Insects

Experimental N. vespilloides animals were the first- to fourth-generation offspring of beetles collected from carrion-baited pitfall traps in a deciduous forest near the University of Ulm, Germany (48° 25′ N, 09° 57′ E). Beetles were maintained in temperature-controlled chambers at 20 °C with a 16:8 h light:dark cycle. Before the experiments, groups of up to five adults of the same sex and family were kept in small plastic containers (10 × 10 cm and 6 cm high) filled with moist peat and fed freshly decapitated mealworms (Tenebrio molitor) twice a week. At the time of experiments all experimental animals were virgin, between 20 and 40 days old and not related to each other within a specific treatment group of an experiment.

General experimental design

All experimental beetles, unless otherwise stated, were handled as follows: non-related males and females were placed pairwise in plastic containers (10 × 10 × 6 cm) half-filled with moist peat and provided with a mouse carcass (10, 20 or >30 g, depending on the experiment; Mäu-Ra Farm, Radensleben, Germany). Once the carcass was buried, the containers were kept in darkness and all following manipulations were performed under red light. After 48 h the beetles and their carcass were transferred to a new container. The old container was provided with a small piece of mouse. As larvae crawl towards the carrion immediately after hatching, this procedure allowed us to determine the time when a pair’s larvae began to hatch. Embryonic development takes, on average, 56 h at 20 °C (ref. 34). We first checked for the presence of larvae 64 h after the adults had been provided with a carcass and every 6 h thereafter. As soon as the first larva was observed, the parents were transferred to a new box and a standardized number of first-instar larvae were placed on or near the carcass to be fed by the adults. The standardization of larval number allowed us to control for effects of brood size on parental physiology or care behaviour71. All experiments were performed in temperature-controlled chambers at 20 °C.

Oviposition and offspring provisioning

To test whether non-feeding mothers oviposit, whereas mothers caring for needy larvae do not engage in egg laying, we randomly assigned experimental females to three different treatment groups. All experimental females were provided with a male partner and a 10-g carcass, but after a specific time the old carcass was replaced by a fresh carcass. This was done to test whether a new carcass triggers oviposition or not. Females of one treatment group (old larvae) were allowed to care for 10 of their larvae for 4 days before we exchanged carcasses (the larvae remained with the female). At this point in their development, larvae are already nutritionally independent18. In the two other treatment groups, carcasses were exchanged after the larvae started to hatch, but only females of one group (young larvae) received 10 freshly hatched larvae to care for; such larvae are needy and are fed by the females. Females of the other group (without larvae) did not receive any larvae and therefore were not able to engage in offspring provisioning. After having exchanged the carcasses, females were left undisturbed for a specific amount of time to give them a chance to discover the new carcass and to produce new eggs. Females usually begin to oviposit about 12 h after carcass detection72 and the mean duration of egg-laying period is about 30 h (refs 34, 73). Forty-eight hours after carcass exchange, beetles along with the new carcass were transferred to new containers and the old containers were searched for eggs. This was done by scanning the peat of the old container manually, as the white eggs are easy to detect in the dark background of the peat. The procedure of transferring the beetles along with their carcasses to a new container and searching for new eggs was repeated a second time with any pairs that had not laid any eggs during the first period. We used a χ2-test to test for differences in oviposition events between treatment groups. Pairwise comparisons were corrected following the Benjamini–Hochberg procedure74. This and all further statistical analysis were done using SPSS 19 (IMB SPSS Statistics, Germany).

