RNA-Seq Reveals That Mitochondrial Genes and Long Non-Coding Rnas May Play Important Roles in the Bivoltine Generations of the N
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Apidologie Original article * The Author(s), 2017. This article is an open access publication DOI: 10.1007/s13592-017-0542-2 RNA-Seq reveals that mitochondrial genes and long non- coding RNAs may play important roles in the bivoltine generations of the non-social Neotropical bee Tetrapedia diversipes Natalia S. ARAUJO, Priscila Karla F. SANTOS, Maria Cristina ARIAS Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Room 320. Rua do Matão, 277—Cidade Universitária, São Paulo, SP 05508-900, Brazil Received 8 March 2017 – Revised 14 August 2017 – Accepted 23 August 2017 Abstract – In animals, voltinism is a result of evolutionary adaptations to environmental conditions. These evolutionary adaptations may profoundly affect the population structure and social organization level. To study the bivoltinism of the solitary bee Tetrapedia diversipes , we performed comparative transcriptomics analyses of foundresses and larvae from the two reproductive generations (G1 and G2) produced per year by this bee. Most of the differentially expressed genes (DEGs) were found between foundresses: 52 DEGs between adults, but only one between the larvae. Among the DEGs in foundresses, 46 were higher expressed in G1 and most of them (38) have no functional annotation defined in the database. Interestingly, mitochondrial genes and long non-coding RNAs were the only type of identified transcripts in the set of upregulated genes. These results highlight the importance of developing studies on non-model species and suggest that maternal genes may be of importance for determining larval diapause in T. diversipes . transcriptome / bivoltinism / diapause / solitary bee / RNA-Seq 1. INTRODUCTION And it may have had profound consequences at the levels of population structure and social orga- The number of reproductive generations pre- nization (Hunt and Amdam 2005). This is espe- sented by a species in a year determines its pattern cially noticeable in species where sociality has of voltinism (Corbet et al. 2006). In insects, the evolved recently, such as in Halictinae bees voltine trait is considered the result of evolution- (Brady et al. 2006). Within the Halictinae, it has ary adaptations to environmental conditions, such been demonstrated that social behavior may be as temperature, humidity, photoperiod, latitude, facultative and directly related to voltine genera- and food resources (Corbet et al. 2006;Altermatt tions (Yanega 1988). Halictus rubicundus fe- 2010; Cardoso and Silveira 2012; Hunt 2012). males, for example, present solitary behavior when individuals are univoltine, but they may become social when individuals are bivoltine Electronic supplementary material The online version of (two generations per year) and enter diapause in this article (https://doi.org/10.1007/s13592-017-0542-2) the second generation (Soucy 2002). Indeed, contains supplementary material, which is available to bivoltinism associated with diapause in one off- authorized users. spring generation is the biological system on ’ Corresponding author: M. Arias, [email protected]. which Hunt s bivoltine ground plan hypothesis Arias, [email protected] on the evolution of insect sociality in temperate Manuscript editor: Klaus Hartfelder climates is based (Hunt and Amdam 2005). N. S. Araujo et al. According to this hypothesis, social behavior for non-model species, as it provides a non- could evolve from solitary species if females from directional and unbiased way of obtaining data the first reproductive generation remain in the nest on the relative frequencies of messenger RNAs and changes in environmental conditions suppress (mRNAs) with no need for species-specific the entry into prepupal diapause of the second probes or a reference genome (Wang et al. 2009). generation (Hunt and Amdam 2005;Hunt2012). Thus, studies of ecological and molecular mecha- 2. MATERIAL AND METHODS nisms involved in voltinism can be important pieces to understand the puzzle of social behavior 2.1. Sample collection in bees and its evolution. In the tropics, bivoltinism has been observed in Wooden trap nests, as described in Alves-dos- different solitary bees (Silveira et al. 2002; Alves- Santos et al. (2002), were placed at the dos-Santos et al. 2007) including Tetrapedia Universidade de São Paulo campus in São Paulo diversipes Klug 1810 (subfamily Apinae, tribe city, Brazil. Foundresses were collected using an Tetrapediini; Michener 2007). This oil-collecting entomological net between 10:00 and 12:00 in bee, native of the Neotropical region, has two front of their nests while constructing. Larvae main reproductive generations during the year, were collected directly from inside the nests that each presenting different developmental times had been completed and closed by the (Alves-dos-Santos et al. 2002). Individuals of the foundresses. All instars (first to fifth) in the non- first generation (G1) show direct development diapause (G1) or prediapause state (G2) were from