Insights into the dynamics of hind leg development in honey bee (<i>Apis mellifera</i> L.) queen and worker larvae - A morphology/differential gene expression analysis

Santos, Carolina Gonçalves; Hartfelder, Klaus

doi:10.1590/S1415-475738320140393

Carolina Gonçalves Santos

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Klaus Hartfelder Send correspondence to Klaus Hartfelder. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brazil. E-mail: klaus@fmrp.usp.br.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Send correspondence to Klaus Hartfelder. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brazil. E-mail: klaus@fmrp.usp.br.

Figures (4)
Tables (3)

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Figure 1
Hind leg imaginal discs development in honey bee larvae. (A–B) In the fourth larval instar (L4,) the disc occupies the peripodial sac (PS and arrow in A). Mesenchymal cells (MC) are observed inside the disc, which is covered by the larval cuticle (C). (C–E) During the feeding stage of the fifth instar (L5F) the discs grow considerably and the leg compartments start to become apparent, but the epithelium (E) remains unchanged. Muscles cells (M) are adjacent to the epithelium where they will establish future connections. (F) In the legs of early spinning stage (L5S) larva, segmentation is more apparent, but the epithelium still remains the same. A, B - L4 queen; C - L5F1 worker; D - L5F2 worker; E - L5F3 worker; F, L5S1 queen.; C, cuticle; E, epidermis; FB, fat body; MC, mesenchymal cells; M, muscle; PS, peripodial sac; Tr, trachea; TG, thoracic ganglion. Scale bars: A, C, D, E, 100 μm; B, 50 μm; F, 200 μm.

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Figure 2
Cell proliferation detection in the hind leg imaginal discs from queens and workers by anti-phospho-histone H3 immunofluorescence (yellow). Cell proliferation was evidenced during all stages analyzed, but it appears to be more prominent during the feeding stage. (A) L5F2 queen; (B) L5F3 queen; (C) L5F2 worker; (D) negative control. Scale bars: A, B, D, 100 μm; C, 50 μm.

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Figure 3
Characteristics of the queen and worker RDA libraries prepared from queen and worker larval hind leg discs. The assembled sequences were grouped into (i) genes similar (E ≤ e⁻⁵)to Drosophila melanogaster, (ii) genes similar to predicted or exclusive transcripts for A. mellifera and other bees represented in the Transcriptome Shotgun Assembly (TSA) database, (iii) genes similar to stem-loop microRNAs (miRBase), (iv) genes with intronic location, (v) genes with a putative UTR location, and (vi) sequences representing unpredicted genes but mapping to the honey bee genome sequence.

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Figure 4
Relative expression levels measured by RT-qPCR of genes selected from the RDA libraries. Each whisker plot represents worker gene expression relative to queens, the latter set at equal to 1. The plot was generated by REST analysis (Pfaffl et al., 2002Pfaffl MW, Horgan GW and Dempfle L (2002) Relative Expression Software Tool (REST) for group-wise comparison and statistical analysis of relative expression results in Real-Time PCR. Nucleic Acids Res 30:e36.). Black asterisks represent genes validated as overexpressed in queens and white ones those overexpressed in workers (random test, REST, p ≤ 0.05).

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Table 1
Primers used in quantitative RT-PCR analyses. Gene names and gene IDs are those attributed in BeeBase Official Gene Set v3.2. Unpredicted genes are named by the genomic scaffold numbers where the respective transcripts were mapped.

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Table 2
Unique sequences assembled from expressed sequence tags of the queen imaginal discs RDA library. Listed are genes with similarity to Drosophila melanogaster genes or stem-loop micro RNAs. The genomic scaffold (Group in Amel_4.5) and predicted CDS (GB number of Official Gene Set v3.2). Gene Ontology terms for Biological Process and Molecular Function are from Flybase and given as inferred from electronic annotation (IEA), inferred from sequence or structural similarity (ISS), inferred from physical interaction, (IPI), or inferred from direct assay (IDA).

