Martin Hasselmann

The gene csd is the primary signal for sexual development in the honeybee and encodes an SR-type protein.

Haplodiploid organisms comprise about 20% of animals. Males develop from unfertilized eggs while females are derived from fertilized eggs. The underlying mechanisms of sex determination, however, appear to be diverse and are poorly understood. We have dissected the complementary sex determiner (csd) locus in the honeybee to understand its molecular basis. In this species, csd acts as the primary sex-determining signal with several alleles segregating in populations. Males are hemizygous and females are heterozygous at this locus; nonreproducing diploid males occur when the locus is homozygous. We have characterized csd by positional cloning and repression analysis. csd alleles are highly variable and no transcription differences were found between sexes. These results establish csd as a primary signal that governs sexual development by its allelic composition. Structural similarity of csd with tra genes of Dipteran insects suggests some functional relation of what would otherwise appear to be unrelated sex-determination mechanisms.

Evidence for the evolutionary nascence of a novel sex determination pathway in honeybees

Sex determination in honeybees (Apis mellifera) is governed by heterozygosity at a single locus harbouring the complementary sex determiner (csd) gene, in contrast to the well-studied sex chromosome system of Drosophila melanogaster. Bees heterozygous at csd are females, whereas homozygotes and hemizygotes (haploid individuals) are males. Although at least 15 different csd alleles are known among natural bee populations, the mechanisms linking allelic interactions to switching of the sexual development programme are still obscure. Here we report a new component of the sex-determining pathway in honeybees, encoded 12 kilobases upstream of csd. The gene feminizer (fem) is the ancestrally conserved progenitor gene from which csd arose and encodes an SR-type protein, harbouring an Arg/Ser-rich domain. Fem shares the same arrangement of Arg/Ser- and proline-rich-domain with the Drosophila principal sex-determining gene transformer (tra), but lacks conserved motifs except for a 30-amino-acid motif that Fem shares only with Tra of another fly, Ceratitis capitata. Like tra, the fem transcript is alternatively spliced. The male-specific splice variant contains a premature stop codon and yields no functional product, whereas the female-specific splice variant encodes the functional protein. We show that RNA interference (RNAi)-induced knockdowns of the female-specific fem splice variant result in male bees, indicating that the fem product is required for entire female development. Furthermore, RNAi-induced knockdowns of female allelic csd transcripts result in the male-specific fem splice variant, suggesting that the fem gene implements the switch of developmental pathways controlled by heterozygosity at csd. Comparative analysis of fem and csd coding sequences from five bee species indicates a recent origin of csd in the honeybee lineage from the fem progenitor and provides evidence for positive selection at csd accompanied by purifying selection at fem. The fem locus in bees uncovers gene duplication and positive selection as evolutionary mechanisms underlying the origin of a novel sex determination pathway.

Genomic signatures of evolutionary transitions from solitary to group living

For bees, many roads lead to social harmony Eusociality, where workers sacrifice their reproductive rights to support the colony, has evolved repeatedly and represents the most evolved form of social evolution in insects. Kapheim et al. looked across the genomes of 10 bee species with varying degrees of sociality to determine the underlying genomic contributions. No one genomic path led to eusociality, but similarities across genomes were seen in features such as increases in gene regulation and methylation. It also seems that selection pressures relaxed after the emergence of complex sociality. Science, this issue p. 1139 Social evolution in bees has followed diverse genomic paths but shares genomic patterns. The evolution of eusociality is one of the major transitions in evolution, but the underlying genomic changes are unknown. We compared the genomes of 10 bee species that vary in social complexity, representing multiple independent transitions in social evolution, and report three major findings. First, many important genes show evidence of neutral evolution as a consequence of relaxed selection with increasing social complexity. Second, there is no single road map to eusociality; independent evolutionary transitions in sociality have independent genetic underpinnings. Third, though clearly independent in detail, these transitions do have similar general features, including an increase in constrained protein evolution accompanied by increases in the potential for gene regulation and decreases in diversity and abundance of transposable elements. Eusociality may arise through different mechanisms each time, but would likely always involve an increase in the complexity of gene networks.

