DOI: 10.13102/sociobiology.v65i4.3428Sociobiology 65(4): 766-769 (October, 2018) Special Issue Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 A Scientifc Note on Validation of Housekeeping Genes for the Primitively Eusocial Bee Euglossa viridissima Friese (Apidae: Euglossini) One of the most crucial factors for studies of gene expression of target genes is the procedure of normalization (Kubista et al., 2006). For this purpose, reference genes (or house-keeping genes) are used, which are assumed to be constitutively expressed and hence their expression is expected to be stable and constant irrespective of developmental stage or any other experimental manipulation. Studies of candidate gene expression, using quantitative real time PCR, often focus on the evaluation of differences in gene expression amongst developmental stages or within a stage amongst different tissues (Lockett et al., 2016). Candidates for suitable reference genes have been identified in several species of the Apidae, e.g. honeybees (Lourenço et al., 2008), bumble bees (Horňáková et al., 2009; Niu et al., 2014), and stingless bees (Dallacqua et al., 2007). However, reference genes have not been described for orchid Abstract Studies on the expression of genes in different contexts are essential to our understanding of the functioning of organisms and their adaptations to the environment. Gene expression studies require steps of normalization, which are done using the stable expression pattern of reference genes. For many different eusocial bees reference genes have been discovered, but not for the primitively eusocial Euglossini bees. We used available genomic resources of Euglossini species and the gene information of Apis mellifera Linnaeus to develop a set of reference genes for the primitive eusocial bee Euglossa viridissima Friese. We tested nine genes, in distinct developmental stages, using three different algorithms, to infer stability of gene expression. The TATA-binding protein (TBP) and 14-3-3 epsilon were the most stable genes across all developmental stages. The strongest deviation in gene expression pattern occurred in pupae, which require a different set of genes for normalizing gene expression. Sociobiology An international journal on social insects S Boff1,2, A Friedel1, A Miertsch1, JJG Quezada-Euán3, RJ Paxton1,4, HMG Lattorff1,4,5 Article History Edited by Cândida Aguiar, UEFS, Brazil Received 09 May 2018 Initial acceptance 08 June 2018 Final acceptance 24 July 2018 Publication date 11 October 2018 Keywords Developmental stage, gene expression, orchid bees, gene normalization. Corresponding author Samuel Boff Facoltá Scienze Agrarie e Alimentari Università Degli Studi di Milano Via Celoria 2, 2133, Milano, Italy. E-Mail: samboff@gmail.com bees, although transcriptome and genome data are available for a few of these species (Woodard et al., 2011; Kapheim et al., 2015; Brand et al., 2017). Here we identified a set of candidate reference genes for the orchid bee Euglossa viridissima Friese based on the transcriptome reference for Euglossa cordata (Linnaeus) (Woodard et al., 2011) and annotated genes of Apis mellifera Linnaeus (Elsik et al., 2015). We tested for stability of gene expression in individuals across different developmental stages (including adults). We choose nine candidate reference genes, namely Ribosomal protein 28S (28S), Actin, Arginine Kinase (ArgK), Elongation Factor 1a (EF-1a), 14-3-3 epsilon, Glutathione S-transferase-1 (Gst1), Inositol 1,4,5-triphosphate receptors (Itpr), Ribosomal protein S18 (Rps18) and TATA-binding protein (TBP). For ArgK we used two different primer pairs, 1 - Institut für Biologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany 2 - Dipartimento di Scienze per gli Alimenti la Nutrizione, l’Ambiente, Università Degli Studi di Milano, Milano, Italy 3 - Departamento de Apicultura Tropical, Campus Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida-Xmatkuil, Mexico 4 - German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany 5 - International Centre of Insect Physiology and Ecology (icipe), Nairobi, Kenya SHORT NOTE Sociobiology 