Name: rna interference in aquatic beetles as a powerful tool for manipulating gene expression at specific developmental time points
Uploaded: 2020-05-29T15:00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points at JoVE.com

We would like to thank Dr. Josh Benoit for his assistance with bioinformatics Hailey Tobler for her help in raising Sunburst Diving Beetles and Tamara Pace for editorial assistance. This research was supported by the National Science Foundation under grants IOS-1456757 and IOS-1856341 to EKB and IOS1557936 to YT.

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The authors have nothing to disclose.

Our goal is that this compilation of methods will make RNAi widely available, especially as this tool remains a powerful synergistic technique to CRISPR/Cas9-based gene editing, with the advantage that it can be applied to the desired developmental stages of studied organisms. To exemplify this strength, we injected dsRNA into embryos and into different larval stages. Injections into eggs affected the development of embryos (Figure 2), injections into the second larval stage had apparent effects on the third larval stage (Figure 4 and Figure 6), and injections into the third larval stage showed effects in the adults (Figure 5). While the exact timing has to be established experimentally, generally, injections take effect within a few days. The success of this process can be affected by the length of the dsRNA sequence. Here, we presented examples using a little over 200 bp to more than 800 bp. As a general rule, sequences between 100 and 600 bp are preferred to limit off-target effects, but sequences up to 1000 bp yield good results22. One question in regard to RNAi is the duration of knockdown that can be achieved through this technique. Since the phenotypes were terminal at each stage, we cannot comment on this issue based on our presented results. However, it has previously been noted that RNAi effects are generally relatively long lived, and that higher concentrations lead to longer lasting knockdowns20.
One limitation of this technique is that it works better for some organisms than for others, and there seems to be no direct way of predicting how well it will work a priori. Nevertheless, it has been found to work well for a large range of different organisms. Within arthropods, this includes arachnids26, crustaceans27, and a variety of insects, with particularly high success rates in beetles28. A further complication is that differences in phenotype severity often occur between individuals despite the application of the same amount of dsRNA. As illustrated in Figure 2B, variation can even occur within an individual. In our RNAi studies targeting different genes involved in T. marmoratus larval eye development, we have frequently found that some eyes are affected more severely than others. This phenomenon may be related to the relatively dense tissue of the eye cluster, with the dsRNA better able to reach some of the units.
For the successful execution of RNAi experiments, it is critical that several parameters are optimized for the target gene. For example, the concentration of the dsRNA and the length of the targeted gene can strongly influence the outcome20. Another critical parameter is how the injections are executed, as this process can greatly influence the survival rate. For embryos, we achieved the best results by targeting the center of the embryo. A well-laid-out plate allows for the injection of 100 or more embryos in a single session. For larvae, it is critical to insert the injection needle between the segments. These injections require more dsRNA, and based on larvae availability, our injection sets here typically only consisted of a few animals at a time. For all injections, it is critical to prevent air from entering the organism.
In some cases, the feedback loops of a gene regulatory network and genetic redundancy can influence the penetrance of RNAi phenotypes, despite consistent knockdowns. This seems to be the case for our behavioral observations of larvae with highly successful knockdowns of a prominent lens protein, Lens318. Although we verified the high efficiency of these knockdowns through qPCR, considerable variation was observed in the associated phenotypes. This result highlights the necessity of properly quantifying RNAi knockdowns (for details on options see22). If there is no clear a priori expectation in regard to the resulting phenotypes, a good way of controlling for the off-target effects of RNAi is to target the same gene with two non-overlapping sequences of dsRNA and to evaluate the results for common phenotypes.
In contrast to gene-editing techniques, RNAi is also a powerful tool for studying lethal genes, and there are two ways to do so. For example, if one is interested in the functional contribution of a gene where loss-of-function early in development is known to be lethal, a functional investigation of such a gene can be achieved by simply allowing the animal to develop normally and then knocking down the gene via RNAi later in development (i.e., in the adult). Alternatively, a gene where complete loss-of-function is known to be lethal can be investigated through a partial knockdown, which can be achieved by injecting a range of dsRNA concentrations. Some of our results show knockdowns of lac2, which are known to be lethal if the cuticle in insects becomes overly soft24. Even the lac2 RNAi beetle depicted in Figure 5 would be unlikely to survive outside laboratory conditions. Another lethal gene is cut, which codes for a transcription factor that is fundamental for cell-fate specification in various organ systems in arthropods and has been linked to glia development in the Drosophila visual system29. Based on our experience with cut RNAi in T. marmoratus embryos, we can evoke informative eye phenotypes in embryos that are able to complete their embryonic eye development (unpublished observations). Here, higher dosages appear to lead to higher lethality rates, while lower doses result in observable and informative phenotypes.
