Research Article: Exploring integument transcriptomes, cuticle ultrastructure, and cuticular hydrocarbons profiles in eusocial and solitary bee species displaying heterochronic adult cuticle maturation

Date Published: March 14, 2019

Publisher: Public Library of Science

Author(s): Tiago Falcon, Daniel G. Pinheiro, Maria Juliana Ferreira-Caliman, Izabel C. C. Turatti, Fabiano C. Pinto de Abreu, Juliana S. Galaschi-Teixeira, Juliana R. Martins, Moysés Elias-Neto, Michelle P. M. Soares, Marcela B. Laure, Vera L. C. Figueiredo, Norberto Peporine Lopes, Zilá L. P. Simões, Carlos A. Garófalo, Márcia M. G. Bitondi, Herman Wijnen.


Differences 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 eusocial Apis mellifera and Frieseomelitta varia, and the solitary Centris 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 in A. mellifera in comparison to the solitary bee. However, this was not confirmed in the comparison with F. varia. The expression profiles of 27 of 119 genes displaying functional attributes related to cuticle formation/differentiation were positively correlated between A. mellifera and F. varia, and negatively or non-correlated with C. analis, suggesting roles in cuticular maturation heterochrony. However, we also found transcript profiles positively correlated between each one of the eusocial species and C. analis. Gene co-expression networks greatly differed between the bee species, but we identified common gene interactions exclusively between the eusocial species. Except for F. 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 in C. 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.

Partial Text

The exoskeleton (cuticle) enables insects to exploit a multitude of ecological habitats, and is central to their evolutionary success and worldwide expansion. It is necessary for muscles attachment, and for protection against predators, injuries, and pathogens [1]. In addition, its thickness is positively correlated with the resistance to some types of insecticides [2]. The exoskeleton is periodically shed and a new, larger one is formed, this characterizing the successive molting episodes that allow for insect growth and development. Its composition is defined by the secretion of products synthesized by the epidermis as well as by the uptake of molecules from other sources, for instances, hemolymph [3]. These products are used for cuticle renewal at each molting episode coordinated by changes in the titer of 20-hydroxyecdysone (20E), the active product of ecdysone hydroxylation. The Ashburner model postulated to explain 20E-induced chromosomal puffs in the larval salivary glands of D. melanogaster have ultimately led to the knowledge of molecular elements regulating molting and metamorphosis [4]. When 20E binds to the heterodimeric receptor consisting of EcR (Ecdysone receptor) and Usp (Ultraspiracle) proteins, its trigger a transcription factor regulatory cascade. Upstream elements of this cascade respond to the high 20E titer that also induces apolysis and initiates molting, whereas most downstream elements are only induced by the subsequent decrease in 20E titer. Binding sites for several of the transcription factors in this cascade were identified in many cuticular protein genes [5], suggesting that they, and other genes involved in cuticle remodeling [6, 7] are indirectly regulated by 20E.

The RNA-seq analysis revealed the set of genes expressed in the integument of three bee species, and also the changes in gene expression as the adult cuticle is deposited and differentiates in a mature and fully functional cuticle. For A. mellifera, for which we have the sequenced genome, the genes expressed in the integument represented 95.07% of the genes in the released genome assembly version 4.5. Similar proportions will likely be found for F. varia and C. analis in the near future, after the sequencing of their respective genomes. Selected genes with potential roles in cuticle formation and maturation were characterized in terms of differential expression profiles. Co-expression networks were reconstructed. In parallel, we examined the ultrastructure of the developing adult cuticle of bee species. Furthermore, the CHC composition of the envelope, the less known cuticle layer, was also characterized. Our data expanded the knowledge on the insect integument. It is our expectation that the obtained data provide a valuable resource for future studies on exoskeleton formation and maturation in insects.

Using RNA-seq analysis of the integument of two eusocial bee species, A. mellifera and F. varia, and a solitary bee, C. analis, we identified genes involved in cuticle (exoskeleton) formation and maturation. The expression profiles of these genes were determined at three developmental time points corresponding to adult cuticle deposition/differentiation at the pharate-adult stage, newly-ecdysed cuticle, and fully developed cuticle of forager bees. TEM analysis of the cuticle at these time points, including other bee species, and CHC profiles determination were performed in addition to the transcriptome analysis. Together, these experimental approaches provided novel data on integument developmet. We also searched for clues in integument gene expression, structure, and CHC profiles that could be consistent with the premise that eusociality might have entailed heterochronic changes in cuticle development, resulting in faster cuticle maturation in the solitary bee, thus allowing flight and forager activities immediately after emergence, and in slow cuticle maturation in the eusocial bees, which benefit from the protected nest environment for a period of time after the emergence. The RNA-seq analysis highlighted differences in gene expression, GO functions, and gene co-expression networks that are consistent with our hypothesis. The results obtained with cuticle ultrastructure analysis in the solitary, primitively eusocial, and facultatively eusocial species in comparison to the eusocial A. mellifera, as well as cuticular n-alkanes absolute quantifications in A. mellifera, F. varia, and C. analis, also revealed interesting differences, which are consistent with cuticle maturation heterochrony. The distance correlation analysis based on RNA-seq data only partially supports our hypothesis. For F. varia, the results of this analysis, as well as the micrographs obtained from cuticle ultrastructure analysis, are in contrast to our hypothesis. This study expands our understanding on the molecular biology and structure of the developing integument, besides highlighting differences in the process of cuticle maturation related to the eusocial/solitary behaviors.

The bee samples that we collected are in agreement with Brazilian laws. A license is not required for exogenous species collection. Native bees were sampled under the register: Ministério do Meio Ambiente—MMA, Instituto Chico Mendes de Conservação da Biodiversidade–ICMBio, Sistema de Autorização e Informação em Biodiversidade–SISBIO, license number: 41883–1; authentication number: 71367685; and license number: 41883–2; authentication number: 39944978.




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