Social insect colonies possess a range of defences which protect them against highly virulent parasites and colony collapse. The host–parasite interaction between honey bees (Apis mellifera) and the ...mite Varroa destructor is unusual, as honey bee colonies are relatively poorly defended against this parasite. The interaction has existed since the mid‐20th Century, when Varroa switched host to parasitize A. mellifera. The combination of a virulent parasite and relatively naïve host means that, without acaricides, honey bee colonies typically die within 3 years of Varroa infestation. A consequence of acaricide use has been a reduced selective pressure for the evolution of Varroa resistance in honey bee colonies. However, in the past 20 years, several natural‐selection‐based breeding programmes have resulted in the evolution of Varroa‐resistant populations. In these populations, the inhibition of Varroa's reproduction is a common trait. Using a high‐density genome‐wide association analysis in a Varroa‐resistant honey bee population, we identify an ecdysone‐induced gene significantly linked to resistance. Ecdysone both initiates metamorphosis in insects and reproduction in Varroa. Previously, using a less dense genetic map and a quantitative trait loci analysis, we have identified Ecdysone‐related genes at resistance loci in an independently evolved resistant population. Varroa cannot biosynthesize ecdysone but can acquire it from its diet. Using qPCR, we are able to link the expression of ecdysone‐linked resistance genes to Varroa's meals and reproduction. If Varroa co‐opts pupal compounds to initiate and time its own reproduction, mutations in the host's ecdysone pathway may represent a key selection tool for honey bee resistance and breeding.
see also the Perspective by Evans
The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. ...The hormones 20‐hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa's reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa's ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH‐like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa's initiation of egg laying.
In an effort to answer a long‐standing question on how hormonal interactions in the honey bee larvae can sustain the parasite Varroa destructor reproduction, the current study focused on differential expression analysis of the genes involved in the Juvenile hormone (JH) and ecdysteroid associated pathways. All the results were discussed considering the V. destructor related reproductive behavior. The results revealed the higher expression in drones relative to workers for the jhamt gene involved in methyl farnesoate (MF) synthesis. MF is the precursor of JH, also an insect pheromone and the JH‐like hormone in Acari. These results suggest a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell sealing, stimulating Varroa's fertility.
Induction of shoots or of somatic embryos is the key step for gaining the morphogenetic potential in sunflower (Helianthus annuus L.), species known as recalcitrant to in vitro regeneration. In the ...immature zygotic embryo derived tissues or in other juvenile tissues resulted from seedlings, the acquisition of the competence for regeneration can be achieved directly by cytokinin treatment or by preconditioning the explants on cytokinin containing medium. In this paper is presented a new type of explant for sunflower in vitro culture, consisting of the apex with primordial leaves, resulted from ungerminated mature zygotic embryo, in which a specific morphogenetic response was triggered by the exogenously applied auxins. Among the auxins tested, indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid are inducers of an organogenetic response, apical/axillary shoots and adventitious buds being regenerated while 2,4-dichlorophenoxyacetic acid, 3,6-dichloro-2-methoxybenzoic acid and 4-amino-3,5,6-trichloropicolinic acid led to somatic embryo formation. Among the auxins tested only 4-amino-3,5,6-trichloropicolinic acid sustains the embryos development up to mature stage. A high amount of sucrose (120 g L-1) supplied during the auxin treatment promotes the maturation of the embryos directly on the induction medium for all tested auxins with embryogenic effect. These findings show that regardless of the type of morphogenetic response aimed in sunflower meristematic tissues resulted from mature embryos, the presence of auxins is mandatory.
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•Naturally virus-infected forager honeybees are useful to determine antiviral activity of plant extracts.•Laurus nobilis extract reduced BQCV virus loads and virus replication.•Plant ...extracts are potential antiviral agents improving honeybee health.