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Table 3
Unique sequences assembled from expressed sequence tags of the worker imaginal discs RDA library with similarity to Drosophila sequences or stem-loops micro RNAs. Listed are the genomic scaffold (Group in Amel_4.5) and predicted CDS (GB number of Official Gene Set 3). Gene Ontology terms for Biological Process and Molecular Function are from Flybase and given as inferred from electronic annotation (IEA), inferred from sequence or structural similarity (ISS), inferred from physical interaction, (IPI), or inferred from direct assay (IDA).

Gene name	Gene ID	Primer sequence	Product size
tenectin	GB53632	5′ TGCGCCCGTTTACGACCAGG 3′ 5′ ACGGGCAAACCCTGAGGTAGCA 3′	176 bp
headcase	GB50117	5′ GCGCTTCGGTAGTACCACGACC 3′ 5′ ACCAGGAACGGGGAATGCGC 3′	164 bp
bowl	GB49901	5′ CGGTGGAGGACCAGTCAGATGAGG 3′ 5′ TGGTCGCTGGATTGGGCCAC 3′	125 bp
NDRG3-like	GB54474	5′ GGCCGCCGCCCTAGAAATGG 3′ 5′ TCGACGCTCGCTATGGAAACG 3′	169 bp
Maternal gene required for meiosis	GB50425	5′ GCTCACGGTACTTGAGCGAC 3′ 5′ GGAGTTGATGGGTGTGGATG 3′	125 bp
Glutaredoxin-like	GB52955	5′ TGGACGAGGTCACCACGATGA 3′ 5′ ACCGTTGCTCCGGCCCCTGT 3′	145 bp
Grainy head	GB46725	5′ CTCTCGACGAGCTCAAATCC 3′ 5′ AACCAACAGTCCCACAGTCC 3′	145 bp
14-3-3ε	GB42560	5′ CCTGATAGAGCATGTCGTCT 3′ 5′ CCTTGCATGTCTGACGTCC 3′	149 bp
Immunoglobulin-like and fibronectin	GB44117	5′ TACCCGACCCATCCTCATTA 3′ 5′ GATAGGGTAGACCGGACACG 3′	150 bp
C-type lectin 1	GB49260	5′ CGGACGTGGAAAAGGGGAACGAAGG 3′ 5′ ACACAAGTTGAACATTCGCGCCG 3′	193 bp
SCAR	GB47014	5′ CCTCCTCCTCCTGGTGAAAC 3′ 5′ GGTGGGGTGGGTGTGGAGTG 3′	128 bp
gustavus	GB55797	5′ CGATACAACACGCGACACAC 3′ 5′ GTACGAACGAGCGAAAAACG 3′	163 bp
troponin	GB55598	5′ TCCGAGAAGGCAGAGTGGCAG 3′ 5′ GCGCGGTTCGGTTGTTGCAC 3′	174 bp
Group 15.19	GB50099	5′ GACGAGACCAGCGACGTTTC 3′ 5′ GATCTTCTTCTTCTTCGTCTTGGTC 3′	120 bp
Group13.5	unpredicted	5′ AGCCGCGATAGAGAGAGGCG 3′ 5′ GGGAAGAAAGGGCGAGGGGG 3′	147 bp
Group3.1	unpredicted	5′ GAGACGTCGGGGTCGTTGTCG 3′ 5′ CCTTCTCGAGGATGTCTCTCTCCC 3′	127 bp
Group2.14	unpredicted	5′ AGACTCGTCCCCTGTCACC 3′ 5′ CTCGACGACTCTTTCTGCTCG 3′	154 bp
Group1.37	unpredicted	5′ TCATAAGCCACGCGAATACC 3′ 5′ AGGCAGACACGTGAATACGC 3′	146 bp
Group2.11	unpredicted	5′ TTGCCGACTTCCCTTACCTAC 3′ 5′ ACCTTGAAAATCCCGGAGAG 3′	124 bp

