Oviparous animals across many taxa have evolved diverse strategies that deter egg predation, providing valuable tests of how natural selection mitigates direct fitness loss. Communal egg laying in nonsocial species minimizes egg predation. However, in cannibalistic species, this very behavior facilitates egg predation by conspecifics (cannibalism). Similarly, toxins and aposematic signaling that deter egg predators are often inefficient against resistant conspecifics. Egg cannibalism can be adaptive, wherein cannibals may benefit through reduced competition and added nutrition, but since it reduces Darwinian fitness, the evolution of anticannibalistic strategies is rife. However, such strategies are likely to be nontoxic because deploying toxins against related individuals would reduce inclusive fitness. Here, we report how D. melanogaster use specific hydrocarbons to chemically mask their eggs from cannibal larvae. Using an integrative approach combining behavioral, sensory, and mass spectrometry methods, we demonstrate that maternally provisioned pheromone 7,11-heptacosadiene (7,11-HD) in the eggshell’s wax layer deters egg cannibalism. Furthermore, we show that 7,11-HD is nontoxic, can mask underlying substrates (for example, yeast) when coated upon them, and its detection requires pickpocket 23 (ppk23) gene function. Finally, using light and electron microscopy, we demonstrate how maternal pheromones leak-proof the egg, consequently concealing it from conspecific larvae. Our data suggest that semiochemicals possibly subserve in deceptive functions across taxa, especially when predators rely on chemical cues to forage, and stimulate further research on deceptive strategies mediated through nonvisual sensory modules. This study thus highlights how integrative approaches can illuminate our understanding on the adaptive significance of deceptive defenses and the mechanisms through which they operate.

Egg-laying species that lack parental care often protect their eggs from predators by laying them in communal groups or by fortifying them with toxins. However, these strategies may backfire when the predators are from the same species (cannibals) since a) there are plenty of available eggs in these sites, b) the cannibals may be resistant to the toxins, and c) poisoning cannibals who may be related would reduce inclusive fitness. Under these circumstances, natural selection should favor anticannibalistic strategies that are likely to be nontoxic. Here, we investigate how fruit flies (Drosophila melanogaster), which oviposit communally, protect their eggs from cannibalism by their own larvae. We show that maternal hydrocarbons incorporated into the egg’s wax layer to make them waterproof interestingly also serve as a mask that conceals their identity from cannibal larvae. In particular, we identify one female sex pheromone that deters cannibalism by forming a layer around the egg to conceal it. We further demonstrate that this pheromone is nontoxic and can mask underlying substrates such as yeast when used as a coating. While deceptive strategies (such as camouflage) deployed to avoid predation are extensively studied from a visual perspective, our findings suggest that deceptive strategies operating through other nonvisual sensory systems might be equally common across taxa.

Funding: Swiss National Science Foundation Grant (grant number 31003A_143939) received by Prof. T.J. Kawecki, supported S.N. and R.K.V. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. European Research Council Starting Grant (grant number 280271) recieved by Y.O.T. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. European Research Council PoC Grant (grant number 768565) recieved by Y.O.T. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft (grant number TH1584/1-1 and TH1584/3-1) received by A.S.T. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Baden Württemberg Stiftung and Zukunftskolleg of the University of Konstanz recieved by A.S.T. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Swiss National Science Foundation R'Equip Grant (grant number 316030_128692) recieved by B.M.H. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we investigate the mechanisms that could deter egg cannibalism in D. melanogaster. To do this, we examine the protective role of the eggshell and its constitutive layers using an integrative approach. We consequently reveal a novel anticannibalistic strategy that is mediated through chemical deception and involves semiochemicals [ 33 ] present in the wax layer of the egg shell, a so-far overlooked strategy that could potentially be widespread across taxa.