65(4): 766-769 (October, 2018) Special Issue 767 ArgK and ArgK_mod. TATA-binding protein (TBP) is often considered stable in gene expression studies and is useful for gene normalization (Yüzbaşioğlu et al., 2010). This gene is related to RNA polymerase II transcription factor. The 14-3-3 epsilon protein is abundantly expressed in the central nervous system (CNS), modulating the activity of a number of kinases and ion channels in humans (Berg et al., 2003). Although the gene 14-3-3 epsilon is linked to several functions in flies (e.g. embryonic hatching, germ cell migration, gonad formation, wing venation and eye development, according to Flybase), this gene was found to be consistently expressed in queens and workers of honeybees (Santos & Hartfelder, 2015) providing evidence for its suitability for gene normalization. Raw reads of the E. cordata transcriptome (Woodard et al., 2011) were mapped against the nucleotide sequences of A. mellifera representing the respective housekeeping genes using CLC Bio Genomics Workbench v7.5 (CLC Bio, Arhus, Denmark). Read mapping was performed using the large gapped read mapping algorithm followed by local realignments. Finally, consensus sequences were extracted and transferred to Primer3 (Untergrasser et al., 2012), which was used with default settings, except that the target product size was set at a maximum of 150 bp. An exception was Actin with 271 bp (see Locke et al., 2012). Primer sequences can be found in Table 1. A QIAxcel capillary electrophoresis system (accuracy to 3 bp) was used to confirm PCR product sizes based on the expected size. RNA was extracted from whole bodied larvae (n = 9) and pupae (n = 8) and the abdomens from adult bees (n = 9) that were collected from14 nests (Department of Apiculture, UADY, México). With exception of larvae, bees were morpho- logically sexed, and only females were used for our analysis. Gene stability was based on RefFinder. This software compares and ranks the results from four different algorithms (BestKeeper, Delta CT, geNorm and Normfinder) and reports stability of the gene based on standard deviations of Ct values (Xie et al., 2012). Since the BestKeeper algorithm uses the original Ct values as input assuming normality of the data (although they are logarithmic in nature), it seems to indicate false differences (Livak & Schmittgen, 2001; Khanlou & Bockstaele, 2012; Chen et al., 2016). Here, we did not use BestKeeper in our analysis. The 10 primers designed to amplify 9 reference genes (Table 1) each produced a single product of the expected size. The PCR efficiency for the primers ranged from 85 - 93.5% (mean ± s.d. = 1.78 ± 0.06). For larvae, the stability value (sv) of Rps18 (1.68) and 14-3-3 epsilon (1.73) were smallest and thus, the most stable reference genes (Fig 1a) indicated by the comprehensive ranking analysis. In pupal samples, ArgK, supported by both pairs of primers, Argk (1.41) and Argk_mod (1.86), and Actin (4.12) were the most stable genes (Fig 1b). In adult samples, the gene Gst1 (1.86) was the most stable, followed by TBP (2.55) (Fig 1c). When larvae, pupae and adults were analysed together, TBP (1.48) was the most stable gene, followed by 14-3-3 epsilon (2.78) (Fig 1d). Table 1. Details of the reference genes designed developed and tested in samples of Euglossa viridissima. Actin, however, was not designed in this study (see Locke et al., 2012 ). bp = base pairs. F = forward primer sequence and R = reverse primer sequence. AT = annealing temperature. Gene name and symbol Amplicon size (bp) Sequence AT Molecular Function Actin 271 F- CGTGCCGATAGTATTCTTG R- CTTCGTCACCAACATAGG 60 Motility, morphological changes, cell division and intracellular movementφ Arginine kinase (Argk) 138 F-CCCAGCTGGTGAATTCATCG R-TCCAAACCAGACAGAGTGCT 60 Arginine kinase activity⊗ Arginine kinase modified (Argk_mod) 143 F-ATGCTCCCGACGCTGAATC R-GGTCAAGATTGCCAAGGGA 60 Arginine kinase activity⊗ Elongation factor F1 (EF1a) 176 F-CCAATTTCTGGTTGGCACGG R-AGAGGAAGACGGAGAGCCTT 58 Catalyzes the delivery of aminoacyl-tRNA to the ribosome in a GTP-dependent manner⊕ 14-3-3 epsilon 118 F-ATAAGGGCGTCGAGAGGAAG