The authors would like to thank Erin Fennern for assisting experiment. This work was funded by the research Council of Norway (180504, 185306 and 191699), PEW Charitable Trust, and the National Institute on Aging (NIA P01 AG22500).

Part 1: RNAi-mediated double gene knockdown
1. dsRNA Synthesis
<ol>
	<li>Design primers of dsRNAs targeting vg, usp and a control gene encoding green fluorescence protein (GFP) which is not in the honey bee genome: primers were designed using online free software Primer3 (<a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a>).</li>
	<li>dsRNA synthesis for vg, usp and GFP: use RiboMax T7 RNA production system from Promega for in vitro transcription.</li>
	<li>dsRNA purification:
	<ol>
		<li>Denaturation and renaturation: heat the dsRNA to 85 &deg;C for 5 min and let it cool down at room temperature for 1 hr.</li>
		<li>DNase I treatment: add 1 &mu;l DNase I into each dsRNA reaction and mix them by flicking the tubes. Incubate for 15 min at 37 &deg;C.</li>
		<li>Add 150 &mu;l nuclease-free water and 750 &mu;l TRizol - LS into each reaction, and gently mix them by inverting the tube. Incubate the samples for 5 min at 30 &deg;C.</li>
		<li>Add 200 &mu;l chloroform into each sample. Mix vigorously for 20 sec. Centrifuge the samples for 15 min at 12,000 x g at 4 &deg;C.</li>
		<li>Transfer the supernatant phase to each new tube and add 500 &mu;l isopropyl alcohol into each tube. Mix them by inverting the tube. Incubate the mixture for 20 min at -20 &deg;C. Centrifuge it for 10 min at 12,000 x g at 4 &deg;C.</li>
		<li>Remove the supernatant and use 1,000 &mu;l 75% ethanol to wash the pellet. After centrifuging the tube for 5 min at 7,500 x g at 4 &deg;C, air dry the dsRNA pellet, and use nuclease free water to dissolve the pellet. In order to ensure the efficacy of gene down-regulation, the dsRNA concentration should be around 9 -10 &mu;g/&mu;l.</li>
	</ol>
	</li>
</ol>
2. dsRNA Abdominal Injection
For double gene knockdown, there are two strategies: 1) single injection: mix dsRNA of two genes and inject the mixture; 2) two-day injection: inject the first dsRNA targeting one gene on the first day, and inject the second dsRNA into the same bees on the second day.
<ol>
	<li>Chill newly emerged bees in a 4 &deg;C refrigerator for 1-2 min. When the bees are immobilized, mount 3-4 bees in parallel on Petri dishes full of solid wax with insect pins crossed between their abdomens and thoraces.</li>
	<li>Chill the bees again in a 4 &deg;C refrigerator. Ensure the bees are completely immobile. It takes about 1-2 min. The bees should look loose. If the bees become curled or contorted, it means they are chilled too long which should be avoided. Chilling too much causes high mortality, but complete immobilization is important as any movement increases the size of the wound and mortality.</li>
	<li>Put a disposable 30 G needle (BD) on a Hamilton micro syringe. Take 3 &mu;l dsRNA and make sure there is no air bubble in the syringe. The needle is inserted to a side of the abdomen to avoid damaging internal organs. Press the syringe plunger slowly to allow the dsRNA to be absorbed. Since dsRNA is very viscous, it takes 2-3 sec to be completely expelled from the syringe. After completely pushing down the plunger, leave the needle in the wound for 4-5 sec. Observe bees for 3-5 sec after injections. If a hemolymph droplet leaks from the wound, discard the bee.</li>