(A) Survival period of freshly hatched larvae held individually without food on agar (mean = 3.4 ± 0.1 days (SE); n = 4 replicate plates, 24 freshly hatched larvae assayed per replicate), demonstrating the surplus nutrition provisioned within an egg for embryonic development well beyond hatching. (B) Proportion of freshly hatched larvae surviving until day four, in absence of food (unfed) and when allowed to feed on two injured eggs (n = 4 replicate plates per treatment, 24 freshly hatched larvae assayed per replicate). Egg-fed larvae had a higher survival than unfed larvae (mean ± SD). (C, D) Larval developmental period (mean ± SD) and larval survival to pupation (mean ± SD), respectively, when reared on standard diet laced with (blue bars) or without (red bars) extract from crushed eggs (n = 4 bottles/diet, egg density: 200 eggs/bottle). On supplemented diet, larvae developed faster (ANOVA: F 1,9 = 9.75, p = 0.014) but survived to a similar extent (ANOVA: F 1,9 = 0.07, p = 0.8). (E) Graphical depiction of the egg shell morphology. The outer three layers—the exochorion, the endochorion, and the inner chorion layer—together form the chorion; the thin wax layer (in black) lies between the chorion and the innermost vitelline membrane that envelops the egg. (F) Proportion (mean ± SE) of intact, injured, dechorinated, and hexane-treated eggs cannibalized when confined in agar vials with second-instar larvae over 12 h (n = 10 vials/treatment, with 5 eggs and 10 larvae per vial). Larvae cannibalized hexane-treated eggs but not dechorinated eggs (ANOVA: F 1,18 = 21.85, p = 0.0002), however, not to the extent of injured eggs (ANOVA: F 1,18 = 7.57, p = 0.0132). Also see S1 Fig . ***p < 0.001, *p < 0.05. Data underlying this figure can be found in S1 Data . NS, not significant

Given that parental care in D. melanogaster is mostly limited to oviposition site selection and egg provisioning [ 31 , 32 ], we hypothesized that parental provisioning in some form protects D. melanogaster eggs from conspecific larvae. Virgin D. melanogaster females can lay nonviable unfertilized eggs, equivalent to trophic eggs laid by other species. However, given that mated D. melanogaster seldom lay such unfertilized eggs, their production more likely represents a “risk–return strategy” (i.e., the cost of producing such unfertile eggs is less than what is risked by aborting them) rather than a strategy that mitigates cannibalism. In several insect species, the eggshell, its pigmentation and patterning, and specific extrachorionic modifications upon it are all known to deter predation [ 8 ]. Drosophila eggs are enveloped within a maternally provisioned eggshell during oogenesis that is composed of three distinct layers ( Fig 1D ): chorion, wax layer, and vitelline membrane, which serve to deter pathogens, prevent dehydration, and facilitate respiration, respectively [ 8 ]. However, the extent to which these eggshell layers play a role in deterring cannibals is not known.

We recently reported predatory cannibalism among D. melanogaster larvae, wherein younger larval instars pack-hunt and consume older conspecific larvae under laboratory conditions [ 26 ]. Surprisingly, despite their predaceous nature, we never observed larvae attacking conspecific eggs, even upon starvation. In nature, D. melanogaster oviposit communally at sites already occupied by conspecific and heterospecific larvae to facilitate social feeding among larvae [ 27 , 28 ]. Although these oviposition sites (decaying fruits) are nutritionally rich, they at times risk desiccation [ 27 ] and immense larval competition [ 29 ], both of which could coerce larvae to seek other food sources such as older conspecifics [ 26 ] and cadavers [ 30 ]. Interestingly, despite availability of several conspecific eggs in their vicinity, larvae never cannibalize them, either for food or for other benefits like reduced competition [ 15 ] and reduced risk of predation by younger larvae [ 26 ]. This observation thus raises important questions on why and how egg cannibalism is averted in this system.

Anticannibalistic strategies that deter egg cannibalism have evolved independently in species across taxa, and convincingly, none of them seem to be toxic. These strategies include laying of nondeveloping eggs (trophic eggs) within clutches by mothers to reduce cannibalism among offspring [ 11 , 21 ], laying of eggs with protective coatings around the egg’s shell [ 22 ], laying eggs on specialized structures (like stalks) [ 23 ], nest guarding [ 24 ], and synchronized egg hatching [ 25 ]. Nevertheless, there are several other communally egg-laying species, including insects, that avoid cannibalizing eggs despite lacking the above strategies [ 15 ]. This prompted us to speculate about the existence of alternative strategies that could modulate this behavior in nature. The understanding of such strategies is crucial, especially to the fields of conservation, epidemiology, and pest management.