Sex Determination in Honeybees: Two Separate Mechanisms Induce and Maintain the Female Pathway

Sex determination in honeybees is realized by the csd and the fem gene that establish and maintain, throughout development, sexual fates via the control of alternative splicing.

Specific developmental gene silencing in the honey bee using a homeobox motif

Manipulating the expression of genes in species that are not currently used as genetic models will provide comparative insights into the evolution of gene functions. However the experimental tools in doing so are limited in species that have not served as models for genetic studies. We have examined the effects of double stranded RNA (dsRNA) in the honey bee, an insect with considerably basic scientific interest. dsRNA derived from a 300 bp stretch of the E30 homeobox motif was injected into honey bee embryos at the anterior pole in the preblastoderm stage. We found that the dsRNA fragment successfully disrupted the protein expression of the target gene throughout the whole embryo. The disruption caused deficient phenotypes similar to known loss of function mutants of Drosophila engrailed, whereas embryos injected with nonsense dsRNA showed no abnormalities. We show that the large size of the honey bee egg (D: 0.3 mm, L: 1.6 mm) and the long preblastoderm stage (11–12 h) can be exploited to generate embryos with partial disruption of gene function, which may provide an elegant alternative to classical chimeric analyses. This is the first report of targeted disruption of gene function in the honey bee, and the results prove that the chosen target gene is a functional ortholog to engrailed in Drosophila.

Rapid Evolution of Immune Proteins in Social Insects

The existence of behavioral traits connected to defense against pathogens manifests the importance of pathogens in the evolution of social insects. However, very little is known about how pathogen pressure has affected the molecular evolution of genes involved in their innate immune system. We have studied the sequence evolution of several immune genes in ants and honeybees. The results show high rates of evolution in both ants and honeybees as measured by the ratio of amino acid changes to silent nucleotide changes, the ratio being clearly higher than in Drosophila immune genes or in nonimmunity genes of bees. This conforms to our expectations based on high pathogen pressure in social insects. The codon-based likelihood method found clear evidence of positive selection only in one ant gene, even though positive selection has earlier been found in both ant and termite immune genes. There is now indication that selection on the amino acid composition of the immune-related genes has been an important part in the fight against pathogens by social insects. However, we cannot distinguish in all the cases whether the high observed d(N)/d(S) ratio results from positive selection within a restricted part of the studied genes or from relaxation of purifying selection associated with effective measures of behaviorally based colony-level defenses.

Evidence for Convergent Nucleotide Evolution and High Allelic Turnover Rates at the complementary sex determiner Gene of Western and Asian Honeybees

Our understanding of the impact of recombination, mutation, genetic drift, and selection on the evolution of a single gene is still limited. Here we investigate the impact of all these evolutionary forces at the complementary sex determiner (csd) gene that evolves under a balancing mode of selection. Females are heterozygous at the csd gene and males are hemizygous; diploid males are lethal and occur when csd is homozygous. Rare alleles thus have a selective advantage, are seldom lost by the effect of genetic drift, and are maintained over extended periods of time when compared with neutral polymorphisms. Here, we report on the analysis of 17, 19, and 15 csd alleles of Apis cerana, Apis dorsata, and Apis mellifera honeybees, respectively. We observed great heterogeneity of synonymous (piS) and nonsynonymous (piN) polymorphisms across the gene, with a consistent peak in exons 6 and 7. We propose that exons 6 and 7 encode the potential specifying domain (csd-PSD) that has accumulated elevated nucleotide polymorphisms over time by balancing selection. We observed no direct evidence that balancing selection favors the accumulation of nonsynonymous changes at csd-PSD (piN/piS ratios are all <1, ranging from 0.6 to 0.95). We observed an excess of shared nonsynonymous changes, which suggest that strong evolutionary constraints are operating at csd-PSD resulting in the independent accumulation of the same nonsynonymous changes in different alleles across species (convergent evolution). Analysis of csd-PSD genealogy revealed relatively short average coalescence times ( approximately 6 Myr), low average synonymous nucleotide diversity (piS < 0.09), and a lack of trans-specific alleles that substantially contrasts with previously analyzed loci under strong balancing selection. We excluded the possibility of a burst of diversification after population bottlenecking and intragenic recombination as explanatory factors, leaving high turnover rates as the explanation for this observation. By comparing observed allele richness and average coalescence times with a simplified model of csd-coalescence, we found that small long-term population sizes (i.e., N(e) < 10(4)), but not high mutation rates, can explain short maintenance times, implicating a strong historical impact of genetic drift on the molecular evolution of highly social honeybees.