	<li>Use different colors to mark their thoraces corresponding to different treatments. At this point if the single injection strategy was used, the bees can be placed back into a colony after 1 hr observation. However, if the two-day injection strategy was used, after being injected with the first dsRNA, the bees should be placed in a cylinder mesh cage with honey supplied on the side and be kept in an incubator at 34 &deg;C and 80% humidity.</li>
	<li>On the second day, follow steps 2.2 and 2.3 to chill the bees in the mesh cages and mount them on wax plates. Perform injections with the second dsRNA as it is described in step 2.4.</li>
	<li>Let the bees recover at room temperature for 1 hr and introduce them into a colony.</li>
</ol>
Note: Knockdown effects can be detected by quantitative RT-PCR (qRT-PCR) and western blotting. In general, knockdown efficacy varies and depends on many factors. In our study, the double gene knockdown can be detected 7 days after single injections and two-day injections.
Part 2: Proboscis extension response (PER) assay
1. Collecting Bees from the Hive
On day 6 after the injections, collect treated bees from the colony without using a smoker. Collect bees in metal mesh cylinder cages. Keep 3 bees in each cage.
2. Mounting Bees for PER
Chill the bees at 4 &deg;C until they are completely immobilized. Mount each bee vertically into a plastic tube specially designed for PER. Keep the abdomen inside the tube by using two small pieces of tape, and keep the head sticking out of the tube. Put the tubes on a rack with two rows of small plastic columns, and assign each tube (bee) with an ID number. Then put treated bees on the racks into an incubator (34 &deg;C and 80% humidity) and keep them in the incubator for 2 hr.
3. Preparing a Series of Sucrose Water Solutions
Prepare 0%, 0.1%, 0.3%, 1%, 3%, 10% and 30% sucrose water solutions and load the solutions into syringes with 15-20 G needles.
4. PER Assay
Touch the bee's antenna with a droplet of 0% solution (water) followed by 0.1%, 0.3%, 1%, 3%, 10% and 30% sucrose water solutions. Test all bees with water first and allow at least two minutes as the interval between each solution is used. Therefore, we usually have at least 20 bees for each trial. If a bee fully extends her proboscis, a positive response is counted.
5. Data Management
Sum up the total positive responses for each individual for gustatory response score (GRS). Perform an appropriate statistical analysis on the data.
Part 3. Gene knockdown validation
Fat bodies are dissected from another set of 7-day old treated bees. A standard TRizol procedure is used for RNA extraction, which is followed by DNase I treatment 24. Vg and usp gene expression is analyzed by a two-step qRT-PCR. Actin is used as a reference gene since it has stable expressions in different tissues of honey bees. Published real-time PCR primers for vg and usp gene are used in this experiment 24. Data are analyzed by using Delta-Delta CT method 25.

