The fru gene specifies male cooperative behaviors in honeybee colonies

Domain: biology
Published: 2025-12-22
Editorial outcome: minor_revision

Abstract
Conspecific individuals can benefit from behavioral interactions, but whether cooperative behaviors require dedicated control in the nervous system is poorly understood. We examine the genetics underlying obligate cooperative male behaviors in honeybee colonies (Apis mellifera) that are deeply hard-wired. We screened for transcription factor genes and found that only the fruitless (fru) BTB zinc-finger gene was restricted to expression in males and in the nervous system. Reporter coexpression revealed Fru-positive neuronal cell populations that are involved in processing and integrating sensory information. Computer-assisted behavioral tracking of male bees with loss-of-function mutations in experimental colonies revealed that fru is specifically required for the rate and/or duration of social approach and feeding behaviors, suggesting that the gene scales bees’ participation in the collective nutrition task. Together, our study establishes a gene-based specification of behavior initiation and sustainment and provides insight into the connection between cooperation and defined neural populations.

Introduction
Behaviors can bind individuals into conspecific groups. In primates and birds, individuals can benefit from cooperative behavioral interactions, as these interactions increase the ability to find food, protect against predators and promote breeding success1,2. In the Western honeybee (Apis mellifera), a classical example of an organism displaying cooperative behaviors, these interactions are innate, not learned and obligate. They are hardwired into the nervous system, making cooperative behavior accessible to genetic and neural manipulations. The behaviors of honeybee colonies are so well controlled that the individuals function together like a single organism or ‘superorganism’ that feeds and sustains itself3,4. This is the basis of the great general ecological success of social insects5–7.

The members of the honeybee colony are not homogenous. A colony that is ready to reproduce consists of hundreds of males, a queen and thousands of usually sterile female worker bees. The cooperative behaviors of males inside a colony are of special interest because they support the reproductive success of a colony. Males do not behaviorally compete with other males over females or food resources in the protected shelter of the colony8,9. Instead, males receive nutrition-rich, predigested food from worker bees during their maturation, which involves a sequence of antennation, begging and trophallaxis behaviors to transfer the food10–12. The trophallaxis behavior plays a central role in the food exchange among colony members, the nutrition of larvae, adult males and queens and the communication about the food status of the colony10. The queen’s behavior involves mating with males in the air outside the colony and the laying of eggs for the remainder of her life. The worker bees instead collect pollen and nectar outside the nest and store them in wax-constructed combs inside the colony. The colonies reproduce asexually via the splitting of colony members (swarming).

How the male-specific cooperative behaviors are molecularly controlled in the nervous system is largely unknown. The male-specific anatomy of the nervous system that can control male-specific behaviors is well documented, but not at the level of neural circuitry. Male identity manifests in male-specific odorant receptor gene expression, male-specific number and anatomy of glomeruli in the primary olfactory processing center (the antennal lobe (AL)), a male-specific smaller mushroom body (MB) harboring 12% fewer neurons and in male-specific larger optical lobes13–18. Compared with worker bees, male-specific neural processing has been detected in the glomeruli of the AL for the queen’s sexual pheromone 9-oxo-(E)-2-decenoic acid (9-ODA) using calcium (Ca) imaging18.

Honeybee males newly evolved their cooperative behaviors, which they display inside the colony, as eusociality evolved during the last 60 million years19,20. However, the evolutionary mechanism that sparked the transition to cooperation is lacking21–24. Whether the control of the many social interactions inside the densely populated colony indeed requires greater neural processing capacity (or even other cognitive abilities; the “social brain hypothesis”) is still under debate, not only for social insects but also for primates25–28.

A point of entry to understand the control of within-colony cooperative behaviors involves genes that hardwire the male-specific nervous system. If such key genes exist, they must be regulated by the well-characterized sex determination cascade (Fig. 1a)29–32. The same versus different complementary sex determiner (Csd) protein variants (expressed from hemizygous or heterozygous csd genotypes) mediate male or female splicing of the feminizer (fem) transcripts, resulting in Fem protein expression only in females (Fig. 1a)30,31. This sex-specific Fem protein activity regulates sex-specific expression of downstream transcription factor genes (TFs) via alternative sex-specific splicing. This involves the conserved doublesex (dsx) and lineage-restricted glubschauge (glu) genes, both of which regulate different aspects of sexual differentiation29,32. However, which TF genes specify the nervous system of males underlying the control of their cooperative behaviors is currently unknown.

Hence, the key questions concerning the control of cooperative behaviors are manifold: How many key genes coordinate the hardwiring in the nervous system? What aspects of cooperative behavioral control are genetically specified in the nervous system? What neural processing abilities are involved? What evolutionary mechanisms underlie the origin of cooperative behaviors?

Thus far, we do not know which genes took on the function of hardwiring the male’s cooperative behaviors in the nervous system. We thus screened for male-specific spliced transcripts in the nervous system33 and found that, in addition to previously known glu and dsx only the fruitless (fru, LOC409022) TF gene has male-specifically spliced transcripts (Supplementary Table 1). Because this Fru BTB zinc finger transcription factor is expressed only in the male nervous system and its homolog in Drosophila melanogaster is involved in hardwiring sexual behaviors34,35, we set out to examine the function of this gene product in manifesting the male’s cooperative behaviors in honeybee colonies.

Results
The male-specific FruM protein is highly spatially and temporally restricted in the nervous system
Our detailed nucleotide sequence transcript analyses revealed that the fru gene in honeybees is transcribed from at least three promoters based on three different transcription start sites. The three transcripts that derive from promoters P1 to P3 encode protein variants with a common Broad-Complex, tramtrack and Bric-à-brac (BTB) domain and three alternative zinc finger (ZnF) domains that are produced via alternative splicing and inclusion of either exons 9A, 9B and 9C (Fig. 1b–d; EMBL-EBI accession number: OZ362630-OZ362641). Only the P1-derived fru transcripts (fruP1) are sex-specifically spliced. In males, the skipping of exon 3 results in male-specific fruP1 transcripts (fruM), which encode 386– to 459-amino-acid-long protein isoforms with three alternative ZnF domains, suggesting that this is a male-specific BTB ZnF transcript, fruM (Fig. 1c). In females, exon 3 is retained, which results in an early translation stop codon of female-specific fruP1 transcripts (fruF) and a predicted 34-amino-acid-long protein that lacks the BTB and ZnF domains (Fig. 1c). To determine whether sex-specific fruP1 transcripts are controlled by the sex determination cascade, we performed RNAi-induced knockdown of the fem gene, a key sex determination regulator31 (Fig. 1a). The fem RNAi-treated females produced male-specific fruP1 transcripts but also female-specific transcripts at larval stage 4 (Fig. 1e and Supplementary Table 2) suggesting a partial shift toward male splicing in response to temporally restricted fem knockdown, which we induced in the embryo31. This result suggests that this male-specific splice regulation requires the absence of female-determining activity from the fem gene, a component of the canonical sex determination pathway30,31,36.

In newly eclosed adult males, sizeable amounts of fruM transcripts are found in the brain and ganglia but not in other tissues, including the thorax muscle, fat body, or gonads (Fig. 1f). This finding suggests a spatial restriction of fruM transcripts to the nervous system. During development, sizeable quantity of fruM transcripts are found from early pupation stages on in the head (Fig. 1g). This finding indicates that expression starts in the nervous system at early pupation, when the adult nervous system is formed37. Next, we developed an antibody against a sequence of the BTB domain. Using confocal light microscopy sections, we found that the anti-Fru antibody labels male but not female pupal brains in spatially characteristic patterns (Fig. 2a, b and Supplementary Movies 1 and 2), suggesting that these male-specific labels derived from P1, but not P2 or P2 transcripts. To examine whether this male-specific expression pattern was specifically derived from FruM protein expression, we generated fruP1− mutants. We deleted the P1 promoter and exons 1 and 2 sequences using the CRISPR/Cas9 method29,32,38 (Fig. 2c), reared homozygous queens carrying this deletion, and confirmed the fruP1-/- genotype at the nucleotide sequence level (Supplementary Fig. S1). These queens produced haploid unfertilized eggs from which our fruP1− male progeny were derived. The fruP1− males lacked the fruM transcript (Supplementary Fig. S2) and did not present consistent male-specific Fru protein labels as observed in staining of the wt males (Fig. 2a and Supplementary Movie 3). These results suggest that the male-specific FruM protein is expressed from the male fruM transcripts, that the anti-Fru antibody labels Fru protein expression, and that the fruP1− mutant is a loss-of-function mutation of fruM.

We found that our anti-Fru labels colocalized with nuclei (Supplementary Fig. S3). These labels were mostly organized in characteristic clusters, which were frequently located in the outer anterior and posterior sections of the male brain (Fig. 2a, b). We also detected such sparse Fru protein labels in the ganglia (Supplementary Fig. S4), suggesting that FruM expression expands to the ventral nerve cord. To determine how many cells express the FruM protein, we counted Fru-labeled nuclei in the adult male midbrain (which consists of approximately 400,000 neurons16) and found that approximately 1800 cells expressed FruM proteins (Fig. 2d). Collectively, these results suggest that the male-specific transcription factor protein FruM is highly spatially and temporally regulated in the developing and adult male nervous systems.

FruM-expressing cells are involved in processing and integrating information from different sensory modalities
As it is unclear where FruM is expressed in the male brain, we inserted the myrGFP/P2A/T2A coding sequence into the fru locus using the CRISPR/Cas9 method29,38,39 so that the fruP1 transcripts expressed both membrane-tethered GFP (myrGFP) and FruM proteins (Fig. 3a, Supplementary Fig. S5). We examined two neural clusters to determine whether the insertions generated robust labeling of Fru-expressing cells. The number of Fru-expressing cells in the fru myrGFP males did not differ from that in the wt males as revealed from anti-Fru antibody staining, suggesting that this genomic insertion did not disrupt the formation of these neurons (Fig. 3b and Supplementary Fig. S6; Cluster I (P=0.54), Cluster II (P=0.33), MWU test). On average, 100% and 96% of the anti-Fru-labeled cells in the two clusters were also positive for GFP, suggesting robust GFP labeling of the Fru-expressing (FruM+) cells (Fig. 3c and Supplementary Table S3).

We detected a number of FruM+ cell populations in brain areas with known functions14,40,41 (Fig. 3d and Supplementary Movies 4–7). We observed FruM+ neurons in periesophageal neuropils (PPs) in the posterior region (Fig. 3d), which are involved in processing mechanosensory and gustatory information. One tract runs medially on each side of the brain, possibly connecting with the superior medial protocerebrum (SMP, Fig. 3d). In the antennal nerve, we identified FruM+ neurons (antennal tracts T5 & T6, Supplementary Movies 4 and 5) that project into the antennal mechanosensory and motor center (AMMC; Supplementary Movies 4 and 5) and possibly into the subesophageal zone (SEZ; Fig. 3d), indicating that mechanosensory and/or gustatory neurons are involved. The olfactory sensory neurons (OSNs) entering the antennal lobe (AL) were not stained (Supplementary Movies 4 and 5). Many glomeruli in the AL (the primary olfactory processing center) exhibit FruM+ in their core region. This includes the male-specific macroglomeruli MG1 and MG2 (AL, Fig. 3d), the latter of which processes the sensory input from the queen pheromone 9-ODA18. The glomerulus core FruM+ circuitry primarily contains processes from local neurons (LNs), because projection neurons (PNs) extending to the mushroom bodies (MBs) were not labeled (Supplementary Movies 4 and 4). The lateral horn, another center of the olfactory pathway, shows some FruM+ fibers (LH, Fig. 3d). Several FruM+ neurons in the middle brain belong to output tracts of the visual system. The anterior optic tract (AOT) has some FruM+ cells that project to the anterior optic tubercle (AOTu, Fig. 3d, Supplementary Movies 2–7). Three optic commissures present FruM+ cells that connect the two optic lobes (inferior and posterior optic commissures, IOC and POC) and the two AOTu (vTUTUT: ventral tubercle-tubercle tract; Supplementary Movies 4 to 7). FruM+ neurons are also observed in the optic lobes with diffuse layers in the medulla, clearly stained fibers in the inner chiasma, and three distinct layers in the lobula (possibly corresponding to layers 1, 3, and 5)42 (Supplementary Movie 8 and 9). In the higher-order processing center, the mushroom body (MB), we detected FruM+ neurons in the calyx (see medial calyx (MC), Fig. 3d). FruM+ neurons were found in the lips (li, which receive projections from the antennal lobe43), in the outer collar zone (co, which receives visual input from the lobula and medulla44) and in the basal ring (br, which receives multisensory input from at least the visual and olfactory systems45; Fig. 3d). Intrinsic MB neurons, including a subpopulation of type I Kenyon cells (KCs) and some type II KCs, are Fru positive. The neurites of type I KCs extend along the peduncle and bifurcate into the medial and vertical lobes of the MB, whereas those of type II KCs project only into the most ventral part of the vertical lobe (VL, Fig. 3d). This manifests as different layers in the VL, which include from rostral to caudal, type I KC input from the basal ring (Supplementary Movies 4 and 5), the collar, the lip (VL, Fig. 3d) and type II KC input (Supplementary Movies 4 and 5). FruM+ neurons are also prominently found in anterior parts of the SMP around the medial lobe and in regions corresponding to the superior lateral protocerebrum (SLP, Fig. 3d), including those that connect the two hemispheres41 (Fig. 3d), which possibly represent a hub for preprocessing input from the MB and possibly other regions of the brain. Collectively, these results suggest that the FruM+ circuitry is involved in the processing of sensory information (olfaction, vision, and possibly gustation and mechanosensation) from the periphery to higher-order processing centers such as the mushroom bodies.