Our protocol not only lists the necessary steps for a researcher to pursue RNAi experiments on T. marmoratus, as illustrated, but also is generally applicable to other organisms, especially aquatic organisms. Among aquatic organisms, there are already several examples within crustaceans such as the water fleas Daphnia30 and shrimp (for a recent review, see reference31). There are ample opportunities among aquatic insects, as they have been estimated to comprise about 6% of all insect diversity, with likely more than 200,000 species32. Furthermore, RNAi has already been performed on water striders that tend to inhabit the surface of aquatic environments33. If no genome is present, then a transcriptome can be assembled de novo. As long as this process reveals contigs of a few hundred nucleotides, dsRNA against specific genes can be designed. Our protocol for immobilizing insects in agarose will likely also be useful for other procedures, especially for soft, malleable, and aquatic organisms. Taken together, RNAi remains a powerful technique for manipulating gene expression in a diverse group of organisms, even when no other molecular and genetic tools are available.

The question of how specific genes contribute to the evolution of diverse traits is an exciting topic in biology. Over the last few decades, much progress has been made in regard to dissecting the genetic underpinnings of developmental processes in a few model organisms, such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the house mouse Mus musculus1. More recently, the invention of powerful gene-editing techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas92 has provided the ability to change the genetic code of non-model organisms (for examples see3,4). As a result, there has been a surge in genetic studies on a variety of organisms that had not previously been approached through molecular techniques. Considering the enormous diversity of our animal kingdom, with many interesting traits or trait variances that are only represented in specific species, this progress has made it an exciting time for evolutionary-developmental biology (&#8220;evo-devo&#8221;) related work. However, genome-editing techniques that are available to non-model organisms are relatively restricted in regard to the developmental time points to which they can be applied, making it challenging to discern the temporal properties related to the role that specific genes play in any trait. In addition, transgenic techniques are often limited to genes that are nonessential for survival (i.e., whose knockout does not result in lethality). Therefore, while gene-editing techniques have started to become popular, there remains a need for effective techniques that are applicable to a variety of different organisms at specific developmental time points and facilitate partial knockdowns (rather than complete loss-of-function). Here, we draw attention to RNA interference (RNAi), a somewhat dated yet powerful gene knockdown technique5 that is particularly valuable as a synergistic approach to gene editing. Specifically, we developed procedures that allow for the application of RNAi to aquatic diving beetles as an example that illustrates the implementation of this technique, from the acquisition of the necessary molecular sequences to the successful injection of double-stranded RNA (dsRNA) into eggs and larvae.
RNAi-based gene knockdown leverages an innate defense mechanism of organisms, in which dsRNA molecules facilitate the silencing of invading nucleic acid sequences, such as viruses and transposons6. In brief, dsRNA is taken up into the cell, where it is cut into 20&#8211;25 nucleotide pieces by the Dicer enzyme. These pieces then activate the formation of the RNA-induced silencing complex (RISC), which inhibits the targeted mRNA by binding to it at specific sites using the guide strand. This process ultimately leads to mRNA degradation and hence interferes with the translation of mRNA into the respective protein6. The RNAi-based gene knockdown technique presented here therefore relies on the injection of dsRNA. For animal models, this technique was originally developed in C. elegans7 and D. melanogaster8 but has since emerged as a powerful functional genetic tool in non-model organisms9,10. Owing to its highly effective nature in some insects, RNAi can even be applied in pest management11.
As a research tool, RNAi has been used to examine how key molecular-developmental pathways function in nontraditional insect models. For example, RNAi in the flour beetle Tribolium&#160;castaneum has been instrumental in determining how deeply conserved genes contribute to specific traits in that beetle, as exemplified for the development of specifically shaped wings12,13,14 and eyes15,16. The techniques that underlie the manipulations in T. castaneum have been well described17 and rely on the ability to immobilize relatively dry eggs and larvae on a sticky surface. Such immobilization however is not possible for the wet developmental forms of aquatic organisms such as the Sunburst Diving Beetle Thermonectus marmoratus. As is the case for many nontraditional model organisms, it lacks an annotated genome. To manipulate gene expression in any organism without a genome, a reasonable and cost-efficient first step is to generate transcriptomes and identify the putative nucleotide sequences of the expressed genes of interest based on sequence similarity with related but more established model organisms, in this case, primarily Tribolium (Coleoptera) and Drosophila genes.