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We have nothing to disclose.

Our study is the first effort to simultaneously knock down two genes in adult insects. Our results show that the two strategies of dsRNA injections (single injection and two-day injection) are effective for the double gene suppression, and simultaneous gene silencing of vg and usp can be tested 6 days after the dsRNA injections into bees 8,9. However, gene knockdown efficacy varies in different insect species depending on transcript level of target gene, protein turnover rates and dsRNA uptak...

RNA interference (RNAi) is RNA based post-transcriptional gene silencing, which occurs in a wide variety of eukaryotic organisms. The process of RNAi is triggered by endogenous or exogenous double-stranded RNA (dsRNA) precursors. The dsRNA activates the ribonuclease protein Dicer which binds and cleaves the dsRNA to small fragments (20-25 bp). Then the small fragments of the dsRNA guide a recognition and cleavage of complementary mRNAs by argonaute proteins, a catalytic component of RNA-induced silencing complex (RISC)1. In mammals, dsRNAs longer than 30 nt, activate an antiviral response (interferon response, IFN) which leads to nonspecific degradation of RNA transcripts 2. However, long dsRNAs have proven to be effective and specific in insects since there is a lack of this IFN 3.
Long dsRNAs have been used for downregulation of target genes in different insect species. Honey bees are one of the pioneer insect organisms in which functions of many important genes in the development and behavior have been revealed by using dsRNA 4,5. Several dsRNA delivery methods have been performed in honey bees: dsRNA feeding efficiently downregulates target gene expression in honey bee larvae 6, whereas dsRNA injection is an effective approach for gene knockdown in honey bee embryos 4 and adult bees 7,8.
Gene knockdown effects exhibited by applying dsRNA to insects are transient and localized. Studies have shown that both abdominal dsRNA injections and thoracic dsRNA injections effectively suppress target gene expression in abdominal fat body cells of insects 9,10. DsRNA is injected into abdominal and thoracic cavities and fat body cells are able to take up the dsRNA from the hemolymph where the cells are bathed 10. However, genes in other organs, such as ovaries and brains, cannot be targeted by either abdominal or thoracic injections. In order to target genes in honey bee brain, brain injection of dsRNA has also been performed, which effectively influences target gene expression in local brain areas 11. Here, we only document abdominal dsRNA injection which is more commonly used in adult honey bees.
RNAi has been primarily used to target a single gene and has been a powerful tool to reveal the gene function. However, any gene is not isolated from others; it is in complex regulatory networks. A key to understand a biological process is to dissect how genes interact with each other, which requires simultaneous manipulations of multiple genes rather than a single gene knockdown. In mammalian cell lines, scientists have succeeded in simultaneously inhibiting two or three genes by using delivery systems 12 or multi-microRNA (miRNA) hairpin designs 13. But in insects, multiple gene knockdowns are still untested. Here, we present different injection strategies which can achieve a double gene knockdown. We target two genes: vitellogenin (vg) which encodes a yolk protein precursor, and ultraspiracle (usp) which encodes a putative receptor for juvenile hormone (JH) and may serve as a transcription factor mediating responses to JH 14 in honey bees. Vg and JH regulate each other in a feedback loop 15 and are involved in honey bee behavioral regulation 9. Using the double gene knockdown, we perturb both Vg and JH pathways, and discover how they jointly affect honey bee behavior and physiology and how vg, usp and JH interact 9.
Gustatory perception is a behavioral predictor for honey bee social behavior 16. In terms of behavioral development, nest bees with high gustatory perception behaviorally mature fast, and usually forage early in life and prefer to collect pollen 16,17. Although regulatory mechanisms underlying gustatory perception are still unclear, studies have shown that gustatory perception is linked to internal energy metabolisms 9, hormonal secretion 18,19 and biogenetic amine pathways 20. Both Vg and JH are important hormonal regulators modulating gustatory perception 7,21. In the laboratory, a variation of gustatory perception in honey bees can be evaluated by testing the proboscis extension response (PER) to different sucrose solutions. Each bee is tested by touching both her antennae with a droplet of water followed by an ascending concentration series of 0.1, 0.3, 1, 3, 10, 30% sucrose. A positive response is noted if a bee fully extends her proboscis when a droplet of water or sucrose is touched to each antenna. Based on the number of positive responses to the solutions, the gustatory perception level of each individual can be determined 16. However, the application of the PER is not limited to measuring gustatory perception. The PER is also an effective method to test the metabolic state of bees such as satiation vs. hunger. The bees with greater responses to sucrose are hungrier in general (Wang and Amdam, unpublished data). Furthermore, the PER paradigm can also be used in associative learning and memory in honey bees. In this case bees will be trained to associate the presence of sucrose water with an odor. When the bees learn the association, only the presence of the odor can evoke a positive proboscis response without rewarding them with the sucrose 22,23. In this video, we show how to perform PER to evaluate gustatory perception which has been connected with vg and usp double knockdown in a previous study 9.

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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.

Both the single-injection and two-day-injection strategies significantly reduced vg (One-way ANOVA, p &lt; 0.001) (Figure 2A) and usp (One-way ANOVA, p &lt; 0.001) (Figure 2B) transcript levels in honey bees six days after the dsRNA was injected. The suppression of usp transcript by using the single injection with dsRNA mixture and the two-day injection with vg first and usp second (vg/usp) was significantly lower than the tw...

Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

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-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 ...

rnai-mediated-double-gene-knockdown-gustatory-perception-measurement

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.8K Views.  Arizona State University. 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.

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

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.

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

Genetics

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Environment

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.

Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity.

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Biology

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

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.

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

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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

Summary

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Chapters in this video

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

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

RNAi Trigger Delivery into Anopheles gambiae Pupae

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

Assessing Agrochemical Risk to Mated Honey Bee Queens

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes