Honey bee promoter sequences for targeted gene expression

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue‐specific and cold shock‐induced gene expression. We identified the promoter sequences of Am‐actin5c, elp2l, Am‐hsp83 and Am‐hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron‐specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.     

Genome‐wide analysis of genes related to ovary activation in worker honey bees

A defining characteristic of eusocial animals is their division of labour into reproductive and nonreproductive specialists. Here, we used a microarray study to identify genes associated with functional sterility in the worker honey bee Apis mellifera. We contrasted gene expression in workers from a functionally sterile wild‐type strain with that in a mutant (anarchist) strain selected for high rates of ovary activation. We identified a small set of genes from the brain (n = 7) and from the abdomen (n = 5) that are correlated in their expression with early stages of ovary activation. Sterile wild‐type workers up‐regulated two unknown genes and a homologue of Drosophila CG6004. By contrast, reproductive anarchist workers up‐regulated genes for the yolk protein vitellogenin, venom peptides and a member of the AdoHycase superfamily, among others. The differentially expressed genes identified are likely to be involved in early differentiation into sterile and reproductive worker phenotypes and may therefore form part of the gene networks associated with the regulation of honey bee worker sterility. Our study may have lacked sufficient power to detect all but a minority of biologically relevant changes taking place; however, the differential expression of vitellogenin and a putative AdoHycase suggests that our screen has captured core reproductive genes and that ovary activation may involve an epigenetic mechanism.     

The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double‐stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient‐1 (Sid‐1) transmembrane channel‐mediated uptake and clathrin‐mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi‐of‐RNAi experiments, we demonstrated the contribution of two different sid‐1‐like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H⁺ ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid‐1‐like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well‐devised reporter system in any future experiments on cellular dsRNA uptake.     

Voxel‐based analysis of the immediate early gene,c‐jun,in the honey bee brain after a sucrose stimulus

Immediate early genes (IEGs) have served as useful markers of brain neuronal activity in mammals, and more recently in insects. The mammalian canonical IEG, c‐jun, is part of regulatory pathways conserved in insects and has been shown to be responsive to alarm pheromone in honey bees. We tested whether c‐jun was responsive in honey bees to another behaviourally relevant stimulus, sucrose, in order to further identify the brain regions involved in sucrose processing. To identify responsive regions, we developed a new method of voxel‐based analysis of c‐jun mRNA expression. We found that c‐jun is expressed in somata throughout the brain. It was rapidly induced in response to sucrose stimuli, and it responded in somata near the antennal and mechanosensory motor centre, mushroom body calices and lateral protocerebrum, which are known to be involved in sucrose processing. c‐jun also responded to sucrose in somata near the lateral suboesophageal ganglion, dorsal optic lobe, ventral optic lobe and dorsal posterior protocerebrum, which had not been previously identified by other methods. These results demonstrate the utility of voxel‐based analysis of mRNA expression in the insect brain.     

Knockdown of genes in the Toll pathway reveals new lethal RNA interference targets for insect pest control

RNA interference (RNAi) is a promising alternative strategy for ecologically friendly pest management. However, the identification of RNAi candidate genes is challenging owing to the absence of laboratory strains and the seasonality of most pest species. Tribolium castaneum is a well‐established model, with a strong and robust RNAi response, which can be used as a high‐throughput screening platform to identify potential RNAi target genes. Recently, the cactus gene was identified as a sensitive RNAi target for pest control. To explore whether the spectrum of promising RNAi targets can be expanded beyond those found by random large‐scale screening, to encompass others identified using targeted knowledge‐based approaches, we constructed a Cactus interaction network. We tested nine genes in this network and found that the delivery of double‐stranded RNA corresponding to fusilli and cactin showed lethal effects. The silencing of cactin resulted in 100% lethality at every developmental stage from the larva to the adult. The knockdown of pelle, Dorsal‐related immunity factor and short gastrulation reduced or even prevented egg hatching in the next generation. The combination of such targets with lethal and parental RNAi effects can now be tested against different pest species in field studies.     