Across most animal taxa, eggs are highly vulnerable to predators because they are immobile, highly nutritious, and defenseless. However, since egg production is costly [ 1 ], losing them to predation greatly reduces Darwinian fitness [ 2 ]. Animals have thus evolved several parent-modulated strategies: camouflage [ 3 , 4 ], communal egg laying [ 5 , 6 ], egg clustering [ 7 ], parental care [ 2 ], chemical defenses (toxins) [ 8 , 9 ], and aposematic signaling [ 10 ] to mitigate this loss of fitness. On the other hand, eggs are not just vulnerable to interspecific predators but are equally at risk of predation from older conspecifics (cannibals), including parents and siblings [ 11 – 13 ]. Egg cannibalism is commonly dismissed as an aberrant behavior, resulting from unnatural breeding conditions. However, mounting evidence has demonstrated its adaptive value in several species, wherein the cannibals increase their fitness through both reduced competition and the supplemented nutrition [ 11 , 14 ]. In support of this argument, egg cannibalism is common even among noncarnivorous species [ 15 ] and has also been shown to have important ecological consequences on population dynamics and stability [ 11 , 16 ]. Nevertheless, egg cannibalism reduces direct fitness to parents and can additionally reduce inclusive fitness if the eggs consumed are genetically related to the cannibals [ 11 ]. Interestingly, most of the aforementioned parent-modulated strategies evolved in response to interspecific egg predators are often ineffective against conspecifics: while cannibals are generally resistant to conspecific toxins and aposematic signals [ 17 ], other strategies like producing surplus eggs and communal egg laying might even facilitate egg cannibalism [ 18 , 19 ]. Additionally, since deploying toxic defenses against conspecifics would further reduce inclusive fitness [ 20 ], natural selection should favor the evolution of anticannibalistic strategies that are likely to be nontoxic.

Results and discussion

Wax layer prevents egg cannibalism by larvae To understand the extent to which D. melanogaster females provision nutrients within their eggs, we assayed larval survival upon hatching in the absence of any food. The first-instar larvae survived for up to five days posthatching in the absence of food (Fig 1A), showing that the nutrients provisioned within an egg are surplus for embryonic development, and they can hence support larval survival well beyond hatching. Feeding two injured conspecific eggs to just-hatched larvae increased their survival (Fig 1B), confirming the previous finding that eggs are nutritious and are thus worth cannibalizing [30]. When egg consumption by second-instar larvae (food deprived for 2 h) was assayed, larvae surprisingly did not cannibalize intact viable eggs (Fig 1F). However, if the eggs presented to the larvae were injured (by pricking), the eggs were immediately consumed (Fig 1F), supporting the results from a recent study that used nonviable eggs [30]. The same larval response was observed when the eggs provided were from an unrelated D. melanogaster strain (panel D in S1 Fig). To rule out the possibility that the eggs are toxic to the larvae, we assayed larval development and survival on standard fly medium laced with crushed eggs. Compared to the control larvae raised on the standard diet, larvae supplemented with crushed eggs developed faster (Fig 1C) and survived equally well (Fig 1D), confirming that the eggs are nontoxic and nutritious. Given that injured eggs were readily cannibalized (Fig 1F), we next tested whether breached eggshells facilitate cannibalism. We sequentially removed the two outer eggshell layers, the chorion membrane and the wax layer (Fig 1E), by treating the eggs with sodium hypochlorite and hexane, respectively [34, 35], and then presented these chemically treated eggs to food-deprived larvae. Indeed, removal of the thin wax layer (4–5 nm, hexane treated) but not the thick chorion layer (840–1,250 nm, dechorinated) [36] made 60% of eggs vulnerable to cannibals (Fig 1F). This refutes a recent report [30] that D. melanogaster larvae cannibalize dechorinated eggs, which we believe is due to the experimental procedure. The dechorinated eggs they use were killed prior to their assays by dyeing with tartrazine NaCl and storing them at 4°C. These treatments could have unintentionally damaged the eggs’ wax layer. In contrast, the consecutive treatment of eggs with sodium hypochlorite and hexane in our experiments had little or no effect on egg hatchability (panel A in S1 Fig), egg’s time to hatching (panel B in S1 Fig), and egg-to-adult viability (panel C in S1 Fig). The experiments above thus suggest that in addition to the primary role of the wax layer in preventing desiccation of the embryo [37], it might also serve to protect eggs from cannibal larvae.