Here, to demonstrate how RNAi can be used on an aquatic organism, we first discuss protocols and software for RNA extraction and transcriptome generation and assemblage, which allow for the identification of specific targeted gene sequences. We then summarize the necessary steps for synthesizing gene-specific dsRNA. Subsequently, we illustrate how eggs can be injected in an aquatic environment and demonstrate incubation protocols for culturing developing embryos. In addition, we show how agarose gel can be used to completely immobilize larvae during the injection process, a technique that is generally useful during various procedures and could be applied to a variety of arthropods. To demonstrate how RNAi can be applied to different developmental stages, we include an example in which we silenced the eye pigmentation gene white in embryos. In addition, we describe an example in which the tanning gene laccase2(lac2) was silenced during both the second larval instar (to affect larvae of the third larval instar) and the third larval instar (to affect adults). Finally, we demonstrate that the injection of a lower concentration of dsRNA leads to partial knockdown, which shows that this technique can also be applied to genes where loss-of-function is known to be lethal.

<table>
	<tbody>
		<tr>
			<td>1. RNA isolation and de novo transcriptome assembly</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>University Stockroom</td>
			<td></td>
			<td>Typically locally available at research institutions</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td>Fisher Scientific</td>
			<td>size dependent</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>RNeasy lipid tissue mini kit (RNA isolation kit)</td>
			<td>Qiagen</td>
			<td>74804</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>spectrophotometer (NnanoDrop)</td>
			<td>Thermo Fisher</td>
			<td>9836674</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.2. De novo transcriptome assembly.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BUSCO.v3</td>
			<td>https://busco.ezlab.org/</td>
			<td></td>
			<td>Any freely available software for assessing trasncriptome completeness can be used.</td>
		</tr>
		<tr>
			<td>CLC Genomics workbench</td>
			<td>Qiagen</td>
			<td>832021</td>
			<td>Other equivalent software packages are also available</td>
		</tr>
		<tr>
			<td>Galaxy workbench</td>
			<td>https://usegalaxy.org/</td>
			<td></td>
			<td>An open source online transcriptome assembly &#38; annotation pipeline</td>
		</tr>
		<tr>
			<td>NCBI BLASTx</td>
			<td>https://blast.ncbi.nlm.nih.gov/Blast.cgi?LINK_LOC=blasthome&#38;PAGE_TYPE=BlastSearch&#38;PROGRAM=blastx</td>
			<td></td>
			<td>An open source online alignment and annotation pipeline</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2. cDNA synthesis, Cloning &#38; Miniprep isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ampicillin sodium salt</td>
			<td>Gibco</td>
			<td>11593-027</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-500</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>Omniscript RT (reverse trasncription) kit</td>
			<td>Qiagen</td>
			<td>205111</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>One Shot&#8482; TOP10 Chemically Competent E. coli</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit</td>
			<td>ThermoFischer</td>
			<td>771471</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>shaker-incubator</td>
			<td>Labnet</td>
			<td>211DS</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>spectrophotometer (NnanoDrop)</td>
			<td>Thermo Fisher</td>
			<td>9836674</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>thermal cycler for PCR</td>
			<td>BioRad</td>
			<td>T100</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>TOPO TA Cloning kit</td>
			<td>Invitrogen</td>
			<td>1845069</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>X-Gal Solution</td>
			<td>Thermo Scientific</td>
			<td>R0941</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.2 PCR amplification &#38; in vitro dsRNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>accuSpin Micro 17R</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Fisher Scientific</td>
			<td>A4094</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>FastTaq DNA Polymerase, dNTPack</td>
			<td>Roche</td>
			<td>13873432</td>
			<td>This kit contains all the reagents necessary for a PCR</td>
		</tr>
		<tr>
			<td>MEGAclear Transcriiption clean up kit</td>
			<td>ThermoFischer</td>
			<td>AM1908</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription kit</td>
			<td>ThermoFischer</td>
			<td>AM1334</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>nuclease-free water</td>
			<td>Fisher Scientific</td>
