We thank Drs. Alison Heffer and Yong Lu for setting up the microinjection apparatus and sharing their invaluable knowledge and experience with insect RNAi. This work was supported by the National Institutes of Health (R01GM113230 to L.P.).

1. Rearing of D. maculatus
NOTE: A breeding colony of D. maculatus was set up in the authors&#39; lab using adults and larvae purchased commercially. The species identity was verified using DNA barcoding53.
<ol>
	<li>To set up a new cage in the lab, spread a thin layer of wood shavings into a medium-size insect cage (30.5 x 19 x 20.3 cm3). Place a ~ 10 x 6 x 3 cm3 chunk of Styrofoam in the cage to let larvae hide for pupation. Add 20 - 50 be...

While a small number of sophisticated model systems (mice, flies, worms) were developed during the 20th century, the 21st century has seen a wave of new animal systems being developed in labs throughout the world. These new systems allow scientists to address comparative, evolutionary questions that cannot be probed using only the &#39;standard&#39; model systems. This deployment of new models requires the rapid development of methods for lab culturing, gene identification, and functional approaches...

In 1998,&#160;Fire and Mello reported that double-stranded RNA (dsRNA) can induce inhibition of gene function in Caenorhabditis elegans1. This response triggered by dsRNA was named RNA interference (RNAi), and such RNAi-mediated gene silencing was reported to be conserved in animals, plants, and fungi2-7. In plants and some animals, RNAi functions systemically, meaning that the effect can spread to other cells/tissues where dsRNA is not directly introduced (reviewed in8-10). Scientists have made use of this endogenous cellular RNAi response by designing dsRNAs to target genes of interest, thereby knocking down gene function without directly manipulating the genome (reviewed in11-14).
RNAi is a powerful tool for functional studies due to the following advantages. First, even with minimal gene sequence information, a gene can be targeted using RNAi. This is especially important for studies of non-model organisms lacking genomic or transcriptomic data. Second, in organisms where the RNAi response is robustly systemic, RNAi-mediated gene knockdown can be performed at almost any developmental stage. This feature is very useful for studying the function of pleiotropic genes. Third, in some cases, RNAi effects spread to the gonads and progeny, such that phenotypes are observed in offspring15,16. This phenomenon, known as parental RNAi (pRNAi), is especially advantageous for genes impacting embryonic development, as numerous offspring produced by a single injected parent can be examined without direct manipulation of eggs. For these reasons, pRNAi is the method of choice. However, if pRNAi is ineffective, for example for genes required for oogenesis, then embryonic RNAi (eRNAi) must be used. Fourth, RNAi can be used to generate the equivalent of an allelic series in that the amount of dsRNA delivered can be varied over a range to produce weak to strong defects. Such a gradation of phenotypes can be helpful for understanding gene function when the gene is involved in a complex process and/or complete loss of function is lethal. Fifth, delivery of dsRNA is generally easy and feasible, especially in animals showing robust systemic RNAi responses. dsRNA can be introduced by microinjection1,5, feeding/ingestion17,18, soaking,19,20 and virus/bacteria-mediated delivery21,22. Sixth, unlike some gene targeting/editing methods, there is no need to screen for organisms carrying the mutation or to carry out genetic crosses to generate homozygotes when using RNAi. Therefore, compared to many other techniques for studying gene function, RNAi is fast, inexpensive, and can be applied for large-scale screens23-25.
The broad utility of RNAi provides means&#160;to carry out functional studies in a wide range of organisms, expanding the range of species available for study beyond the traditional model systems for which genetic tools have been developed. For example, studies using non-model systems are required to give insights into the evolution of genes and gene networks by comparing the functions of orthologs from species representing different development modes or exhibiting distinct morphological features26-29. These types of studies will provide a better understanding of biological diversity, with impacts for both applied and basic research.
