Farnesoic acid O-methyl transferase FAMeT isoformsConserved traits and gene expression patterns related to caste differentiation in the stingless bee Melipona scutellaris.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 67:97–106 (2008) Farnesoic Acid O-Methyl Transferase (FAMeT) Isoforms: Conserved Traits and Gene Expression Patterns Related to Caste Differentiation in the Stingless Bee, Melipona scutellaris Carlos U. Vieira,1* Ana M. Bonetti,1 Zilá L.P. Simões,2 Andréa Q. Maranhão,3 Christiane S. Costa,3 Maria Cristina R. Costa,4 Ana Carolina S. Siquieroli,1 and Francis M.F. Nunes5* Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silico analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity. Arch. Insect Biochem. Physiol. 67:97–106, 2008. © 2007 Wiley-Liss, Inc. KEYWORDS: farnesoic acid O-methyl transferase; caste differentiation; microexon; M. scutellaris INTRODUCTION In arthropods, the mevalonate pathway is responsible for the production of juvenile hormone (JH), a group of sesquiterpenoid compounds with numerous regulatory functions in tissues, developmental stages, and physiological processes (Bellés et al., 2005). In insects, the hydrolysis of farnesyl pyrophosphate results in farnesol, which is oxidized into farnesal and then into farnesoic acid (FA) by a dehydrogenase. Decapod crustaceans produce methyl farnesoate (MF), the immediate precursor to the structurally similar insect JH III, by the mandibular organ that is homologous to the insect corpora allata, the site of JH biosynthesis (Chang, 1993). 1 Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Brasil 2 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brasil 3 Laboratório de Biologia Molecular, CEL-IB, Universidade de Brasília, Brasília, Brasil 4 Curso de Medicina, Universidade de Ribeirão Preto, Ribeirão Preto, Brasil 5 Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brasil Contract grant sponsor: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Contract grant numbers: 99/00719-6; 05/03926-5; Contract grant sponsor: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Contract grant numbers 154827/2006-1; 142034/03-7. *Correspondence to: Carlos U. Vieira, Laboratório de Genética 2E33, Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Campus Umuarama, 38400-902, Uberlândia, MG, Brasil. E-mail: firstname.lastname@example.org; or to: Francis M.F. Nunes, Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brasil. E-mail: email@example.com Received 1 February 2006; Accepted 22 August 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20224 Published online in Wiley InterScience (www.interscience.wiley.com) 98 Vieira et al. In insects, the process of JH biosynthesis was studied in Bombyx mori, where JH acid methyltransferase (BmJHAMT), which contains a conserved Sadenosyl-L-methionine (SAM) binding motif, converts FA and juvenoid acids directly into juvenoid methyl ester cognates such as methyl farnesoate (MF) and JH I, II, and III (Shinoda and Itoyama, 2003). Crustacean farnesoic acid O-methyl transferase (FAMeT, also known as FAOMeT and FAMTase) even without SAM binding motif (Silva Gunawardene et al., 2001; Shinoda and Itoyama, 2003), catalyzes the conversion of FA into MF (Silva Gunawardene et al., 2002). Although enzyme assays have shown that BmJHAMT and crustacean FAMeT can convert FA into MF, these proteins share only about 11% similarity (Shinoda and Itoyama, 2003). Despite the homology between BmJHAMT and Drosophila melanogaster CG17330 gene, other insect sequences share more similarities to crustacean FAMeTs such as the orthologs observed in D. melanogaster CG10527 and Apis mellifera (NCBI-GenBank/ UniGene Dm.11449 and Ame.5015, respectively). Here, we cloned and sequenced two crustaceanlike FAMeT in Melipona scutellaris (Hymenoptera, Apidae) and analyzed gene expression profiles during different developmental phases, in the context of caste differentiation and JH metabolism. Treatment of Larvae With JH III Larvae (3th instar) were topically treated with 0.5 µg of JH