Wax-layer composition and parental role in its formation Next, to understand the mechanism underlying the wax layer’s anticannibalistic function, we extracted this layer from fertilized eggs and analyzed its biochemical composition using gas chromatography hyphenated with mass spectrometry (GC-MS) and high-resolution atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry (APPI FT-ICR MS) [38]. We used these advanced mass analyzers with high accuracy (sub-parts–million mass accuracy) and resolving power, mainly to mine for low-molecular–weight compounds (for example, toxins). However, in addition, we aimed to establish methods that could unambiguously assign elemental formulas to metabolites for better characterization, especially in life sciences (see Materials and Methods). We identified 13 compounds (linear alkenes, alkadienes, and sterols) that were mostly known cuticular hydrocarbons (pheromones) of adult D. melanogaster [39] (Figs 2A and S2 and S1 Table), which are mainly known to be synthesized by specialized epicuticular cells called the “oenocytes” (oe) [39]. Below, we focus on four pheromones (7,11-heptacosadiene [7,11-HD]; 7,11-nonacosadiene [7,11-ND]; 7-tricoscene [7-T]; and 11-cis-vaccenyl acetate [cVA]), given that they are sex-specific, have known functions, and are commercially synthesizable. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Maternally provisioned hydrocarbons confer protection. (A) Hydrocarbon profile of the wax layer in D. melanogaster eggs. (B) Relative amount (SNR) of four known sex-specific D. melanogaster pheromones (7-T, cVA, 7,11-HD, and 7,11-ND) present in the wax layer of eggs laid by the four oe mutant crosses. The relative amounts of pheromones were calculated from mass spectra acquired by high-resolution 10 T APPI-FT-ICR-MS of hexane extracts of wax layers (S2 Table). (C) Vulnerability of eggs laid by the four oe mutant crosses to larval cannibals, assayed as the proportion (mean ± SD) of these eggs cannibalized when confined with second-instar larvae (n = 7 replicate vials). Only eggs laid by the two crosses with oe− mothers were cannibalized, to extents that only differed marginally (ANOVA: F 1,12 = 3.66, p = 0.08). (D) Proportion (mean ± SE) of hexane-treated eggs that were cannibalized when perfumed with commercially synthesized 7-T, cVA, 7,11-HD, and 7,11-ND at different concentrations (n = 3 replicate vials/pheromone concentration, with 5 eggs and 10 larvae per vial). Least-squares means contrast of 7,11-HD versus other pheromones was different (F 1,35 = 17.2, p = 0.0002). Also, see S2, S3 and S4 Figs. Data underlying this figure can be found in S1 Data. APPI FT-ICR MS, atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry; a.u., arbitrary units; cVa, 11-cis-vaccenyl acetate; oe, oenocyte; SNR, signal-to-noise ratio; 7-T, 7-tricoscene; 7,11-HD, 7,11-heptacosadiene; 7,11-ND, 7,11-nonacosadiene https://doi.org/10.1371/journal.pbio.2006012.g002 The pheromone profile we detected in the wax layer was similar to the pheromones already known to be deposited by adult flies (both sexes) on egg-laying sites to facilitate aggregation [40, 41]. Furthermore, these pheromones are also known to be present in the reproductive tract of mated females [42]. Thus, to exclude the possibility of potential cross-contamination of our samples by adult flies, we analyzed the hexane washes of the outer chorion layer and that of dechorinated eggs and compared their hydrocarbon profiles. Most of the pheromones detected on the outer chorion (layer exposed to environment and female reproductive tract) were also found after dechorination (in the wax layer) (panel A in S3 Fig). The male pheromone cVA produced by the male’s ejaculatory bulb [43] was an exception; it was abundant only on the chorion but greatly reduced upon dechorination, suggesting that eggs acquire cVA postchoriogenesis either from the environment or from male ejaculate within the female reproductive tract [42] (panel A in S3 Fig). To further ascertain that these pheromones are indeed present in the wax layer, we analyzed three successive hexane washes of the dechorinated eggs and detected these pheromones at progressively decreasing concentrations across the washes, possibly reflecting the compact nature of the wax layer [37] (panel B in S3 Fig). Interestingly, the presence of such pheromones within the eggshell having other physiological functions has been previously reported in insects and nematodes [44, 45]. For over three decades, the wax layer has been considered to be synthesized by the follicle cells that surround the oocytes, exclusively based on electron microscopy observation of lipid endosomes within the follicle cells during oogenesis and their eventual deposition onto the vitelline membrane of the egg [46, 47]. Most of the hydrocarbons we detect in the wax layer are so far only known to be synthesized in the oes [39]; for example, the biosynthesis of dienes like 7,11-HD and 7,11-ND (female-specific pheromones) requires the enzymatic action of a specific desaturase desatF (Fad2) in the oes [48]. However, transcriptional data (microarray and RNA-sequencing [RNA-seq] data from FlyAtlas and FlyBase, respectively) show that this gene is not expressed in the ovary. Thus, further empirical investigations are necessary to clarify the role of follicle cells in the synthesis of the wax layer. The presence of hydrocarbons specific to both sexes in the wax layer motivated us to track the parental origin of these pheromones using mutant flies with ablated oes (oe−) [39]. Males and females with or without ablated oes were crossed, and the wax-layer composition of their eggs was analyzed. The label-free, semiquantitatively established hydrocarbon profile of eggs appears to be directed by the cuticular hydrocarbon composition of the parental cross [39] they were laid by; eggs of oe− parents had fewer hydrocarbons than those of oe+ parents (Fig 2B and S2 Table, and panel B in S4 Fig). The eggs parented by oe+ males and oe− females had reduced female-specific hydrocarbons—for example, 7,11-HD and 7,11-ND—compared to male-specific hydrocarbons—for example, 7-T. Its reciprocal cross had reduced male-specific hydrocarbon (7-T) compared to the female-specific hydrocarbons. However, the reason as to why cVA, the male pheromone of non-oe origin, was also reduced in the eggs laid by this cross is unclear. The presence of hydrocarbons corresponding to oe− parents at low concentrations in our samples could possibly be due to residual hydrocarbons produced prior to or during oe ablation. Thus, it seems that both parents contribute towards provisioning the pheromonal content of the wax layer. This suggests that wax-layer synthesis is likely to involve transportation of maternal and paternal hydrocarbons from the oes and deposited seminal fluid, respectively, to the ovary during oogenesis. Nevertheless, the existence of such transport mechanisms involving lipophorin molecules has been speculated in D. melanogaster [31] and other insects [45].

