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. 2019 Mar 21;17(3):e3000171.
doi: 10.1371/journal.pbio.3000171. eCollection 2019 Mar.

A genetic switch for worker nutrition-mediated traits in honeybees

Annika Roth  1 , Christina Vleurinck  1 , Oksana Netschitailo  1 , Vivien Bauer  1 , Marianne Otte  1 , Osman Kaftanoglu  2 , Robert E Page  2   3 , Martin Beye  1
Affiliations

A genetic switch for worker nutrition-mediated traits in honeybees

Annika Roth et al. PLoS Biol. .

Abstract

Highly social insects are characterized by caste dimorphism, with distinct size differences of reproductive organs between fertile queens and the more or less sterile workers. An abundance of nutrition or instruction via diet-specific compounds has been proposed as explanations for the nutrition-driven queen and worker polyphenism. Here, we further explored these models in the honeybee (Apis mellifera) using worker nutrition rearing and a novel mutational screening approach using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. The worker nutrition-driven size reduction of reproductive organs was restricted to the female sex, suggesting input from the sex determination pathway. Genetic screens on the sex determination genes in genetic females for size polyphenism revealed that doublesex (dsx) mutants display size-reduced reproductive organs irrespective of the sexual morphology of the organ tissue. In contrast, feminizer (fem) mutants lost the response to worker nutrition-driven size control. The first morphological worker mutants in honeybees demonstrate that the response to nutrition relies on a genetic program that is switched "ON" by the fem gene. Thus, the genetic instruction provided by the fem gene provides an entry point to genetically dissect the underlying processes that implement the size polyphenism.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Reproductive organ and head phenotypes of females and males reared on worker nutrition in the laboratory and in the colony.
Scale bar = 1 mm.
Fig 2
Fig 2. Examples of FL and nucleotide sequence analyses of the targeted genomic sites of single bees using the efficient CRISPR/Cas9 method.
FL analysis is presented on the left, and the nucleotide sequences are presented on the right for single bees. Examples of WT alleles and mutated sequences are shown. The cleavage site of the Cas9 protein is indicated with arrows. The PAM site (the essential targeting component for CRISPR/Cas9) is underlined in the nucleotide sequence. Dashes indicate deletions. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; FL, fragment length; mut, mutated sequences; PAM, Protospacer adjacent motif; WT, wild type.
Fig 3
Fig 3. Size polyphenism of gonads in genetic females at larval stage 5 that were double mutants for the fem gene.
(a) Model of the known components of the sex-determining pathway in honeybees with nutritional differences in females. (b) Gonad development at larval stage 5. (Right) A pair of large gonads (male type) from fem sgRNA2-treated genetic females reared on worker nutrition. The gonads display densely packed layers of folded testioles, similar to those observed in haploid males (WT males). (Left) Pairs of small gonads (female type) from WT workers and genetic female bees reared on worker nutrition. A WT large queen ovary from a queen reared in a colony on queen nutrition. A large WT testis of a haploid male manually reared on worker nutrition. (c) Male dsx (dsxM) and female dsx (dsxF) transcripts in mutated genetic females with male phenotypes (fem-sgRNA1 or fem-sgRNA2). Male and female transcripts were separately amplified by RT-PCR [64], and the male and female fragments of each single bee were resolved via agarose gel electrophoresis. Numbers indicate different control and mutated bees. (d) Deduced amino acid sequences from sequenced amplicons of the fem gene at the designated CRISPR/Cas9 cleavage sites for the four worker nutrition-reared genetic female larvae with large gonads of the male type. Stars indicate premature translation stop codons. Numbers indicate different mutated bees. Scale bars, 1 mm. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; dsxF, female dsx; dsxM, male dsx; RT-PCR, reverse transcription PCR; sgRNA, single guide RNA; WT, wild type.
Fig 4
Fig 4. Size polyphenism of the reproductive organs in genetic female double mutants for the dsx gene.
Pictures of the head and internal reproductive organs of mutated and WT control bees are shown on the left, while the genotypes at the dsx locus with the deduced amino acid sequences are displayed on the right. Mutated and control genetic females and males were reared on worker nutrition. Queens were reared on the queen diet in a colony (we cannot mimic queen rearing in the laboratory). The WT amino acid sequence is shown above the detected alleles for comparison. (a, b) WT genetic female reared on queen nutrition (RJ) in the colony. (c, d) WT genetic females manually reared on worker nutrition. (e–l) Genetic females reared on worker nutrition that were double mutants for dsx via the dsx-sgRNA6 (note that a small part of the worker bee head 17–39 [picture i] is missing due to the dissection process). (m–p) Genetic females reared on worker nutrition that were double mutants for dsx via the dsx-sgRNA2. (q, r) Genetic males manually reared on worker nutrition. Organs were stained with aceto-orcein (reddish coloring) to facilitate the dissection process. Testis tissues are marked with arrows. Scale bar, 1 mm. Dashes in the sequence indicate deletions, and stars illustrate early translational stop codons. RJ, royal jelly; WT, wild type.
Fig 5
Fig 5. The role of the sex-determining genes fem and dsx in size polyphenism.
(a) Schematic presentation of the mutant effects of fem and dsx gene on size polyphenism. Genetic female bees reared on worker nutrition produce only small reproductive organs. Genetic females with a mutant fem gene show no small size polyphenism of reproductive organs. Genetic females that have a mutated dsx (operating downstream of fem) do show size polyphenism of the intersex reproductive organ and male-like gonads. Thus, we conclude that the fem gene is required for the small size polyphenism. Crosses mark the genes that we compromised using CRISPR/Cas9-induced mutations. (b) The role of the fem gene for caste development. The gene products of the sex determination pathway (Fem, DsxF, DsxM) are shown in red (female) and blue (male) boxes. The nutrition-mediated process is shown in pink. Arrows indicate regulatory relationships. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9.
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Grants and funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft (http://www.dfg.de/) to MB (BE 2194). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.