Isoform specific roles of Broad‐Complex in larval development in Leptinotarsa decemlineata

Broad‐Complex (BrC) is a downstream target of both 20‐hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1–Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double‐stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6 < Z3 < Z4. When silencing all BrC isoforms at the last instar period, most of the RNAi larvae did not normally pupate or emerge as adults. Separately silencing each of the five zinc finger domains revealed that approximately 70% of the Z1 RNAi larvae remained as prepupae, around 60% of the Z6 RNAi specimens formed aberrant prepupae or pupae and about 60% of the Z2 RNAi beetles became deformed pupae. After removal of the old exuviae, these deformed larvae in which either Z1, Z2 or Z6 was depleted possessed adult prothorax and mesothorax, developing antenna, mouthparts and wing discs. Moreover, less than 50% of the resultant pupae finally emerged as adults when either of Z1, Z2 or Z6 was knocked down. Therefore, our findings reveal, for the first time, that the two roles of BrC in insect groups (ie directing morphogenetic changes during juvenile development and regulating larval–pupal–adult metamorphosis) are played by different BrC isoforms in Leptinotarsa decemlineata.     

Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6‐year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long‐chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa‐tolerant honey bees.     

Division of labour in honey bees: age‐ and task‐related changes in the expression of octopamine receptor genes

The honey bee (Apis mellifera L.) has developed into an important ethological model organism for social behaviour and behavioural plasticity. Bees perform a complex age‐dependent division of labour with the most pronounced behavioural differences occurring between in‐hive bees and foragers. Whereas nurse bees, for example, stay inside the hive and provide the larvae with food, foragers leave the hive to collect pollen and nectar for the entire colony. The biogenic amine octopamine appears to play a major role in division of labour but the molecular mechanisms involved are unknown. We here investigated the role of two characterized octopamine receptors in honey bee division of labour. AmOctαR1 codes for a Ca²⁺‐linked octopamine receptor. AmOctβR3/4 codes for a cyclic adenosine monophosphate‐coupled octopamine receptor. Messenger RNA expression of AmOctαR1 in different brain neuropils correlates with social task, whereas expression of AmOctβR3/4 changes with age rather than with social role per se. Our results for the first time link the regulatory role of octopamine in division of labour to specific receptors and brain regions. They are an important step forward in our understanding of complex behavioural organization in social groups.     

The nuclear hormone receptor E75A regulates vitellogenin gene (Al‐Vg) expression in the mirid bug Apolygus lucorum

Apolygus lucorum is the predominant pest of Bacillus thuringiensis (Bt) cotton in China. 20‐hydroxyecdysone (20E) plays a key role in the reproduction of this insect. To better understand the mechanism underlying 20E‐regulated reproduction, the nuclear hormone receptor E75 isoform‐A of Ap. lucorum (Al‐E75A) was cloned and its expression analysed. A 2241‐bp sequence of Al‐E75A cDNA encoded an open reading frame of a polypeptide with a predicted molecular mass of 69.04 kDa. Al‐E75A mRNA was detected in female adult stages of Ap. lucorum with peak expression in 7‐day‐old animals. Al‐E75A was also expressed in several tissues, particularly in the fat body and ovary. A 3.2 kb Al‐E75A mRNA was detected in all tissues by Northern blot. The fecundity and longevity were significantly decreased in female adults treated with Al‐E75A small interfering RNA. The rates of egg incubation rates were considerably lower in the RNA interference‐treated animals compared to the untreated controls. In order to investigate the molecular mechanism underlying the effects described above, vitellogenin (Al‐Vg) was selected for further investigation. The expression pattern of Al‐Vg was similar to that of Al‐E75A and was up‐regulated by 20E. After knockdown of Al‐E75A, the expression profile of Al‐Vg and the protein levels were down‐regulated. These findings suggest that Al‐E75A plays a crucial role in the regulation of Al‐Vg expression in Ap. lucorum.     

Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family

Antimicrobial peptides (defensins) are effectors of the immune system. Herein, we describe a novel Ixodes ricinus defensin gene(s), analyse its structure and compare it with other known antimicrobial peptides from different tick species. For the first time, an intron/exon structure is discovered in a hard-tick defensin gene. The intron/exon genomic organization of the gene is similar to the organization in Ornithodoros moubata, but not to that of the intronless defensins of Dermacentor variabilis and Ixodes scapularis. The analysis of the deduced amino acid sequences of different recombinants from the I. ricinus cDNA library reveals the presence of two defensin isoforms with three amino acid substitutions. Whether or not these substitutions affect the biological properties of the peptides is currently unknown. The expression of the defensin gene is strongly induced in the tick midgut after infection with Borrelia burgdorferi.