Pronounced differences of recombination activity at the sex determination locus of the honeybee, a locus under strong balancing selection.

Recombination decreases the association of linked nucleotide sites and can influence levels of polymorphism in natural populations. When coupled with selection, recombination may relax potential conflict among linked genes, a concept that has played a central role in research on the evolution of recombination. The sex determination locus (SDL) of the honeybee is an informative example for exploring the combined forces of recombination, selection, and linkage on sequence evolution. Balancing selection at SDL is very strong and homozygous individuals at SDL are eliminated by worker bees. The recombination rate is increased up to four times that of the genomewide average in the region surrounding SDL. Analysis of nucleotide diversity (π) reveals a sevenfold increase of polymorphism within the sex determination gene complementary sex determiner (csd) that rapidly declines within 45 kb to levels of genomewide estimates. Although no recombination was observed within SDL, which contains csd, analyses of heterogeneity, shared polymorphic sites, and linkage disequilibrium (LD) show that recombination has contributed to the evolution of the 5′ part of some csd sequences. Gene conversion, however, has not obviously contributed to the evolution of csd sequences. The local control of recombination appears to be related to SDL function and mode of selection. The homogenizing force of recombination is reduced within SDL, which preserves allelic differences and specificity, while the increase of recombination activity around SDL relaxes conflict between SDL and linked genes.

Origin of a function by tandem gene duplication limits the evolutionary capability of its sister copy

The most remarkable outcome of a gene duplication event is the evolution of a novel function. Little information exists on how the rise of a novel function affects the evolution of its paralogous sister gene copy, however. We studied the evolution of the feminizer (fem) gene from which the gene complementary sex determiner (csd) recently derived by tandem duplication within the honey bee (Apis) lineage. Previous studies showed that fem retained its sex determination function, whereas the rise of csd established a new primary signal of sex determination. We observed a specific reduction of nonsynonymous to synonymous substitution ratios in Apis to non-Apis fem. We found a contrasting pattern at two other genetically linked genes, suggesting that hitchhiking effects to csd, the locus under balancing selection, is not the cause of this evolutionary pattern. We also excluded higher synonymous substitution rates by relative rate testing. These results imply that stronger purifying selection is operating at the fem gene in the presence of csd. We propose that csds new function interferes with the function of Fem protein, resulting in molecular constraints and limited evolvability of fem in the Apis lineage. Elevated silent nucleotide polymorphism in fem relative to the genome-wide average suggests that genetic linkage to the csd gene maintained more nucleotide variation in todays population. Our findings provide evidence that csd functionally and genetically interferes with fem, suggesting that a newly evolved gene and its functions can limit the evolutionary capability of other genes in the genome.

Fine scale mapping in the sex locus region of the honey bee (Apis mellifera)

Isolating an unknown gene with fine‐scale mapping is possible in a ‘non‐model’ organism. Sex determination in honey bees consists of a single locus (sex locus) with several complementary alleles. Diploid females are heterozygous at the sex locus, whereas haploid males arise from unfertilized eggs and are hemizygous. The construction of specific inbred crosses facilitates fine scale mapping in the sex locus region of the honey bee. The high recombination rate in the honey bee reduces the physical distance between markers compared with model organisms and facilitates a novel gene isolation strategy based on step‐wise creation of new markers within small physical distances. We show that distances less than 25 kb can be efficiently mapped with a mapping population of only 1000 individuals. The procedure described here will accelerate the mapping, analysis and isolation of honey bee genes.