA results in increased D. melanogaster resistance. I am having difficulty understanding how the experiment was performed. The Methods section does not provide any specifics of the apparatus used in the assay. Is it possible that the D. melanogaster are simply further away from the food if there is DA present? This question assumes the assay was performed in a cylindrical tube. Was it performed on a Petri dish where distance of the fly from the food would not be an issue? Please clarify and comment.

12) In the Methods section, under Behavioural assay, the authors state they used a mix of 3:1 octanoic to hexanoic acid. M. citrifolia contains highly volatile compounds that confer toxicity (Legal 1994), with the two most abundant being octanoic acid (58%) and hexanoic acid (19.24%) (Farine 1996). Octanoic acid is the most toxic compound in the ripe fruit (Legal 1994), which D. sechellia has been able to overcome by the development of both larval and adult tolerance mechanisms (R'Kha 1991, Legal 1992, Amlou 1997, Amlou 1998, Jones 1998, Jones 2001, Jones 2004). Given these observations, the ratio provided was on par. Please cite these papers so that readers are aware of the proper use of concentrations.

13) Figure 3–figure supplement 1 shows D. sechellia fed Morinda diet compared to 11 Drosophila siblings. More than one D. sechellia line should be tested if compared to multiple siblings. Additionally, stating what species the siblings are would be beneficial to the figure. When I looked up the reference (Markow 2009) the 11 species being referred to are:

a) D. ananassae (14024-0371.13), D. erecta (14021- 0224.01), D. melanogaster (14021-0231.36), D. mojavensis (15081-1352.22), D. persimilis (14011-0111.49), D. pseudoobscura (14011-0121.94), D. sechellia (14021- 0248.25), D. simulans (14021-0251.195), D. virilis (15010-1051.87), D. willistoni (14030-0811.24), D. yakuba (14021-0261.01). All cultures were maintained at 24° C on a 12-h ⁄ 12-h light-dark cycle.

Earlier, the text uses the term “sibling species” only in reference to D. simulans, which is a sister to D. sechellia and D. mauritiana. This figure now uses the term for flies across the genus Drosophila. Please change the language to reflect the change in what flies are being discussed and add some text about why comparing to other species outside of the melanogaster subgroup is important. Additionally, please indicate which of the grey bars D. sechellia is from the Markow study.

14) Figure 4–figure supplement 2: sensory bristles contain a histogram of sternopleural bristles of D. melanogaster wild-type and D. sechellia. Please add in DGRP-357 to the histogram to draw the parallel between the two lines.

15) In the Methods section, under “Ovariole staining and immunofluorescence,” please define ON as overnight as it has not yet been defined in text.

16) The Materials and methods states that these experiments utilized three strain of D. sechellia. Were the Catsup gene sequences in all three of these strains? If so, do all three carry catsup alleles with a 45-bp deletion and the various non-synonymous mutations? If that is the case, it would suggest that these changes may have been present in the ancestor of present-day D. sechellia as it was evolving specialization on morinda. It is also possible that D. sechellia switched to morinda first as a breeding site, which then relaxed selection on Catsup, allowing it to accumulate the large deletion on non-synonymous mutations. It might be possible to examine details of molecular variation in and around Catsup to infer the age of the deletion, as well as to detect evidence of a recent selective sweep at this gene. If the alternative scenario I suggest (relaxed selection) had occurred, one would not expect to find evidence of a selective sweep.

eLife. 2014 Dec 9;3:e03785. doi: 10.7554/eLife.03785.022

Author response

1) Please provide data to show that a morinda diet does not affect egg-laying in Drosophila melanogaster. Particularly as previous work shows that contact with the ripe fruit kills any member of the D. melanogaster complex with the exception of D. sechellia, which is attracted by and prefers it (Legal and Plawecki 1995).

As pointed out by the reviewers, morinda fruit has toxic effects on D. melanogaster, what hindered us to maintain a permanent culture of D. melanogaster in the fruit. For the purpose of this manuscript, we fed D. melanogaster a morinda diet for as long as each experiment required, which was not detrimental for the flies_ this fact has been clarified in the revised Materials and Methods and the citation suggested has been added.

Figure 1B shows that morinda does not significantly affect the relative production of eggs in D. melanogaster wild type Berlin compared to a standard diet. We have included the actual data of egg-production in a standard diet versus a morinda diet for D. melanogaster wild type Berlin and for one additional D. melanogaster line (Canton-S) in a supplementary figure (Figure1–figure supplement 1). Results show that morinda has no effect or slightly inhibits egg-production in D. melanogaster wild type Berlin and D. melanogaster Canton-S, respectively.