FruM+ circuitry has a male-specific anatomy
To understand whether the FruM+ circuitry has a male-specific anatomy that can hardwire male-specific behaviors, we generated fruP1myrGFP female workers and compared the labeled structures with those of fruP1myrGFP males. In females, we note these labeled cells as myrGFPP1+ because the GFP is generated from the same P1 transcript as in males, but these cells do not express the male FruM protein. At the gross level, myrGFPP1+ and FruM+ circuitries in females and males were similar, but neural populations involved in olfactory visual information processing showed notable differences (arrows and arrowheads in Fig. 4 and Supplementary Movies 6, 7, 11 and 13). The antennal nerve is labeled in both male and female worker bees (arrow, Fig. 4a and Supplementary Movie 6, 11 and 13). In workers, a small number (approximately a dozen) of glomeruli on the ventro-medial side of the AL had myrGFPP1+ labels both in the core and cortex (arrow, Fig. 4b), whereas in males FruM+ labels were consistently restricted to the core (arrowhead, Fig. 4b). This female-specific labeling of the cortex suggests that OSNs in the antennal nerve are FruM+ negative in males but some are myrGFPP1+ positive in females. This is supported by a more restricted labeling of the nerve in males than in females (arrows, Fig. 4a: note that the antennal nerve contains approximately 4 times more neurons in males than in female workers14). Hence, sensory neurons from the antennal nerve that bypass the AL and glomeruli on the ventral side (antennal tracts T5 & T6) towards the AMMC and potentially the subesophageal zone (SEZ) were labeled in males but also in female worker bees. The set of worker-specific labeled glomeruli may correspond to the T3b cluster, which may be involved in the processing of cuticular hydrocarbon cues14,46,47. FruM+ circuitry is present in the core of the macroglomeruli, which are male-specific structures not found in workers (MG1, Fig. 4b and Supplementary Movies 6, 11 and 13). The FruM+ circuitry of the male visual system has an extra male-specific tract not found in the myrGFPP1+ circuitry of workers (large arrow, Fig. 4c and Supplementary Movies 7). This male-specific tract projects from the lobula (LO) to the posterior lateral protocerebrum (PLP). However, two other labeled tracts in the same area are shared between males and worker bees (small arrows, Fig. 4c). In the central complex, a few labeled myrGFPP1+ fibers were found in the noduli (NO) of female worker bees, but corresponding FruM+ fibers in males were absent in that region (arrows, Fig. 4d and Supplementary Movies 6, 11 and 13)48. We conclude from these comparisons that the FruM+ circuit has a male-specific identity that manifests at the gross level in the anatomy of the antennal nerve, macroglomeruli, and innervation of the core/cortex of the glomeruli and optic tracts.

The initiation and sustainment of social feeding behavior dysfunction in fruP1− males
Sexual maturation of males depends on protein-enriched nutrition which the males receive from the worker bees via trophallaxis behavior. This process requires a sequence of behaviors with the option to quit at a particular step. The male approaches and initiates contact with the worker bee’s head with its antennae while displaying begging behavior. This behavior eventually leads to trophallaxis behavior, in which the male extends its proboscis to the donor worker bee’s mandibles to receive liquid food via mouth-to-mouth transfer10,11. The (nurse) worker bees thereby provide honey liquid from the expandable part of their gut (“honey” stomach), which is enriched with proteins from the hypopharyngeal glands10,12. The components of these proteins are derived from digested and metabolized pollen, which males can hardly digest10,12.

To determine whether the male-specific FruM transcription factor protein hardwires neural circuits that control the social feeding behaviors, we studied loss-of-function fruP1− males in small experimental colonies. Twenty fruP1− males and wt males, together with 460 worker bees and a queen, were assembled on combs (Fig. 5a). These combs contained the same amount of pollen and honey stored at the same location in the cells (Supplementary Fig. S7). The behavior of each male was tracked using unique two-dimensional barcode labels and our computer-based Bee Behavioral Annotation System (BBAS)49 (Fig. 5a).

We observed that the rate of begging behavior was markedly and significantly reduced by approximately twofold in fruP1− males compared with wt males (P=0.004, MWU test; Fig. 5b). Moreover, the rate of trophallaxis behavior was reduced by more than twofold on average in fruP1− versus wt males (P=0.005, MWU test; Fig. 5b). We found that the duration of trophallaxis behavior was slightly but significantly reduced in fruP1− compared with wt males (P=0.027, MWU test; Fig. 5c). However, the movement patterns during the execution of begging and trophallaxis behaviors were stereotypic and unaffected, suggesting that the initiation of begging and trophallaxis and the sustainment of trophallaxis behaviors were specifically impaired in fruP1− males (Supplementary Movies 14–17).

Begging behavior requires that a male approach another colony member. To understand whether the male’s approaching behavior is actively controlled and innately specified via the FruM protein or whether it arises passively from random locomotion-driven encounters, we examined the rate at which males approached with their antennae the abdomens and thoraxes of worker bees or males, a behavior that does not lead to begging behavior. We observed that these other social contacts were also substantially reduced irrespective of whether the approached bee was a worker (P=0.004, MWU test) or a male (P=0.015, MWU test; Fig. 5d; Supplementary Movies 18–21). This result suggests that approaching behavior is impaired in fruP1− males and is innately specified. The rates of other male behaviors, such as self-cleaning behavior (P=0.21, MWU test; Fig. 5e), resting behavior (P=0.3, MWU test; Fig. 5f), and locomotion behavior (P=0.94, MWU test; Fig. 5g), were not affected in fruP1− versus wt males, suggesting that social feeding behaviors were particularly impaired (Supplementary Movies 22–25). Collectively, these results suggest that the initiation of approaching, begging and trophallaxis behaviors and the sustainment of trophallaxis were specifically impaired in the fruP1− males.

Other male-specific within-colony behaviors are also impaired
Surprisingly, we observed that fruP1− males repeatedly entered the pollen-containing cells (average 17 times per hour), whereas wt males never did so (P<0.001, MWU test; Fig. 6a). The fruP1− and wt males did not enter the honey cells (no difference: P=0.79, MWU test; Fig. 6a), a behavior which has been reported for older males50. Our fruP1− and wt males sporadically entered the empty cells, but the rates did not differ (P>0.96, MWU test; Fig. 6a). The movements of fruP1− males into pollen cells followed the same stereotypic pattern as those of wt males that entered an empty cell (which often involved bouts of repeated cell entries (Supplementary Table S4 and Supplementary Movies 26–29)). These results suggest that the choice of which cell type is entered is disrupted in fruP1− males. The increase in the entering of pollen cells cannot be explained by higher visiting rates to the pollen stores because the pollen area visits were lower in fruP1− than in wt males (P=0.002, MWU test; Fig. 6b). Because the entering of pollen cells in fruP1− males lasted for more than 2 s (Fig. 6c), we investigated whether the fruP1− males consumed pollen, a food source they can hardly digest10,12. No sizeable amount of pollen grains was found in the midguts and hindguts of fruP1− males (Supplementary Fig. S8), suggesting that the entering of pollen cells do not result in a consumption of pollen.

Males do not perform any behavioral task inside the colony. They also do not compete with other males over food sources or females8. Hence, males are usually located on the periphery and in the honey areas rather than in the densely populated center of the colony, in which the brood areas and pollen stores are found51. We examined whether the location of the males relative to the food stores is also innately programmed via FruM. We found that the fruP1− spent more time in the pollen and empty areas but less time in the honey area than wt males (Fig. 6d), suggesting that the allocation behaviors towards food stores were impaired in fruP1− mutants. Together, these findings suggest that the allocation towards the food stores and the choice of which food is visited in the cells are innately programmed and involve FruM.

Sex pheromone-related behaviors, but not general sensorimotor functions are impaired in fruP1− males
Males mate with queens outside the colony during a mating flight. Virgin queens release a sex pheromone (9-oxo-(E)-2-decenoic acid (9-ODA)), which attracts males during these flights52,53. To understand whether the FruM protein also contributes to sexual behavior in the nervous system, we examined changes in locomotion behavior in response to the sex pheromone 9-ODA54 in a behavioral Petri dish assay. Compared with wt males, fruP1− males exhibited reduced locomotion in response to 9-ODA (P=0.011, MWU test; Fig. 7a), indicating that sexual behavior is also impaired in fruP1− males.

To exclude the possibility that the dysfunction of sexual and within-colony behaviors in fruP1− males results from a general impairment of sensorimotor functions (such as a general dysfunction of neuronal processing or moving abilities), we quantified behavioral responses to olfactory, gustatory, mechanical, or visual stimuli. We found that the locomotion responses to the repellent benzaldehyde (P=0.84, MWU test; Fig. 7b), light (P=0.75, MWU test; Fig. 7c), and contact with worker bees (P=0.66, MWU test; Fig. 7d) did not differ between fruP1− and wt males. The locomotion behaviors of fruP1− and wt males in the absence of these stimuli also did not differ (P=0.3, MWU test; Fig. 7e). Both groups also responded with proboscis extension to honey (P=0.51, Fisher’s exact test; Supplementary Table 5). Moreover, impaired viability cannot explain the dysfunction of male-specific behaviors because the fruP1− mutation did not influence the survival rate of adult males (log-rank test, Chi2=0.22, df=1, P=0.64; Supplementary Fig. 9). The behavioral dysfunctions also cannot be explained by a malfunction and malformation of external and internal structures at the gross level because the external head, antennae, abdomen and leg morphology as well as the reproductive organ anatomy of fruP1− males had a wt phenotype (Fig. 7f). These results suggest that general sensorimotor functions are intact in fruP1− males. Hence, the impairment of general sensorimotor functions cannot explain the dysfunction of male-specific behaviors in the mutants.

Cuticular hydrocarbons (CHCs) can provide important cues for social interactions among colony members55–57. To examine whether fruP1− mutation altered CHC profiles, we examined the CHC profiles via gas chromatography‒mass spectrometry (GC‒MS) analysis. We observed that all the compounds were present in fruP1− and wt males and that all the compound classes were equally represented between fruP1− and wt males (Supplementary Fig. 10). However, there was a significant difference in the overall CHC composition between the mutant and wt males (Supplementary Fig. 10). Next, we examined whether the individual CHC composition profiles of fruP1− and wt males produced separate clusters in two-dimensional multidimensional scaling (MDS) space (Supplementary Fig. 11). We found that the individual profiles partially overlapped between the groups, suggesting that the CHC are unlikely to have triggered a behavioral difference between the groups of mutant and wt bees.

The gross neuropil structure and odor processing features of the AL are not impaired in fruP1− males
How can the FruM protein specify these male behaviors? We propose that the FruM protein specifies connectivity or functional features in the nervous system that determine behavioral control. To understand the role of the FruM protein in the programming of behaviors, we examined chemosensory receptor abundance, neuropil anatomy, and odor processing in fruP1− males.

We found no changes in the expression of odorant receptor (OR), odorant binding protein (OBP), gustatory receptor (GR), or chemosensory protein (CSP) genes in the antennae of fruP1− versus wt males using the RNAseq method (Supplementary Fig. 12; EMBL-EBI accession number: PRJEB100586). This result suggests that the repertoire of chemosensory proteins involved in odor detection is not impaired in fruP1− males. This includes the Or11 gene, which expresses a key odorant receptor protein involved in the detection of the queen pheromone 9-ODA15, a gene that is highly male-specifically expressed (>25-fold higher expression in males than in worker bees). Our optical sections of anti-synapsin-labeled midbrains revealed no gross anatomical differences in the neuropil structure or neuron bundles between fruP1− and wt males (Supplementary Movies 30 and 31).

Higher-resolution analysis of the AL, the first-order olfactory center, was performed. Four male-specific enlarged and structured glomeruli (MGs) were found in fruP1− males that were in the same location as those in wt males (Fig. 8a)14,18,58. These results suggest that the gross brain anatomy and the sex-dimorphic structure of the AL were not malformed in fruP1− males during development. Finally, we examined whether neural processing features were impaired in adulthood using in vivo Ca2+ imaging of the AL. Odor information detected by OSNs in the antenna is translated into a glomerulus-specific pattern of neural activity in the AL18,47. For the queen pheromone 9-ODA, the activity in fruP1− males was confined to MG2, as in wt males (Fig. 8b). The response intensity did not differ between fruP1− and wt males (P=0.46, 2-way repeated-measures ANOVA; 9-ODA fruP1− vs. 9-ODA wt males: P=0.36, Sidak’s multiple comparison test; Fig. 8c, d). The responses in MG2 were specific to the 9-ODA application in both cases (P<0.0001, Tukey posthoc tests, 9-ODA vs. control, P<0.0001; Fig. 8c, d). These observations suggest that 9-ODA odor information processing was not impaired in fruP1− males in this first-order processing center. For other odorants, such as 1-hexanol (1-6ol), the repellent benzaldehyde, and the alarm pheromone component isopentyl acetate (IPA), we also detected no signal intensity or pattern differences between fruP1− and wt males (P=0.49, 2-way repeated-measures ANOVA; Fig. 8b, e), suggesting no dysfunction in fruP1− males. Collectively, these results suggest that neither a dysregulated neuropil connectivity structure and chemoreceptor protein repertoire nor dysfunctional primary olfactory processing can explain, at this gross level, impaired behavior in fruP1− males.

Methods
Honeybee source
Honeybee colonies (Apis mellifera) were located in the beeyard or in the containment at the Heinrich-Heine University Düsseldorf, Germany. The genetically manipulated honeybees were maintained together with worker bees in small mating nucleus hives (Segeberger nucs), which we kept in a secure containment so that genetically manipulated animals could not escape into nature.

RNAi mediated knockdown procedure
Female eggs were injected with fem siRNAs, and larvae were reared to 4th instar (L4) inducing a temporally restricted knockdown as described in ref. 31.

CRISPR/Cas9 experimental procedures
Target sites for the sgRNAs were identified using Benchling software (Biology Software, 2017, https://benchling.com). The sgRNAs for the deletion were fruP1 sgRNA 6 (GATACCCCCCACGACATTTC) 855bp upstream of the designated P1 transcript start, and sgRNA 12 (GGCTGCTGTGCACGCTTAGA) 232bp downstream of the translational start codon (Fig. 1C). The deletion rate was 14%. The sgRNA for the genomic insertion was fru sgRNA 1 (GTGTTTGGCGCATCGTTACC) 22bp downstream of the start codon. sgRNAs were synthesized29.