Viral diseases are one of the multiple factors associated with honeybee colony losses. Apart from their innate immune system, including the RNAi machinery, honeybees can use secondary plant metabolites to reduce or fully cure pathogen infections. Here, we tested the antiviral potential of Laurus nobilis leaf ethanolic extracts on forager honeybees naturally infected with BQCV (Black queen cell virus). Total viral loads were reduced even at the lowest concentration tested (1mg/ml). Higher extract concentrations (≥5mg/ml) significantly reduced virus replication. Measuring vitellogenin gene expression as an indicator for transcript homeostasis revealed constant RNA levels before and after treatment, suggesting that its expression was not impacted by the L. nobilis treatment. In conclusion, plant secondary metabolites can reduce virus loads and virus replication in naturally infected honeybees.
As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome ...integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of
gene may induce a 'mutator' phenotype and microsatellite instability (MSI), as is demonstrated in
knock-out and silenced lines of
. The goal of this study was to screen for 'mutator' phenotypes in somatic hybrids between potato cvs. 'Delikat' and 'Désirée' and MMR deficient
transformed using antisense (AS) or dominant negative mutant (DN)
2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with 'mutator' phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene
II, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species.
Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four ...economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.
Somatic hybridization based on protoplast fusion is a biotechnological tool enabling multiple resistance genes or traits to be transferred from wild relatives into crops. A key step for successful ...regeneration of hybrids is the selection of the heterokaryons. Selection can be done on specific culture media, by using intrinsic cellular or fluorescent markers, or specific traits of the fusion products, such as vigorous growth. More vigorous growth is one of the most common used criteria, since it does not require the use of mutants and allows faster selection of putative somatic hybrids. However, vigorous growth does not occur in all fusion combinations and often additional approaches have to be used (molecular markers, flow cytometry, chromosome counts, etc.) which are more costly and time consuming. There is, therefore, a need for the development of a faster, simpler, more cost-effective and reliable method for the selection of the heterospecific fusion products. Our study reports the effective use of the reporter gene
gfp
to confirm the vigorous growth as a reproducible selection for the somatic hybrids between
Solanum tuberosum
dihaploid line and the wild species
S. chacoense
. In vitro shoots of transgenic
S. chacoense
expressing the reporter gene
gfp
and a wild type dihaploid line of potato were used to isolate the mesophyll protoplasts. Electrofusion of mesophyll protoplasts and standard culture methods were applied. The
S. chacoense
gfp
+ protoplasts were unable to regenerate and when parental protoplast and putative somatic hybrid cultures were monitored, the GFP was expressed and visualized microscopically only in hybrid regenerants showing vigorous growth. The results confirmed that vigorous growth can be used to select the fusion products between these two species. This strategy might be applied to other fusion combinations to improve somatic hybrid selection.
Sunflower, as one of the most important oil-producing crops, represents an important target for genetic improvement through gene transfer or somatic hybridization. Unfortunately, sunflower is ...recognized as recalcitrant to in vitro culture. The aim of our paper was to improve sunflower protoplast regeneration. Three cultivars (Romanian hybrids) and one inbred line were used for protoplast isolation from etiolated hypocotyls. Isolated protoplasts were embedded in alginate disks and cultured in two plating densities, using two culture regimes as indicated by previous authors. Plating efficiency, callus development and plant regeneration were evaluated as well as old callus histology. In cv. ‘Select’, the effects of 1:50 haemoglobin and 1 mM spermidine were assayed on asymmetric division and/or plating efficiency. Plant regeneration from hypocotyl protoplasts was achieved for two cvs., ‘Florom 328’ and ‘Turbo’, with the former proving once more its totipotency. The best culture regime proved to be as recommended by Krasnyanski and Menczel (1993), but the best density in the culture medium was the highest ever tested, 8 × 105 pp ml−1. Moreover, the histology of old green compact protoplast-derived callus revealed a very well organized structure suggesting senescence. In the non-responsive cv. ‘Select’, haemoglobin was found to stimulate protoplast asymmetric division and the development of heart-shaped embryo-like structures, while spermidine stimulated overall protoplast plating efficiency.
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