Maternal sex pheromone 7,11-HD deters egg cannibalism The deterrent effect of hydrocarbons present on the egg surface towards cannibals has been previously speculated about in the coccinellid Adalia bipunctata [49]. However, our system allowed us to ascertain the deterrent role of sex-specific hydrocarbons in egg cannibalism. For this, we first assayed the vulnerability of eggs laid by the above four crosses to cannibal larvae. Conspicuously, larvae only cannibalized eggs with oe− motherhood (Fig 2C). The eggs from the three oe− mutant crosses had similar egg-to-adult viability (slightly less than eggs from the oe+ control), thus excluding nonviability of eggs with oe− motherhood (panel E in S1 Fig). We next independently verified this deterrent role of hydrocarbons by assaying the vulnerability of hexane-washed eggs perfumed with four commercially synthesized hydrocarbons found most abundantly on the egg (7-T, cVA, 7,11-HD, and 7,11-ND; panel A in S4 Fig) to cannibal larvae. Since the actual concentration of the pheromones in the wax layer could not be determined (because of the differential solubility of the wax layer in hexane), the synthetic pheromones were applied and tested at several (serially diluted) concentrations. Interestingly, larvae only refrained from cannibalizing eggs that were perfumed with the female pheromone 7,11-HD (Fig 2D), even when present at very low concentrations. However, the other female pheromone 7,11-ND we used, despite being structurally very similar to 7,11-HD (with just two additional carbon atoms), failed to deter cannibalism (Fig 2D). Given that a) the eggs of male oe+ and female oe− with 7,11-HD levels slightly lower than oe+ controls become vulnerable to cannibalism and b) eggs perfumed with various concentrations of 7,11-HD remain protected, these results strongly suggest that the deterrent function of 7,11-HD is dose independent. These findings suggest that 7,11-HD, rather than the overall wax layer, deters egg cannibalism; nevertheless, the synergistic effect of other hydrocarbons and chemicals we detect within the wax layer (S2 Fig and S1 Table) cannot be ruled out.