			<td>AM9932</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Detailed specific protocol is provided with the kit</td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>BP333-500</td>
			<td>Make 3M working solution</td>
		</tr>
		<tr>
			<td>Spectrophotometer (NnanoDrop)</td>
			<td>Thermo Fisher</td>
			<td>9836674</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3. Collecting and preparing early stage T. marmoratus embryos for dsRNA injections</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>agarose</td>
			<td>Fisher Scientific</td>
			<td>9012-36-6</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>distilled water</td>
			<td>Fisher Scientific</td>
			<td>9180</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>forceps (Dumon #4 Biology)</td>
			<td>Fine Science Tools</td>
			<td>11242-40</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>glass cavity dish (3 well-dish)</td>
			<td>Fisher Scientific</td>
			<td>50-243-43</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>microwave</td>
			<td>Welbilt</td>
			<td>turn-table</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>natural hair paintbrush</td>
			<td>Amazon</td>
			<td></td>
			<td>Any fine brush will do</td>
		</tr>
		<tr>
			<td>P1000 micro-pipetter</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Microsocope Central</td>
			<td>10446293</td>
			<td>Any good stereomicroscope will work</td>
		</tr>
		<tr>
			<td>transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>21-200-109</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4. dsRNA micro-injections in early stage T. marmoratus embryos</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>digital camera</td>
			<td>Edmund optics (Qimaging)</td>
			<td>Retiga 2000R</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Fisher Scientific</td>
			<td>A4094</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>food dye</td>
			<td>Kroger</td>
			<td></td>
			<td>Any available food dye should work fine</td>
		</tr>
		<tr>
			<td>humidity chamber</td>
			<td></td>
			<td></td>
			<td>take any plastic box with a lid, sterilize it with 70% ethanol and let it dry</td>
		</tr>
		<tr>
			<td>incubator</td>
			<td>Labline</td>
			<td>203</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>injection buffer</td>
			<td></td>
			<td></td>
			<td>Prepared for 1 mL following reference #22 : Mix 10 &#181;L of 0.1 M sodium phosphate buffer, 100 &#181;L of 0.5 M potassium chloride solution, 100 &#181;L of food dye and 790 &#181;L of double-distilled water. Store in 4 &#176;C.</td>
		</tr>
		<tr>
			<td>intracellular Microinjection Systems (Picospritzer)</td>
			<td>Parker</td>
			<td>052-0500-900</td>
			<td>Currently the model III is available, but older models work also</td>
		</tr>
		<tr>
			<td>micro needle holder</td>
			<td>A-M Systems</td>
			<td>672441</td>
			<td>Other products would work equally well</td>
		</tr>
		<tr>
			<td>microinjection needles (1.2 mm x 0.68 mm, 4 inches)</td>
			<td>A-M Systems</td>
			<td>603000</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-024-R</td>
			<td>Any quality micromanipulator will work</td>
		</tr>
		<tr>
			<td>monosodium phosphate</td>
			<td>Fischer scientific</td>
			<td>7558-80-7</td>
			<td>To make 10 mL of 1 M working solution: Add 1.2 g of monosodium phosphate powder to 10 mL of Double distilled water and mix until clear solution is obtained</td>
		</tr>
		<tr>
			<td>P-1000 Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Puller settings:Heat 575, Pull 60, Velocity 75 Delay 110, Pressure 700.</td>
		</tr>
		<tr>
			<td>P10 micro-pipetter</td>
			<td>Gilson</td>
			<td>F144802</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>P1000 micro-pipetter</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Fischer scientific</td>
			<td>7447-40-7</td>
			<td>To make 100 mL of 0.5 M potassium chloride solution: Add 3.73 g of potassium chloride crystals to 100 mL of double distilled water and mix until clear solution is obtained.</td>
		</tr>
		<tr>
			<td>sodium phosphate buffer (0.1M)</td>
			<td></td>
			<td></td>
			<td>To make 10 mL of 0.1 M of this buffer: Mix 8.5 mL of 1 M sodium phosphate dibasic solution with 1.5 mL of 1 M monosodium phosphate solution. Check the pH with a pH meter and adjust accordingly to pH 7.6 at room temprature.</td>
		</tr>
		<tr>
			<td>sodium phosphate dibasic</td>
			<td>Fischer scientific</td>
			<td>7558-79-4</td>
			<td>To make 10 mL of 1 M working solution: Add 1.42 g of sodium phosphate dibasic powder to 10 mL of Double distilled water and mix until clear solution is obtained</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Microsocope Central</td>
			<td>10446293</td>
			<td>Any good stereomicroscope will work</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5. Preparing T. marmoratus larvae for dsRNA injections</td>