2) Please provide DAPI visualization of the ovaries to either confirm or deny apoptosis as a possible culprit for the depressed oviposition in D. sechellia when on standard media. Alternatively, some other method of detecting apoptosis, like TUNEL or activated caspase staining could be used to determine whether an oogenesis checkpoint has been triggered. Subsequently, demonstrating that apoptosis is no longer present when the flies are raised on Morinda fruit is also necessary. This is required because stressors can cause egg chambers in the ovaries to be eliminated by apoptosis at oogenesis checkpoints in region-2/3 of the germarium or stage 7/8 egg chambers (the mid-oogenesis checkpoint) (Drummond-Barbosa 2001; McCall 2004), which could be a possible explanation for the deceased egg numbers.

Following the reviewers suggestion, we have checked for apoptosis occurring in D. sechellia ovaries, staining live ovaries with the vital dye acridine orange, following the protocol described in Arama and Steller (2006) (reference has been included therein). We found massive apoptosis occurring at stage S7/S8 cysts (mid-oogenesis checkpoint) and to a lesser extent in region-2/3 of the germarium, in D. sechellia fed a standard diet; confirming apoptosis as a possible culprit for the depressed oviposition on standard media. On the other hand, D. sechellia flies fed a morinda diet showed very few apoptotic cysts, in line with the stimulatory effect of morinda in egg-production. Corresponding images of the stained ovaries have been included in a supplementary figure (Figure 1–figure supplement 2). Additionally, we show that supplementation of a standard diet with l-DOPA is sufficient to reduce apoptosis in D. sechellia ovaries, while depleting morinda of l-DOPA by pre-treatment of the fruit with COMT prevents the anti-apoptotic effect of the morinda diet. We have quantified these effects and included the data in the revised Figure 2. In sum, these results further confirm the requirement of l-DOPA present in morinda fruit for an optimal reproduction of D. sechellia.

3) Please test whether heteroallelic D. melanogaster fly lines carrying Catsupln270del and the null Catsup1 allele, show egg depression that can be rescued by feeding the flies l-DOPA (as with the D. sechellia experiments). This is a very important experiment that would complete the story.

Figure 4I shows that heteroallelic D. melanogaster fly lines carrying CatsupIn270Del and the null Catsup1 allele show egg-depression compared to D. melanogaster wild type Berlin and to the parental lines. We have added new data showing that supplementation of a standard diet with l-DOPA significantly increases egg-production in D. melanogaster heteroallelic CatsupIn270Del/Catsup1 flies (Figure 4–figure supplement 4), as it does for D. sechellia (Figure 2A).

4) The paper states that no D. melanogaster wild-type eggs hatched in Morinda pulp but 13.5% of DGRP-357 eggs do. Could this be a strain difference in D. melanogaster lines? This issue could be addressed by testing a few other D. melanogaster lines or by genetic rescue.

To rule out strain differences in D. melanogaster eggs on the ability to hatch in morinda fruit, we tested 3 further D. melanogaster wild type stains (Canton-S, Oregon-R and a wild line D. melanogaster pferta, caught in a local winery); one additional D. melanogaster line carrying the CatsupIn270Del allele (DGRP-437) and two D. sechellia sister species D. simulans and D. mauritiana. The data obtained confirms that eggs carrying CatsupIn270Del allele are able to hatch to living larvae 1 and even molt up to larvae 3 in morinda, while no D. melanogaster wild type egg made it alive to larvae 1; although some embryos completed development and hatched, dying in their way out of the chorion. Results have been graphed in a supplementary figure (Figure 5–figure supplement 1).

5) At the end of the Results and Discussion please present a clear and complete description of the evolutionary scenario by which D. sechellia first became tolerant to and then dependent on morinda fruit. Even if the scenario includes some speculations, it is important to see the whole thing put together.