Response of larval sensory receptors to 7,11-HD Next, we attempted to identify the larval sensory receptors associated with this 7,11-HD–mediated deterrence effect. In adult D. melanogaster, 7,11-HD mediates mate recognition [39], maintains the species barrier with D. simulans [50], and is used to mark sites suitable for mating and oviposition [51]. It was recently reported [52] that in D. melanogaster, 7,11-HD detection is dependent on the joined action of the three receptor genes pickpocket 23 (ppk23), ppk25, and ppk29 that are necessary for the function of gustatory neurons located in their forelegs [53, 54]. However, whether and how larvae respond to 7,11-HD remains elusive [55]. Anticipating that the role of ppk23 in 7,11-HD detection may be conserved in larvae, we tested whether egg cannibalism is promoted when ppk23 is mutated. For this, egg cannibalism by ppk23 mutant larvae was assayed when eggs were either dechorinated or hexane washed. Indeed, ppk23 mutant larvae cannibalized eggs from both treatments (Fig 3D). In a subsequent assay, ppk23 mutant larvae also cannibalized hexane-washed eggs that were perfumed with 7,11-HD (panel A in S5 Fig), thus suggesting that the function of ppk23 required for hydrocarbon detection is conserved across the developmental stages and, interestingly, modulates different behaviors in larvae and adults. Given that larvae might concurrently sense the wax layer as an aversive cue through other sensory pathways (olfactory or gustatory), we further assayed whether larvae that are mutant for gustatory receptor (Gr33a), odorant coreceptor (Orco), and ionotropic receptor (Ir25a) cannibalized dechorinated eggs. All tested mutants and their respective control (wild-type) larvae abstained from cannibalizing dechorinated eggs but cannibalized eggs treated with hexane (panels B–D in S5 Fig). All in all, these results suggest that the deterrent effect of the wax layer was not mediated by the function of the classical set of chemosensory systems (Gr, Or, and Ir) but is limited to the ppk-dependent sensory system. However, since our understanding of how larvae detect their gustatory and pheromonal environment at present is rather limited [55, 56], further work is necessary to identify the ppk23-dependent neuronal circuits that respond to 7,11-HD to regulate egg cannibalism. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Maternal hydrocarbons form a waterproof layer that prevents leakage of eggs. (A) Egg contents leaking out of hexane-washed eggs but not from dechorinated or intact eggs when allowed to stand on an agar surface for over 25 min (n = 10 eggs/treatment). (B) Egg contents leak through the vitelline membrane in the form of tiny droplets; bar = 100 μm. (C) Cryo-electron scanning of an egg leak showing egg contents permeating through the vitelline membrane; bar = 20 μm. (D) Proportion (mean ± SD) of dechorinated (dark bars) and hexane-washed (light bars) eggs cannibalized by Canton S and ppk23 mutant larvae (ANOVA: n = 4 replicates, 10 eggs/replicate). (E) Larvae do not differentiate injured (open triangle) and intact (closed triangle) eggs in a simple choice assay and do not get attracted to injured eggs (n = 5 replicate plates; ANOVA, F 1,8 = 0.78, p = 0.404). (F) Larvae cannibalize only dechorinated eggs coated with egg contents of injured eggs (closed circle) but not dechorinated eggs (open circle); (mean ± SE; n = 5 replicate vials/treatment; ANOVA, F 1,8 = 129.2, p < 0.0001). (G) Larval feeding (mean ± SD) is significantly reduced when yeast droplets are coated with 7,11-HD than when coated with the solvent alone (hexane); feeding is, however, completely restored when the 7,11-HD layer is pricked (n = 8 replicate Petri plates/treatment). Furthermore, 7,11-HD coated on yeast droplets does not inhibit feeding in ppk23 mutant larvae (mean ± SD) and is not different than when coated with solvent alone (hexane). However, feeding on yeast is further facilitated when the 7,11-HD layer is pricked (ANOVA; n = 8 replicate Petri plates/treatment). Also see S6 and S7 Figs. ***p < 0.0001, **p < 0.001, †p < 0.1. Data underlying this figure can be found in S1 Data. NS, not significant; ppk23, pickpocket 23; 7,11-HD, 7,11-heptacosadiene https://doi.org/10.1371/journal.pbio.2006012.g003