		</tr>
		<tr>
			<td>agarose</td>
			<td>Fisher Scientific</td>
			<td>9012-36-6</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>forceps (Dumon #4 Biology)</td>
			<td>Fine Science Tools</td>
			<td>11242-40</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>Products from other vendors would work equally well</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6. dsRNA micro-injections in T. marmoratus larvae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>injection buffer (10x)</td>
			<td></td>
			<td></td>
			<td>See section 4</td>
		</tr>
		<tr>
			<td>microinjection needles (1.2 mm x 0.68 mm, 4 inches)</td>
			<td>A-M Systems</td>
			<td>603000</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>microinjection syringe</td>
			<td>A-M Systems</td>
			<td>603000</td>
			<td>Other models would work equally well</td>
		</tr>
		<tr>
			<td>micro needle holder</td>
			<td>A-M Systems</td>
			<td>672441</td>
			<td>Other products would work equally well</td>
		</tr>
		<tr>
			<td>P-1000 Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Puller settings:Heat 575, Pull 60, Velocity 75 Delay 110, Pressure 700.</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Microsocope Central</td>
			<td>10446293</td>
			<td>Any good stereomicroscope will work</td>
		</tr>
	</tbody>
</table>

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.

Using the protocol described above, we knocked down three different genes, namely, white, laccase2 (lac2), and Lens3 (Table 1), at a variety of different developmental stages of the Sunburst Diving Beetle T. marmoratus. We performed RNAi in T. marmoratus by injecting dsRNA at a very early stage during embryogenesis (Figure 1A). As some of the embryos do not survive the process and turn necrotic (Figure 1B), they need to be removed to keep the remaining embryos healthy. Exemplified here are the injections of dsRNA against the white gene. This gene is well known in Drosophila as one of three ATP-binding cassette (ABC) transporters involved in the uptake and storage of the precursors of eye pigment23. Accordingly, its loss-of-function results in an unpigmented, white-eye phenotype. Our results show that the injections of dsRNA targeting the orthologous white gene in T. marmoratus embryos leads to the loss of eye pigmentation in newly emerging larvae. In this case, wild type larvae are characterized by heavily pigmented eyes, and the RNAi knockdown of white leads to various levels of reduction and even complete elimination of eye pigment. Overall, we observed at least some reduction in eye coloration in 34% of surviving embryos (n = 35). Figure 2A compares a control individual and an individual with slightly lighter eye color. Figure 2B illustrates a more severe knockdown in an individual, in which the more ventral eyes (Eyes 2&#8211;5) of the cluster are completely unpigmented, whereas the dorsal eyes still show some pigmentation. These differences highlight how the efficiency of the knockdown can vary regionally, which is possibly related to differences in the efficacy of dsRNA penetration in dense tissue. Another individual shows essentially complete pigment loss in all eyes (Figure 2C).
To investigate how well RNAi works at the larval stage of T. marmoratus, we injected dsRNA targeting the tannic gene lac2 into second instar larvae a few days before they were due to molt into third instars (Figure 3) and evaluated the effect on the cuticular coloration of third instar larvae. Lac2 is a type of phenoloxidase that oxidatively conjugates proteins to make them insoluble, harder, and darker. Knockdown in the flour beetle T. castaneum has been shown to lead to lighter colored individuals in low doses but is considered lethal in high doses24. Figure 4 illustrates that this treatment also leads to lighter colored Sunburst Diving Beetle larvae. Specifically, in this experiment, 75% of the surviving injected larvae (n = 12) had reduced pigmentation (compared to 0% in the control group). Figure 4A shows an individual with relatively mild depigmentation, whereas Figure 4C illustrates the head of a T. marmoratus larva in which the dark coloration of the cuticle is nearly absent. Depigmentation was particularly evident for the central dark patterning that is typical for these larvae, whereas this pattern remained clearly visible in a control-injected individual (Figure 4B). In addition, a lightening of the tail trachea was observed, as depicted in Figure 4D. In the case where lac2 dsRNA was injected into third instar larvae, lighter adult individuals were obtained (Figure 5). Note that the wings of the knockdown beetle are somewhat deformed, likely due to its unusual softness.