Female eggs were collected from wild type queens every 90 min and injected directly with 53-mm injection needles (Hilgenberg, Malsfeld, Germany)29,79. We injected 375 ng/μl of Cas9 protein (EnGen Cas9 NLS, S. pyogenes, #M0646, New England Biolabs), sgRNA 1 and donor DNA were at a molar ratio of 1:1:1. The myrGFP-P2A-T2A coding sequence was synthesized with fru homologous arms (Supplementary Fig. 5; Eurofins, Ebersberg, Germany). The homologous arms (each 250bp long) corresponding to the intron 1/2 and exon 2 [National Center for Biotechnology Information (NCBI); gene ID: 409022; reference Sequence: NC_037646; Assembly: Amel_HAv3.1 (GCF_003254395.2)]. In the inserted sequence, we modified the PAM site of the fru sgRNA 1 target site39. Queens were reared, and genotypes were determined via PCR amplification, sized determination, and sequencing39,64,79. Mutant queens were treated twice with CO2 (on day 9 and day 10) to stimulate oviposition of unfertilized, male eggs. Queens were introduced into small colonies and nucs (Holtermann, Germany).

Immunohistology and image acquisition procedure
We developed for the honeybee an antibody against Fru protein involving part of the BTB domain. The protein for immunization (Seqlab, Göttingen, Germany) in rabbits was 98 amino acid long (VSQHLLPMFLKTAEALQIRGLTDNSVNNKTEEKSPSPEPETQTGIRHTESPNLQPPPEKRKRKASGSYDVSLSGPPSERFMSDSQTSSQCSYKSSPPV) and is present in all protein isoforms (Fig. 1b–d). Anti-Fru antibodies were purified from the serum using HiTrap NHS-activated HP columns (GE Healthcare Life Sciences, Freiburg, Germany). For whole mounts, brains were dissected in ice-cold honeybee saline (130 mM NaCl, 5 mM KCl, 4 mM MgCl2, 5 mM CaCl2, 15 mM Hepes, 25 mM glucose, 150 mM sucrose, pH 7.2) and fixed in 4% ice-cold formaldehyde (Roth, Karlsruhe, Germany) in phosphate-buffered saline (PBS, pH 7.2) at 4°C for at least 24 hours. The brains were washed 3×10 min in PBS, 1×10 min in PBS containing 2% Triton X-100 (PBS-T) and 2×10 min in 0.2% PBS-T. All washing steps were performed at room temperature on a shaker. Brains were blocked at room temperature for 1 h in 0.2% PBS-T containing 2% normal goat serum (NGS), and incubated with 0.2 units of Alexa Fluor 568 phalloidin (Molecular Probes, A-12380, Eugene, USA) and rabbit-anti-Fruitless antibody (1:800 to 1:2000) in blocking solution for 4 days at 4°C. In case of fru myrGFP male brains, chicken-anti-GFP antibody (1:1000; Rockland Immunochemicals, Inc., Limerick, PA, USA) was added. Next the brains were washed 3×1 h and incubated with goat-anti-rabbit secondary antibody (1:250 to 1:800; Fisher Scientific, Schwerte, Germany) in 0.2% PBS-T with 2% NGS for 2 days at 4°C on a shaker. In case of fru myrGFP male brains, goat-anti-chicken antibody (1:250; Fisher Scientific, Schwerte, Germany) was added. After washing 4×5 min in PBS, the brains were dehydrated in an isopropanol series (10, 30, 50, 70, 90% isopropanol in PBS and 2×100% isopropanol, 5 min each step) and subsequently cleared in methyl salicylate (MS; 99%; Sigma Aldrich, Steinheim, Germany) before mounting in fresh MS and storing at 4°C in the dark until imaging.

For cryosections, the dissected brains were fixed in 4% ice-cold formaldehyde (Roth, Karlsruhe, Germany) in phosphate-buffered saline (PBS, pH 7.2) with 8% sucrose at room temperature (RT) for 90 min. Fixed brains were washed 3×10 min in PBS at RT, followed by 10% (30 min) and 20% (30 min), and 30% sucrose buffered PBS solutions (overnight). Brains were frozen in tissue imbedding media on dry ice. Cryosections (10 μm and 20 μm) were washed 3×10 min at RT in PBS-T followed by overnight anti-Fru antibody staining (1:10000) at 4°C using PBS-T and 0.1 % bovine serum albumin (BSA). After washing 3×10 min u at RT in 0.1% PBS-T, the cryosections were incubated with the secondary antibody (donkey-anti-rabbit Cy3 (1:200) in 0.1% BSA, 0.1% PBS-T for 90 min and replaced 0.1% BSA, 0.1% PBS-T with dye Hoechst 34580 (100 ng/ml) for 30 min at RT. Cryosections were washed 3×10 min and embedded into glycerol-propyl-gallate. For detailed anatomical information, immunostaining of antennal lobes was performed after calcium imaging experiment (Fig. 8a). Dissected brains were immediately immersed in cold 1% zinc formaldehyde in PBS80 and kept overnight at 4°C. Brains were then washed six times in PBS (10 min each), permeabilized using 1% PBS-T for 30 min, and pre-incubated for 3 h in 1% BSA, 0.3% PBS-T. The brains were then incubated in 0.1% BSA 0.3% PBS-T, and mouse monoclonal anti-SYNORF1 (DHSB, #3C11, US) at 1:100 dilution for 7 days. Brains were then washed 6 times in 0.3% PBS-T and secondary anti-mouse antibody (1:200) coupled to Alexa 555 (Thermofisher, #A-21147, France) for 5 days. Brains were then washed in PBS, dehydrated in an ascending ethanol series (30% to 100%), cleared and finally mounted in MS.

Confocal images of whole mounts were captured on a Leica TSC SP8 STED 3X (Leica Microsystems, Wetzlar, Germany). Optical sections for all z-stacks were taken at μm intervals with a resolution of 512×512 pixels. We generated image z-stacks of one hemisphere of midbrains and ganglia. In addition, we generated tile scans of whole brains, which were merged using the processing tool Mosaic Merge of LAS X (Leica Application Suite X 3.0.0, Leica Microsystems CMS, Wetzlar, Germany) with a 20x objective (multi/NA 0.75). Additionally, z-stacks of selected brain regions were imaged using a 40x objective (water/NA 1.10).

Cryosections were imaged with a laser-scanning confocal microscope (Zeiss LSM510, Carl Zeiss Microscopy, Jena, Germany). For antennal lobe anatomical details after imaging, lobes were scanned using a laser-scanning confocal microscope (Zeiss LSM700) with a W Plan-Apochromat 20x/NA 1.0. Images were processed using FIJI (ImageJ 1.53c; Wayne Rasband, National Institutes of Health, USA).

DNA and RNA preparation and PCR procedures
Genomic DNA was isolated using innuPREP DNA Mini Kit (Analytik Jena, Jena, Germany). Induced mutations were characterized by PCR amplification using high fidelity polymerase (Phusion High-Fidelity DNA Polymerase (Thermo Scientific)81. Amplicons of different progeny (haploid males) were cloned and double strand Sanger sequenced (Eurofins, Ebersberg, Germany) using at least three different clones per sample. RNA was isolated using a Trizol-based protocol33. First-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). P1 transcript was amplified using oligonucleotide primers SK60/SK61 (SK60: AACTGATCCTCCTTCCGTGCTGCG, SK61: AGTGGTTCCTGATGTGCGTCACGA) for female P1 transcripts and SK73 & SK61 (SK73: TCGCGATGCTACGTCAACTGTAGG) for male fru P1 transcripts (Eurofins Genomics, Ebersberg, Germany). Semiquantitative RT-PCR amplifications were run under non-saturating conditions and in technical triplicates for each sample using elongation factor 1α (ef1α) gene for semiquantitative adjustment across samples (GATATCGCCCTGTGGAAGTTC (for 632 bp) or GTGATCGGCCACGTTGATTC (for 800 bp) together with GCTGCTGGAGCGAATGTTAC)32. Amplicons were resolved by agarose gel electrophoresis and stained with ethidium bromide. The uncropped gel pictures are presented in the Source Data file or as a Supplementary Fig. The 5’ and the 3’ of the various fru transcripts were identified by RACE experiments using mRNAs from 2 to 4-day old male and female pupal heads31. Other intron/exon structure was determined by mapping the sequences derived from various RT-PCRs to the genomic sequence [National Center for Biotechnology Information (NCBI) Assembly: Amel_HAv3.1 (GCF_003254395.2)]. Sequenced data of the transcripts are available under EMBL-EBI accession number: OZ362630-OZ362641.

For the RNAseq we generated three pools of wt and three pools of fruP1− male antennae (each pool with antennae from 5 individuals). RNA was initially isolated using Trizol, and further purified using RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany)33. Total RNA samples were quantified (Qubit RNA HS Assay), and quality was accessed using the Fragment Analyzer and the “Total RNA Standard Sensitivity Assay” (Agilent Technologies). Library preparation was performed according to the manufacturer’s protocol using the “VAHTS Universal RNA Seq Library Prep Kit for Illumina® V6 with mRNA capture module version 7.0” (Vazyme Biotech Co.). 500 ng total RNA was used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation, and library amplification. Bead-purified libraries were normalized and finally sequenced on the HiSeq 3000/4000 system (Illumina Inc.) with a read setup of SR 1×150 bp. The bcl2fastq2 tool (v2.20.0.422) was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing. 4–12 million single-end reads with a length of 150 bp (EMBL-EBI accession number: PRJEB100586) were mapped to the Apis mellifera transcriptome (NCBI Assembly Amel_HAv3.1_rna.fna) using the kallisto software tool82. Estimated read counts were normalized using the transcripts per kilobase million (TPM) method83. Differences in gene expression were calculated using DESeq284. Genes were differentially expressed (DEGs) if adjusted P-values (Padj) were <0.05 (Wald test) and log2 fold change was greater than 1.5.

Behavioral analysis in colonies and petri dishes
Capped male brood from fruP1−/− and wt queen colonies were placed in separate nucs in an incubator at 34°C along with capped wild-type worker brood frames and a comb containing honey and pollen. fruP1− and wt adult males were color-coded to keep track of the age. The adult workers were removed from the nuclei each morning.

For the within-colony experiments, newly emerged (0–24 h old) fruP1− males, wt males, and worker bees were each tagged with unique 2D barcodes. 20 fruP1−, 20 wt males, 460 worker bees, and a queen were transferred to comb containing the same amount of honey and pollen in same location in each replicate64 (Supplementary Fig. S7). Each of the two pollen store areas contained 15 g of pollen (“Echter Deutscher Spezial Blütenpollen”, Werner-Seip-Biozentrum GmbH & Co. KG, Butzbach, Germany), which we added to the cells together with 25 μl of sugar syrup (“Ambrosia Futtersirup”, Nordzucker AG, Braunschweig, Germany). The two honey store areas consisted of 225 cells, each cell filled with 200 μl of sugar syrup. This small colony was kept overnight at 34°C, and tracking was run lasted for 24 h at RT. We performed two tracking experiments (August 25th and September 8th, 2021) on males that were 2–5 days old. Tags were computer-based tracked using the Bee Behavioral Annotation System (BBAS49). Bee trajectories were not continuous, and gaps resulting from single missing frames, were linearly interpolated64. To measure duration (minutes spent in area/hour) and number of visits (visits/hour) in the different work areas and the walking distance (meters/hour), we used our C++ and Java programs scripts64. We thereby included only hours in which the detection rate was more than 10%. Only bees that had more than 12 of those hours per day were included in the analysis. To follow the behavioral repertoire of male bees in the colony, the tracking data were overlaid on the video using VirtualDub software (VirtualDub-1.9.11, virtualdub.org)64. This allowed us to continuously track individually tagged males throughout the observation period, even when the barcode was not automatically recognized. Behavior of fruP1− and wt males was examined for 15-min periods between noon and 3.30 p.m. The examinations were randomized and blinded in respect to the genotype. Individuals who rested for more than 40% of the time during the observation period (possibly a sleeping period) were not included in the analysis. Male behaviors tend to follow stereotypical patterns and were categorized according to a previous study8. If a male did not move for at least 5 s, this was defined as resting behavior. If a male was disturbed by other bees and moved to a new spot, but rested again within 10 s, we counted this as same resting behavior event.

Self-cleaning behaviors include (i) head cleaning (the male uses its forelegs to brush the head), and (ii) body cleaning (the male uses its hindlegs to brush its abdomen). We also observed that males entered with their heads the cells on the comb. In some cases, this occurred repeatedly in short bouts of cell entering activities. Duration of entering a cell was defined as the time the head was in the cell. The males were actively interacting with other colony members. The male contacted other colony members either using their antennae and/or proboscis. In the case of begging behavior a male contacted a worker’s head with either its antennae or its proboscis. If the male contacted the abdomen or the thorax of either a worker or a male, we defined this as other social contact behavior. During trophallaxis behavior male’s glossa protruded between the worker’s mandibles to obtain the liquid food. Thereby, the male’s forelegs often tapped the worker and the male moved its head forward.

General sensorimotor functions of males were examined in petri dishes as previously reported64. The following changes were applied. A rectangular petri dish (12 cm2) with 5 mm (diameter) ventilation holes was used. Males were introduced into the arena 5 min prior to the start of the test. Males were older than 2 days. Locomotion responses to odorants and worker bees were quantified by calculating the differences in line crossings between control condition (2 min) and the treatment (2 min) for each male. To quantify the odor response, we introduced an odorant into the arena via a filter paper (75 mm, grade 413; VWR, International GmbH, Darmstadt, Germany) through a ventilation hole. For the 9-oxo-2-decenoic acid (9-ODA) response, the locomotion behavior with respect to control filter paper (1 μl isopropanol) and 9-ODA filter paper (1 μl of 9-ODA; 50 μg/μl, dissolved in isopropanol) was examined. Filter papers were only introduced into the arena after the isopropanol had evaporated for 1 minute. The benzaldehyde response has been previously described64. To quantify the male’s locomotion response to social contacts, we introduced into the arena two worker bees to the male and compared line crossings 2 min before and 2 min after the introduction. Petri dishes and papers were changed after each bee was tested. We used a camera (60 fps, Full HD, 44100 Hz; Casio Exilim Pro EX-F1) to count line crossings in the video recordings obtained using VSDC Free Video Editor (Multilab LLC). The proboscis extension response (PER) was examined using honey that was presented to antennae of immobilized males85. Whether a male responded or not was examined every two 2 hours for a maximum of four times. All examinations were randomized and blinded in respect to the genotype.