Being the largest animal group on the planet, insects provide a great opportunity to explore the mechanisms underlying diversity. Additionally, insects are generally small, have short life cycles, high fecundity, and are easy to rear in the lab. In the past two decades, RNAi has been successfully applied in insects spanning orders, including Diptera (true flies)5, Lepidoptera (butterflies and moths)30, Coleoptera (beetles)16,31, Hymenoptera (sawflies, wasps, ants and bees)32, Hemiptera (true bugs), Isoptera (termites)34, Blattodea (cockroaches)35, Orthoptera (crickets, grasshoppers, locusts, and katydids)36and Phthiraptera (lice)37. Successful application of RNAi has provided functional data for studies of patterning in early embryogenesis (anterior-posterior axis32, dorsal-ventral axis28, segmentation26,38), sex determination39,40, chitin/cuticle biosynthesis41, ecdysone signaling42, social behavior43, and more. RNAi methods developed for different insect species may be of additional benefit in that they are likely to be useful for pest control (reviewed in44-46). RNAi effects will be gene-specific as well as species-specific, as long as non-conserved regions are chosen for targeting. For beneficial insect species like honeybees and silkworms, targeting genes vital for the survival of viruses or parasites to control infection may provide a novel strategy to protect these species47,48.
Dermestes maculatus (D. maculatus), common name hide beetle, is distributed worldwide except for Antarctica. As a holometabolous insect, the D. maculatus life cycle includes embryonic, larval, pupal, and adult stages (Figure 1). Because it feeds on flesh, D. maculatus is used in museums to skeletonize dead animals and forensic entomologists can use it to estimate time of death49,50. D. maculatus feeds on animal products including carcasses, dried meat, cheese, and the pupae/cocoons of other insects and thus causes damage to households, stored food, and the silk, cheese, and meat industries 51,52. Applying RNAi in this beetle could provide an efficient and environmentally friendly way to minimize its economic impact. Our lab has used D. maculatus as a new model insect to study segmentation53. In addition to being amenable to lab rearing, D. maculatus is of interest for basic research as it is an intermediate-germ developer, making it a useful species to study the transition between short- and long-germ development.
<img alt="Figure 1" src="/files/ftp_upload/54976/54976fig1.jpg" /> 
Figure 1: Life Cycle of D. maculatus. Photographs of D. maculatus at different life stages, as indicated. The life cycle from egg to adult takes three weeks at 30 &#176;C but longer at lower temperatures. (A, F) Freshly laid embryos are white to light yellow and oval, approximately 1.5 mm in length. Embryogenesis takes ~55 hr at 30 &#176;C. (B, C and G) Larvae have dark pigmented stripes and are covered with setae. Larvae go through several instars depending on the environment and their length can extend up to over 1 cm. (D, H) Young pupae are light yellow. Pupation takes ~ 5 - 7 days at 30 &#176;C. (E, I) Shortly after eclosion, dark pigmentation appears over the adult beetle body. Adults can live up to several months and one female can lay hundreds of embryos over her lifetime. <a href="http://cloudflare.jove.com/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Previously, we showed that RNAi is effective in knocking down gene function in D. maculatus53. Here our experience rearing D. maculatus colonies in the laboratory is shared along with step-by-step protocols for both embryonic and parental RNAi set-up, injection, post-injection care, and phenotypic analysis. The dsRNA-mediated gene knockdown and analysis methods introduced here not only provide detailed information for addressing questions in D. maculatus, but also have potential significance for applying RNAi in other non-model beetle/insect species.