JH III and methyl geranate profile during a breeding cycle

To gain insight into the pheromonal and hormonal profiles of the female during an entire breeding cycle (∼9 days), N. vespilloides females (N=387) were randomly assigned to two treatment groups (‘with larvae’, ‘without larvae’), each consisting of 10 subgroups (see Supplementary Table 1 for sample size of each subgroup). Each subgroup represents a day in the breeding cycle (day 0–day 9). Experimental females were provided with a male partner and a 10-g mouse carcass. Pairs from the treatment group ‘with larvae’ were allowed to care for 10 of their own and therefore related larvae; in the treatment group ‘without larvae’, larvae were withheld from their parents to force females to resume egg laying. Each female was subjected once to volatile (‘headspace analysis’, see below) and haemolymph collection (see below) at a pre-designated time. Females of the subgroup ‘day 0’ were sampled before carcass introduction. The females of the next groups (‘day 1’ and ‘day 2’) were sampled and 48 h after they had been provided with a carcass. Females of the subgroups ‘day 3’ were sampled at the time of larval hatching, which is not necessarily 72 h after carcass provisioning, as egg laying and consequently larval hatching does not occur in perfect synchrony between different breeding pairs. Females of all subsequent subgroups were sampled 24 h (day 4), 48 h (day 5) and so on, after the larvae of the respective female had hatched (for example, if larvae of a female of the subgroups ‘day 7’ hatched at 06:00, sampling took place 96 h later at 06:00). We chose this non-repeated measurement design as it is very likely that haemolymph collection has an effect on subsequent female behaviour and physiology. Therefore, all beetles were freeze-killed after haemolymph collection.

After headspace sampling (see below), each female was weighed and all her haemolymph was collected. Haemolymph samples were obtained by piercing the intersegmental membrane between pro- and mesothorax with a fine cannula and aspirating the oozed haemolymph into a calibrated glass capillary. After quantifying the volume, the haemolymph was transferred to a 1.5-ml cone-shaped glass vial by blowing nitrogen through the capillary. An amount of 100 μl hexane containing 20 ng of vernolic acid methyl ester (Sigma-Aldrich, Deisenhofen, Germany) as an internal standard was added to the vial. The mixture was vortexed for 30 s and the clear hexane phase was transferred to a new vial, concentrated to a volume of 25 μl and subjected to size exclusion high performance liquid chromatography (SE-HPLC) for cleaning. SE-HPLC settings and all further steps for quantifying JH III, including gas chromatography-mass spectrometry analysis and calibration, were performed following the protocol described in ref. 75. We used Gaussian GLMs to test for effects of treatment and day on methyl geranate emission and JH level and a Pearson correlation to test for relationship between pheromone and hormone levels. As we measured the absolute amount of methyl geranate emitted by an individual, we also used the absolute amount of JH per individual for statistical analyses. However, as studies usually provide hormone titres, we additionally calculated JH titres (ng JH III per μl haemolymph; Supplementary Fig. 1). Furthermore, we calculated the proportion of females that resumed egg laying. Because females were freeze-killed subsequent to the treatment, we could only consider females whose larvae had already hatched (day 4 to day 9). To determine whether a female had resumed egg laying, we counted the number of larvae in the treatment group with larvae; in the treatment group without larvae, females were transferred every 48 h to a new box and the old box was checked daily for newly hatched larvae.

RNA isolation

RNA was extracted from females that had been caring for 10 larvae for 24 h (corresponds to the treatment group ‘with larvae, day 4’, Fig. 1b) and females whose larvae had hatched 24 h earlier, but were not allowed to care for their larvae (corresponds to the treatment group ‘without larvae, day 4’; Fig. 1b; each N=3). Individual beetles (a single female per biological replicate per treatment) were frozen in liquid nitrogen and ground with a mortar and pestle, and total RNA was extracted from a fraction of the powdered material, using TriReagent (Molecular Research Centre, Cincinnati, OH, USA) and further purified using the DirectZol Kit (Zyme Research) following the manufacturers’ guidelines. The integrity of the RNA was verified using an Agilent 2,100 Bioanalyzer and a RNA 6,000 Nano Kit (Agilent Technologies, Palo Alto, CA). The quantity of RNA was determined using a Nanodrop ND-1,000 UV/Vis spectrophotometer (Thermo Scientific).