Anatomical analysis procedures
Images of the gross anatomy were taken using a binocular (SteREO Discovery.V12, Zeiss) with an attached camera (Axiocam 208 color, Zeiss) and 3D software (ZEN blue, Zeiss). Reproductive organs (from 1 to 2 days old males) or midgut and hindgut (from 2-day old males kept on a pollen/honey comb) were dissected and imaged using a binocular (S8 APO, Leica) with an attached camera (UI-1240LE-C-HQ, IDS Imaging Development Systems) and the uEye Cockpit software (IDS). Content of the gut was characterized by dispersing the midgut and hindgut in 80 μl H2O. The number of pollen grains in 40 μl of this suspension was counted under a microscope (Axiovert 25 CFL, Zeiss) using microscope slides.

Cuticular hydrocarbon analysis procedures
Surface lipids were extracted from individual bees by submerging bodies in 0.5 ml of n-hexane for 1 min. Washes were analyzed by GC/MS using a HP 5890 II gas chromatograph coupled to a HP 5972 mass spectrometer (Hewlett Packard, Palo Alto, California, USA), equipped with a DB-5MS column (30 m, 0.25 μm film thickness, 0.25 mm ID), with splitless injection. The GC oven was programmed from 60 to 300°C at 10°C/min followed by 15 min at 300°C. Peaks were called using a standardized integration threshold in ChemStation Software (Agilent Technologies, Santa Clara, California, USA). Integrated ion currents (peak areas) of all linear and methyl-branched hydrocarbons (CHC) were used for downstream analysis.

in vivo calcium imaging procedures
Eclosed mutant males were maintained in hives for a minimum of 8 days and a maximum of 15 days. On the day of the experiment, the males were chilled on ice for 5 min just prior to the experiment until they stopped moving. Then, they were prepared following the standard preparation used to image the ventral part of the honey bee brain86. Glands as well as trachea were removed to expose the brain, and the pool was filled with ringer solution (in mM: NaCl, 130; KCl, 6; MgCl2, 4; CaCl2, 5; sucrose, 160; glucose, 25; Hepes, 10; pH 6.7, 500 mOsmol; all chemicals from Sigma-Aldrich, France), to avoid desiccation of the brain surface. For staining, the saline solution was gently removed, and the brain was bathed with 20 µL of dye solution (10 µg Oregon Green 488 BAPTA-2 AM dissolved with 4 µl Pluronic F-127, 20% in dimethyl sulfoxide, all from Molecular Probes, Invitrogen). The bee was left for 45 min in a humid and dark place, and then the brain was rinsed again thoroughly with saline solution in order to remove extracellular dye.

A T.I.L.L. Photonics imaging system (Martinsried, Germany) was used to perform in vivo optical recordings, as described elsewhere87,88. An epifluorescence microscope (Olympus BX51WI) was used to record activity in the AL using a 10× water-immersion objective (Olympus, UMPlanFL; NA 0.3). Oregon Green was excited using 488 nm monochromatic light (T.I.L.L. Polychrom IV). Fluorescence light was separated by a 505 nm dichroic filter and a long-pass 515 nm emission filter, and recorded with a 640×480 pixels 12-bit monochrome CCD camera (T.I.L.L. Imago) cooled to −12°C with 4×4 binning on chip. Each measurement consisted of 100 frames recorded at a rate of 5 Hz (integration time for each frame ~50 ms). To test the odorant stimuli, a constant airstream was directed from a distance of 1 cm to the male’s antennae, and stimuli were given at the 15th frame for 1 second (5 frames correspond to 1 second). For each odorant stimulus (all obtained from Sigma-Aldrich, France), 5 µL of the solution was deposited on a filter paper inserted in a Pasteur pipette. We tested a small set of 3 volatile odorant stimuli known from previous work to trigger strong neural activity in workers: 1-hexanol (1-6ol), isopentyl acetate (IPA), and benzaldehyde. We also recorded responses to 9-ODA at a concentration of 50 µg/µL. As control stimulus, a pipette containing the solvent, isopropanol (iso), or a clean piece of filter paper was used. Each odorant stimulus was presented twice in a pseudo-randomized order. Imaging data were processed and analysed using custom-made software written in IDL 6.0 (Research Systems, Boulder, CO). First, the relative fluorescence changes were calculated as ΔF/F = (F–F0)/F0 by taking as reference background F0 the average of three frames just before the odorant stimulation (frames 9–11). Possible irregularities of lamp illumination and bleaching were corrected by subtracting the median pixel value of each frame from every single pixel of the corresponding frame. Finally, the two spatial dimensions were filtered with a Gaussian filter of window size 7×7 pixels for noise reduction. A biphasic calcium signal was observed in all recordings. For the quantification of response intensity, a mask was precisely drawn in order to exclude regions outside of the imaged structure, and the response intensity was calculated by averaging the intensity of all pixels located within the unmasked area and the two presentations of each odorant. The intensity of the odor-induced response was obtained by averaging three consecutive frames at the end of the odor presentation (frames 19–21) and subtracting the average of 3 frames during the negative component of the signal (frames 49–51). All results are displayed as means over individuals ± SEM.

