ABSTRACT
Sex-specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female-specific modification system whereas little success was reported on male-specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene-based, female-specific lethality system has been established based on sex-specific alternative splicing factors and a female-specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male-specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis-specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta-tubulin 4 gene (Bmß4) were introduced using piggybac-based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmß4p showed precisely testis-specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmß4p might be used as distinct regulatory elements in directing testis-specific gene expression. Identification of these testis-specific promoters not only contributes to a better understanding of testis-specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.
Subject(s)
Bombyx/genetics , Green Fluorescent Proteins/genetics , Insect Proteins/genetics , Transgenes , Animals , Animals, Genetically Modified , Base Sequence , Bombyx/growth & development , Bombyx/metabolism , DNA Transposable Elements , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/metabolism , Insect Proteins/metabolism , Larva/genetics , Larva/growth & development , Larva/metabolism , Male , Promoter Regions, Genetic , Testis/chemistry , Testis/growth & development , Testis/metabolismABSTRACT
Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori.
Subject(s)
Bombyx/genetics , Ecdysterone/metabolism , Green Fluorescent Proteins/genetics , Promoter Regions, Genetic , Vitellogenins/genetics , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/growth & development , Animals, Genetically Modified/metabolism , Bombyx/growth & development , Bombyx/metabolism , Female , Genes, Reporter , Green Fluorescent Proteins/metabolism , Larva/genetics , Larva/metabolism , Male , Pupa/genetics , Pupa/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Vitellogenins/metabolismABSTRACT
Atropine is commonly a used pre anesthetic medication. A 22-year-old female with history of unexplained recurrent syncope during electrophysiology developed inducible ventricular arrhythmias when 0.5 mg of atropine was injected intravenously to improve this Wenckebach. There is a significant change in the autonomic influence on the heart prior to idiopathic ventricular tachycardia and this seems to result mainly from decreased vagal activity.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Atropine/adverse effects , Tachycardia, Ventricular/chemically induced , Arrhythmias, Cardiac/physiopathology , Electrophysiological Phenomena , Female , Heart/physiopathology , Humans , Syncope/physiopathology , Tachycardia, Ventricular/physiopathology , Young AdultABSTRACT
Five organophosphates (OPs) (chlorpyrifos, chlorpyrifos methyl, fenthion, malathion, and temephos), 3 pyrethroids (bifenthrin, cypermethrin, and permethrin), 1 phenyl pyrazole (fipronil), 2 microbial pesticides (Bacillus thuringiensis serovar. israelensis [B.t.i.] and Bacillus sphaericus), and 3 insect growth regulators (IGRs) (diflubenzuron, methoprene, and pyriproxyfen) were evaluated against field-collected Culex quinquefasciatus larvae from urban Dhaka, Bangladesh. The LC90 values of all OPs, except for temephos (LC90 = 0.0096 ppm), were high, ranging from 0.13 ppm (fenthion) to 2.882 ppm (chlorpyrifos methyl). Pyrethroid LC90 values were 0.021 ppm (bifenthrin), 0.00061 (cypermethrin), and 0.017 ppm (permethrin). Fipronil exhibited a superior activity with LC90 value of 0.000896 ppm. Technical powders of B.t.i. and B. sphaericus (VectoBac TP and VectoLex TP) were considered highly effective against the Cx. quinquefasciatus larvae. The IGRs also were effective with pyriproxyfen (LC90 = 0.0011 ppm), being 3 times and 47 times more active than diflubenzuron (LC90 = 0.0034 ppm) and methoprene (LC90 = 0.052 ppm), respectively. In general, toxicity ranking of chemicals and microbials tested was phenyl pyrazole > IGRs > pyrethroids > microbials > OPs.