Illumina sequencing

Transcriptome sequencing of each of the six RNA samples (two different female samples with three biological replicates each) was performed with RNA fragmented to an average of 150 nucleotides. Sequencing was carried out by the Max Planck Genome Centre Cologne (MPGCC) on an Illumina HiSeq2500 Genome Analyzer platform using paired-end (2 × 100 bp) read technology. This yielded ∼25–28 million reads for each of the six samples, respectively. Quality control measures, including the filtering of high-quality reads based on the score given in fastq files, removal of reads containing primer/adaptor sequences and trimming of read length, were carried out using CLC Genomics Workbench v6.5 (http://www.clcbio.com).

Transcriptome assembly and annotation

The de novo transcriptome assembly was carried out with the same software, combining all of the six RNAseq samples, and selecting the presumed optimal consensus transcriptome as described in ref. 76. Any conflicts among the individual bases were resolved by choosing the base with highest frequency. Contigs shorter than 250 bp were removed from the final analysis. The resulting final de novo reference transcriptome assembly (backbone) of N. vespilloides contained 55,934 contigs with a N50 contig size of 954 bp and a maximum contig length of 18,454 bp. The transcriptome was annotated using BLAST, Gene Ontology and InterProScan searches using BLAST2GO PRO v2.6.1 (www.blast2go.de)77 as described in ref. 76.

Digital gene expression analysis

Digital gene expression analysis was carried out by using QSeq Software (DNAStar Inc.) to remap the Illumina reads from all six samples onto the reference transcriptome and then counting the sequences to estimate expression levels, using previously described parameters for read mapping and normalization76. Biases in the sequence data sets and different transcript sizes were corrected using the RPKM algorithm (reads per kilobase of transcript per million mapped reads) to obtain correct estimates for relative expression levels. To control for the effect of global normalization using the RPKM method, we also analysed a number of highly conserved housekeeping genes, including several genes encoding ribosomal proteins (rpl3, rpl5, rpl9, rpl22e, rps3a, rps5, rps8, rps18 and rps27), elongation factor 1alpha and eukaryotic translation initiation factors 4 and 5. The overall expression levels across samples and treatments for these housekeeping genes was lower than 1.3-fold between samples (based on log2 transformed RPKM values), indicating they were not differentially expressed. Transcript abundances of genes of the different treatment groups were compared using a Student’s t-test with multiple testing correction using FDR (Benjamini and Hochberg) as implemented in the QSeq Software.

Pyriproxyfen treatment

PPN is a potent JH analogue, which mimics the effects of JH but is metabolically more stable. We applied 5 μg PPN (Sigma-Aldrich, Taufkirchen, Germany) dissolved in 2 μl acetone topically on the intersegmental membrane between pro- and mesothorax of female beetles. The control group was treated with 2 μl acetone only. Afterwards females were provided with a 10-g mouse carcass in a box filled with peat to trigger oviposition. After 24 h, the PPN/acetone procedure was repeated. Two days after carcass provisioning the parents were removed and newly hatched larvae counted until hatching ceased. The total amount of applied PPN was similar to the amount used in other studies26,29. To test whether the treatment (PPN versus control) had an effect on the number of offspring produced, we performed a Poisson GLM.

Effect of brood size on methyl geranate emission

Pairs of N. vespilloides beetles were provided with a 20-g mouse carcass and allowed to care for 1, 5, 10 or 20 larvae, respectively. After caring for larvae for 24 h, the number of surviving larvae was counted and the females were subjected to headspace analyses. To be able to provide each experimental pair with the planned number of larvae, we additionally provided a large number of N. vespilloides pairs with a 10-g mouse carcass, and use these pairs as ‘larvae donors’78. A Gaussian GLM was performed with the number of larvae as fixed factor and the amount of methyl geranate as dependent variable.