III (Sigma, cat. number J2000-synthetic) dissolved in 1 µL acetone (analytical grade, Merck), according to Bonetti et al. (1995). The larvae were kept at 31°C and 80% relative humidity provided by means of a saturated solution of KCl in a desiccator (ASTM, 2002). Larvae used as control were treated with acetone only, and maintained in the same conditions described above. Samples from both groups were collected 1, 2, 3, and 4 h after treatment and stored at –80°C for RNA extraction. Detailed RT-PCR conditions and primer sequences are described below. RNA Extraction and cDNA Synthesis Total RNA was isolated using the TRIzol (Invitrogen) protocol following the manufacturer’s recommendations. The material was resuspended in 0.1% DEPC (diethylpyrocarbonate) treated water, quantified at 260 nm, and its integrity analyzed in 1% agarose gel. To avoid contamination with genomic DNA, the samples were treated with DNase I RNase Free (Sigma). One microgram of each RNA preparation was reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen) and 2 pmol of oligo dT(15), according to the manufacturer’s instructions. MATERIALS AND METHODS Biological Material Amplification of a Putative Melipona scutellaris FAMeT (MsFAMeT) Brood cells containing larvae and pupae of M. scutellaris were obtained from colonies maintained in the Meliponary Uberlândia, MG, Brazil, and transported to the Laboratory of Genetics (Federal University of Uberlândia). The females from the brood cells were staged as follows: white-eyed pupa (Pw), pink-eyed pupa (Pp), brown-eyed pupa (Pb), black-eyed pupa (Pd), and black-eyed pupa with pigmented body (Pdm). Third instar larvae (L3), of unknown sex and caste, were identified according to Dias et al. (2001). All samples were immediately stored at –80°C for RNA extraction. The RT-PCR reactions were carried out in separate tubes, maintaining the same proportions of all reagents and keeping the primer annealing temperature at 55°C for both Actin and MsFAMeT. The kinetic curve indicates 35 cycles as the best number to avoid saturation in PCR reactions to amplify MsFAMeT and Actin (data not shown). One microliter of each cDNA sample was subjected to PCR amplification with a denaturing at 95°C for 5 min, followed by 35 cycles of: 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and a final step of 5 min at 72°C, using 200 µM dNTP, 2 mM Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. FAMeT Isoforms in Melipona scutellaris of MgCl2, 6 pmol of each primer, 1.5 U Taq DNA Polimerase, and buffer 1× (Invitrogen). The MsFAMeT primers were designed on the sequence of Apis mellifera deposited in the GenBank (accession number CK628812; Nunes et al., 2004). In A. mellifera, these primers were used to amplify a 404 base pairs (bp) fragment. Actin primers, designed on the sequence of A. mellifera (accession number AB023025.1), were used as an internal control to show the integrity of the RNA samples and as a housekeeping gene to normalize the relative levels of MsFAMeT transcription, as described by Bitondi et al. (2006). The primer sequences used in the PCR were: MsFAMeT-F: 5′ATGCGTGGATTTTGGATAAGAT-3′, MsFAMeT-R: 5′-CCACCCCAAGCTACATAACAAA-3′, Actin-F: 5′AGCTATGAACTTCCAGATGGT-3′, and Actin-R: 5′CCACATCTGTTGGAAGGT-3′. Assembly of the Entire Coding Region (CDS) of the MsFAMeT The complete CDS of the MsFAMeT was acquired by overlapping one sequence of M. scutellaris fat body 3′ library (unreported data) and 5′ end sequences using CAP3 Contig Assembly Program (Huang and Madan, 1999). The 5′end sequences were obtained by the sequencing of different fragments of the gene using MsFAMeT-F, MsFAMeT-R, and 5′UTR-F (5′-GCTCGTCGCAGTACAAATTG-3′, designed on the 5′ untranslated region of A. mellifera FAMeT predicted gene, accession number XM_623204.2) primers. The purified fragments were inserted into a pGEM®-T Easy Vector System I (Promega), according to the manufacturer’s protocol. Positive clones were sequenced in both directions in MegaBace™ 1000 (Molecular Dynamics, Amersham Life Sciences), performed by the dideoxy sequencing method using M13-forward and M13-reverse universal primers according to the manufacturer’s instructions. Developmental Expression Analysis by RT-PCR The cDNA generated by RT-PCR during the pupal phase and also those obtained after JH treatArchives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. 