Statistical analyses procedures
Mann–Whitney U test, one-sample Wilcoxon and log-rank tests were performed using the SPSS 29 and Systat 13 software. Testing was performed two-sided. The compositional similarity of CHC profiles of individual bees was visualized by nonmetric multidimensional scaling (n-MDS) and analyzed using one-way analysis of similarities (ANOSIM, with pairwise tests) using Primer v.6 software89. ANOSIM is a non-parametric permutation statistic that tests whether a factor affects the rank order of pairwise similarities in a similarity matrix (here Bray–Curtis similarities of CHC composition between individual bees). The resulting R value (−1<R<1) indicates the degree of separation between treatments (complete separation: R=1, no separation: R=0). To identify compounds that contributed most strongly to overall dissimilarity between treatment groups, we used the SIMPER module, also in Primer v.6. n-MDS. ANOSIM and PRIMER analyses were based on standardized (percentage), square root transformed GC/MS integrated ion currents (peak areas). For calcium imaging recordings, odor response intensities were compared with ANOVA for repeated measurements, using odorants as within-group factors. A Dunnett posthoc test was applied to compare the intensity of the response to each stimulus with a common reference, the air control. All tests were performed with GraphPad Prism V7.00 and R (version 4.4.0, www.r-project.org).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Köhnen et al presents molecular, morphological, and behavioural evidence for the involvement of the fru gene in shaping the feeding behaviour of male honey bees. This study makes a valuable contribution to our understanding of the molecular underpinnings of social behaviour. The ms is well written, clearly presenting the results of elegant experiments and concluding with balanced, insightful discussion. I have only a few minor suggestions, as outlined below:\nIntroduction\nLines 71-72: This paragraph should not begin with “however,” because this word typically contrasts with a statement just made, and here the contrast is with a point from the previous paragraph (again in line 102). More importantly, the ms emphasizes the lack of information regarding the molecular mechanisms controlling cooperative behaviours in honey bee males. The behaviours described so far primarily focus on the male-worker interaction where males receive food -a one-way interaction that provides an immediate benefit only to the males. Please also highlight the reciprocal aspect by stressing the indirect benefits that workers (and the colony as a whole) gain from trophallaxis, thereby presenting a clearer view of the studied “cooperative” behaviour.\nLine 77: The phrase “Compared with worker bees” is unclear. Could the authors clarify whether males possess the pathway while workers do not? Please provide more details.\nResults\nLine 115 (and figure 1): Are P1-P3 designated as promoters or as transcriptional start sites? Please clarify their functional roles based on the specific nucleotide sequences or motifs used to define or identify these elements. Additionally, Figure 1c is lacking the female protein representation, which is expected to be strikingly different to that of males. In lines 122-123 include the information on the expected peptide size of the female counterpart.\nFigure 1e: Are the levels of ef1α in wt males exactly the same to those in females?\nLines 431-434: I recommend softening this statement, as the type and extent of the morphological analyses conducted do not provide sufficient support for such a strong conclusion.\nDiscussion\nLines 532-536: This idea has been reiterated throughout the manuscript -initially in the Introduction, followed by the Results, and now in the Discussion. I recommend elaborating on it more thoroughly in the Introduction, particularly emphasizing its benefits for the colony as a whole, as previously suggested.\nLines 616-619: Has the fru gene been specifically identified as associated with social evolution in any of the 4 papers cited by the authors? If so, do these studies propose any common or divergent mechanisms of action for the gene?\nOverall, the ms would benefit from punctuation and spelling editing (e.g. lines 87, 327).", "reviewer_2": "The manuscript titled “The fru gene specifies male cooperative behaviors in honeybee colonies” presents compelling and novel findings that significantly advance our understanding of the genetic control underlying social behaviors in honeybee colonies. This research provides insightful data that builds upon existing knowledge of social behavior in insects, with a particular focus on the role of genetics in shaping these behaviors. By exploring the fruitless (fru) ortholog in Apis mellifera, the authors uncover essential facets of male development and interaction within the hive.\nTheir investigation reveals that the fru gene expresses male-specific products during the late developmental stages of the nervous system, highlighting a critical window in which genetic influences can shape behavioral outcomes. This expression is particularly noteworthy because it suggests that the fru gene is active at key points in male maturation, potentially influencing the development of behaviors necessary for reproductive success and social integration within the colony. These findings align with observations in Drosophila and other dipterans, where the fru gene has also been implicated in regulating mating behaviors, demonstrating a conserved mechanism across different species.\nHowever, the manuscript goes beyond mere correlation and establishes a direct link between the male-specific fru products and various behavioral paradigms within honeybee colonies. Notably, these male-enriched fru products are shown to be essential not only for modulating male courting behaviors but also for regulating social feeding behaviors that are vital for the colony's overall function. This dual role of the fru gene suggests a complex interplay between mating and cooperative social interactions, providing a deeper understanding of how evolutionary pressures may have shaped these behaviors to promote colony survival.\nThe research illustrates how the expression of male-specific fru products is crucial for drones to exhibit typical behaviors such as normal rates of begging for food, the duration of trophallaxis—an important process in which food is exchanged among colony members—and the dynamics of interactions with worker bees. These interactions are critical, as they help to maintain the nutritional health of the colony and ensure a cohesive social structure. The results underscore the fundamental importance of these behaviors in the daily functioning of the hive, emphasizing the role of males beyond reproduction.\nA particularly striking aspect of the study is the documentation of the consequences of manipulating fru gene expression. The authors provide compelling evidence that drones with a specific disruption in the expression of the fruM isoform exhibit a dramatic two- to threefold decrease in the execution of essential social behaviors, such as begging and trophallaxis. This finding provides robust support for the hypothesis that the fru gene is a key regulator of male-specific behaviors that facilitate not only individual reproductive opportunities but also the overall health and efficiency of the colony.\nIn summary, this manuscript significantly enriches our understanding of the genetic underpinnings of social behavior in honeybees, illuminating the intricate connections between genetics, behavior, and social structure within colonies. The implications of this research extend beyond honeybees, potentially informing studies on behavioral genetics in other social insects and adding depth to our comprehension of the evolutionary biology of complex social systems. By elucidating the multifaceted role of the fru gene in male-specific behaviors, this work sets the stage for future investigations into the genetic and molecular mechanisms that orchestrate social interactions in eusocial species.\nTo further improve the manuscript, we propose minor revisions and request the authors to address the following points:\nPrevious studies in Drosophila have demonstrated that male expression of the fruitless gene is not only necessary for male courtship behaviors but is also sufficient to induce male-like behaviors in otherwise normal females (Demir & Dickson, 2005, Cell). This significant finding led to the conclusion that the fru gene plays a central role in specifying various aspects of complex innate behaviors. Given this context, we are interested in understanding why the authors did not incorporate gain-of-function experiments—specifically, the expression of fruM in the brains of female workers through methods such as deleting the female-specific exon—to rigorously test whether fruM is sufficient to activate male-like cooperative behaviors in otherwise normal female workers.\nThis omission is particularly intriguing as it presents an opportunity to explore the sufficiency of fruM in reshaping behavioral phenotypes traditionally associated with male Drosophila. By examining the potential to induce male-like behaviors in females, the results of such experiments could significantly enhance our understanding of how fruM integrates with existing neural circuits involved in cooperative behavior. Furthermore, additional insights could clarify the genetic and neurobiological underpinnings of sex-specific behaviors, potentially revealing broader implications for understanding the evolution of behavioral traits across species.\nGeneral Comments and Suggestions for Revision:\n1. Title Revision: We propose changing the title from “The fru gene specifies male cooperative behaviors in honeybee colonies” to “The fru gene contributes to male cooperative behaviors in honeybee colonies” to better reflect the data, which demonstrate necessity but not sufficiency.\n2. Missing Gain-of-Function Evidence: The authors provide strong loss-of-function data using CRISPR/Cas9. However, gain-of-function experiments (e.g., ectopic expression of fruM in females by deleting the female-specific exon) would be crucial to test sufficiency. We request that the authors comment on why this was not attempted.\n3. Reference to Previous Work:\n• The authors should cite and compare their findings on fru gene structure and splicing isoforms in Apis mellifera with Nasonia vitripennis (Bertossa et al., 2009, Mol Biol Evol. PMID: 19349644).\n• Additionally, they should reference Siwicki & Kravitz (2009, Curr Opin Neurobiol) for background on fru and dsx in Drosophila behavior.\n• Consider also Gailey et al. (2006, PNAS, PMID: 16319090) regarding the evolutionary conservation of fruitless function.\n4. Clarification on Expression and Antibody Specificity: The manuscript notes that the antibody detects both male-specific FruM and a common isoform. The authors should clarify if the common isoform is undetectable or weakly expressed in both sexes, and whether P2/P3 promoters are active in the brain.\n5. Discussion of Additional Genetic Contributors: While the paper attributes behavioral phenotypes to fruM loss, the observed partial phenotypes suggest other genes likely act in parallel. The authors should discuss candidate genes or regulatory pathways that might also contribute.\n6. Potential Role of dsx: Drosophila studies show that dsxM and fruM act in concert to establish male courtship behavior. The authors should comment on why they did not knock down dsxM alone or in combination with fruM.\n7. Clarification of RNAi and Developmental Timing:\n• The authors should explain why fruM mRNA was detected in fem RNAi-treated female larvae alongside substantial female-specific fru transcripts.\n• They should also provide the developmental timeline (egg to L4 larva), duration of RNAi effect, and siRNA sequences used.\n8. RT-PCR Band Size Discrepancy: In Fig. 1 panels e and f, fruM and ef1a bands differ in size. The authors should clarify this, providing primer sequences and amplicon locations.\n9. Clarify Methodological Details:\n• sgRNA design and efficiency for CRISPR deletions should be reported.\n• Clarify ambiguous phrases such as “Numbers: larval and pupal stages,” and rephrase statements like “it is unclear where FruM is expressed” (when prior data showed expression).\n10. Gene-Centric Language: Several statements over-attribute behavioral control directly to a single gene. For example:\n• “FruM specifies decision processes…” should be changed to “FruM facilitates decision processes…”\n• “…programs decision processes…” should be softened to “contributes to the neural basis of…”\n• These revisions would avoid an overly reductionist interpretation and better reflect the multifactorial nature of behavior.\n11. Abstract and Introduction Revisions:\n• Line 35: Replace “whether” with “how” — “but how cooperative behaviors require…”\n• Lines 39–40: The claim about screening transcription factors lacks supporting data. Either delete this sentence or clarify that fru was identified based on prior evidence or candidate selection.\n• Clarify inconsistencies between the stated screen (lines 102–105) and the cited reference (Vleurinck et al., 2016, Ref. 33), which does not report male-specific splicing of fru.\n12. Male Behavior Description: Please provide a clear list of innate and learned male cooperative behaviors in Apis, such as:\n• Drone congregation behavior\n• Aggregation pheromone production\n• Trophallaxis\n• Heat production and clustering\n• Courtship flight patterns\n13. Specific Phrase Suggestions:\n• Line 530: “FruM facilitates decision processes…”\n• Line 540: “FruM is required for the emergence of aspects of innate behavior…”\n• Line 548: Replace “specifies” with “contributes to”\n• Line 620: “We identified a gene that contributes to decision processes…”\nIn summary, we support the publication of this important work with minor revisions, pending clarification of the above points and the inclusion of appropriate references and discussion. The integration of neuroanatomy, genetics, and behavior is commendable, and with additional detail and balance, this manuscript will be a significant contribution to the field.", "reviewer_3": "This study contains several important findings that will advance our understanding on the neural basis of cooperative social behavior. Most importantly, results show that the male-specific isoform of the honeybee fruitless gene (FruM) is involved in the regulation of male behaviors through its neural functions. Although the function of FruM on male-specific behaviors has been well characterized in the common fruit fly Drosophila melanogaster and its sister species, whether fruitless is important for sex-specific behaviors outside dipterans has been ambiguous. Consequently, the genetic origins of sexually dimorphic behaviors in insects have been unclear.\nThis manuscript is significant beyond honeybee or even insects, because the data suggest that a dedicated transcriptional regulation is a generalized mechanism that specifies sexual dimorphism of neural circuits and behaviors across phyla. In mammals, neurons that express sex hormone receptors (such as estrogen receptor, progesterone receptor, etc) play critical roles in regulating various aspects of reproductive behaviors. Evidence presented in this study generalizes this rule to Hymenopterans, which will draw attentions of wide range of neuroscientists and evolutionary biologists.\nTheir conclusions are based on high-quality data and careful observations. Successful creation of genome-edited honeybees is a technical accomplishment on its own. The genetic mutation of fru provide convincing evidence that FruM is required for appropriate expression of male honeybee behaviors. The nervous system-specific yet restricted expression of FruM suggests that its main function is the regulation of neural processes. Behaviors of male mutants show intriguing differences from wild type males in social feeding and positions in the nest. Lastly, the functional imaging experiments show that FruM influences male behavior not by altering the sensory systems, but likely through central processing of sensory information. Overall, their multidisciplinary characterization of fru honeybee mutants has established a new entry point for investigating the neural basis of sexually dimorphic behavior in social insects.\nAlthough I believe this manuscript should be published promptly, I would like to make a few editorial suggestions.\nFirst, quantitative analysis on the anatomical data can strengthen their conclusions. Movies and images of the fruP1myrGFP allele (Fig. 3 and 4, Movie 4-7) are annotated only qualitatively in the text. Some of their claims are not immediately obvious to untrained eyes as green fluorescence seems to be faintly present across the brain. For instance, the fluorescence in the male antennal lobe (including in the macroglomeruli) is unclear, and it is even harder to recognize any difference between the worker bee antennal lobe. Likewise, the stated male-specific labeling of a tract in the lobula and lateral protocerebrum is not obvious because it is unclear where the corresponding tract in female brain is.\nMy suggestions are as follows:\n1) show the GFP staining of a wild type bee brain as a negative control. Faint but broad signals of anti-GFP could be due to the background staining. A negative control specimen will help clarify where the allele-specific signals exist.\n2) Quantify the fluorescence intensity of targeted neuropils where sexually dimorphic labeling were observed, such as ventromedial antennal lobe or the lobula/lateral protocerebrum. Phalloidin staining could serve as a normalizing signal.\n3) Annotate structures of interest in the movies, so that readers know where to pay attention to. For example, Movie 7 supposedly shows fru-expressing antennal nerves only in workers (lines 279-280), but it is impossible to tell where the nerve is. This statement is also confusing since the text says that both males and workers have fru-expressing antennal fibers (lines 275-277).\nSecond, I would like to see the signals of anti-FruM and fruP1myrGFP in the posterior end of the brain. Fig. 3d is the only image of the posterior side, and movies appear to stop at around the middle of the brain. Studies in Drosophila have shown that posterior medial part of the brain contains cell types that control courtship behavior. It is interesting to address if a cluster of FruM neurons is present in the male honeybee brain or not.