egg to adults, and they emerge within a sampled. Individuals were immediately frozen in few weeks during the hot and wet months. In liquid nitrogen. G1 samples were collected from contrast, individuals from the second generation November to December (mean temperature (G2) halt their development in the fifth larval 26.4 °C; mean humidity 62.2%) and G2’sfrom instar during the cold and dry season and emerge March to beginning of July (mean temperature as adults only after a diapause period (Camillo 24 °C; mean humidity 59.2%). The larvae were 2005; Alves-dos-Santos et al. 2006). Due to this not sexed prior to pooling for RNA extraction. diapause, the developmental time of G2 individ- uals may be four times longer in comparison to 2.2. RNA extraction and sequencing G1 (Alves-dos-Santos et al. 2002). In addition to this interesting developmental aspect, T. diversipes Total RNA was extracted from the whole body is an attractive species to study because (i) it easily using the RNeasy® Kit (Qiagen) and following nidifies in trap nests (Camillo 2005; Alves-dos- the manufacturer’s protocol. RNA quality was Santos et al. 2006; Menezes et al. 2012; Neves verified by the sequencing facility (Macrogen) et al. 2012; Rocha-Filho and Garófalo 2015), (ii) using a Bionalyzer® system, and results were it has already characterized molecular markers interpreted as discussed in Winnebeck et al. (Arias et al. 2016), and (iii) its genome is currently (2010). Nine foundresses and nine larvae from being sequenced. In combination, all these charac- each generation were selected for RNA sequenc- teristics make T. diversipes a promising emerging ing. Samples were divided into three replicates, model for developmental and evolutionary studies each containing the RNA of three individuals on bees in tropical climates. from the same developmental stage. This sam- In this exploratory study, we report the com- pling approach was adopted to improve gene plete transcriptomes of foundress females and identification and differential expression analyses non-diapause larvae of T. diversipes using next- (Hart et al. 2013; Lin et al. 2016). Although the generation sequencing (RNA-Seq) and these data larval samples contained all developmental were used to search for major differences in the stages, the larvae in each replication were pooled expression profiles of the two reproductive gener- according to their instar—one pool of larvae from ations. This sequencing approach has become the first to fourth instar; one from second to fifth; widely used in gene expression studies, especially and one consisting of fifth instar larvae only. This Genes involved in bivoltinism approach was adopted to focus on the identifica- database (version February 2016; Suzek et al. tion of major differences between the two- 2015). Transcripts with significant blast hits (E generation groups without confounding effects value < 1e−5) against possible contaminants from the larval developmental instars. Altogether, (plants, fungus, mites, and bacteria) in UniRef 12 samples were sequenced, 6 from G1 (3 pools were removed from the final dataset and were also of adults and 3 of larvae) and 6 from G2 (3 pools used to identify other contaminants based on clus- of adults and 3 of larvae), using a HiSeq2000® ter analysis from Corset, as described in Araujo sequencer (Illumina). Paired-end reads of 100 bp et al. (2016). Quality assessments of the final were sequenced, and about 50 million paired assembly were performed with QUAST (version reads were obtained per sample. Sequencing and 4.0; Gurevich et al. 2013), BUSCO (version 2; library preparation were done by Macrogen Simão et al. 2015), and Qualimap (version 2.2; (South Korea). García-Alcalde et al. 2012). 2.3. Transcriptome assembly 2.4. Differential expression analysis Quality assessment of the reads was performed The Trinity script was used to automate the using the FastQC program (version 0.11.2; differential expression analyses using the Bowtie2 Andrews 2010) before and after cleaning. The program (version 2.2.5; Langmead and Salzberg FASTX Toolkit (version 0.0.14; Hannon Lab 2012) to realign all cleaned libraries to the final 2009) was used to trim the first 14 bp of all reads transcriptome; the RSEM program (version because of the initial GC bias (Hansen et al. 1.2.22; Li and Dewey 2011) to count the realigned 2010). Low-quality bases (phred score below 30) reads; and the edgeR package (version 3.14.0; and small reads (less than 31 bp) were removed by Robinson et al. 2009) for the statistical analyses the SeqyClean program (version 1.9.3; (minimum FDR p value < 1e−5). Differential Zhbannikov 2013). expression analyses were performed independent- Prior to assembly, the data from each develop- ly for each developmental stage (adult and larva) mental stage and reproductive generation were by comparing the two reproductive generations digitally normalized (20× coverage) to increase (G1 × G2). assembly efficiency using the Brown et al. (2012) protocol incorporated in Trinity (version 3. RESULTS 2.0.6, Grabherr et al. 2013). Normalized data were independently assembled de novo using Trinity 3.1.