99 ment (as described above) were analyzed by electrophoresis in 2.5% agarose gel stained with ethidium bromide. The bands’ optical densities (OD) were analyzed and quantified using version 2.0 of the Image Master™ VDS Software from Pharmacia Biosciences. The MsFAMeT/Actin mRNA ratio was used to assess the relative expression of the MsFAMeT gene in each sample. Bioinformatics Analysis Sequence homology searches were performed by BLAST algorithms (Altschul et al., 1990) to compare nucleotide and amino acid against the GenBank and the Baylor A. mellifera Genome Assembly Data version 4.0 (http://www.hgsc.bcm.tmc.edu/projects/ honeybee/). ClustalW (Thompson et al., 1994) were used for multiple FAMeTs ortholog alignments. Conserved motifs were analyzed with Prosite, release 20.2 (Falquet et al., 2002) and homologous domains were analyzed with ProDom, release 2005.1 (Servant et al., 2002). The NetPhos 2.0 server (Blom et al., 1999) was used to analyze post-translational modifications, and the Compute pI/Mw tool was used to calculate the theoretical isoelectric point (pI) and molecular weight (Mw) (Bjellqvist et al., 1993). RESULTS In Silico Sequence Analysis and Microexon Detection Reverse transcription using total RNA from M. scutellaris pupa, followed by electrophoresis of PCR products generated by specific primers (MsFAMeTF and MsFAMeT-R) revealed two cDNA fragments around 400 bp (Fig. 1A), which were cloned and sequenced. The sequences showed 401 and 374 nucleotides (nt). The alignment of these two sequences revealed that the 374 nt sequence lacks 27 nt. The additional 27 nt is a microexon that displays an open reading frame (ORF) of nine amino acids (ICVGGHDRY) as predicted in the A. mellifera XP_623207.1. Based on these results, we 100 Vieira et al. Fig. 1. Expression and sequences of the alternative isoforms of MsFAMeT of M. scutellaris, demonstrating the splicing of a 27 nucleotide microexon. A: RT-PCR products derived from MsFAMeT mRNA in a 2.5% agarose gel stained with ethidium bromide. M = 100 bp marker (Invitrogen); 1 and 2 = amplicons of white-eyed pupa (Pw) and brown-eyed pupa (Pb) workers, respectively. B: Alignment of isoforms 1 and 2 showing the 27 nucleotide gap. C: Nucleotide sequence and deduced amino acids of MsFAMeT gene. The nucleotides of the splice region are underlined and the correspondent amino acids are shaded in grey. can specify two isoforms for MsFAMet, where isoform 1 has the microexon and isoform 2 does not (Fig. 1B). We noted that the inclusion of 27 nt causes a unique amino acid modification preceding ICVGGHDRY, changing an Aspartate (D) in isoform 2 by an Alanine (A) in isoform 1 (Fig. 1C). Assembly and Molecular Characterization of Entire CDS of the MsFAMeT All sequences were assembled using CAP3 resulting in a cDNA contig containing the entire CDS of the MsFAMeT, including the microexon. The Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. FAMeT Isoforms in Melipona scutellaris ORFs of MsFAMeT isoform 1 and 2 are constituted by 777 and 750 nt, respectively, encoding 258 and 249 amino acids (aa) residues. The estimated Mw for MsFAMeTs were 27,653 (isoform 1) and 26,696 (isoform 2), and the calculated pI was 4.53 (isoform 1) and 4.46 (isoform 2). The lengths of the cDNA and deduced amino acid sequences and the estimated Mw of the MsFAMeTs are similar to those obtained for other arthropods FAMeTs: Cancer pagurus (275 aa; 31,114, accession number AAR00732.1), Homarus americanus (276 aa; 31,463, accession number AAA67080.1), Metapenaeus ensis (280 aa; 32,000, accession number AAK28535.1), and Panulirus interruptus (275 aa; 30,973, accession number AAF65551.1). Motif analysis of MsFAMeT isoform 1 