\nThird, the discussion on the FruM circuitry (line 585-609) can include a brief comparison between the Drosophila fru circuit. In Drosophila, sexually dimorphic neurons are mostly in the central interneurons but some sensory neurons do express FruM. This seems to be a notable difference in the honeybees (line 279), leaving a question over the genetic mechanism of antennae sexual dimorphism (ref. 14). Classic works on the Drosophila fru circuit (Yu et al., Curr. Biol. 20:1602-1614 (2010); Cachero et al., Curr. Biol. 20:1589-1601 (2010)) can provide discussion points.\nFourth, the relevance of the discussion over the decision processes (lines 555-583) is unclear. The authors' conclusion that \"FruM specifies decision process in the nervous system\" (line 548) is based on behavioral phenotypes on the cooperative feeding behaviors (Fig. 5). While other innate behaviors and global response to odors (Fig. 8) are largely unchanged, relatively limited types of odors tested and the lack of glomerular resolution of the imaging experiment leave it possible that response to uncharacterized, behaviorally relevant olfactory cues are indeed affected in fru mutants. If this is the case, the effects on cooperative feeding might be caused simply by the loss of relevant sensory inputs, rather than “decision process” as discussed in this subsection. This discussion could be saved for future studies in which the relationship between fru and action choice during social interaction in honeybees is better characterized.\nHowever, the overall scientific merit of the manuscript remains very high. I strongly recommend that this work becomes broadly available as soon as points raised above are addressed.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "The responses to the REVIEWERS’ COMMENTS\nWe thank the reviewers for their time and efforts and their helpful comments. They further\nimproved the ms!\nReviewer #1 (Remarks to the Author):\nThe manuscript by Köhnen et al presents molecular, morphological, and behavioural\nevidence for the involvement of the fru gene in shaping the feeding behaviour of male honey\nbees. This study makes a valuable contribution to our understanding of the molecular\nunderpinnings of social behaviour. The ms is well written, clearly presenting the results of\nelegant experiments and concluding with balanced, insightful discussion. I have only a few\nminor suggestions, as outlined below:\nIntroduction\nLines 71-72: This paragraph should not begin with “however,” because this word typically\ncontrasts with a statement just made, and here the contrast is with a point from the previous\nparagraph (again in line 102).\nResponse: We deleted “however” in line 71 and replaced “however” with “thus far” in line 102\n(now line 104).\nMore importantly, the ms emphasizes the lack of information regarding the molecular\nmechanisms controlling cooperative behaviours in honey bee males. The behaviours\ndescribed so far primarily focus on the male-worker interaction where males receive food -a\none-way interaction that provides an immediate benefit only to the males. Please also\nhighlight the reciprocal aspect by stressing the indirect benefits that workers (and the colony\nas a whole) gain from trophallaxis, thereby presenting a clearer view of the studied\n“cooperative” behaviour.\nResponse: Yes, we agree. We now inserted a sentence stating the importance of trophallaxis\nbehaviors for the colony. Line 66: “The trophallaxis behavior plays a central role in the food\nexchange among colony members, the nutrition of larvae, adult males and queens and the\ncommunication about the food status of the colony 1. “\nLine 77: The phrase “Compared with worker bees” is unclear. Could the authors clarify\nwhether males possess the pathway while workers do not? Please provide more details.\nResponse: we rephrased the sentence to clarify the content, now line 75: “Male identity that\ndiffer from worker bees and queens manifests in male-specific odorant receptor gene\nexpression, male-specific number and anatomy of glomeruli in the primary olfactory\nprocessing center (the antennal lobe (AL)), a male-specific smaller mushroom body (MB)\nharboring 12% fewer neurons and in male-specific larger optical lobes 2-7.”\nResults\nLine 115 (and figure 1): Are P1-P3 designated as promoters or as transcriptional start sites?\nPlease clarify their functional roles based on the specific nucleotide sequences or motifs\nused to define or identify these elements.\ntranscript nucleotide sequence analyses revealed that the fru gene in honeybee is\ntranscribed from at least three promoters based on three different transcription start sites“.\nWe can only state their existence and that the P1 derived transcript is nervous system\nspecific expressed that produce a sex-specific splice product. This P1 derived transcript is\ninvolved in the phenotypes which we described in this study.\nAdditionally, Figure 1c is lacking the female protein representation, which is expected to be\nstrikingly different to that of males. In lines 122-123 include the information on the expected\npeptide size of the female counterpart.\nResponse: we are not so sure if this protein is indeed produced. Cellular mechanisms are\noperating that will inhibit that such small protein is produced from a long transcript with an\nearly translation stop codon (for example the nonsense-mediated mRNA decay mechanism)\nTo further clarify this issue, we now provided additional information in Fig 1 c and added the\nexpected sizes in line 126 (“ 34-amino-acid-long protein…” ). We now also schematically\npresent in Figure 1 C this small sized peptide. We state in the figure legend line 175: “Note,\nthat if a female protein is produced it will only have 34 amino acids.”\nFigure 1e: Are the levels of ef1α in wt males exactly the same to those in females?\nResponse: Oh, sorry we used different primer pairs (over the years) to amplify and the ef1α\nfor the males in Figure 1e was somehow not included. We now added the wt male data of\nef1α in Figure 1e and stated in the figure legend that different primer pairs were used.\nWe inserted in line 182. (figure legend): “Please note that the ef1α RT-PCRs in (e) versus (f,\ng) generated different amplicon sizes because different oligonucleotide primer pairs were\nused.”\nLines 431-434: I recommend softening this statement, as the type and extent of the\nmorphological analyses conducted do not provide sufficient support for such a strong\nconclusion.\nResponse: we softened the statement from\n“Malfunction and the malformation of external and internal structures also cannot explain the\nobserved behavioral dysfunction because the external head, antennae, abdomen and leg\nmorphology as well as the reproductive organ anatomy of fruP1- males had a wt phenotype\n(Fig. 7f). “\nInto (now line 444):\n“These behavioral dysfunctions also cannot be explained by a malfunction and malformation\nof external and internal structures at the gross level because the external head, antennae,\nabdomen and leg morphology as well as the reproductive organ anatomy of fruP1- males had\na wt phenotype (Fig. 7f)”\nLines 532-536: This idea has been reiterated throughout the manuscript -initially in the\nIntroduction, followed by the Results, and now in the Discussion. I recommend elaborating\non it more thoroughly in the Introduction, particularly emphasizing its benefits for the colony\nas a whole, as previously suggested.\nResponse: beside the changes in the introduction (see above) we now provided more\narguments concerning the benefits for the colony in the discussion.\nWe inserted (now line 546): “These trophallactic interactions among colony members thereby\ncommunicate the food status of the colony 1. From these cooperative behavioral interactions,\nthe colony benefits as a whole thereby increasing its reproductive success.\nLines 616-619: Has the fru gene been specifically identified as associated with social\nevolution in any of the 4 papers cited by the authors? If so, do these studies propose any\ncommon or divergent mechanisms of action for the gene?\nResponse: We were not able to identify the fru gene in these data sets. We better\nemphasized the focus of this study as compared to the excellent previous studies.\nLine 632:\n“In this study, we identified a single gene by functional perturbation that programs initiation\nand sustainment processes of within-colony cooperative behaviors.”\nWe provided further information in the discussion about how fru function possibly evolved at\nthe molecular level.\nLine 652:\n“Rather the comparisons suggest that the connectivity and functional properties of FruM+\ncircuitry diverged in the species that possibly involve the evolution of other downstream\ncontrols of Fru protein target genes. “\nOverall, the ms would benefit from punctuation and spelling editing (e.g. lines 87, 327).\nResponse: We carefully edited the entire ms.\nReviewer #2 (Remarks to the Author):\nThe manuscript titled “The fru gene specifies male cooperative behaviors in honeybee\ncolonies” presents compelling and novel findings that significantly advance our\nunderstanding of the genetic control underlying social behaviors in honeybee colonies. This\nresearch provides insightful data that builds upon existing knowledge of social behavior in\ninsects, with a particular focus on the role of genetics in shaping these behaviors. By\n3\nexploring the fruitless (fru) ortholog in Apis mellifera, the authors uncover essential facets of\nmale development and interaction within the hive.\nTheir investigation reveals that the fru gene expresses male-specific products during the late\ndevelopmental stages of the nervous system, highlighting a critical window in which genetic\ninfluences can shape behavioral outcomes. This expression is particularly noteworthy\nbecause it suggests that the fru gene is active at key points in male maturation, potentially\ninfluencing the development of behaviors necessary for reproductive success and social\nintegration within the colony. These findings align with observations in Drosophila and other\ndipterans, where the fru gene has also been implicated in regulating mating behaviors,\ndemonstrating a conserved mechanism across different species.\nHowever, the manuscript goes beyond mere correlation and establishes a direct link between\nthe male-specific fru products and various behavioral paradigms within honeybee colonies.\nNotably, these male-enriched fru products are shown to be essential not only for modulating\nmale courting behaviors but also for regulating social feeding behaviors that are vital for the\ncolony's overall function. This dual role of the fru gene suggests a complex interplay between\nmating and cooperative social interactions, providing a deeper understanding of how\nevolutionary pressures may have shaped these behaviors to promote colony survival.\nThe research illustrates how the expression of male-specific fru products is crucial for drones\nto exhibit typical behaviors such as normal rates of begging for food, the duration of\ntrophallaxis—an important process in which food is exchanged among colony members—\nand the dynamics of interactions with worker bees. These interactions are critical, as they\nhelp to maintain the nutritional health of the colony and ensure a cohesive social structure.\nThe results underscore the fundamental importance of these behaviors in the daily\nfunctioning of the hive, emphasizing the role of males beyond reproduction.\nA particularly striking aspect of the study is the documentation of the consequences of\nmanipulating fru gene expression. The authors provide compelling evidence that drones with\na specific disruption in the expression of the fruM isoform exhibit a dramatic two- to threefold\ndecrease in the execution of essential social behaviors, such as begging and trophallaxis.\nThis finding provides robust support for the hypothesis that the fru gene is a key regulator of\nmale-specific behaviors that facilitate not only individual reproductive opportunities but also\nthe overall health and efficiency of the colony.\nIn summary, this manuscript significantly enriches our understanding of the genetic\nunderpinnings of social behavior in honeybees, illuminating the intricate connections\nbetween genetics, behavior, and social structure within colonies. The implications of this\nresearch extend beyond honeybees, potentially informing studies on behavioral genetics in\nother social insects and adding depth to our comprehension of the evolutionary biology of\ncomplex social systems. By elucidating the multifaceted role of the fru gene in male-specific\nbehaviors, this work sets the stage for future investigations into the genetic and molecular\nmechanisms that orchestrate social interactions in eusocial species.\nTo further improve the manuscript, we propose minor revisions and request the authors to\naddress the following points:\nPrevious studies in Drosophila have demonstrated that male expression of the fruitless gene\nis not only necessary for male courtship behaviors but is also sufficient to induce male-like\nbehaviors in otherwise normal females (Demir & Dickson, 2005, Cell). This significant finding\nled to the conclusion that the fru gene plays a central role in specifying various aspects of\ncomplex innate behaviors. Given this context, we are interested in understanding why the\nauthors did not incorporate gain-of-function experiments—specifically, the expression of fruM\nin the brains of female workers through methods such as deleting the female-specific exon—\nto rigorously test whether fruM is sufficient to activate male-like cooperative behaviors in\notherwise normal female workers.\nResponse: Such an experiment is not the focus of this study, which is clearly stated in the\nintroduction (see below). We wanted to identity genes that are involved and required for the\nsocial within colony behaviors. We do not state anything about a master regulator function.\nThe experiment conducted by Demir and Dickson had the goal to answer the question if thr\n4\nfru gene is a master regulator of behavioral specification, which requires an essential and\nsufficient analysis. Such master regulators have been identified in developmental programs.\nThey were key to understand the organization of the body plan formation. And it was an open\nquestion whether such master regulator also exists for behavioral specifications.\nTaken from the introduction defining the goal of this study.\n“Thus far, we do not know which genes took on the roles in hardwiring the male’s\ncooperative behaviors in the nervous system. We thus screened for male-specific spliced\ntranscripts in the nervous system 8 and found that, in addition to previously known glu and\ndsx, only the fruitless (fru) TF gene has male-specifically spliced transcripts. Because this\nFru BTB zinc finger transcription factor is expressed only in the male nervous system and its\nhomolog in Drosophila melanogaster is involved in hardwiring sexual behaviors 9, 10, we set\nout to examine the function of this gene product in manifesting the male’s cooperative\nbehaviors in honeybee colonies.”\nThis omission is particularly intriguing as it presents an opportunity to explore the sufficiency\nof fruM in reshaping behavioral phenotypes traditionally associated with male Drosophila. By\nexamining the potential to induce male-like behaviors in females, the results of such\nexperiments could significantly enhance our understanding of how fruM integrates with\nexisting neural circuits involved in cooperative behavior. Furthermore, additional insights\ncould clarify the genetic and neurobiological underpinnings of sex-specific behaviors,\npotentially revealing broader implications for understanding the evolution of behavioral traits\nacross species.\nResponse: The goal of this study is to identify a transcription factor which is essential for the\nspecification and expression of the male specific behavior in the colony. Hence, this what we\npresented in this manuscript. No doubt this is an interesting question which deserve an extra\nstudy as the one from Demir and Dickson.\nGeneral Comments and Suggestions for Revision:\n1. Title Revision: We propose changing the title from “The fru gene specifies male\ncooperative behaviors in honeybee colonies” to “The fru gene contributes to male\ncooperative behaviors in honeybee colonies” to better reflect the data, which demonstrate\nnecessity but not sufficiency.\nResponse: Thank you for your efforts to improve the manuscript. However, your title\nsuggestion does not cover what we want to present as the focus of this study. Our study\nrevealed that the fru gene is required or essential for the specification of some aspects of\nsocial within colony behaviors. The title is a short summary of that. The word specification is\nactually used to reflect this partial effect. Not all behavior is “controlled” by this gene. And\nspecification is used to reflect that the gene specifies during development the capacity of the\nadult male to express behavior. Contribution does not cover the latter mechanism. Similarly,\nimagine a development study in which someone would state in the title “The X gene\ncontribute to antennae”. The process is missing.\n2. Missing Gain-of-Function Evidence: The authors provide strong loss-of-function data using\nCRISPR/Cas9. However, gain-of-function experiments (e.g., ectopic expression of fruM in\nfemales by deleting the female-specific exon) would be crucial to test sufficiency. We request\nthat the authors comment on why this was not attempted.\nare interested to find transcription factors that are essential for the specification of the within\ncolony behaviors.\n3. Reference to Previous Work:\n• The authors should cite and compare their findings on fru gene structure and splicing\nisoforms in Apis mellifera with Nasonia vitripennis (Bertossa et al., 2009, Mol Biol Evol.\nPMID: 19349644).\n• Additionally, they should reference Siwicki & Kravitz (2009, Curr Opin Neurobiol) for\nbackground on fru and dsx in Drosophila behavior.\n• Consider also Gailey et al. (2006, PNAS, PMID: 16319090) regarding the evolutionary\nconservation of fruitless function.\nResponse: thank you for letting us know about other publications which we may have\nmissed. After re-evaluating those and comparing those with the ones we have cited and most\nimportantly, the focus of our study (please take into account that we have a restriction of 70\ncitations) we finally came to the conclusion that we prefer the publications cited. Here the\nreasons. Our evolutionary analysis did not cover “gene structure” as a phenotype. As you\nknow, such feature can evolve fast. Instead we included those studies which showed\nevidence that fru gene has a conserved function in specifying sex-specific behaviors, which\nis the very focus of this study. We cited here the relevant publications. We also examine\ncooperative behavior and not general social behavior. Interesting, some authors refer to\nsexual behavior as social behavior. The latter is an interesting topic on its own.