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Material and Method
Mosquito rearing
Mosquitoes used in all experiments were Aedes aegypti, Rockefeller strain. Mosquito larvae were raised in distilled H2O (~200 larvae per 1.5 liter tray) and fed pestle-ground TetraMin® fish food (Tetra Holding Inc.). Pupae were transferred to a 400 ml tray prior to adult emergence. Adults were fed 10% sucrose in H2O. Both larvae and adults were raised at 28°C in a 16 hr light 8 hr dark cycle
Abdominal ligation assays
Female Ae. aegypti were collected and anesthetized using either CO2, or by cold within 30 minutes of adult emergence. Ligations were performed by severing the head and adjacent 1/3 portion of the thorax, which includes the CA. 3M Vetbond™ tissue adhesive was used to seal the open wound, and affix the thorax/abdomen to a glass slide. Specimens were treated with hormone dissolved in acetone as specified in text. Sample groups (5-8 specimens each) were incubated at 28°C in moisture chambers for times specified in text, and then placed in 150 µl TRIzol® (Invitrogen) and stored at -80°C
Tissue dissection
Tissues extracted and sample sizes are indicated in text with the following notes:Thorax included wings and legs. Ovaries included the two most posterior segments of the abdomen. Fat body included everything in the abdomen section minus the midgut and ovaries. All specimens were cold anesthetized prior to dissection. All tissue sample groups were placed in 150 µl TRIzol® and stored at -80°C
RNAi
Template DNA for dsRNA synthesis: A 603 bp region of the AaKr-h1 transcript (Figure 3) was PCR amplified using Ae. aegypti cDNA template and primers with added Pst1 and Xho1 restriction site sequences (see primers below). Reaction mixture: 50 µl PCR Supermix High Fidelity (Invitrogen); 0.5 µl each primer (see primers), 25 pmol/µl;1.0 µl cDNA. PCR program: 3 min @ 94°C; 40 cycles of 30 sec @ 94°C, 30 sec @ 55°C, 1 min @ 72°C; 5 min @ 72°C. The resulting DNA fragment was cloned into a LITMUS 28i plasmid (New England Biolabs) between Pst1 and Xho1 restriction sites.The insert was flanked by two inverted T7 promoters on LITMUS 28i. Template DNA for AaKr-h1 dsRNA, with T7 promoters at each end, was obtained by PCR using a minimal T7 primer. Template DNA for Mal dsRNA (described in text) was produced in the same fashion using 2.0 µl (50 ng/µl) of supplied LITMUS 28iMal plasmid (New England Biolabs). The expected 768 bp (Kr-h1 dsRNA template) and 962 bp (Mal dsRNA template) DNA fragments were confirmed on a 1% agarose gel. The quantity of both templates was insufficient for dsRNA synthesis, so PCR products were purified using QIAquick PCR Purification (Qiagen), and the products were used as template for multiple PCR amplifications to generate needed quantities of template DNA. Reaction mixture and thermocycler program were the same as previous PCR, using 1.0 µl of each template (Kr-h1 dsRNA template, 30 ng/µl; Mal dsRNA template, 60 ng/µl). A MiniElute PCR Purification Kit (Qiagen) was used to purify and concentrate products of multiple PCR reactions to concentrations and quantities sufficient for dsRNA synthesis. Template DNA sequencing: The template DNA for Kr-h1 dsRNA was cloned into a pCR®4-TOPO plasmid (TOPO TA Cloning® Kit for Sequencing, Invitrogen) and sent to Virginia Bioinformatics Institute for DNA sequencing using M13 forward and reverse primers. Chromas Lite 2.01 (Technelysium) was used to interpret sequences from the chromatogram result files. The sequence was compared with the putative transcript for AaKr-h1 in Vectorbase (ID# AAEL002390) using Kalign and found to differ by two bases over the 603 bp region of homology. dsRNA synthesis: MEGAscript® RNAi Kit (Ambion, #1626) was used for synthesis of dsRNA, using template DNA following the manufacturer’s protocol. Product quality and size were confirmed on 1% agarose gel, and expected bands at~730 bp (Kr-h1dsRNA) and ~930 bp (MaldsRNA) were observed (Figure 4). Ethanol precipitation was use to concentrate final dsRNA products to ~3 µg/µl, in 0.1X PBS. dsRNA injections: Newly emerged female Ae. aegypti were injected with 0.6 µg dsRNA in 200 nl 0.1X PBS between 2 hr and 4 hr post-emergence using a Nanoject II nanoliter injector (Drummond Scientific Company). Mosquitoes were cold anesthetized, and injections were administered in the side of the thorax. Mosquitoes were then raised at 28°C in a 16 hr light / 8 hr dark cycle for the specified analysis time
RNA extraction / cDNA synthesis
All tissue and abdomen samples were stored in 150 µl TRIzol at -80°C prior to RNA extraction. Samples were homogenized in 150 µl TRIzol in Eppendorf tubes using a plastic pestle. TRIzol was then added to 1.0 ml total volume, and manufacturer’s protocol was followed for RNA extraction. The resulting purified RNA pellet was reconstituted in 40 µl nuclease-free H2O. Concentration was determined by measuring the A260 with a spectrophotometer (1A260 = 40 ng/µl). RNA quality was confirmed by running 1 µg per lane on a 1% agarose gel, and verifying two distinct ribosomal RNA bands. For all samples, 1 µg RNA was used for cDNA synthesis as follows: Residual genomic DNA was removed using Deoxyribonuclease I, Amplification Grade (Invitrogen) following the manufacturer’s protocol. Reverse transcription was performed using the Omniscript RT Kit (QIAGEN) following manufacturer’s protocol. Final reaction volume of 20 µl was diluted to 80 µl for subsequent qRT-PCR reactions
qRT-PCR and analysis
All primers were checked for acceptable amplification efficiency (m ≤ 3.5) and single amplification product (single melting curve peak) in serial cDNA dilutions and gene-specific DNA concentration standards. DNA concentration standards were serial 10-fold dilutions of PCR product, using Ae. aegypti cDNA template and gene-specific primer (see qRT-PCR primers) purified with PureLink™ PCR Purification Kit (Invitrogen). All qRT-PCR reactions used SYBR® GreenER qPCR Supermix for ABI PRISM (Invitrogen) per manufacturer’s protocol, with 2 µl sample cDNA or gene-specific DNA standard. Reactions were performed on an ABI PRISM 7300 (Applied Biosystems) Thermal program for all reactions was 2 min @ 50°C; 10 min @ 95°C; 40 cycles of 15 sec @ 95°C, 1 min @ 60°C. Dissociation step was included and the melting curve checked for single peak in all reactions
Abstract
Acknowledgements
List of Figures / Tables
Introduction
Juvenile hormone
Met, br, and Kr-h1: genes in JH signaling
The Aedes aegypti mosquito
Research goals
Material and Methods
Mosquito rearing
Abdominal ligation assays
Tissue dissection
RNAi
RNA extraction / cDNA synthesis
qRT-PCR and analysis
Primers
Results
Part 1. Identifying JH response genes in newly emerged female Ae. aegypti
What genes are differentially expressed in response to a JH mimic in abdominal ligation assays?
What are the temporal expression profiles of the identified genes in response to the JH mimic?
Do the mRNA profiles of these genes in adult females correlate well with the endogenous JH titers?
Part II: Further characterization of AaKr-h1
Is AaKr-h1 a homolog of Kr-h1 discovered in D. melenogaster, T. castaneum,and A. mellifera?
Does AaKr-h1 specifically respond to juvenile hormone in the abdominal ligation assays?
What is the function of AaKr-h1 in newly-emerged females in response to JH?. 17
Conclusion and Discussion JH target genes
AaKr-h1 role in JH regulation
Future work
Figures / Tables
References