revealed three sites for casein kinase II phosphorylation, one for tyrosine kinase phosphorylation, five for Nmyristoylation, one for Asn-glycosylation, and one for ATP/GTP-binding. These results corroborate the phosphorylation sites found in MsFAMeT isoform 1 for Serine (n = 5), Threonine (n = 2), and Tyrosine (n = 6) predicted by NetPhos 2.0 Server, using a score >0.6 as a cut-off. Comparisons using rps- and cdart-BLAST for conservation and architecture of protein domains revealed that the C-terminal portion of MsFAMeT isoform 1 shares similarity with the two consecutive DM9 domains (residues 121 to 185, e-value = 6e-18 and residues 189 to 255, e-value = 9e-20). The DM9 domain (smart00696) is described as repeats found in Drosophila proteins, however without determined function. Search for homologous domains using isoform 1 revealed identity with two protein families: (1) PD293778 (e-value = 1e-47) from residues 120 to 255, corresponding to the region of the DM9 repeat domains described for Drosophila melanogaster, and (2) PD125360 (e-value = 4e-30) from residues 1 to 91, corresponding to the region of the CF domain. The CF domain in the first half of the Nterminal of MsFAMeT has not yet been reported in any database, but its characteristics have been described for crustacean FAMeTs by Holford et al. (2004). The family PD293778 is related to “acid omethyltransferase” (NorMD quality value = 1.019) Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. 101 and the family PD125360 is related to “metal-binding” proteins (NorMD quality value = 0.547). The NorMD value is computed for every ProDom family and scores >0.4 indicate that the alignment is of high quality. The microexon does not codify for characteristic FAMeT domains. The sequences reported in this study have been deposited in the GenBank database [accession numbers AM493718 (isoform 1) and AM493719 (isoform 2)]. Comparative Sequence Analyses BLASTN analyses of MsFAMeT isoform 1 against Expressed Sequence Tags (ESTs) database (dbESTNCBI/GenBank) showed that the best matches corresponded to A. mellifera sequences produced in different cDNA libraries, namely: (1) BI509433 (95% identity; e-value = 5e-134) generated from a Normalized/Subtracted Library prepared from dissected brains of adult worker bees of various ages and various behavioral groups; (2) DB729592 (94% identity; e-value = 2e-133) RIKEN full-length enriched cDNA library from head; (3) BI946505 (94% identity; e-value = 2e-115) generated from different parts of the brain of adult workers using the 5′-library strategy, the unique match annotated as “similar to farnesoic acid O-methyltransferase”; (4) CK633120 (93% identity; e-value = 1e-100) generated from whole body of worker pupae. Similar results were obtained when BLASTN of MsFAMeT isoform 2 was performed against dbEST. The sequences referred to above are grouped by UniGene Ame.5015. Deduced amino acids sequences for MsFAMeTs showed strong similarity to the Group5.20 (e-value = 1e-122 for both isoforms) of the A. mellifera genome version 4.0 when analyzed by TBLASTN. BLASTP analysis showed strong similarity among MsFAMeT protein with A. mellifera XP_623207.1 (e-value = 1e-123) and correlated data compiled under LOC412543–Entrez Gene/NCBI (GeneID: 412543) for A. mellifera, which contains three predicted amino acid sequences from the same gene. Multiple alignments among amino acid sequences of MsFAMeT, the predicted insect FAMeTs (A. mellifera, D. melanogaster, Anopheles gambiae, 102 Vieira et al. MsFAMeT Gene Expression Aedes aegypti, and Tribolium castaneum) and crustacean FAMeTs demonstrated that the nine spliced amino acid microexons occur only in bees. For protein similarities searches in the public databases were used the first 107 amino acids residues of MsFAMeT showing higher degree of conservation with corresponding enzymes in Insecta, from 73% similarity with A. gambiae (1e-32) until 59% with D. pseudoobscura (1e-26). This same region shares around 46–54% similarity with Crustacea (9e-19 to 3e-15) confirming the results obtained by Holford et al. (2004) that pointed out the N-terminal of arthropod FAMeTs as the most conserved portion of the protein (Fig. 2). Given the difficulty in classifying M. scutellaris queens and workers in the larval stages, the expression during the immature phases is not shown. The PCR protocol was used to analyze the MsFAMeT gene expression during representatives stages of workers’ and queens’ development. For cDNA synthesis, one microgram of RNA was used for both workers and queens, in independent experiments. MsFAMeT transcripts occurred throughout pupal development of both castes. In queens, only traces of alternative isoform 1 could be detected in Pw, Pp, and Pb phases, and in workers Fig. 2. N-terminal alignments of MsFAMeT isoform 1 and the following arthropods FAMeTs: Apis mellifera (XP_623207.1), Aedes aegypti (ABF18366.1), Anopheles gambiae (XP_318631.3), Drosophila melanogaster (NP_611544.1), Drosophila pseudoobscura (EAL26493.1), Tribolium castaneum (XP_974395.1), Fenneropenaeus merguiensis (ABA86957.1), Penaeus monodon (ABA86955.1), Litopenaeus vannamei (AAZ22180.1), Cherax quadricarinatus (ABA86959.1), Marsupenaeus japonicus (BAE78496.1), Homarus americanus (AAA67080.1), Metapenaeus ensis (AAK28535.1), Cancer pagurus (AAR00732.1), Thenus orientalis (ABA86962.1), Scylla serrata (ABA86952.1), Portunus pelagicus (AAZ40197.1). Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. FAMeT Isoforms in Melipona scutellaris 103 Here we have cloned and sequenced two FAMeT isoforms in M. scutellaris, which are very similar to ESTs, predicted and experimentally validated sequences in A. mellifera and other insects. However, these enzymes are only moderately conserved in the evolution of arthropods considering the sequence as a whole, but higher similarities are found when the domains are analyzed. In crustaceans, FAMeT is involved in the biosynthesis of methyl farnesoate, which has a role in development, morphogenesis, and reproduction (Silva Gunawardene et al., 2001, 2002, 2003; Ruddell et al., 2003; Holford et al., 2004). In these organisms, FAMeT lacks the signature SAM binding motif present in the JHAMT enzyme of B. mori, indicating that they are not orthologs (Shinoda and Itoyama, 2003). So far, crustacean FAMeTs are the only known proteins with two CF domains in tandem. These domains correspond to approximately 95% of the enzyme’s primary sequence, consisting of conserved hydrophobic residues that may contribute to the binding of farnesoic acid (Holford et al., 2004). We found a strong identity between part of the MsFAMeT sequence and these domains, and there- fore represent the first record of a CF domain in Hymenoptera. The similarities among deduced amino acid sequence and predicted molecular weight of all studied arthropods FAMeTs indicate that they are orthologs, including MsFAMeT. Sequence analyses of the predicted DNA RefSeq relative to A. mellifera FAMeT (GeneID: 412543) indicated the presence of three possible mRNAs, two of which we experimentally found in M. scutellaris. Multiple isoforms of FAMeT are common as recently described by Kuballa et al. (2007) for crustacean species. Nucleotide analysis of the MsFAMeT isoforms indicated that the second band (or isoform 2) was generated by an excision of a microexon. There is a paucity of reports about microexons in the literature. In insects, such splicing has been described for fasciclin I (McAllister et al., 1992), ultrabithorax (O’Connor et al. 1988; Artero et al., 1992), calcium channel alpha1 subunit (Smith et al., 1996; Gallo et al., 2002), and troponin T (Benoist et al., 1998) in the Drosophilidae. Volfovsky et al. (2003) detected only 4 and 14 internal microexons in the Caenorhabditis elegans and D. melanogaster genome, respectively, against 170 in the human genome, indicating that it is an unusual event in invertebrates. Analyses of the protein sequence of MsFAMeT revealed multiple, high-scoring sites for potential phosphorylation side chains. These results are