Deuterium labelling experiment

To test whether the production of JH III and methyl geranate is physiologically linked via the mevalonate pathway33 (Fig. 2), we injected a deuterium-labelled geranyl pyrophosphate precursor32,79 ((E)-3,7-dimethylocta-2,6-dien-1-yl-2-d diphosphate, [2-2H]-GPP, kindly provided by W. Boland, Max-Planck-Institute for Chemical Ecology, Jena, Germany; see Supplementary Fig. 2) into the body cavity of brood-caring N. vespilloides females. Geranyl pyrophosphate is a well-known metabolic precursor of JH III33. Pairs of beetles were provided with a 10-g mouse carcass and left undisturbed until we started to control for larval hatching. After the beetles had cared for their larvae for up to 13 h, the female was removed and subjected to [2-2H]-GPP injection. We injected 10 μg [2-2H]-GPP per female diluted in 1 μl distilled water either into the abdomen, into the intersegmental membrane between pro- and mesothorax, or into the ventral cervical membrane using a pointed glass microcapillary, which was mounted to a micromanipulator and connected to a model 2010 Nanoliter Injector (World Precision Instruments, Berlin, Germany). After injection, females were returned to their respective brood and male partner and were subjected to headspace analyses as described below between 2 and 6 h later. The incorporation of the deuterium-labelled geranyl pyrophosphate into methyl geranate was concluded from the increase in the abundance of diagnostic ions of methyl geranate80.

Gas chromatography coupled with electroantennographic detection

The electrophysiological response of N. vespilloides male antennae towards methyl geranate was tested using GC-EAD. The same GC-EAD set-up as in ref. 81 was used, but the GC was equipped with a DB-5 capillary column (30 m × 0.25 mm i.d., 25 μm film thickness, J & W Scientific, Folsom, CA, USA) and the oven temperature was raised from 50 °C to 220 °C at a rate of 10 °C per min. The final temperature was held for 2 min. The responses of three male antennae were tested to 50 ng of synthetic methyl geranate (100 ng μl−1 pentane, 1 μl injected, Split FID:EAD=1:1, mixture of isomers, Santa Cruz Biothechnology, USA). The antennae were cut at their base and at the tip. The excised antenna was mounted between two glass electrodes filled with insect Ringer solution (see ref. 81 for further details).

Copulation behaviour and natural methyl geranate emission

Mating behaviour of N. vespilloides males in two different breeding contexts was analysed by observing them continuously for 90 min. Experimental N. vespilloides pairs were provided with a 10-g mouse carcass and divided in two different treatment groups. In the first treatment group, pairs were allowed to care for 10 of their own larvae and in the second treatment group larvae were withheld from their parents. Observation took place 48 h after larval hatching. During observation we recorded whether they copulated or not, and if they did, how often they engaged in copulations. At the end of each observation period the females were directly subjected to headspace analyses to quantify methyl geranate emission. To test whether male copulation behaviour (yes/no) or the number of copulations was/were dependent on the amount of methyl geranate a female emitted, a logistic regression and a Poisson GLM was performed.

Bioassay with synthetic methyl geranate

To test the effect of synthetic methyl geranate on male copulation behaviour, we used triangular shaped pieces (4 × 4 × 5 mm, 1 mm thick) of Supelco Molded Thermogreen LB-2 silicone septa (Sigma-Aldrich, Taufkirchen, Germany), which were either conditioned for 7 h in 1 ml n-hexane (control) or a solution of 10 mg synthetic methyl geranate diluted in 1 ml n-hexane. Subsequently the silicon pieces were left under a fume hood for 15 h to allow the solvent to evaporate. Headspace analyses (N=7) of the silicone pieces revealed that after this procedure they emitted a relatively constant amount of methyl geranate (430 ng±57.8 ng per 20 min) over a long time period. The silicon pieces were attached to females that were breeding with a male at a 10-g carcass for about 120 h, but were not allowed to care for larvae (corresponds to the treatment group ‘without larvae, day 5’. These females thus emitted no or only trace amounts of their own methyl geranate; Fig. 1d). After fixing the silicon pieces on a female’s pronotum with superglue, the female and the male and their carcass were transferred to a small plastic container (7 × 3 × 3 cm) with a plaster floor and observed continuously for 60 min. During observation, the following parameters were recorded: the number of encounters between male and female, whether they copulated or not and how often they engaged in copulations. Recording started after observation of the first encounter. To test whether the treatment (methyl geranate versus control) had an effect on the occurrence (yes/no) or number of copulations, we performed a χ2-test and a Poisson GLM, respectively.