Group	GB	Blast X (CG)	miR base	Molecular function	Biological process
Group1.3	GB50376	CG33197		metal ion binding^IEA	apoptotic process, embryo development, muscle organ development, regulation of gene expression, compound eye photoreceptor cell differentiation
Group1.40	GB49260	CG43164		unknown	unknown
Group3.4	GB49076	CG8671		unknown	dsRNA transport
Group3.8	GB55797	CG2944 gustavus		protein binding^IPI	dorsal appendage formation, germ cell development, oocyte anterior/posterior axis specification
Group3.9	GB53632	CG13648 tenectin		integrin binding^IDA	embryonic hindgut morphogenesis, imaginal disc-derived wing morphogenesis, epithelial tube morphogenesis
Group3.15	GB47014	CG4636 SCAR		protein binding^IPI, actin binding^IEA	actin cytoskeleton organization, cell adhesion mediated by integrin, compound eye morphogenesis
Group4.1	GB49650	CG30497		unknown	unknown
Group4.1	GB50425	CG45477 maternal gene required for meiosis		chromatin binding^IDA, sequence-specific DNA binding^IDA, metal ion binding^IEA	female meiotic division, regulation of chromatin organization, regulation of transcription from RNA polymerase II promoter
Group5.15	GB47845	CG2302 Nicotinic Acetylcholine Receptor alpha 7E		acetylcholine receptor activity^IDA	cation transport
Group6.10	GB54474	CG15669 Misexpression suppressor of KsR2		unknown	unknown
Group6.14	GB52211	CG5215 Zinc-finger protein at 72D		mRNA binding^ISS, zinc ion binding^IEA	phagocytosis, mRNA splicing, via spliceosome
Group7.9	GB42560	CG31196 14-3-3ε		protein binding ^IPI, protein heterodimerization activity^IPI	determination of adult lifespan, DNA damage checkpoint, germarium-derived oocyte fate determination, regulation of mitosis; wing disc dorsal/ventral pattern formation
Group10.23	GB48399	CG6890 Tollo		transmembrane receptor activity^ISS	innate immune response in mucosa, peripheral nervous system neuron development, regulation of protein glycosylation
Group10.25	GB52624	CG17077 pointed		protein binding^IPI, repressing transcription factor binding^IPI	anatomical structure development; localization; post-embryonic organ morphogenesis; anterior/posterior axis specification
Group11.16	GB47245	CG33141 sticks and stones		protein binding^IPI	actin cytoskeleton organization, compound eye morphogenesis, myoblast fusion, nephrocyte filtration, spermatid development
Group11.18	GB44976	CG14029 vrille		RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity involved in negative regulation of transcription^IDA	circadian rhythm; bristle morphogenesis; imaginal disc-derived wing hair organization
Group11.18	GB44972	CG17484 Adherens junction protein p120		cadherin binding^IEA	adherens junction maintenance; cell adhesion; compound eye morphogenesis
Group13.12	GB49901	CG10021 brother of odd with entrails limited		metal ion binding ^IEA; nucleic acid binding ^IEA, sequence-specific DNA binding RNA polymerase II transcription factor activity^ISS	imaginal disc-derived leg morphogenesis, lateral inhibition, embryonic foregut morphogenesis, embryonic hindgut morphogenesis
Group13.12	GB49872	CG6831 rhea		integrin binding^IPI, protein binding^IPI	apposition of dorsal and ventral imaginal disc-derived wing surfaces, border follicle cell migration, cell adhesion, muscle attachment, regulation of cell shape
Group14.14	GB52743	CG10847 encore		nucleic acid binding ^IEA	germarium-derived oocyte fate determination; cystoblast division; oogenesis; germarium-derived egg chamber formation
Group14.15	GB41625	CG13676		chitin binding^IEA	chitin metabolic process
Group15.19	GB50099	CG33521		zinc ion binding^IEA	unknown
Group15.19	GB50117	CG15532 headcase		unknown	RNA interference; axon extension involved in development; neurogenesis
Group16.2	GB54355	CG5654 ypsilon schachtel		DNA binding^IEA	mRNA splicing, via spliceosome, oogenesis, regulation of transcription, DNA-template
Group16.8	GB45968	CG4145 Collagen type IV		extracellular matrix structural constituent^IEA	cardiac muscle cell development, oviduct morphogenesis
Group1.20			osa-MIR5149
Group1.37			sbi-MIR396c
Group3.3			mmu-mir-466f-3
Group8.21			sbi-MIR396c
Group16.2			smo-MIR1082a

