Name: maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions
Uploaded: 2018-08-31T14:00:00.000+00:00
Duration: 7 min 20 s
Description: Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Vorremmo ringraziare Michelle Moon (Vanderbilt University) per assistenza con la fotografia. Vanderbilt University fornito supporto per PA e SSLB è supportato da DGE-1445197.

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	<li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum.:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum.: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> BioEssays. 28, 747-755 (2006).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphid Ecology: An Optimization Approach. , (1985).</li><li>Consortium, T. I. A. 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L., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+adaptations+toward+plant+toxins+in+milkweed-herbivores+systems+-+a+review.">Insect adaptations toward plant toxins in milkweed-herbivores systems - a review.</a> Entomologia Experimentalis et Applicata. 58, 579-610 (2018).</li><li>Rothschild, M., von Euw, J., Reichstein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+glycosides+in+the+oleander+aphid,+Aphis+nerii.">Cardiac glycosides in the oleander aphid, Aphis nerii.</a> Journal of Insect Physiology. 16, 1141-1145 (1970).</li><li>Harrison, J. S., Mondor, E. 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Lungamente è stato riconosciuto che l'aposematico a. nerii può fornire le comprensioni i modelli e meccanismi di resistenza per le difese della pianta e sequestro in particolare chimica18,37. Un numero di risorse genomiche è recentemente emerse per a. nerii10, offrendo nuove opportunità per studi di genomica funzionale ed ecologici che utilizzano a. nerii come un modello. Delineiamo protocolli di base nella c...

Gli afidi sono piccoli insetti hemimetabolous che colonizzano su famiglie di piante diverse in tutto il mondo. Sono distintivi per diverse funzioni, più in particolare i cicli di vita complessi che coinvolgono partenogenesi ciclica e discreti polyphenisms e loro simbiosi nutrizionali obbligano con endosimbionti batterici o lievito che forniscono le sostanze nutrienti mancanti da la loro dieta di pianta sap1. Mentre la maggior parte gli afidi sono specialisti di pianta ospite, alcune specie generalista sono parassiti delle colture importanti, infliggendo notevoli danni economici sulle colture sia direttamente o tramite gli agenti patogeni e virus hanno vector2. La pubblicazione del primo genoma afide nel 2010, l'afide di pisello Acyrthosiphon pisum3, segnò un'importante pietra miliare nello studio della biologia dell'afide perché ha fornito le risorse genomiche per questioni dell'insetto adattamenti agli erbivori stili di vita, comprese quelle che potrebbero portare a una migliore di strategie di controllo4. Da quel momento, ulteriori risorse genomiche hanno accumulato con la pubblicazione di un genoma con annotazioni per la soia afide glycines Aphis5e pubblicamente disponibile intero genoma risorse per un'altra specie di tre-afide (Myzus Cerasi (afide nero ciliegia), Myzus persicae (afide peach-patata), Rhopalosiphum padi (afide ciliegia di uccello-avena)6. Sono disponibili anche per un numero di altre specie di afide prezioso de novo Transcrittomica risorse (e.g.,Aphis gossypii (afide del cotone)7, Sitobion avenae (afide grano)8, Cinara pinitabulaeformis (afide pino)9, Aphis nerii (afide del milkweed-oleander)10).
Gli afidi hanno anche fatto duraturo contributo alla nostra comprensione delle interazioni pianta-insetto e l'ecologia della vita su piante11. Un'area dove gli afidi hanno dato contributi particolarmente importanti è nella nostra comprensione dell'ecologia chimica delle interazioni pianta dell'ospite. Adattamenti di esprimere diversi insetti erbivori per superare le difese della pianta e alcuni anche cooptare le difese della pianta per il proprio beneficio12,13,14. Ad esempio, l'afide del milkweed-oleander, Aphis nerii, è un brillante giallo, dilagante afide trovato nelle regioni temperate e tropicali in tutto il mondo che colonizza su piante appartenenti alla famiglia del milkweed (Apocynaceae). Piante della famiglia delle Apocynaceae si sono evolute diverse difese chimiche, tra cui lattice latteo e glicosidi cardiaci, noti come complessi, che legano il trasportatore di cationi Na, K-atpasi e sono efficaci deterrenti a generalista erbivori15, 16. specialisti milkweed express varie modalità di resistenza ai complessi, e alcuni in modo selettivo o passivamente si accumulano o modificare complessi nei loro tessuti come un mezzo per scoraggiare la predazione o per altri benefici17. A. nerii è pensato per sequestrare complessi in questo modo, anche se i meccanismi e i vantaggi funzionali rimangono poco chiari10,18.
Date le risorse genomiche a portata di mano, a. nerii fornisce un eccellente modello sperimentale per lo studio dei meccanismi molecolari e genetici coinvolti nelle interazioni chemio-ecologico tra piante tossiche e loro specialista erbivori. Vale la pena notare che, mentre alcuni dei primi studi di a. nerii incentrato sul sequestro di complessi19, da quel momento, gli studi di a. nerii hanno fornito le comprensioni in una vasta gamma di domande evolutive ed ecologiche, compresa la struttura genetica di insetti invasivi20 e l'interazione tra bottom up e top-down regolamento l'erbivoro densità21. A. nerii è così un buon candidato come un modello sperimentale per un insieme particolarmente vasto di studi delle interazioni insetto-pianta. Critico per il successo di qualsiasi studio con a. nerii è la cultura attenta dell'afide popolazioni, che comprende la coltura delle piante da cui dipendono gli afidi, come pure una generazione efficiente di dati di alta qualità - omic. Il nostro obiettivo è quello di guidare il lettore attraverso entrambi. Descritte di seguito sono i metodi per la generazione e la manutenzione delle colture vegetali e afide in serra e laboratorio, DNA ed estrazioni RNA, analisi dei microsatelliti, de novo del trascrittoma assemblaggio e annotazione, trascrittoma analisi di espressione differenziale e la verifica di qPCR di geni differenzialmente espressi. Mentre questi metodi vengono scritti per a. nerii, i metodi di coltura, estrazione e analisi generali possono estendersi ad una varietà di specie di afidi.

<table><tbody><tr><td>Sun Gro Fafard Germination Mix</td><td>Hummert International</td><td>10-0952-2</td><td></td></tr><tr><td>Sun Gro Fafard 3B/ Metro Mix</td><td>Hummert International</td><td>10-0951-2</td><td></td></tr><tr><td>2&quot; x 4&quot; Round Standard Pot</td><td>Anderson Pots</td><td>1503</td><td></td></tr><tr><td>DreamTaq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>EP0701</td><td></td></tr><tr><td>Trizol</td><td>ThermoFisher Scientific</td><td>15596026</td><td></td></tr><tr><td>SuperScript&amp;reg; III First-Strand Synthesis kit</td><td>ThermoFisher Scientific</td><td>18080051</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>ThermoFisher Scientific</td><td>4367659</td><td></td></tr></tbody></table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

Gli afidi sono eccellenti modelli sperimentali per una varietà di domande biologiche che vanno dall'evoluzione delle simbiosi e lo sviluppo di polyphenisms a domande che circondano interazioni dell'insetto con le loro piante ospite. Risorse genomiche sono disponibili per diverse specie di afidi, e i progressi compiuti il sequenziamento di nuova generazione, trascrittomica studi sono stati estesi a organismi non-modello che non dispongono di genomi. Inoltre, afide culture possono essere raccolti dal campo e allevate in laboratorio per l'uso negli esperimenti organismi e molecolari per colmare il divario tra gli studi ecologici e genetici. Infine, molti afidi possono essere mantenuti in laboratorio sulle loro piante ospite preferito in perpetui, partenogenetiche cicli di vita consentendo confronti di riprodursi asessualmente genotipi. Aphis nerii, l'afide del milkweed-oleander, fornisce un modello per studiare le interazioni insetto con piante tossiche utilizzando esperimenti sia organismi che molecolari. Metodi per la generazione e la manutenzione delle colture vegetali e afide nell'espressione differenziale di serra e laboratorio, estrazioni di DNA e RNA, l'analisi del microsatellite, de novo del trascrittoma assemblaggio e annotazione, trascrittoma analisi e la verifica di qPCR di geni differenzialmente espressi sono descritte e discusse qui.

Colture della pianta: Semi prenderà circa due-quattro settimane, a seconda della stagione, per diventare grande abbastanza per essere ri-vaso (Figura 1A). Re-potted piantine porterà un altro due-quattro settimane per aumentare a una dimensione ottima per le culture di afide (Figura 1B).
Culture afide:...

Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

L'afide Aphis nerii colonizza sulle piante altamente difeso nella famiglia dogbane (Apocyanaceae) e offre numerose opportunità per studiare le interazioni pianta-insetto. Qui, presentiamo una serie di protocolli per la manutenzione di impianti e afide, culture e la generazione e l'analisi di molecolare e dati - omic per a. nerii.

Mantenimento di colture biologiche e misurazione dell'espressione genica in Aphis nerii: un sistema Non-modello per le interazioni pianta-insetto

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

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Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

7.5K Views.  Vanderbilt University. L'afide Aphis nerii colonizza sulle piante altamente difeso nella famiglia dogbane (Apocyanaceae) e offre numerose opportunità per studiare le interazioni pianta-insetto. Qui, presentiamo una serie di protocolli per la manutenzione di impianti e afide, culture e la generazione e l'analisi di molecolare e dati - omic per a. nerii.

Video: Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

Consegna degli acidi nucleici attraverso Embrione microiniezione in tutto il mondo agricolo parassita,<em&gt; Capitata Ceratitis</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Ricerca

Genetica

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

Individuazione di condizioni critiche per Immunostaining nel pisello afide embrioni: l&#39;aumento del tessuto Permeabilità e diminuire la colorazione di fondo

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Biologia dello sviluppo

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Allevamento e iniezione di<em&gt; Manduca sexta</em&gt; Le larve per valutare virulenza batterica

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Immunologia e infezioni

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Allevamento di massa e studi molecolari negli insetti parassiti Tortricidae

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

Ambiente

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi mediato doppia Gene atterramento e gustative Percezione misura di api mellifere (<em&gt; Apis mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuroscienze

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

Coltivare e manipolare geneticamente i nematodi entomopatogeni

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Espressione transitoria e Localizzazione cellulare di proteine ​​ricombinanti in cellule in coltura per insetti

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Biologia

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Mantenimento di Aedes aegypti zanzare infettate con Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

Iniezioni pupali e adulte per l'editing genico RNAi e CRISPR a Nasonia al vetripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Mantenimento di colture biologiche e misurazione dell'espressione genica in Aphis nerii: un sistema Non-modello per le interazioni pianta-insetto

Riepilogo

Esplora Altri Video

Capitoli in questo video

Consegna degli acidi nucleici attraverso Embrione microiniezione in tutto il mondo agricolo parassita,

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,

Individuazione di condizioni critiche per Immunostaining nel pisello afide embrioni: l'aumento del tessuto Permeabilità e diminuire la colorazione di fondo

Allevamento e iniezione di

Allevamento di massa e studi molecolari negli insetti parassiti Tortricidae

RNAi mediato doppia Gene atterramento e gustative Percezione misura di api mellifere (

Coltivare e manipolare geneticamente i nematodi entomopatogeni

Espressione transitoria e Localizzazione cellulare di proteine ricombinanti in cellule in coltura per insetti

Mantenimento di Aedes aegypti zanzare infettate con Wolbachia

Iniezioni pupali e adulte per l'editing genico RNAi e CRISPR a Nasonia al vetripennis

Mantenimento di colture biologiche e misurazione dell'espressione genica in Aphis nerii: un sistema Non-modello per le interazioni pianta-insetto

Riepilogo

Esplora Altri Video

Capitoli in questo video

Consegna degli acidi nucleici attraverso Embrione microiniezione in tutto il mondo agricolo parassita,

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,

Individuazione di condizioni critiche per Immunostaining nel pisello afide embrioni: l'aumento del tessuto Permeabilità e diminuire la colorazione di fondo

Allevamento e iniezione di

Allevamento di massa e studi molecolari negli insetti parassiti Tortricidae

RNAi mediato doppia Gene atterramento e gustative Percezione misura di api mellifere (

Coltivare e manipolare geneticamente i nematodi entomopatogeni

Espressione transitoria e Localizzazione cellulare di proteine ​​ricombinanti in cellule in coltura per insetti

Mantenimento di Aedes aegypti zanzare infettate con Wolbachia

Iniezioni pupali e adulte per l'editing genico RNAi e CRISPR a Nasonia al vetripennis

Espressione transitoria e Localizzazione cellulare di proteine ricombinanti in cellule in coltura per insetti