Discovery of a CI-inducing Wolbachia and its associated fitness costs in the biological control agent Aphytis melinus DeBach (Hymenoptera: Aphelinidae)

Wolbachia is an endosymbiotic bacterium that infects a large percentage of arthropods and can affect the fitness of its host. Here we verified for the first time that the biological control agent Aphytis melinus DeBach is infected with a Wolbachia that causes complete cytoplasmic incompatibility, and conducted an insectary and field survey to determine the infection frequency. A. melinus appears to suffer fitness costs associated with infection based on measurements of longevity and fecundity. We also quantified the Wolbachia titers of A. melinus reared at different temperatures and found that, although not completely cured, increased temperature resulted in a significant reduction in the number of Wolbachia copies found in an individual wasp. Implications of our results for biological control are discussed.     

Fitness components of Aphytis melinus (Hymenoptera: Aphelinidae) reared in five California insectaries

Shipments of 50,000 commercially reared Aphytis melinus DeBach were obtained from each of the five insectaries that produce and sell this parasitoid to citrus growers in California for control of California red scale, Aonidiella aurantii (Maskell). Shipments were received from each insectary every 2 mo over a period of a year to assess variability in quality through time and between insectaries. As indices of quality, we assessed the percentage of live parasitoids (both sexes) 1, 3, 7, 14, and 28 d after receipt of the shipment, shipment sex ratio, and the size of female wasps. We found a fair amount of variation in the percentage of A. melinus that were alive on different sampling days. Despite the fact that all insectaries rear A. melinus in temperature controlled rooms and all of our studies were done at 22°C, wasp mortality occurred more quickly in the colder months of November, January through February, and March. Similar trends were observed with sex ratios; many of the insectaries had male-biased sex ratios in the colder months, especially January through February. Wasp size varied significantly for each of the insectaries throughout the year, with the summer months of July through August yielding significantly smaller females than other months. Collectively our results have important implications for biological control on citrus in California.     

Freeze-crack technique to study epidermal development in zebrafish using differential interference contrast microscopy and fluorescent markers

The zebrafish is a model organism used to study organogenesis during vertebrate development; however epidermis development has been the focus of only a few studies. Thus, new methodologies to highlight and study epidermal cells could be valuable to deepen our understanding of skin development. Large-scale mutagenic screenings have already identified many zebrafish mutants, which are models for human developmental diseases, however only four epidermis mutants have been isolated. Novel screening techniques are needed to improve this collection. We designed and tested a novel freeze-crack technique to obtain, fix, and stain epidermal cells from 5 days postfertilization zebrafish larvae. Using commercially available fluorescent markers and differential interference contrast (DIC) microscopy, we were able to label and highlight subcellular structures such as microridges, cell boundaries, nuclei, and the Golgi complex from epidermis cells. Acquiring and processing epidermis samples from 15 to 75 larvae takes about 2-4 h, respectively. Therefore this method could be used as part of large-scale screenings. In addition, we present a more extensive protocol for antibody staining, which could be employed for more specific studies.     

Epiphytic orchids in tropical dry forests of Yucatan,Mexico – Species occurrence,abundance and correlations with host tree characteristics and environmental conditions

Tropical dry forests have been less studied in terms of their resident epiphyte flora compared to wet forests. We studied five species of epiphytic orchids in two dry forest fragments differing in tree composition, stature and rainfall regime. We compared the vertical distribution within the host tree, epiphyte-host associations and seasonal variation in microclimatic conditions in a tropical dry deciduous (Celestún) and a semi-deciduous forest (Kaxil-Kiuic) of the Yucatan Peninsula, Mexico, during the wet, early dry and dry seasons. Light, vapor pressure deficit, air temperature, and dew were measured on two heights (1.5 and 3.5 m) of the host with the highest abundance of orchids. Surprisingly, orchid abundance was higher in the Celestún deciduous forest, the site with low precipitation. High epiphyte abundance in the middle canopy stratum of the hosts in both forests was arguably related to a favorable combination of micro-environmental factors. In both forests, about 90% of all orchids grew on a single host tree species. Although bark roughness and the area of the substrate were the most important host characteristics that influenced the abundance of orchids in the Celestún deciduous forest, this did not explain this preference. Climatic variation was greater among seasons than between microenvironments in the host trees. The most abundant species, Encyclia nematocaulon, had a great capacity to occupy different strata in both forests, even in tree tops with very large micro-environmental fluctuations during the year.     

High resilience to extreme climatic changes in the CAM epiphyte Tillandsia utriculata L. (Bromeliaceae)

Climate change is expected to increase the frequency of extreme climatic events, yet few studies have addressed the capacity of plant species to deal with such events. Species that are widespread are predicted to be highly plastic and able to acclimate to highly changing conditions. To study the plasticity in physiological responses of the widely distributed epiphyte Tillandsia utriculata, we transplanted individuals from a coastal scrub and broadleaf evergreen forest to a similar coastal scrub site and forest. After a 45-day acclimation, the plants were moved to a semi-controlled greenhouse at each site, and then subjected to a 20-day drought. Physiological variables were measured during the acclimation and the drought. The individuals of scrub and forest populations had similar relative water content and carbon assimilation in the contrasting conditions of the two transplantation sites despite the high discrepancy between the environments at their original site. Electron transport rates were higher in individuals from the scrub population. Electron transport rates were also higher than estimated from carbon assimilation, suggesting that photorespiration was present. The individuals of the coastal scrub population had a higher capacity to dissipate excess energy this way. The relative distance index of plasticity was high overall, indicating that some traits are highly plastic (titratable acidity, carbon assimilation) in order to maintain the stability of others (maximum quantum yield F_v/F_m and relative water content). We conclude that T. utriculata is a highly plastic species with a high capacity to tolerate extreme environmental changes over a short time.     

TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons

9-fluorenylmethoxycarbonyl (Fmoc) and 4,4′-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr behaves oppositely. Based on these chemical characteristics, we have now devised TrimerDimer, a novel codon-based saturation mutagenesis approach that removes redundant and stop codons during the assembly of degenerate oligonucleotides. In this approach, five DMTr-protected trinucleotide phosphoramidites (dTGG, dATG, dTTT, dTAT and dTGC) and five Fmoc-protected dinucleotide phosphoramidites (dAA, dTT, dAT, dGC and dCG) react simultaneously with a starting oligonucleotide growing on a solid support. The Fmoc group is then removed and the incorporated dimers react with a mixture of three DMTr-protected monomer phosphoramidites (dC, dA and dG) to produce 15 trinucleotides: dCAA, dAAA, dGAA, dCTT, dATT, dGTT, dCAT, dAAT, dGAT, dCGC, dAGC, dGGC, dCCG, dACG and dGCG. After one mutagenic cycle, 20 codons are generated encoding the 20 natural amino acids. TrimerDimer was tested by randomizing the four contiguous codons that encode amino acids L64–G67 of an engineered, nonfluorescent GFP protein. Sequencing of 89 nonfluorescent mutant clones and isolation of two fluorescent mutants confirmed the principle.     

N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens

The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance-inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S. meliloti to induce resistance depends on the production of the quorum-sensing molecule, oxo-C14-HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis-Salmonella model, we also demonstrate that the application of N-acyl-homoserine lactones-producing bacteria could be a successful strategy to prevent plant-originated infections with human pathogens.