Group	GB	Blast X (CG)	miR base	Molecular function	Biological process
Group1.40	GB49260	CG43164		unknown	unknown
Group2.19	GB55598	CG7178 wings up A		unknown	cardiac muscle tissue development, nervous system development, nuclear division
Group3.8	GB55828	CG9160 NADH dehydrogenase (ubiquinone) acyl carrier protein		ACP phosphopantetheine attachment site binding involved in fatty acid biosynthetic process^ISA	fatty acid biosynthetic process, mitochondrial electron transport, NADH to ubiquinone
Group4.13	GB44117	CG12676 echinoid		protein binding^IPI	dorsal appendage formation, imaginal disc-derived wing morphogenesis, sensory organ development, morphogenesis of an epithelium, negative regulation of epidermal growth factor receptor signaling pathway
Group5.9	GB46725	CG42311 grainy head		sequence-specific DNA binding transcription factor activity^IDA, protein homodimerization activity^IPI	chitin-based cuticle development, epithelial cell morphogenesis, nervous system development, open tracheal system development
Group5.11	GB42054	CG5670 Na pump α subunit		sodium:potassium-exchanging ATPase activity^IMP, ATP binding^IEA	adult locomotory behavior, determination of adult lifespan, neuromuscular process, cation transport, sensory perception of sound
Group6.23	GB52955	CG31559		electron carrier activity^IEA, protein disulfide oxidoreductase activity^IEA	cell redox homeostasis
Group8.21	GB51653	CG17927 Myosin heavy chain		actin-dependent ATPase activity^IDA, protein homodimerization activity^IDA, structural constituent of muscle^IMP	adult somatic muscle development, locomotion, muscle organ development, myofibril assembly
Group10.24	GB54239	CG11352 jim		metal ion binding^IEA, sequence-specific DNA binding transcription factor activity^ISS	dendrite morphogenesis, regulation of chromatin silencing
Group10.26	GB50974	CG30084 Z band alternatively spliced PDZ-motif protein 52		muscle alpha-actinin binding^IPI, protein binding^IPI	muscle structure development, myofibril assembly
Group11.18	GB44972	CG17484 Adherens junction protein p120		cadherin binding^IEA	adherens junction maintenance, cell adhesion, compound eye morphogenesis
Group12.13	GB40670	CG10573 knockout		unknown	motor neuron axon guidance
Group12.5	GB40240	CG2184 Myosin light chain 2		calcium ion binding^IEA	flight, muscle system process, myofibril assembly
Group14.13	GB54192	CG4651 Ribosomal protein L13		structural constituent of ribosome^IDA	centrosome duplication, mitotic spindle elongation
Group15.19	GB50117	CG15532 headcase		unknown	axon extension, imaginal disc-derived wing morphogenesis, RNA interference, neurogenesis
Group1.1			aly-MIR408
Group1.35			oan-mir-153-1:
Group2.19			mtr-MIR2608
Group8.16			mtr-MIR2676f:
Group15.19			aly-MIR169j
GroupUn98			mmu-mir-6240:	GroupUn98

[1] Send correspondence to Klaus Hartfelder. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brazil. E-mail: klaus@fmrp.usp.br.

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Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis