Exploring integument transcriptomes, cuticle ultrastructure, and cuticular hydrocarbons profiles in eusocial and solitary bee species displaying heterochronic adult cuticle maturation
Authors: Falcon Tiago , Pinheiro Daniel G. , Ferreira-Caliman Maria Juliana , Turatti Izabel C. C. , Pinto de Abreu Fabiano C. , Galaschi-Teixeira Juliana S. , Martins Juliana R. , Elias-Neto Moysés , Soares Michelle P. M. , Soares Michelle P. M. , Laure Marcela B. , Figueiredo Vera L. C. , Lopes Norberto Peporine , Simões Zilá L. P. , Garófalo Carlos A. , Bitondi Márcia M. G. 
Published: October 2018

AbstractDifferences in the timing of exoskeleton melanization and sclerotization are evident when comparing eusocial and solitary bees. This cuticular maturation heterochrony may be associated with life style, considering that eusocial bees remain protected inside the nest for many days after emergence, while the solitary bees immediately start outside activities. To address this issue, we characterized gene expression using large-scale RNA sequencing (RNA-seq), and quantified cuticular hydrocarbon (CHC) through gas chromatography-mass spectrometry in comparative studies of the integument (cuticle plus its underlying epidermis) of two eusocial and a solitary bee species. In addition, we used transmission electron microscopy (TEM) for studying the developing cuticle of these and other three bee species also differing in life style. We found 13,200, 55,209 and 30,161 transcript types in the integument of the eusocialApis melliferaandFrieseomelitta varia, and the solitaryCentris analis, respectively. In general, structural cuticle proteins and chitin-related genes were upregulated in pharate-adults and newly-emerged bees whereas transcripts for odorant binding proteins, cytochrome P450 and antioxidant proteins were overrepresented in foragers. Consistent with our hypothesis, a distance correlation analysis based on the differentially expressed genes suggested delayed cuticle maturation inA. melliferain comparison to the solitary bee. However, this was not confirmed in the comparison withF. varia. The expression profiles of 27 of 119 genes displaying functional attributes related to cuticle formation/differentiation were positively correlated betweenA. melliferaandF. varia, and negatively or non-correlated withC. analis, suggesting roles in cuticular maturation heterochrony. However, we also found transcript profiles positively correlated between each one of the eusocial species andC. analis. Gene co-expression networks greatly differed between the bee species, but we identified common gene interactions exclusively between the eusocial species. Except forF. varia, the TEM analysis is consistent with cuticle development timing adapted to the social or solitary life style. In support to our hypothesis, the absolute quantities of n-alkanes and unsaturated CHCs were significantly higher in foragers than in the earlier developmental phases of the eusocial bees, but did not discriminate newly-emerged from foragers inC. analis. By highlighting differences in integument gene expression, cuticle ultrastructure, and CHC profiles between eusocial and solitary bees, our data provided insights into the process of heterochronic cuticle maturation associated to the way of life.Author SummaryFrom our previous observation that bees with distinct habits of life, eusocial and solitary, exhibit different degrees of cuticle melanization and sclerotization at the emergence, we decided to analyze the genetic signatures and ultrastructure of the integument, as well as the CHC profiles that could be involved in cuticle maturation. The expression patterns of certain genes involved in the melanization/sclerotization pathway, chitin metabolism, cuticle structure, and also regulators of cuticle renewal and tanning, in addition to other genes, might be grounded the slow process of cuticle maturation in the eusocial bees in comparison to the solitary ones. The electron micrographs revealed differences in the timing of cuticle deposition for the eusocial and solitary species. Among the identified CHCs, the proportions and quantities of n-alkanes in the developing cuticle are consistent with the faster cuticular maturation in the solitary bee, thus supporting our hypothesis.