\nTo further improve the ms we better specified what we evolutionary compared here. We\ninserted (underlined) line 635 “The fru gene also plays a role in hardwiring mating behaviors\nin honeybees as in other insects. They all use the same mechanism of sex-specific fruP1\ntranscript splice regulation which suggests a deep conservation of fru’s role in specifying\nsexual behaviors 11-14. “\n4. Clarification on Expression and Antibody Specificity: The manuscript notes that the\nantibody detects both male-specific FruM and a common isoform. The authors should clarify\nif the common isoform is undetectable or weakly expressed in both sexes, and whether\nP2/P3 promoters are active in the brain.\nResponse: Our focus is to examine the male specific program and thus we are interested in\nthe male specific expressed Fru protein. Our immunostainings used our developed Anti-Fru\nantibody. They showed a male-specific labeling pattern suggesting that these labels derived\nfrom P1 transcript. We functionally tested that by deleting P1 promoter region. Indeed, this\nAnti-Fru labels were lost in the knockouts. Hence, we conclude that these male-specific\nspecific labels are derived from P1 transcript expression. This also suggests specificity of our\nAB. Whether there is a weak P2/P3 derived expression in both sexes that are near the\ndetection level using our Anti-Fru antibody is hard to tell. Anyway, the common fru proteins\nwhich are common to both sexes cannot specify a male-specific behavior. And the male-\nspecific behavior is the focus of this study.\nWe improved the ms in that respect. We added in line 144: “…suggesting that these male-\nspecific labels derived from P1 and not P2 or P2 transcripts.“\nLine 152: “These results suggest that the male-specific FruM protein is expressed from the\nmale fruM transcripts, that the anti-Fru antibody labels Fru protein expression and that the\nfruP1- mutant is a loss-of-function mutation of fruM.”\n6\n5. Discussion of Additional Genetic Contributors: While the paper attributes behavioral\nphenotypes to fruM loss, the observed partial phenotypes suggest other genes likely act in\nparallel. The authors should discuss candidate genes or regulatory pathways that might also\ncontribute.\nResponse: We think that this is too speculative to highlight possible other factors given\n12,000 genes of the honeybee genome and to discuss whether they act in parallel.\n6. Potential Role of dsx: Drosophila studies show that dsxM and fruM act in concert to\nestablish male courtship behavior. The authors should comment on why they did not knock\ndown dsxM alone or in combination with fruM.\nResponse: These are very interesting experiments which we would love to do in the future.\nAgain, our study has a focus, which we clearly stated in the introduction, which is the\nfollowing:\n“Because this Fru BTB zinc finger transcription factor is expressed only in the male nervous\nsystem and its homolog in Drosophila melanogaster is involved in hardwiring sexual\nbehaviors 9, 10, we set out to examine the function of this gene product in manifesting the\nmale’s cooperative behaviors in honeybee colonies.”\n7. Clarification of RNAi and Developmental Timing:\n• The authors should explain why fruM mRNA was detected in fem RNAi-treated female\nlarvae alongside substantial female-specific fru transcripts.\nResponse: We improved this section and added further information. We showed by\nperforming this experiment that the P1 fru gene is regulated by the sex determination\ncascade. The effect is partial as suggested because knockdown is only temporally induced\n(see 15, 16).\n• They should also provide the developmental timeline (egg to L4 larva), duration of RNAi\neffect, and siRNA sequences used.\nResponse: The details of the experimental procedure on fem knockdown has been described\nin previous studies, which can be found here 15, 16. The Gempe et al paper 2009 showed that\nan early downregulation of the transcript via RNAi in the embryo resume at larval stage 5\n(see Figure 4 in Gempe et al.). At this stage we see resumed transcripts in fem RNAi\nfemales (maintained via a positive feedback loop as also shown in the Gempe et al study)\nthat induce only a partial switch at the level of the fru gene (this study).\nThe changes we have made to improve this section: We added in line 129 the following\ninformation (underlined):\n“The fem RNAi-treated females produced male-specific fruP1 transcripts but also female-\nspecific transcripts at larval stage 4 (Fig. 1e; Supplementary Table 2) suggesting a partial\nshift toward male splicing in response to temporally restricted fem knockdown which we\ninduced in the embryo 15”.\nWe added in the material section (underlined).\n” Female eggs were injected with fem siRNAs and larvae were reared to 4th instar (L4)\ninducing a temporally restricted knockdown as described 15.”\n7\n8. RT-PCR Band Size Discrepancy: In Fig. 1 panels e and f, fruM and ef1a bands differ in\nsize. The authors should clarify this, providing primer sequences and amplicon locations.\nResponse: Sorry for this confusion. We used different primer pairs for the amplification in\nfigure 1 e versus f/g. This has historical reasons. We changed the primer pair over the\nyears to smaller amplicons for various reasons. We now added the primer pairs and stated\nthat these are different primer pairs. The different amplicons do not compromise the\nconclusion.\nWe improve the manuscript. We added a sentence in Figure 1 legend line 182:\n“Please note that the ef1α RT-PCRs in (e) versus (f, g) generated different amplicon sizes\nbecause different oligonucleotide primer pairs were used.”\n9. Clarify Methodological Details:\n• sgRNA design and efficiency for CRISPR deletions should be reported.\nResponse: The sequences and how we designed the sequences have been reported in the\nmaterial and method section and the relevant citation. The deletion efficiency is not added in\nline 12 : “The deletion rate was 14%.”\n• Clarify ambiguous phrases such as “Numbers: larval and pupal stages,”\nResponse: we improved thus phrase. “Numbers above gel lanes represent the larval and\npupal stages.”\nand rephrase statements like “it is unclear where FruM is expressed” (when prior data\nshowed expression).\nResponse: Hm. We refer to where it is expressed not if it is expressed in the brain. This is\ncorrect.: “As it is unclear where FruM is expressed in the male brain…”\n10. Gene-Centric Language: Several statements over-attribute behavioral control directly to a\nsingle gene. For example:\nResponse: We hopefully clarified this point by now given the above explanation. We together\nagree that a gene cannot control such behavior. We already have devoted a whole chapter\non how such gene can specify the behaviors. Please see line 482 on for further information.\n“How can the FruM protein specify these male behaviors? We propose that the FruM protein\nspecifies connectivity or functional features in the nervous system that determine behavioral\ncontrol. To understand the role of the FruM protein in the programming of behaviors, we\nexamined chemosensory receptor abundance, neuropil anatomy and odor processing in\nfruP1- males. … “\nOur work asks the question how the behavioral capacity is established through genetic\nspecification. Tthe word “contribution” is misleading as it provides in our understanding no\nlink to an underlying process. (For example: gene X contribute to antennae is not a helpful\ndescription of a developmental process).\n• “FruM specifies decision processes…” should be changed to “FruM facilitates decision processes…”\nnervous system that regulate motor program elements of cooperative behavior”\n• “…programs decision processes…” should be softened to “contributes to the neural basis\nof…”\nResponse: We changed as above: ”…programs initiation and sustainment processes of\nwithin-colony cooperative behaviors.”\n• These revisions would avoid an overly reductionist interpretation and better reflect the\nmultifactorial nature of behavior.\n11. Abstract and Introduction Revisions:\n• Line 35: Replace “whether” with “how” — “but how cooperative behaviors require…”\nResponse: we ask whether there is a dedicated program for the cooperative behavior. This is\ncorrect. This is the question we ask (see above. Also other researcher asked that questions\nfor example in the case of the fru gene.\n• Lines 39–40: The claim about screening transcription factors lacks supporting data. Either\ndelete this sentence or clarify that fru was identified based on prior evidence or candidate\nselection.\n• Clarify inconsistencies between the stated screen (lines 102–105) and the cited reference\n(Vleurinck et al., 2016, Ref. 33), which does not report male-specific splicing of fru.\nResponse: This is all correctly stated. We understand that we need to provide further\ninformation. Vleurinck et al., 2016, Ref. 33 describes the genome-wide search for sex-\nspecific spliced transcripts using the Spanki software program and the generated RNAseq\ndata set of the pupal brain (which is in detail described in the above citation). We now\nprovide a detailed list of genes that we identified as sex-specific spliced and highlight those\nthat were transcription factors (TFs) (now supplementary data table 1).\nChanges: we added a supplementary table with the list of sex-specific spliced genes\ndescribed in Ref. 33 and marked the subset of genes that encode transcription factor protein\n(line 106).\nThe title of the table: “List of sex-specific spliced genes in the pupal brain and the subset of genes\nthat encode a transcription factor “\n12. Male Behavior Description: Please provide a clear list of innate and learned male\ncooperative behaviors in Apis, such as:\n• Drone congregation behavior\n• Aggregation pheromone production\n• Trophallaxis\n• Heat production and clustering\n• Courtship flight patterns\nresearch question. Key publications that we cited and that describe our within colony\nbehavioral phenotypes are those 1, 17.\n13. Specific Phrase Suggestions:\n• Line 530: “FruM facilitates decision processes…”\n• Line 540: “FruM is required for the emergence of aspects of innate behavior…”\n• Line 548: Replace “specifies” with “contributes to”\n• Line 620: “We identified a gene that contributes to decision processes…”\nResponse: We suggest (see above) above that the wording “contribution” is not what we\nwant to state here. It is hard to imagine how a gene can contribute to a behavior. It is the\nspecification of the nervous system. We hope that the reviewer is by now convinced that this\nat least an alternative. It is about the specification of the nervous system. We replaced\n“decision” with “initiation and sustainment”.\nIn summary, we support the publication of this important work with minor revisions, pending\nclarification of the above points and the inclusion of appropriate references and discussion.\nResponses: We added relevant citations in places, added more information and sharpened\nour discussion in the places indicated.\nThe integration of neuroanatomy, genetics, and behavior is commendable, and with\nadditional detail and balance, this manuscript will be a significant contribution to the field.\nReviewer #3 (Remarks to the Author):\nThis study contains several important findings that will advance our understanding on the\nneural basis of cooperative social behavior. Most importantly, results show that the male-\nspecific isoform of the honeybee fruitless gene (FruM) is involved in the regulation of male\nbehaviors through its neural functions. Although the function of FruM on male-specific\nbehaviors has been well characterized in the common fruit fly Drosophila melanogaster and\nits sister species, whether fruitless is important for sex-specific behaviors outside dipterans\nhas been ambiguous. Consequently, the genetic origins of sexually dimorphic behaviors in\ninsects have been unclear.\nThis manuscript is significant beyond honeybee or even insects, because the data suggest\nthat a dedicated transcriptional regulation is a generalized mechanism that specifies sexual\ndimorphism of neural circuits and behaviors across phyla. In mammals, neurons that express\nsex hormone receptors (such as estrogen receptor, progesterone receptor, etc) play critical\nroles in regulating various aspects of reproductive behaviors. Evidence presented in this\nstudy generalizes this rule to Hymenopterans, which will draw attentions of wide range of\nneuroscientists and evolutionary biologists.\nTheir conclusions are based on high-quality data and careful observations. Successful\ncreation of genome-edited honeybees is a technical accomplishment on its own. The genetic\nmutation of fru provide convincing evidence that FruM is required for appropriate expression\n10\nof male honeybee behaviors. The nervous system-specific yet restricted expression of FruM\nsuggests that its main function is the regulation of neural processes. Behaviors of male\nmutants show intriguing differences from wild type males in social feeding and positions in\nthe nest. Lastly, the functional imaging experiments show that FruM influences male\nbehavior not by altering the sensory systems, but likely through central processing of sensory\ninformation. Overall, their multidisciplinary characterization of fru honeybee mutants has\nestablished a new entry point for investigating the neural basis of sexually dimorphic\nbehavior in social insects.\nAlthough I believe this manuscript should be published promptly, I would like to make a few\neditorial suggestions.\nFirst, quantitative analysis on the anatomical data can strengthen their conclusions. Movies\nand images of the fruP1myrGFP allele (Fig. 3 and 4, Movie 4-7) are annotated only qualitatively\nin the text. Some of their claims are not immediately obvious to untrained eyes as green\nfluorescence seems to be faintly present across the brain. For instance, the fluorescence in\nthe male antennal lobe (including in the macroglomeruli) is unclear, and it is even harder to\nrecognize any difference between the worker bee antennal lobe. Likewise, the stated male-\nspecific labeling of a tract in the lobula and lateral protocerebrum is not obvious because it is\nunclear where the corresponding tract in female brain is.\nMy suggestions are as follows:\n1) show the GFP staining of a wild type bee brain as a negative control. Faint but broad\nsignals of anti-GFP could be due to the background staining. A negative control specimen\nwill help clarify where the allele-specific signals exist.\nResponse: This is a very helpful idea. The changes we did. We now included these controls\nas a movie (Supplementary movie 12 and 13) and added this male negative control directly\nin Figure 4 in order to show the specificity also of the faint signals. As you can see the anti-\nGFP staining generated a nearly black background in the negative control (see below). We\nalso reorganized Figure 4 to better present the sex-dimorphic structures. We added the\nrelevant information to the movie and figure 4 legend, line 312: “Anti-GFP staining of wt\nmales were used as negative control.”\n2) Quantify the fluorescence intensity of targeted neuropils where sexually dimorphic labeling\nwere observed, such as ventromedial antennal lobe or the lobula/lateral protocerebrum.\nPhalloidin staining could serve as a normalizing signal.\nRepsonse: This is our first step of labeling such structure in bees and social insects in\ngeneral. It is an initial step and at this stage our goal is to qualitatively describe differences\nbetween the sexes. The negative control establishes a nearly black background confirming\nthat the labels were specific. We did the following: (i) improved the presentation of the sex\ndimorphic structures in the figure 4 to support our claim, (ii) presented the negative control in\nFigure 4.\nOur current analysis is not at high spatial resolution. The goal at this step is to only broadly\ndescribes the pattern. In a next and future step, we would like to quantify differences at high\nresolution. However, this requires more genetic engineering of the bees than the current\nknockin. We would like to employ amplifications of GFP protein expressing using a genetic\nbinary system such as a Gal4/UAS or Q. Hence, at this first step, we would like to stay with\nqualitative and structural differences.\nThe changes we have made to improve the ms:\nimproved the arrow and arrowhead presentation and description in order to better mark the\nstructures. We now also marked these structures in supplementary movies 6, 7, 11 and 13.\nWe rewrote the paragraph in the main ms to more clearly describe the differences.\nLine 285 on: “The antennal nerve is labeled in both male and female worker bees (arrow,\nFig. 4a, Supplementary Movie 6, 11 and 13). In workers, a small number (approximately a\ndozen) of glomeruli on the ventro-medial side of the AL had myrGFPP1+ labels both in the\ncore and cortex (arrow, Fig. 4b), whereas in males, FruM+ labels were consistently restricted\nto the core (arrowhead, Fig. 4b). This female-specific labeling of the cortex suggests that\nOSNs in the antennal nerve are FruM+ negative in males and myrGFPP1+ positive in females.\nThis is supported by a more restricted labeling of the nerve in males than in females (arrows,\nFig. 4a: note, that the antennal nerve contains approximately 4 times more neurons in males\nthan in female workers 3). Hence, sensory neurons from the antennal nerve that bypass the\nantennal lobe (AL) and glomeruli on the ventral side (antennal tracts T5 &T6) towards the\nantennal mechanosensory and motor center (AMMC) and potentially the subesophageal\nzone (SEZ) were labeled in males but also in female worker bees. The set of worker-specific\nlabeled glomeruli may correspond to the T3b cluster and could be involved in the processing\nof information derived from cuticular hydrocarbon perception 3, 18, 19. FruM+ circuitry is present\nin enlarged macroglomeruli in the core, which are male-specific structures that are absent in\nworkers (MG1, Fig. 4b, Supplementary Movie 6, 11 and 13). The FruM+ circuitry of the male\nvisual system has a male-specific tract (large arrow, Fig. 4c, Supplementary Movie 7) that\nprojects from the lobula (LO) to the posterior lateral protocerebrum (PLP) but shares two\nother tracts with the myrGFPP1+ circuitry of worker bees in the same area (small arrows, Fig.