E. D. Tanaka

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.

Deep Sequencing of Organ- and Stage-Specific microRNAs in the Evolutionarily Basal Insect Blattella germanica (L.) (Dictyoptera, Blattellidae)

Background microRNAs (miRNAs) have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum) have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs. Methodology/Principal Findings Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates. Conclusions/Significance Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.

Exoskeleton formation in Apis mellifera: Cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development

Cuticular hydrocarbons (CHCs) are abundant in the superficial cuticular layer (envelope) of insects where they play roles as structural, anti-desiccation and semiochemical compounds. Many studies have investigated the CHC composition in the adult insects. However, studies on the profiles of these compounds during cuticle formation and differentiation are scarce and restrict to specific stages of a few insect species. We characterized the CHCs developmental profiles in the honeybee workers during an entire molting cycle (from pupal-to-adult ecdyses) and in mature adults (forager bees). Gas chromatography/mass spectrometry (GC/MS) analysis revealed remarkable differences in the relative quantities of CHCs, thus discriminating pupae, developing and newly-ecdysed adults, and foragers from each other. In parallel, the honeybee genome database was searched for predicted gene models using known amino acid sequences of insect enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases) as queries in BLASTP analysis. The expression levels of six desaturase genes and ten elongase genes potentially involved in CHC biosynthesis were determined by reverse transcription and real time polymerase chain reaction (RT-qPCR) in the developing integument (cuticle and subjacent epidermis). Aiming to predict roles for these genes in CHC biosynthesis, the developmental profiles of CHCs and desaturase/elongase transcript levels were evaluated using Spearman correlation coefficient. This analysis pointed to differential roles for these gene products in the biosynthesis of certain CHC classes. Based on the assumption that homologous proteins may share a similar function, phylogenetic trees were reconstructed as an additional strategy to predict functions and evolutionary relationships of the honeybee desaturases and elongases. Together, these approaches highlighted the molecular complexity underlying the formation of the lesser known layer of the cuticular exoskeleton, the envelope.

Dicer-1 is a key enzyme in the regulation of oogenesis in panoistic ovaries

In insects, the action of microRNAs (miRNAs) on oogenesis has been explored only in dipterans, which possess meroistic ovaries, a highly modified ovarian type. Here we study miRNA function in the most primitive, panoistic type of ovaries using the phylogenetically basal insect Blattella germanica (Dictyoptera, Blattellidae) as model.

MicroRNA signatures characterizing caste-independent ovarian activity in queen and worker honeybees (Apis mellifera L.).

Queen and worker honeybees differ profoundly in reproductive capacity. The queen of this complex society, with 200 highly active ovarioles in each ovary, is the fertile caste, whereas the workers have approximately 20 ovarioles as a result of receiving a different diet during larval development. In a regular queenright colony, the workers have inactive ovaries and do not reproduce. However, if the queen is sensed to be absent, some of the workers activate their ovaries, producing viable haploid eggs that develop into males. Here, a deep‐sequenced ovary transcriptome library of reproductive workers was used as supporting data to assess the dynamic expression of the regulatory molecules and microRNAs (miRNAs) of reproductive and nonreproductive honeybee females. In this library, most of the differentially expressed miRNAs are related to ovary physiology or oogenesis. When we quantified the dynamic expression of 19 miRNAs in the active and inactive worker ovaries and compared their expression in the ovaries of virgin and mated queens, we noted that some miRNAs (miR‐1, miR‐31a, miR‐13b, miR‐125, let‐7 RNA, miR‐100, miR‐276, miR‐12, miR‐263a, miR‐306, miR‐317, miR‐92a and miR‐9a) could be used to identify reproductive and nonreproductive statuses independent of caste. Furthermore, integrative gene networks suggested that some candidate miRNAs function in the process of ovary activation in worker bees.

Sequence and expression pattern of the germ line marker vasa in honey bees and stingless bees

Queens and workers of social insects differ in the rates of egg laying. Using genomic information we determined the sequence of vasa, a highly conserved gene specific to the germ line of metazoans, for the honey bee and four stingless bees. The vasa sequence of social bees differed from that of other insects in two motifs. By RT-PCR we confirmed the germ line specificity of Amvasa expression in honey bees. In situ hybridization on ovarioles showed that Amvasa is expressed throughout the germarium, except for the transition zone beneath the terminal filament. A diffuse vasa signal was also seen in terminal filaments suggesting the presence of germ line cells. Oocytes showed elevated levels of Amvasa transcripts in the lower germarium and after follicles became segregated. In previtellogenic follicles, Amvasa transcription was detected in the trophocytes, which appear to supply its mRNA to the growing oocyte. A similar picture was obtained for ovarioles of the stingless bee Melipona quadrifasciata, except that Amvasa expression was higher in the oocytes of previtellogenic follicles. The social bees differ in this respect from Drosophila, the model system for insect oogenesis, suggesting that changes in the sequence and expression pattern of vasa may have occurred during social evolution.