consistent with a previous report that identified several potential phosphorylation sites related to FAMeT function in lobster (Holford et al., 2004) and shrimp (Silva Gunawardene et al., 2002). We Fig. 3. Expression of MsFAMeT gene in queens and workers during pupal development. The alternative transcripts, isoforms 1 and 2, are constitutively expressed in workers. In queens, a modulation of isoform 1 is observed. Pw = white-eyed pupa, Pp = pink-eyed pupa, Pb = brown-eyed pupa, Pd = black-eyed pupa, Pdm = black-eyed pupa with pigmented body. Actin (accession number AB023025.1) was used as a constitutive gene. the two isoforms seem to be constitutively expressed, at least in the stages studied (Fig. 3). The effect of JH III on MsFAMeT expression was analyzed in L3 (larval phase). JH III caused downregulation only in the MsFAMeT isoform 2 transcript until 4 h after treatment (P < 0.05, Student’s t-test, Fig. 4). DISCUSSION Archives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. 104 Vieira et al. Fig. 4. Rapid downregulation in the MsFAMeT isoform 2 expression after topical application of JH III. Third instar larvae were treated with 0.5 µg of JH III/µL in acetone solution. Control samples were treated only with acetone. The expression of MsFAMeT isoforms 1 and 2 was analyzed by semiquantitative RT-PCR (A) and the bands’ optical densities (OD) were quantified separately (B). Actin (accession number AB023025.1) was used as an internal control to normalize the relative levels of MsFAMeT transcription. The results were obtained from the three independent experiments. The application of JH III did not significantly affect the expression of isoform 1 of MsFAMeT. The asterisks (**) indicate a significant difference (P < 0.05, Student’s t-test) between the expression of the isoform 2 transcripts of MsFAMeT, before (control) and after JH III treatment. even found a tyrosine phosphorylation site in the MsFAMeT microexon. These molecular data agree with Benoist et al. (1998), who suggested that posttranslational regulation was associated with products generated by developmentally regulated alternative splicing in troponin T. MsFAMeT transcripts detected during pupal development of M. scutellaris castes showed expression fluctuations and, in some stages, isoform 1 is less expressed. The topical treatment of L3 with JH III inhibited expression of the isoform 2 transcript, indicating that the gene is modulated by JH. The expression of MsFAMeT isoform 2 throughout pupal development of M. scutellaris is coherent with basal levels of JH circulating as observed in A. mellifera (around 10 ng/mL, Elekonich et al., 2003) and M. scutellaris (around 20 ng/mL, Viera CU, unreported data) in the same period. In accordance, Bonetti et al. (2006) found, based on the cellular ultrastructure of corpora allata, signs of synthesis activity in both pupal castes of Melipona quadrifasciata. The dynamic of JH metabolism is crucial during the entire life cycle, justifying the presence of their catabolic and degradation enzymes. The diminished levels of expression of isoform 1 observed in the initial stages of pupal development in queens when compared with the same phases in workers permitted us to suggest that it is a marker of caste differentiation in M. scutellaris. This proposition is according to an alternative funcArchives of Insect Biochemistry and Physiology February 2008 doi: 10.1002/arch. FAMeT Isoforms in Melipona scutellaris tion for MsFAMeT as proposed by Silva Gunawardene et al. (2002) in shrimp. The alternative isoforms of MsFAMeT mRNA showed a singular trait, a microexon splicing, and conserved traits among arthropods orthologs. Moreover, they provide new insights for elucidating female plasticity events associated with differential gene expression in queen/worker differentiation and JH metabolism in the stingless bee, M. scutellaris. Bjellqvist B, Hughes GJ, Pasquali C, Paquet N, Ravier F, Sanchez JC, Frutiger S, Hochstrasser D. 1993. 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