The text has been changed accordingly. “Our results are compatible with an evolutionary scenario in which an original Catsup allele carrying a 45-bp deletion and six non-synonymous mutations was present in the ancestor of present-day D. sechellia (Figure 4–figure supplement 5), probably showing differentially low expression of CATSUP compared to D. melanogaster and the sister species D. simulans and D. mauritiana (see Figure 4E and Figure 4–figure supplement 1); what caused diminished egg production and concomitantly enhanced early survival in the fruit. A second event, further elevating DA (see Figure 4F), would have redeemed adult D. sechellia from the toxic effects of morinda acids allowing them to oviposit in ripe fruits, benefiting from morinda stimulating effect on egg production and from the lack of siblings competition, rendering D. sechellia dependent on morinda. This dependency would have, in turn, shaped the super specialization of the chemosensory system (6, 7, 11) present in today’s D. sechellia morinda obligate specialist.”

Minor comments to which we request a constructive response:

1) The authors state that some D. sechellia in Figure 1 were raised on a Morinda diet (pulp from the fruit collected from the greenhouse). On its native islands, D. sechellia specializes on the ripe fruit of Morinda citrifolia (Louis and David 1986, Lachaise et al. 1998), which is native from the region of the Indian Ocean and is not present in Africa (Lecomte 1924, Jones 2005). How many generations was the stock raised on this media? If more than one, is it possible that you have forced selection pressures on the fly lines to be octanoic acid resistant? Additionally, are they allowed to only feed on ripe fruit, or rotten as well?

D. sechellia flies used in this work were originally obtained from the Drosophila Species Stock Center (UC, San Diego) and kept as a stock in standard Drosophila media. A subculture of this was used to establish a stock in morinda fruit; only ripe fruits were used; which was raised for more than 10 generations. To rule out a forced selection pressure on D. sechellia to be resistant to octanoic acid, we compared their survival in morinda fruit to that of D. sechellia flies from a new re-ordered stock of the same strain line (14021-0248.25). Results show no difference in survival to morinda between the new and old experimental stock, discarding an artificial adaptation to morinda acids in our experimental D. sechellia stocks. This fact has been clarified in the revised Material and methods.

2) The Methods state that 3 different lines of D. sechellia were acquired from the species stock center. However, I cannot see which lines were used for what figures. Could you please clarify how many lines were tested, and what lines were used for the experiments? Was only one line tested, or was an outbred population bred? Having multiple lines to make the result generalizable is important. At least one additional D. melanogaster line would be preferred as well in order to make the overall claims of the paper more generalizable.

We have tested one additional D. sechellia line and one additional D. melanogaster line for the main experiments, and have clearly stated in each revised figure legend which line of D. sechellia or D. melanogaster was used in each case.

3) The authors state several times that genes involved in biogenic amine dopamine (DA) is “differentially expressed when compared to generalist Drosophila siblings” followed by two citations. I found the need to look at the citations, as the language of the sentence confused me. It could be referring to D. sechellia's relationship to D. melanogaster, D. simulans, or D. mauritiana (or some combination thereof). Please make this clear.

The text has been clarified accordingly “Moreover, genes involved in DA differentiation have been shown differentially expressed in D. sechellia compared to the generalists species D. melanogaster and the sister species Drosophila simulans (Dworkin and Jones, 2009, Wurmser et al., 2011).”

4) “We screened for genes that differ in expression between D. sechellia and its generalist sister species, D. simulans. We also screened for genes that are differentially expressed in D. sechellia when these flies chose their preferred host vs. when they were forced onto other food. D. sechellia increases expression of genes involved with oogenesis and fatty acid metabolism when on its host.”

Two peers are cited. Please provide values and the comparisons (species wise) from the papers cited here.

The findings of Dworkin and Jones 2009 have been described in the revised Introduction and cited therein: “In turn, morinda stimulates egg production, and D. sechellia clearly prefers to oviposit in medium containing morinda carboxylic acids. On its host, D. sechellia increases expression of genes involved with oogenesis and fatty acid metabolism (Dworkin and Jones 2009). Thus, we here examined the dependence of Drosophila sechellia on morinda, for optimal reproduction.”

The findings of Wurmser et al 2011 have been described in the revised Results and cited therein: “In a screen for genes that differ in expression between D. sechellia and four geographically distinct populations of its generalist sister species D. simulans, Wurmser et al showed lower expression of the DA catabolic enzyme (Dopamine-N-acetyltransferase)…”

5) The authors state that fresh and overripe Morinda contains l-DOPA in equal amounts. Please show this data in supplement to confirm to readers that the state of the fruit being used (ripe/overripe) is not skewing the results.

The data of unripe and overripe fruits has been included in the text, showing similar values.