Maternal pheromones prevent leakage of egg contents Earlier studies have reported the water proofing nature of an egg’s wax layer [37] that can be compromised using organic solvents [57] to facilitate eggshell permeability (especially during histological preparations). We therefore next morphologically examined the surface of wax-layer–deprived eggs to understand why they become vulnerable to cannibal larvae and found that they extrude egg contents through their vitelline membranes. Fine fluid droplets appeared on the surface of hexane-treated eggs after about 20–25 min (Fig 3B). However, such droplets were not present on dechorinated or intact eggs (Fig 3A–3C). Cryo-scanning electron microscopy of the egg surface confirmed the droplets to be egg contents permeating through the vitelline membrane (Fig 3C; panels A–D in S7 Fig). Interestingly, in most eggs, these droplets stabilize (i.e., they do not completely drain the egg) and persist as such for several hours. This suggests that eggs might have repair mechanisms or that the extruded material could be blocking the permeating sites. To further ascertain the extent to which maternal and paternal pheromones in the wax layer contribute towards leak-proofing an egg, we examined dechorinated eggs of the four oe mutant crosses (described earlier) for leakage under both light and electron microscopy. We found that only eggs with oe− mutant motherhood that had reduced female pheromones were leaky, suggesting the involvement of maternal pheromones present in the wax layer in preventing egg leakage (panel A in S6 Fig; panels E–H in S7 Fig). However, when hexane-washed eggs perfumed with 7-T, cVA, 7,11-HD, and 7,11-ND were examined for leakage using a similar setup, we found that all eggs leaked irrespective of the added pheromone (panel B in S6 Fig). We nevertheless observed some differences among the eggs perfumed by the four hydrocarbons in a) the extent to which they leaked and b) the persistence of the leak. However, we were unable to quantify these differences empirically, and thus, despite demonstrating a strong link between maternally provisioned hydrocarbons and egg leakage, this assay could not independently tag 7,11-HD to the leak-proofing mechanism. This could also be possibly attributed to the arbitrary concentration of 7,11-HD we use or because perfuming fails to completely remodel the wax-layer structure artificially in the absence of other synergetic pheromones.

Leaked egg content provides gustatory cues The above finding that altering the wax-layer composition through either chemical treatment or genetic manipulation leads to extrusion of egg contents through the vitelline membrane led us to test whether this leaking egg content makes eggs vulnerable to cannibals. We first assayed larval movement in the presence of intact and injured eggs in agar-lined Petri plates to quantify changes in foraging patterns. However, unlike cannibalistic aggregation occurring around injured larva that we have previously reported [26], groups of injured eggs did not elicit larval aggregation, and the injured eggs were only cannibalized when they were accidentally encountered (Fig 3E). However, 30% of the generally invulnerable dechorinated eggs (Fig 1F) succumbed to cannibals when smeared with egg content leaking from injured eggs (Fig 3F). These results imply that larval recognition and cannibalism of conspecific eggs relies on specific gustatory cues emanating from leaking egg content. Nevertheless, this does not rule out the possibility that leaky or injured eggs might additionally release other stress/injury responsive signals that act as cues, facilitating larval detection.

7,11-HD layer can mask sensory cues Since several experiments above suggested that the protective role of 7,11-HD is dose independent (Figs 1F, 2C and 3F), we speculated that the wax layer in general and 7,11-HD in particular form a physical layer that masks or conceals the egg’s nutrient content. To validate our hypothesis, we tested whether drops of yeast that are attractive to larvae lose their ability to do so when coated externally with a layer of 7,11-HD. We thus assayed larval detection and feeding of such 7,11-HD–coated yeast droplets that were dyed to score larval feeding. Larval feeding on yeast coated with 7,11-HD was much lower than in the control treatment in which the yeast was coated with the solvent hexane (Fig 3G). Interestingly, when the 7,11-HD layer on the yeast was damaged with a needle (analogous to injuring an egg), larval feeding was no longer reduced despite the presence of 7,11-HD (Fig 3G). ppk23 mutant larvae, in contrast, showed no reduction of larval feeding on yeast droplets coated with 7,11-HD (Fig 3G). These results suggest that 7,11-HD forms a physical layer that prevents emanation of cues from substances (yeast or egg) it envelops. However, damage to this layer compromises this effect, even though the same quantity of 7,11-HD remains on the surface.