R-CACGGGAGCAGATGTTTGTC 60 Protein binding, Protein heterodimerization activity⊗ glutathione S-transferase-1 (Gst1) 141 F-TCCAAAGAAACGTGCACTCG R-GCCGTGCCAATACTTTCGAA 58 DDT-dehydrochlorinase activity and glutathione transferase activity⊗ Inositol 1,4,5-triphosphate receptor (Itpr) 85 F-TGGGTGACACTCCTTCTTCA R-TGTCGTGCGTTTCATCAAGA 58 Inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity⊗ Ribosomal Protein S18 (Rps18) 87 F-ATTCTTCGTGTCATGGGCAC R-GTACCGACGACCTACACCTT 60 RNA binding and structural constituent of ribosome⊗ Ribosomal protein (28S) 139 F-CTCCCCTAGTAGAACGTCGC R-CGACCTGATACCGTACGAGT 60 rRNA endonuclease activity and rRNA N-glycosylase activity∗ TATA-binding protein (TBP) 150 F-GGGTGAAGAAGATGGTGCAG R-ACACCGACCAAATCTCCAGG 60 General RNA polymerase II transcription factor⊗ Information source: *Gene Ontology (http://geneontology.org/), FlyBase (http://flybase.org/), φHennessey et al., 1993, ⊕Marygold et al., 2017). S Boff, A Friedel, A Miertsch, JJG Quezada-Euán, RJ Paxton, HMG Lattorff – Housekeeping genes for Euglossa768 Out of nine potential endogenous genes, we suggest two (TBP and 14-3-3 epsilon) to be used for controlling gene normalization and for assessing relative gene expression of target genes. It is important to state that another set of genes (TBP and Rps18) was considered stable for immature samples. This may imply that alternatively, Rps18 has reasonable applicability to analyze gene expression in non-adult bees. Although, groups of most stable genes varied amongst different developmental stages, the genes TBP and 14-3- 3 epsilon were among the three most stable in three of four (larvae, adult and all samples analyzed together) sets that we tested (see Fig 1). Thus, it might be of benefit to use these housekeeping genes to study gene expression in primitively eusocial species like Euglossini bees, especially because recent studies showed that there are major dissimilarities between ages and environment (Lockett et al., 2016), in different tissues (e.g. brain and abdomen), and between solitary and eusocial bee species with respect to transcriptional regulation (Jones et al., 2017; Kapheim et al., 2015). Fig 1. Stability values (Geomean ranking values) of 9 reference genes according to RefFinder. The most stable genes, those with lower values, are listed on the left and the least stable genes on the right. (a) larvae, (b) pupae, (c) adult (abdomen of females) and (d) all of individuals combined. Epsilon = 14-3-3 epsilon; Argkm = Argk_mod. Acknowledgement We thank anonymous referees, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and the program Science without Borders Brazil (Ciências sem Fronteiras) for financial support (SB). Supplementary Material DOI: 10.13102/sociobiology.v65i4.3428.s2233 Link: http://periodicos.uefs.br/index.php/sociobiology/rt/ suppFiles/3428/0 Authors’ Contribution SB, RJP conceived and designed the study; JJQE provided samples. SB, HMGL designed the primers. SB, AF, AM performed experiments and analysis; SB, HMGL analyzed the results. SB, HMGL led the writing. All authors read, discussed and approved the final manuscript. Most stable genes Least stable genes a b c 0 2 4 6 8 10 Rp s1 8 Ep sil on Tb p Ac tin 28 S EF 1a Gs t1 Itp r Ar gk Ar gk m G eo m ea n of ra nk in g va lu es 0 2 4 6 8 10 12 Ar gk Ar gk m Ac tin Rp s1 8 Tb p EF 1a Itp r Gs t1 Ep sil on 28 S G eo m ea n of ra nk in g va lu es 0 1 2 3 4 5 6 7 8 9 Gs t1 Tb p Ep sil on Ac tin Rp s1 8 Ar gk m Ar gk EF 1a 28 S Itp r G eo m ea n of ra nk in g va lu es 0 2 4 6 8 10 Tb p Ep sil on Ac tin EF 1a Gs t1 Itp r Ar gk _m od Ar gk Rp s1 8 28 S G eo m ea n of ra nk in g va lu es Most stable genes Least stable genes d Ar gk mTB P TB P TB P TB P Sociobiology 65(4): 766-769 (October, 2018) Special Issue 769 References Berg, D., Holzmann, C., & Riess, O. (2003). 14-3-3 proteins in the nervous system. Nature Reviews Neuroscience, 4: 752. doi: 10.1038/nrn1197 Brand, P., Saleh, N., Pan, H., Li, C., Kapheim, K.M., & Ramírez, S.R. (2017). The nuclear and mitochondrial genomes of the facultatively eusocial orchid bee Euglossa dilemma. 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