The development of an egg-soaking method for delivering dsRNAs into spider mites.

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

Selection of Reference Genes for RT-qPCR Analysis in the Hawthorn Spider Mite, Amphitetranychus viennensis (Acarina: Tetranychidae), Under Acaricide Treatments.

Hawthorn spider mite, Amphitetranychus viennensis Zacher, one of the most damaging arthropod pests for Rosaceaous fruit trees and ornamentals, has developed resistance to most of the commercially available acaricides. To understand the molecular basis of acaricide resistance, a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) following the MIQE (minimum information for publication of quantitative real time PCR experiments) guidelines is needed. In this study, we screened for the internal references in A. viennensis to study in acaricide resistance. In total, 10 candidate reference genes, including EF1A, 28S rRNA, 18S rRNA, α-tubulin, Actin3, RPS9, GAPDH, V-ATPase B, RPL13, and V-ATPase A, were assessed under the treatments of four commonly used acaricides with distinct mode-of-actions (MOAs). Based on the Insecticide Resistance Action Committee MOA classification, avermectin, bifenazate, spirodiclofen, and fenpropathrin belong to group 6, 20D, 23, and 3A, respectively. The expression profiles of these candidate genes were evaluated using geNorm, Normfinder, BestKeeper, and ∆Ct methods, respectively. Eventually, different sets of reference genes were recommended for each acaricide according to RefFinder, a comprehensive platform integrating all four above-mentioned algorithms. Specifically, the top three recommendations were 1) 28S, V-ATPase A, and Actin 3 for avermectin, 2) GAPDH, RPS9, and 28S for bifenazate, 3) Actin 3, V-ATPase B, and α-tubulin for spirodiclofen, and 4) Actin 3, α-tubulin, and V-ATPase A for fenpropathrin. Although unique sets of genes are proposed for each acaricide, α-tubulin, EF1A, and GAPDH are the most consistently stably expressed reference genes when A. viennensis was challenged chemically. Our findings lay the foundation for the study of acaricide resistance in the phytophagous mites in general, and in the hawthorn spider mite, A. viennensis, in particular.

A double-taper optical fiber-based radiation wave other than evanescent wave in all-fiber immunofluorescence biosensor for quantitative detection of Escherichia coli O157:H7.

Cylindrical or taper-and-cylinder combination optical fiber probe based on evanescent wave has been widely used for immunofluorescence biosensor to detect various analytes. In this study, in contrast to the contradiction between penetration depth and analyte diameter of optical fiber probe-based evanescent wave, we demonstrate that double-taper optical fiber used in a radiation wave-based all-fiber immunofluorescence biosensor (RWAIB) can detect micron-scale analytes using Escherichia coli O157:H7 as representative target. Finite-difference time-domain method was used to compare the properties of evanescent wave and radiation wave (RW). Ray-tracing model was formulated to optimize the taper geometry of the probe. Based on a commercial multi-mode fiber, a double-taper probe was fabricated and connected with biosensor through a "ferrule connector" optical fiber connector. The RWAIB configuration was accomplished using commercial multi-mode fibers and fiber-based devices according to the "all-fiber" method. The standard sample tests revealed that the sensitivity of the proposed technique for E. coli O157:H7 detection was 10(3) cfu · mL(-1). Quantitation could be achieved within the concentration range of 10(3) cfu · mL(-1) to 107 cfu · mL(-1). No non-specific recognition to ten kinds of food-borne pathogens was observed. The results demonstrated that based on the double-taper optical fiber RWAIB can be used for the quantitative detection of micron-scale targets, and RW sensing is an alternative for traditional evanescent wave sensing during the fabrication of fiber-optic biosensors.