6) Figure 2D demonstrates that depleting levels of l-DOPA in Morinda by using COMT hinders egg production stimulation. To further convey this synthetic alteration of the Morinda being the same as standardized food, please provide >S8/<S8 ratios as in Figure 2B.

We have repeated the experiment feeding D. sechellia morinda or morinda pre-treaded to COMT and quantified >S8/<S8, confirming that depleting levels of l-DOPA in morinda by pre-incubation with COMT hinders stimulation of oogenesis, as quantified by >S8/<S8. The data has been included as a graph in the revised Figure 2–figure supplement 1.

7) Figure 3A demonstrates that eggs of D. sechellia are larger in size when compared to those of sibling species. Please state what siblings are being compared to. Additionally, the eggs of D. sechellia fed Morinda are 3-fold larger than wild-type D. melanogaster. It is unclear to me what media the D. melanogaster lines are being fed in this experiment; banana food or standardized media? Given that the food substrate (and many other factors) is so important for these experiments, please explicitly write exactly what media each species is on, temperature, humidity, number of flies per vial/bottle, etc.

The text stating which siblings of D. sechellia are being compared to has been changed accordingly in the revised results: “Eggs of D. sechellia are characteristically large compared to those of sibling species D. melanogaster, D. simulans, Drosophila ananassae, Drosophila erecta, Drosophila mojavensis, Drosophila persimilis Drosophila pseudoobscura, Drosophila virilis, Drosophila willistoni and Drosophila yakuba (Figure 3A and Figure 3–figure supplement 1)”.

The feeding media of D. melanogaster to which D. sechellia egg size is being compared to has been explicitly written in the revised Results “In accordance, we observed D. sechellia eggs to be 45% larger in size compared to eggs of D. melanogaster wild type Berlin (Figure 3A), in a standard diet condition. Upon being fed a morinda diet, eggs of D. sechellia increased in volume two-fold compared to eggs of conspecifics fed standard medium (Figure 3A and Figure 3–figure supplement 1); the resulting eggs had an almost three-fold larger volume than did D. melanogaster eggs in standard conditions (Figure 3A)”.

Breeding conditions (food substrate, temperature, humidity, number of flies per vial/bottle, etc.) have been clearly stated in the revised Materials and methods and figure legends.

8) Figure 3C shows a Western blot of total protein whole-fly extracts for TH-PLE, Catsup, and ∝-tubulin. To make the claims more generalizable, please provide one additional strain for both D. melanogaster and D. sechellia. While it may be beyond the scope of the paper, it would be interesting to provide the other sister species on the Western blot as well (D. simulans, D. mauritiana).

We have performed a Western blot of total protein whole-fly extracts for TH-PLE, CATSUP, and ∝-TUBULIN in one additional strain of D. melanogaster, one additional strain of D. sechellia, one strain of each of the sister species D. simulans and D. mauritiana, and one D. melanogaster stain carrying CatsupIn270Del allele. The Western blot image and the relative quantification of proteins have been included in a supplementary figure (Figure 4–figure supplement 1).

9) Consider performing an LD50 assay with this DGRP line as this could be very useful to show that Catsup deficiencies could confer resistance to an otherwise toxic fruit, demonstrating multiple roles for this gene.

A survival analysis of DRGP-357 flies in morinda fruit showed no statistically difference with that of D. melanogaster, suggesting that further genes might be involved in the adult resistance of D. sechellia to morinda.

10) Figure 5 demonstrates the accumulation of TH-PLE in D. sechellia and D. melanogaster oocytes using antibody staining. D-E shows Catsup expression in nurse cells of D. melanogaster. If possible, accompanying images of D. sechellia and DGRP-357 ovaries should accompany the panels in this figure to further confirm Catsup expression changes.

The corresponding images of D. sechellia and DGRP-357 ovaries showing a reduced CATSUP expression in the nurse cells and in the secretory cells of the spermatheca have been added to the revised Figure 5.

11) Figure 6 shows that DA contributes to the behavioral resistance to Morinda, specifically to octanoic and hexanoic acid. Increased DA results in increased D. melanogaster resistance. I am having difficulty understanding how the experiment was performed. The Methods section does not provide any specifics of the apparatus used in the assay. Is it possible that the D. melanogaster are simply further away from the food if there is DA present? This question assumes the assay was performed in a cylindrical tube. Was it performed on a Petri dish where distance of the fly from the food would not be an issue? Please clarify and comment.

The chamber used is sufficiently small (35 mm diameter x 50 mm high) to assure a uniform exposure to the acids. Additionally, we have supplied the flies with a drop of fresh yeast paste to force the attracted flies to get in contact to the agar containing the mixture of morinda acids. Details on the methodology utilized have been clarified in the revised Materials and methods section.

12) In the Methods section, under Behavioural assay, the authors state they used a mix of 3:1 octanoic to hexanoic acid. M. citrifolia contains highly volatile compounds that confer toxicity (Legal 1994), with the two most abundant being octanoic acid (58%) and hexanoic acid (19.24%) (Farine 1996). Octanoic acid is the most toxic compound in the ripe fruit (Legal 1994), which D. sechellia has been able to overcome by the development of both larval and adult tolerance mechanisms (R'Kha 1991, Legal 1992, Amlou 1997, Amlou 1998, Jones 1998, Jones 2001, Jones 2004). Given these observations, the ratio provided was on par. Please cite these papers so that readers are aware of the proper use of concentrations.

The facts and papers mentioned have been cited in the revised Materials and methods.

13) Figure 3–figure supplement 1 shows D. sechellia fed Morinda diet compared to 11 Drosophila siblings. More than one D. sechellia line should be tested if compared to multiple siblings. Additionally, stating what species the siblings are would be beneficial to the figure. When I looked up the reference (Markow 2009) the 11 species being referred to are: a) D. ananassae (14024-0371.13), D. erecta (14021- 0224.01), D. melanogaster (14021-0231.36), D. mojavensis (15081-1352.22), D. persimilis (14011-0111.49), D. pseudoobscura (14011-0121.94), D. sechellia (14021- 0248.25), D. simulans (14021-0251.195), D. virilis (15010-1051.87), D. willistoni (14030-0811.24), D. yakuba (14021-0261.01). All cultures were maintained at 24° C on a 12-h ⁄ 12-h light-dark cycle.

Earlier, the text uses the term “sibling species” only in reference to D. simulans, which is a sister to D. sechellia and D. mauritiana. This figure now uses the term for flies across the genus Drosophila. Please change the language to reflect the change in what flies are being discussed and add some text about why comparing to other species outside of the melanogaster subgroup is important. Additionally, please indicate which of the grey bars D. sechellia is from the Markow study.

Data from one additional D. sechellia line as well as the names of the 11 Drosophila siblings showed in the figure have been explicitly mentioned in the revised Figure 3–figure supplement 2 and its legend. The D. sechellia line from the Markow et al. study has been indicated. The language has been changed accordingly in the revised Results to refer to sister species (D. simulans and D. mauritiana) or “sibling species” (the Drosophila species here named).

14) Figure 4–figure supplement 2: sensory bristles contain a histogram of sternopleural bristles of D. melanogaster wild-type and D. sechellia. Please add in DGRP-357 to the histogram to draw the parallel between the two lines.

We have added two additional D. sechellia lines, one additional D. melanogaster wild type line and two D. melanogaster lines carrying CatsupIn270Del allele to the histogram of sternopleural bristles shown in the revised Figure 4–figure supplement 3. A graph showing the mode of sensory bristles for each species has been added to the same revised figure to aid comparison between species.

15) In the Methods section, under “Ovariole staining and immunofluorescence,” please define ON as overnight as it has not yet been defined in text.

ON has been defined as overnight in the revised Materials and methods.

16) The Materials and methods states that these experiments utilized three strain of D. sechellia. Were the Catsup gene sequences in all three of these strains? If so, do all three carry catsup alleles with a 45-bp deletion and the various non-synonymous mutations? If that is the case, it would suggest that these changes may have been present in the ancestor of present-day D. sechellia as it was evolving specialization on morinda. It is also possible that D. sechellia switched to morinda first as a breeding site, which then relaxed selection on Catsup, allowing it to accumulate the large deletion on non-synonymous mutations. It might be possible to examine details of molecular variation in and around Catsup to infer the age of the deletion, as well as to detect evidence of a recent selective sweep at this gene. If the alternative scenario I suggest (relaxed selection) had occurred, one would not expect to find evidence of a selective sweep.

We cloned Catsup from five D. sechellia strains original from two different original geographical locations. All five strains carry Catsup alleles with a 45-bp deletion and five conserved non-synonymous mutations (out of a total of nine); suggesting that these changes may have been present in the ancestor of present-day D. sechellia as it was evolving specialization on morinda. This result is discussed in the revised Discussion.

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