RESUMEN
During the honeybee larval stage, queens develop larger brains than workers, with morphological differentiation appearing at the fourth larval phase (L4), just after a boost in nutritional difference both prospective females experience. The molecular promoters of this caste-specific brain development are already ongoing in previous larval phases. Transcriptomic analyses revealed a set of differentially expressed genes in the L3 brains of queens and workers, which represents the early molecular response to differential feeding females receive during larval development. Three genes of this set, hex70b, hex70c and hex110, are more highly transcribed in the brain of workers than in queens. The microRNAs miR-34, miR-210 and miR-317 are in higher levels in the queens' brain at the same phase of larval development. Here, we tested the hypothesis that the brain of workers expresses higher levels of hexamerins than that of queens during key phases of larval development and that this differential hexamerin genes expression is further enhanced by the repressing activity of miR-34, miR-210 and miR-317. Our transcriptional analyses showed that hex70b, hex70c and hex110 genes are differentially expressed in the brain of L3 and L4 larval phases of honeybee queens and workers. In silico reconstructed miRNA-mRNA interaction networks were validated using luciferase assays, which showed miR-34 and miR-210 negatively regulate hex70b and hex110 genes by directly and redundantly binding their 3'UTR (untranslated region) sequences. Taken together, our results suggest that miR-34 and miR-210 act together promoting differential brain development in honeybee castes by downregulating the expression of the putative antineurogenic hexamerin genes hex70b and hex110.
Asunto(s)
Abejas , Encéfalo/crecimiento & desarrollo , Proteínas de Insectos/genética , MicroARNs , Animales , Abejas/genética , Abejas/crecimiento & desarrollo , Femenino , Larva/genética , Larva/crecimiento & desarrollo , MicroARNs/genética , Estudios ProspectivosRESUMEN
Apis mellifera adult workers feature more developed key brain regions than queens, which allows them to cope with the broad range of duties they need to perform in a colony. However, at the end of larval development, the brain of queens is largely more developed than that of workers. Major morphogenetic changes take place after metamorphosis that shift caste-specific brain development. Here, we tested the hypothesis that this phenomenon is hormonally governed and involves differential gene expression. Our molecular screening approach revealed a set of differentially expressed genes in Pp (first pharate-adult phase) brains between castes mainly coding for tissue remodelling and energy-converting proteins (e.g. hex 70a and ATPsynß). An in-depth qPCR analysis of the transcriptional behaviour during pupal and pharate-adult developmental stage in both castes and in response to artificially augmented hormone titres of 18 genes/variants revealed that: i. subtle differences in hormone titres between castes might be responsible for the differential expression of the EcR and insulin/insulin-like signalling (IIS) pathway genes; ii. the morphogenetic activity of the IIS in brain development must be mediated by ILP-2, iii. which together with the tum, mnb and caspase system, can constitute the molecular effectors of the caste-specific opposing brain developmental trajectories.
Asunto(s)
Abejas , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estadios del Ciclo de Vida/fisiología , Animales , Abejas/genética , Abejas/metabolismo , Abejas/fisiología , Expresión Génica , Perfilación de la Expresión Génica , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva , Metamorfosis Biológica , Morfogénesis , Hormonas Peptídicas/metabolismo , Pupa , Transducción de SeñalRESUMEN
Ftz-f1 is an orphan member of the nuclear hormone receptor superfamily. A 20-hydroxyecdysone pulse allows ftz-f1 gene expression, which then regulates the activity of downstream genes involved in major developmental progression events. In honeybees, the expression of genes like vitellogenin (vg), prophenoloxidase and juvenile hormone-esterase during late pharate-adult development is known to be hormonally controlled in both queens and workers by increasing juvenile hormone (JH) titres in the presence of declining levels of ecdysteroids. Since Ftz-f1 is known for mediating intracellular JH signalling, we hypothesized that ftz-f1 could mediate JH action during the pharate-adult development of honeybees, thus controlling the expression of these genes. Here, we show that ftz-f1 has caste-specific transcription profiles during this developmental period, with a peak coinciding with the increase in JH titre, and that its expression is upregulated by JH and downregulated by ecdysteroids. RNAi-mediated knock down of ftz-f1 showed that the expression of genes essential for adult development (e.g. vg and cuticular genes) depends on ftz-f1 expression. Finally, a double-repressor hypothesis-inspired vg gene knock-down experiment suggests the existence of a positive molecular loop between JH, ftz-f1 and vg.
Asunto(s)
Abejas/metabolismo , Factores de Transcripción Fushi Tarazu/metabolismo , Regulación del Desarrollo de la Expresión Génica , Animales , Abejas/crecimiento & desarrollo , Proteínas de Insectos/metabolismo , Hormonas Juveniles/metabolismo , Fenotipo , Interferencia de ARN , Vitelogeninas/metabolismoRESUMEN
Hexamerins and prophenoloxidases (PPOs) proteins are members of the arthropod-haemocyanin superfamily. In contrast to haemocyanin and PPO, hexamerins do not bind oxygen, but mainly play a role as storage proteins that supply amino acids for insect metamorphosis. We identified seven genes encoding hexamerins, three encoding PPOs, and one hexamerin pseudogene in the genome of the parasitoid wasp Nasonia vitripennis. A phylogenetic analysis of hexamerins and PPOs from this wasp and related proteins from other insect orders suggests an essentially order-specific radiation of hexamerins. Temporal and spatial transcriptional profiles of N. vitripennis hexamerins suggest that they have physiological functions other than metamorphosis, which are arguably coupled with its lifestyle.
Asunto(s)
Catecol Oxidasa/genética , Precursores Enzimáticos/genética , Evolución Molecular , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Filogenia , Avispas/genética , Animales , Teorema de Bayes , Biología Computacional , Cartilla de ADN/genética , Componentes del Gen , Perfilación de la Expresión Génica , Modelos Genéticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la EspecieRESUMEN
Two hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) are key regulators of insect development including the differentiation of the alternative caste phenotypes of social insects. In addition, JH plays a different role in adult honey bees, acting as a 'behavioural pacemaker'. The functional receptor for 20E is a heterodimer consisting of the ecdysone receptor and ultraspiracle (USP) whereas the identity of the JH receptor remains unknown. We have cloned and sequenced a cDNA encoding Apis mellifera ultraspiracle (AMUSP) and examined its responses to JH. A rapid, but transient up-regulation of the AMUSP messenger is observed in the fat bodies of both queens and workers. AMusp appears to be a single copy gene that produces two transcripts ( approximately 4 and approximately 5 kb) that are differentially expressed in the animal's body. The predicted AMUSP protein shows greater sequence similarity to its orthologues from the vertebrate-crab-tick-locust group than to the dipteran-lepidopteran group. These characteristics and the rapid up-regulation by JH suggest that some of the USP functions in the honey bee may depend on ligand binding.
Asunto(s)
Abejas/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hormonas Juveniles/farmacología , Filogenia , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Abejas/fisiología , Northern Blotting , Southern Blotting , Análisis por Conglomerados , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Datos de Secuencia Molecular , Receptores de Esteroides/metabolismo , Análisis de Secuencia de ADN , Predominio Social , Factores de Transcripción/metabolismoRESUMEN
The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.