Экспериментальные и биоинформатика Протокол Секвенирование РНК Анализы ФОТОПЕРИОДИЧЕСКИХ диапаузы в Азиатско тигровый комар,<em> Кусака бело-пёстрый</em
Summary
RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.
Abstract
Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.
Introduction
Rapid advances in next-generation sequencing (NGS) technologies are providing exciting opportunities to probe the molecular underpinnings of a wide range of genetically complex ecological adaptations in a broad diversity of non-model organisms1–3. This approach is extremely powerful because it establishes a basis for population and functional genomics studies of organisms with an especially interesting and/or well-described ecology or evolutionary history, as well as organisms of practical concern, such as agricultural pests and disease vectors. Thus, NGS technologies are leading to rapid advances in the fields of ecology and have the potential to address problems such as understanding the mechanistic bases of biological responses to rapid contemporary climate change4, the spread of invasive species5, and host-pathogen interactions6,7.
The extraordinary potential of NGS technologies for addressing basic and applied questions in ecology and evolutionary biology is in part due to the fact that these approaches can be applied to any organism at a moderate cost that is feasible for most research laboratories. Furthermore, these approaches provide genome-wide information without the requirement of a priori genetic resources such as a microarray chip or complete genome sequence. Nevertheless, to maximize the productivity of NGS experiments requires careful consideration of experimental design including issues such as the developmental timing and tissue-specificity of RNA sampling. Furthermore, the technical skills required to analyze the massive amounts of data produced by these experiments, often up to several hundred million DNA sequence reads, has been a particular challenge and has limited the widespread implementation of NGS approaches.
Recent RNA-Seq studies on the transcriptional bases of diapause in the invasive and medically important mosquito Aedes albopictus provide a useful example of some of the experimental protocols that can be employed to successfully apply NGS technology to studying the molecular basis of a complex ecological adaptation in a non-model organism8–10. A. albopictus is a highly invasive species that is native to Asia but has recently invaded North America, South America, Europe, and Africa11,12. Like many temperate insects, temperate populations of A. albopictus survive through winter by entering a type of dormancy referred to as photoperiodic diapause. In A. albopictus, exposure of pupal and adult females to short (autumnal) day lengths leads to the production of diapause eggs in which embryological development is completed, but the pharate larva inside the chorion of the egg enters a developmental arrest that renders the egg refractory to hatching stimulus15–17. Diapause eggs are more desiccation resistant5,18 and contain more total lipids19 than non-diapause eggs. Photoperiodic diapause in A. albopictus is thus a maternally controlled, adaptive phenotypic plasticity that is essential for surviving the harsh conditions of winter in temperate environments. Despite the well-understood ecological significance of photoperiodic diapause in a wide range of insects20,21, the molecular basis of this crucial adaptation is not well characterized in any insect22. In organisms such as A. albopictus that undergo an embryonic diapause at the pharate larval stage, it remains a particularly compelling challenge to understand how the photoperiodic signal received by the mother is passed to the offspring and persists through the course of embryonic development to cause arrest at the pharate larval stage.
This protocol describes mosquito rearing, experimental design and bioinformatics analyses for NGS experiments (transcriptome sequencing) performed to elucidate transcriptional components of photoperiodic diapause in A. albopictus. This protocol can be used for additional studies of diapause in A. albopictus, can be adapted to investigate diapause in other closely related species such as other aedine mosquitoes that undergo egg diapause23, and is also more generally relevant to employing NGS approaches to study the transcriptional bases of any complex adaptation in any insect.
Protocol
Representative Results
Discussion
Этот протокол представляет методы, чтобы обнаружить дифференциально выраженные гены из-за photoperiodically индуцированной диапаузы в А. albopictus. Протокол имеет важное значение в том, что она уникально сочетает в себе комаров воспитание и методы биоинформатики, чтобы сделать все экспериме?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Institutes of Health grant 5R21AI081041-02 and Georgetown University.
Materials
| Incubator – Model 818 | Thermo-Scientific | 3751 | 120V |
| Controlled environment room | Thermax Scientific | N/A | Walk-in controlled environment room built to custom specifications by Thermax Scientific Products. A larger alternative to an incubator. http://thermmax.com/ |
| Cool Fluorescent bulb | Philips | 392183 | 4 Watt |
| Petri Dish 100mm x 20mm | Fisher | 08-772-E | |
| Filter Paper 20.5cm | Fisher | 09-803-6J | |
| 9.5L Bucket | Plastican | Bway Products | http://www.bwayproducts.com/sites/portal/plastic-products/plastic-open-head-pails/117 |
| Utility Fabric-Mosquito Netting White | Joann | 10173292 | http://www.joann.com/utility-fabric-mosquito-netting-white/10173292.html |
| Orthopedic stockings | Albahealth | 23650-040 | product no. 081420 |
| Organic Raisins | Newman's Own | UPC: 884284040255 | |
| Oviposition cups (brown) | Fisher Scientific | 03-007-52 | The product is actually an amber 125 mL bottle that we saw the top off of. |
| Recycled Paper Towels | Seventh Generation | 30BPT120 | |
| Modular Mates Square Tupperware Set | Tupperware | http://order.tupperware.com/pls/htprod_www/coe$www.add_items | |
| Glass Grinder | Corning Incorporated | 7727-2 | These Tenbroeck tissue grinders break the eggs and release RNA into the TRI Reagent. |
| TRI Reagent | Sigma Aldrich | T9424 | Apply 1ml TRI Reagent per 50-100mg of tissue. Caution – this reagent is toxic. |
| TURBO DNA-free | Ambion/Life Technologies | AM1907 | This kit generates greater yield than traditional DNase treatment followed by phenol/chloroform cleanup, and it is simpler to use. |
| RNaseZap | Ambion/Life Technologies | AM9782 | Apply liberally on the bench surfaces and any equipment that might be in contact with the RNA samples. The solution is slightly alkaline/corrosive, can cause irritation and is harmful when swallowed. |
| 2100 Bioanalyzer | Agilent Technologies | G2939AA | Place up to 12 RNA samples on one chip. |
| Hemotek Membrane Feeder | Hemotek | 5W1 | This system provides 5 feeding stations that can be used simultaneously. Includes PS5 Power Unit and Power cord; 5 FUI Feeders + Meal Reservoirs and O-rings; Plastic Plugs, Hemotek collagen feeding membrane; Temperature setting tool; and Plug extracting tool. The company's mailing address is: Hemotek Ltd; Unit 5 Union Court; Alan Ramsbottom Way; Great Harwood; Lancashire, UK; BB6 7FD; tel: +44 1254 889 307. |
| Digital Thermometer and Probe | Hemotek | MT3KFU | MicroT3 thermometer and KFU probe. This is used to set the temperature of each FUI feeding unit. |
| Chicken Whole Blood, non-sterile with Sodium Citrate | Pel-Freez Biologicals | 33130-1 | The 500 ml of blood were frozen and stored in 20 ml aliquots at -80 degrees C for up to 1 year. Thaw blood at room temperature for at least 1 h before using. |
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