\n4c). In the central complex, a few labeled myrGFPP1+ fibers were found in the noduli (NO) of\nfemale worker bees, but no FruM+ cells were found in males (arrows, Fig. 4d, Supplementary\nMovie 6, 11 and 13) 20. We conclude from these comparisons that the FruM+ circuit has a\nmale-specific identity that manifests at the gross level in the anatomy of the antennal nerve,\nmacroglomeruli, and innervation of the core/cortex of the glomeruli and optic tracts.”\n3) Annotate structures of interest in the movies, so that readers know where to pay attention\nto. For example, Movie 7 supposedly shows fru-expressing antennal nerves only in workers\n(lines 279-280), but it is impossible to tell where the nerve is. This statement is also\nconfusing since the text says that both males and workers have fru-expressing antennal\nfibers (lines 275-277).\nResponse: Yes, they were not easy to find sorry. We now carefully labeled these structures\nin the movies and explained these labels in the movie descriptions (now movies 6, 7, 11 and\n13). We rewrote this entire paragraph (see above) to better explain and delete confusing\nstatements.\nSecond, I would like to see the signals of anti-FruM and fruP1myrGFP in the posterior end of the\nbrain. Fig. 3d is the only image of the posterior side, and movies appear to stop at around the\nmiddle of the brain. Studies in Drosophila have shown that posterior medial part of the brain\ncontains cell types that control courtship behavior. It is interesting to address if a cluster of\nFruM neurons is present in the male honeybee brain or not.\nResponse: It appears that we needed a 2nd round of stacks with higher laser intensity to\nmake these posterior parts visible. Sorry for not presenting them. We now present these\nstacks as extra movies (Supplementary Movie 7 and 9; posterior region). We were not able\nto characterize the cluster of FruM neurons, the ones the reviewer mentioned. We now\nstated about these additional movies in the main text.\nbetween the Drosophila fru circuit. In Drosophila, sexually dimorphic neurons are mostly in\nthe central interneurons but some sensory neurons do express FruM. This seems to be a\nnotable difference in the honeybees (line 279), leaving a question over the genetic\nmechanism of antennae sexual dimorphism (ref. 14). Classic works on the Drosophila fru\ncircuit (Yu et al., Curr. Biol. 20:1602-1614 (2010); Cachero et al., Curr. Biol. 20:1589-1601\n(2010)) can provide discussion points.\nResponse: We now added a brief comparison to D. melanogaster in the last paragraph of the\ndiscussion and cited the relevant studies. The reviewer is correct: there is a notable\ndifference to the fruit fly: In honeybee males FruM is not expressed in the OSNs that enter the\nglomeruli (see explanation and Fig. 4 b). FruM is also not required for the sexual dimorphic\ndevelopment of the male-specific enlarged macrogloremruli (Fig. 8a) and for the processing\nof queen's pheromone information at the level of the macroglomeruli MG 2 (Fig. 8b to d).\nLine 640 we now wrote: “. In D. melanogaster FruM+ expressing OSNs, which detect female\npheromones, project into three male-specific enlarged glomeruli 21-23. The formation of these\nsexual dimorphic glomeruli depends in the fruit fly on the male-specific activity of Fru protein\n21-23. To the contrary, FruM in honeybee males is not expressed in the OSNs that enter the\nglomeruli (Fig. 4a, b), is not required for the sexual dimorphic development of the male-\nspecific enlarged macrogloremruli (Fig. 8a) and for the processing of queen's pheromone\ninformation at the level of the macroglomeruli MG 2 (Fig. 8b to d).”\nFourth, the relevance of the discussion over the decision processes (lines 555-583) is\nunclear. The authors' conclusion that \"FruM specifies decision process in the nervous\nsystem\" (line 548) is based on behavioral phenotypes on the cooperative feeding behaviors\n(Fig. 5). While other innate behaviors and global response to odors (Fig. 8) are largely\nunchanged, relatively limited types of odors tested and the lack of glomerular resolution of\nthe imaging experiment leave it possible that response to uncharacterized, behaviorally\nrelevant olfactory cues are indeed affected in fru mutants. If this is the case, the effects on\ncooperative feeding might be caused simply by the loss of relevant sensory inputs, rather\nthan “decision process” as discussed in this subsection. This discussion could be saved for\nfuture studies in which the relationship between fru and action choice during social\ninteraction in honeybees is better characterized.\nResponse: We agree that this could be misleading. Our intension was to anchor the\ndiscussion around the behavioral phenotype in some places. The rate/duration impairment is\nn interesting phenotype to understand the innate social task organization in the colony. We\nnow deleted the words \"decision process\" and replaced it with the words \"initiation and\nduration \" (or related). Accordingly, we rewrote the sentences to more clearly distinguish\nbetween the behavioral phenotype and the control driven by the nervous system. This\nrequired rewritings of the discussion section.\nThis includes:\nLine 553: “Specifically, we found that fruP1 transcripts are required for the rate of\napproaching, begging and trophallaxis behaviors, suggesting that the initiation of three\nsequences of the social feeding behavior are innately programmed by FruM. The duration of\ntrophallaxis behavior also requires the FruM protein, suggesting that there is a neural basis\nfor a sustainment process that controls the duration of social feeding. The choice of whether\nmales contact pollen food sources in the cells also requires fruP1 transcripts, indicating that\nFruM protein programs repression of the cell entering for the pollen stimulus. This repression\nof pollen interacting behaviors reduce social conflicts among colony members over the\ncollectively used food stores. Overall, we showed that FruM specifies imitation and\nsustainment processes in the nervous system that regulate motor program elements of\ncooperative behaviors.”\nLine 574: “This specification of the rate and duration aspects has profound consequence in\nthe colony as it innately scales the bee's participation in the social feeding task.\nWe now better structured the discussion and more clearly stated the two possibilities where\nthe rate impairment comes from. Line 600: “The FruM protein is required for initiation and\nsustainment of social feeding behaviors and is spatially restricted expressed in the nervous\nsystem suggesting that the FruM-expressing circuitry is involved in controlling these aspects\nof behaviors. This FruM+ circuitry is involved in processing olfactory, gustatory and\nmechanosensory information, suggesting a possible role of different sensory modalities in\nthe control of these within-colony behaviors 3, 7, 18, 19, 24, 25. FruM+ circuitries are also found in\nhigher-order processing centers—the mushroom bodies—indicating that the circuitry is\npossibly involved in evaluating and integrating sensory information for behavioral decision\nprocesses 24-28.”\nHowever, the overall scientific merit of the manuscript remains very high. I strongly\nrecommend that this work becomes broadly available as soon as points raised above are\naddressed.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReferences:\n1. Crailsheim K. Trophallactic interactions in the adult honeybee (Apis mellifera L.). Apidologie\n29, 97-112 (1998).\n2. Jain R, Brockmann A. Sex-specific molecular specialization and activity rhythm-dependent\ngene expression in honey bee antennae. J Exp Biol 223, (2020).\n3. Mariette J, Carcaud J, Sandoz JC. The neuroethology of olfactory sex communication in the\nhoneybee Apis mellifera L. Cell Tissue Res 383, 177-194 (2021).\n4. Wanner KW, Nichols AS, Walden KK, Brockmann A, Luetje CW, Robertson HM. A honey bee\nodorant receptor for the queen substance 9-oxo-2-decenoic acid. Proceedings of the National\nAcademy of Sciences of the United States of America 104, 14383-14388 (2007).\n5. Witthöft W. Absolute Anzahl und Verteilung der Zellen im Hirn der Honigbiene. Zeitschrift für\nMorphologie der Tiere 61, 160-184 (1967).\n6. Marco Antonio DS, Hartfelder K. Toward an Understanding of divergent compound eye\ndevelopment in drones and workers of the honeybee (Apis mellifera L.): A correlative analysis\nof morphology and gene erxpression. J Exp Zool B Mol Dev Evol 328, 139-156 (2017).\n14\n7. Sandoz JC. Odour-evoked responses to queen pheromone components and to plant odours\nusing optical imaging in the antennal lobe of the honey bee drone Apis mellifera L. J Exp Biol\n209, 3587-3598 (2006).\n8. Vleurinck C, Raub S, Sturgill D, Oliver B, Beye M. Linking genes and brain development of\nhoneybee workers: a whole-transcriptome approach. PLoS One 11, e0157980 (2016).\n9. Rings A, Goodwin SF. To court or not to court - a multimodal sensory decision in Drosophila\nmales. Curr Opin Insect Sci 35, 48-53 (2019).\n10. Kimura K, Hachiya T, Koganezawa M, Tazawa T, Yamamoto D. Fruitless and doublesex\ncoordinate to generate male-specific neurons that can initiate courtship. Neuron 59, 759-769\n(2008).\n11. Basrur NS, et al. Fruitless mutant male mosquitoes gain attraction to human odor. Elife 9,\n(2020).\n12. Ryner LC, et al. Control of male sexual behavior and sexual orientation in Drosophila by the\nfruitless gene. Cell 87, 1079-1089 (1996).\n13. Ueno M, Nakata M, Kaneko Y, Iwami M, Takayanagi-Kiya S, Kiya T. fruitless is sex-\ndifferentially spliced and is important for the courtship behavior and development of silkmoth\nBombyx mori. Insect Biochem Mol Biol 159, 103989 (2023).\n14. Meier N, Kappeli SC, Hediger Niessen M, Billeter JC, Goodwin SF, Bopp D. Genetic control of\ncourtship behavior in the housefly: evidence for a conserved bifurcation of the sex-determining\npathway. PLoS One 8, e62476 (2013).\n15. Gempe T, Hasselmann M, Schiott M, Hause G, Otte M, Beye M. Sex determination in\nhoneybees: two separate mechanisms induce and maintain the female pathway. PLoS Biol 7,\ne1000222 (2009).\n16. Hasselmann M, Gempe T, Schiott M, Nunes-Silva CG, Otte M, Beye M. Evidence for the\nevolutionary nascence of a novel sex determination pathway in honeybees. Nature 454, 519-\n522 (2008).\n17. Ohtani T. Behavior repertoire of adult drone honeybee within observation hives. Jour Fac Sci\nHokkaido Univ SerVI, Zool 19, 706-721 (1974).\n18. Galizia CG, Sachse S, Rappert A, Menzel R. The glomerular code for odor representation is\nspecies specific in the honeybee Apis mellifera. Nat Neurosci 2, 473-478 (1999).\n19. Paoli M, Galizia GC. Olfactory coding in honeybees. Cell Tissue Res 383, 35-58 (2021).\n20. Kaiser A, et al. A three-dimensional atlas of the honeybee central complex, associated\nneuropils and peptidergic layers of the central body. J Comp Neurol 530, 2416-2438 (2022).\n21. Stockinger P, Kvitsiani D, Rotkopf S, Tirián L, Dickson BJ. Neural circuitry that governs\nmale courtship behavior. Cell 121, 795-807 (2005).\n22. Cachero S, Ostrovsky AD, Yu JY, Dickson BJ, Jefferis GS. Sexual dimorphism in the fly brain.\nCurr Biol 20, 1589-1601 (2010).\n23. Yu JY, Kanai MI, Demir E, Jefferis GS, Dickson BJ. Cellular organization of the neural circuit\nthat drives Drosophila courtship behavior. Curr Biol 20, 1602-1614 (2010).\n24. Habenstein J, Grubel K, Pfeiffer K, Rossler W. 3D atlas of cerebral neuropils with previously\nunknown demarcations in the honey bee brain. J Comp Neurol 531, 1163-1183 (2023).\n25. Strausfeld NJ. Organization of the honey bee mushroom body: representation of the calyx\nwithin the vertical and gamma lobes. J Comp Neurol 450, 4-33 (2002).\n26. Arican C, Schmitt FJ, Rossler W, Strube-Bloss MF, Nawrot MP. The mushroom body output\nencodes behavioral decision during sensory-motor transformation. Curr Biol 33, 4217-4224\ne4214 (2023).\n27. Strube-Bloss MF, Rossler W. Multimodal integration and stimulus categorization in putative\nmushroom body output neurons of the honeybee. R Soc Open Sci 5, 171785 (2018).\n28. Noyes NC, Davis RL. Innate and learned odor-guided behaviors utilize distinct molecular\nsignaling pathways in a shared dopaminergic circuit. Cell Reports 42, 112026 (2023).\n16"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have fully addressed all of the suggestions and concerns raised in the earlier review.", "reviewer_2": "I am satisfied with the reply of the authors and the modifications to the draft.", "reviewer_3": "The revision of the manuscript by Kohnen et al. addresses my concerns in satisfactory manner. I must admit that the labeling pattern of fruP1-GFP is not necessarily as convincing as the description of the manuscript (for example, the labeling in the core regions of the antennal glomeruli is very faint). However, I acknowledge that the creation of this genetic allele is an accomplishment on its own, which opens an avenue for further characterization of fru-expressing neural circuits in honeybees. I am happy with the qualitative description of fruP1-GFP labeling pattern at this stage.\nThe modification in Result and Discussion sections made the message of the paper more straightforward. However, I find the text sometimes difficult to read due to issues in syntax and punctation. I don’t think a reviewer should serve as a proofreader, but I strongly recommend that the authors use a professional English proofreading service before the manuscript is published. Some errors are relatively easy to read through, but others compromise the clarity of the manuscript, which is a regrettable disservice to this otherwise exciting manuscript.\nI managed to note only the following examples. These are by no means comprehensive.\nLines 139-140: “Next, we developed an anti-Fru antibody against a sequence of the BTB domain.” “anti-Fru antibody against” is an awkward expression. Something like the following would be better”\n“Next, we developed an antibody against a sequence of the Fru BTB domain.”\nLine 344: “The behavior of each male was behaviorally tracked using…” The expression is redundant. It should be changed to:\n“The behavior of each male was tracked using…”\nLines 591-592: “…worker bees within the same age class perform also other tasks…” The location of adverb (also) is incorrect. It should be changed to:\n“…worker bees within the same age class also perform other tasks…”\nLines 595-596: “…a large fraction is informed about the food status that can perform task informed decisions just from the local interactions.” This is an awkward sentence. The following is a suggestion:\n“…a large fraction is informed about the food status that can choose appropriate tasks based only on the local interactions.”\nLines 644-646: “In D. melanogaster FruM+ expressing OSNs, which detect female pheromones, project into three male-specific enlarged glomeruli 76-78.” This sentence is difficult to understand, but I assume authors mean:\n“In D. melanogaster, FruM-expressing OSNs, which detect female pheromones, project into three glomeruli that are enlarged specifically in males”\nLines 651-653: “These differences revealed that FruM's role diverged in the two species suggesting notable entry points to dissect honeybee confined specifications of the circuitry and cooperative behaviors.” The lack of appropriate punctations makes this sentence difficult to understand. It should be changed to something like the following:\n“These differences revealed that FruM's role diverged in the two species, suggesting notable entry points to dissect honeybee-confined specifications of the circuitry and cooperative behaviors”.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: Indeed, the reviewer is correct: there were still several places to improve the ms.\nWe did another round of careful editing. We followed the suggestions below.\nLines 139-140: “Next, we developed an anti-Fru antibody against a sequence of the BTB\ndomain.” “anti-Fru antibody against” is an awkward expression. Something like the following\nwould be better”\n“Next, we developed an antibody against a sequence of the Fru BTB domain.”\nResponse: We deleted “anti-Fru”\nLine 344: “The behavior of each male was behaviorally tracked using…” The expression is\nredundant. It should be changed to:\n“The behavior of each male was tracked using…”\nResponse: we followed these changes\nLines 591-592: “…worker bees within the same age class perform also other tasks…” The\nlocation of adverb (also) is incorrect. It should be changed to:\n“…worker bees within the same age class also perform other tasks…”\nResponse: Oh sorry, thanks: we changed it.\nLines 595-596: “…a large fraction is informed about the food status that can perform task\ninformed decisions just from the local interactions.” This is an awkward sentence. The\nfollowing is a suggestion:\n“…a large fraction is informed about the food status that can choose appropriate tasks based\nonly on the local interactions.”\nResponse: OK: we improved the entire paragraph. We realized that this paragraph was still\nhard to understand.\nWe changed into: “If a large fraction of colony members participates in social feeding, a large\nfraction is informed about the food status. As a consequence, a large fraction of colony\nmembers can adjust their task decisions to colony need 69, when encountering task-related\nstimuli.”\nLines 644-646: “In D. melanogaster FruM+ expressing OSNs, which detect female\npheromones, project into three male-specific enlarged glomeruli 76-78.” This sentence is\ndifficult to understand, but I assume authors mean:\n“In D. melanogaster, FruM-expressing OSNs, which detect female pheromones, project into\nthree glomeruli that are enlarged specifically in males”\nResponse: thanks for your suggestion, which we used this wording to improve the ms.\nLines 651-653: “These differences revealed that FruM's role diverged in the two species\nsuggesting notable entry points to dissect honeybee confined specifications of the circuitry\nand cooperative behaviors.” The lack of appropriate punctations makes this sentence difficult\nto understand. It should be changed to something like the following:\n“These differences revealed that FruM's role diverged in the two species, suggesting notable\nentry points to dissect honeybee-confined specifications of the circuitry and cooperative\nbehaviors”.\nResponse: many thanks: we followed this suggestion."}]

Decision letter