In addition to altering morphological traits, it is possible to use RNAi to target genes that affect behavior. To demonstrate this, we performed RNAi against a key lens protein coding gene, Lens318, and injected it into second instar larvae to affect the optical properties of third instar larval eyes. Any effects on the lens observed here are likely because the eyes of T. marmoratus larvae undergo major eye growth at this transition, which also involves major optical changes of the lens25. RNAi knockdown in this experiment was highly efficient. Verification through qPCR showed a 100% success rate; out of 13 tested individuals, 12 were knocked down to less than 10% of the expression level of control individuals, with the remaining individual having an expression level of 17% of the control level (unpublished observation). At the phenotypic level, only some individuals were severely handicapped or incapable of prey capture, as is illustrated in Figure 6 for an individual that repeatedly approached its prey from a very close distance but consistently overshot it.
Table 1: Primer sequences and amplicons for white, lac2, and Lens3 proteins. <a href="https://cloudflare.jove.com/files/ftp_upload/61477/Table1.xlsx" target="_blank">Please click here to download this table.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61477/61477fig01.jpg" /> 
Figure 1: Illustration of embryo injections.&#160;(A) Dechorionated embryos lined up on an agarose plate and injected near their center using a microinjection needle filled with dsRNA and food-dye-containing injection buffer. The scale bar represents 500 &#956;m. (B) An individual with a more severe knockdown. Injected embryos are kept in a humidity chamber and monitored daily to score phenotypes and remove dead individuals (which turn brown). The scale bar represents 5 mm. <a href="https://www.jove.com/files/ftp_upload/61477/61477fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61477/61477fig02.jpg" /> 
Figure 2: Example of fully developed embryos that were injected with dsRNA against white.&#160;(A) Comparison of an individual with a reduction in eye pigmentation (left) and a control-injected individual (right). E1&#8211;E6 refer to Eyes 1&#8211;6. (B) An individual with a more severe knockdown phenotype, which illustrates that, at times, some of the eyes within the cluster are more severely affected by the knockdown than others. (C) Individual with a nearly complete loss of eye pigmentation. The scale bar represents 200 &#956;m; Panels B and C are represented at the same scale. <a href="https://www.jove.com/files/ftp_upload/61477/61477fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61477/61477fig03.jpg" /> 
Figure 3: Illustration of larval injections.&#160;(A) Several larvae immobilized by embedding in 2% agarose with their tail spiracles left clear of any agarose. (B) Microelectrode containing the injection solution placed so that its tip can penetrate the fine membrane between two segments. (C) Blue injection dye visible at the injection site after the injection. Scale bars represent 1 cm. <a href="https://www.jove.com/files/ftp_upload/61477/61477fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61477/61477fig04.jpg" /> 
Figure 4: RNAi for laccase2 applied to second instar larvae resulting in reduced cuticle coloration in third instar larvae.&#160;(A) Relatively mild loss of coloration in a lac2 RNAi individual (bottom) when compared with a control-injected individual (top). The scale bar represents 5 mm. (B) Head of a control individual showing the characteristic dark colored pattern at the center of the head. (C) Relatively severe knockdown of lac2 leading to a nearly complete loss of central head coloration. (D) Loss of coloration in the major tail cuticle of a lac2 RNAi individual (bottom) when compared with a control-injected individual (top). Scale bars in B&#8210;D represent 1 mm. <a href="https://www.jove.com/files/ftp_upload/61477/61477fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61477/61477fig05.jpg" /> 
Figure 5: RNAi for laccase2 applied to a third instar larva resulting in reduced cuticle coloration in an adult.&#160;The knockdown individual (left) is also characterized by very soft elytra when compared with the control (right). The scale bar represents 5 mm. <a href="https://www.jove.com/files/ftp_upload/61477/61477fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61477/61477fig06.jpg" /> 
Figure 6: Knockdown of a major lens protein leading to deficiencies in prey capture.&#160;(A&#8211;C). Three examples wherein a Sunburst Diving Beetle larva, in which optical deficits were induced through RNAi, was unable to capture prey (mosquito larvae). Representative still images were selected from a video recording of characteristic prey captures. (D) Control larva catching prey. All scale bars represent 2 cm. <a href="https://www.jove.com/files/ftp_upload/61477/61477fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points at JoVE.com

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

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 ...

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