Effect of breeding partner on methyl geranate emission

This experiment aimed to test the effect of the sex of the breeding partner on methyl geranate emission. Two females were kept together with a male for 48 h, so to that both females were inseminated. Afterwards the females (without the males) were transferred to a new container (20 × 20 × 6 cm) and provided with a large carcass (>30 g) to induce cooperative breeding. A 30 g carcass was prodvided because cooperative same sex brood care usually only occurs on large carcasses82. As soon as the first larva had hatched the respective female pair was provided with up to 30 first instar larvae to care for. Additionally, male-female pairs were established and subjected to the same procedure. As in previous experiments, we also provided a large number of N. vespilloides pairs with a 10 g mouse carcass to use them as ‘larvae donor’ in case a pair did not have enough of their own larvae to add78. After 24 h of larval care all females were subjected to individual headspace analyses and then weighed. A Gaussian GLM was used with the sex of the breeding partner as the fixed factor and the amount of methyl geranate emitted as dependent variable. To verify that in the female–female dyads both females performed brood care, we observed their feeding behaviour. In all tested cases, both females were feeding larvae. Furthermore, there was no difference in the weight between females breeding with a female and those breeding a male partner (Gaussian GLM, N=28, F 1,26 =0.01, P=0.92), which is a good indicator that all female had equal access to the carrion resource.

Headspace analyses

To quantify methyl geranate emission, females were put singly into a glass jar (inner diameter 3 cm) and left undisturbed to accumulate the volatiles emitted by the beetles. Before use, all glasses were silanized with 5% dimethyldichlorosilane solution in toluene to increase their hydrophobicity and prevent adherence of chemical substances. Each glass jar was provided with a moist, Soxhlet-cleaned ball of cotton wool to provide sufficient humidity for the beetles. After an accumulation time of 20 min, air was sucked through the jar at a rate of 200 ml min−1 for 5 min using a membrane pump (DC 12/16FK, Fürgut, Aichstetten, Germany). Incoming air was cleaned by an activated charcoal filter (50 mg, Supelco, Bellefonte, PA, USA). The effluent air stream passed through the glass jar and the emitted volatiles were trapped on a thermal desorption filter filled with a mixture of Tenax-TA and Carbotrap as described in ref. 83. For quantification, 20 ng of methyl undecanoate (dissolved in 1 μl methanol) were applied to each adsorbent filter as internal standard. A calibration curve was created by analysing known amounts (5–100 ng) of synthetic methyl geranate that were applied to the adsorbent filter together with 20 ng of the internal standard.

Headspace samples from the first experiment (Methyl geranate and JH profile during a breeding cycle) were thermally desorbed (8 min at 300 °C) using a Shimadzu TD20 automated thermal desorption system connected to a Shimadzu GC 2010 gas chromatograph coupled to a QP2010 plus mass spectrometer. The GC was equipped with a non-polar capillary column (BPX-5, 30 m length, 0.25 mm i.d., 0.25 μm film thickness, SGE Analytical Science, Milton Keynes, UK) and helium was used as carrier gas (50 cm s−1 linear velocity). The oven programme started at 50 °C and was raised at a rate of 5 °C min−1 to 200 °C, and then at a rate of 15 °C min−1 to 280 °C that was held for 10 min. The mass spectrometer was run in electron impact mode (70 eV) and set to a scan range from 35 to 450 m/z. Headspace samples of all other experiments were analysed using an Agilent Technologies 7820A gas chromatograph equipped with a non-polar capillary column (DB-5, 30 m length, 0.25 mm i.d., 0.25 μm film thickness, J & W Scientific, Folsom, CA, USA) and hydrogen as carrier gas (40 cm s−1 linear velocity). Adsorbent filter were introduced via ChromatoProbe kit (CPAV6890, Aviv Analytical Ltd, Israel) and thermally desorbed for 1 min at 310 °C. GC temperature settings were the same as described above.