<table border="0" cellpadding="0" cellspacing="0" width="642">
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		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dermestes maculatus live beetles</td>
			<td style="width:140px;">Our lab or Carolina Biological Supply</td>
			<td style="width:119px;">#144168</td>
			<td style="width:167px;">Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wet cat food</td>
			<td style="width:140px;">Fancy Feast</td>
			<td style="width:119px;"></td>
			<td>Chunks of meat with gravy. Can buy at most pet food and grocery stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry dog food</td>
			<td style="width:140px;">Purina Puppy Chow</td>
			<td style="width:119px;"></td>
			<td>Can buy at most pet food and grocery stores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect cage (size medium, 30.5x19x20.3 cm)</td>
			<td style="width:140px;">Exo Terra</td>
			<td>PT2260</td>
			<td>For colony maintenance. Can use larger cage if needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect cage (size mini, 17.8x10.2x12.7 cm)</td>
			<td style="width:140px;">Exo Terra</td>
			<td>PT2250</td>
			<td>For embryo collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">89038-968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cotton ball</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">22-456-883</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Megascript T7 transcription kit</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">AM1334</td>
			<td>For 40 reactions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pneumatic pump</td>
			<td style="width:140px;">WPI</td>
			<td style="width:119px;">PV830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Capillary holder</td>
			<td style="width:140px;">WPI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:140px;">NARISHIGE</td>
			<td style="width:119px;">MN-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black filter paper (90 mm)</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">28342-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Food coloring (green)</td>
			<td>McCormick</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Borosilicate glass capillary</td>
			<td style="width:140px;">Hilgenberg</td>
			<td style="width:119px;">1406119</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle puller (micropipette puller)</td>
			<td style="width:140px;">Sutter Instrument Co.</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope glass slide</td>
			<td>WorldWide Life Sciences Division</td>
			<td style="width:119px;">41351157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sealing film (Parafilm M)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">13-374-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Model 801 Syringe (10 &#181;l )</td>
			<td style="width:140px;">Hamilton</td>
			<td style="width:119px;">7642-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle (32-gauge)&#160;</td>
			<td style="width:140px;">Hamilton</td>
			<td style="width:119px;">7762-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fixation Solution (Pampel&#39;s)</td>
			<td>BioQuip Products, Inc.</td>
			<td style="width:119px;">1184C</td>
			<td>Toxic, needs to be handled in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep (DUMONT #5)</td>
			<td style="width:140px;">Fine Science Tools</td>
			<td style="width:119px;">11252-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover slip (24X50 mm, No. 1.5)</td>
			<td style="width:140px;">Globe Scientific</td>
			<td style="width:119px;">1415-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Femtotips Microloader pipette tip</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">E5242956003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscopy for embryo injection</td>
			<td style="width:140px;">Leica</td>
			<td style="width:119px;">M420</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscopy for larval phenotypic visualization</td>
			<td style="width:140px;">Zeiss</td>
			<td colspan="2">SteREO Discover. V12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DIC microscopy</td>
			<td style="width:140px;">Zeiss</td>
			<td style="width:119px;">AXIO Imager. M1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The authors&#39; lab has used RNAi technology to study the functional evolution of genes regulating segmentation in insects53,55. While all insects are segmented, the genes regulating this process appear to have diverged during insect radiations26,38,56-63. Genetic screens in Drosophila identified a set of nine pair-rule segmentation genes that are responsible for promoting the formation of body segments64-70. Here, the ortholog of one of these ge...

Watch this Scientific Journal Video about Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus at JoVE.com

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.

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 ...

The overall goal of this procedure is to explore gene function in Dermestes maculatus, a new insect model system, which represents the diverse clade of beetles, and facilitates the study of basic and applied research questions. This method can help answer key questions in the fields of developmental biology, evolutionary biology and beyond, by making it possible to test gene function in Dermestes embryos and adults. The main advantage of this technique is that RNA interference can be used to target any gene, based only on sequence information, even in the absence of a full genome sequence.Methods introduced here not only provide an approach to study gene function in Dermestes. They can also be applied to develop new insect model systems, and potentially to control insect pests. To prepare a rearing cage for a D.maculatus, first spread a thin layer of wood shavings into a medium sized insect cage.Place a section of styrofoam into the cage to allow larvae an environment, in which to pupate. Add twenty to fifty adult beetles or late instar larvae to the cage. Place wet cat food into a Petri dish or weigh boat, as a food source.Cover the cage with a mesh cloth and place it into an incubator. Before starting embryo collection, first check the cage carefully for any embryos present and remove them. Once the cage is clear, place a stretched cotton ball into the cage and incubate the colony at 30 degrees Celsius.After the appropriate time window, collect the cotton ball, and remove any adults present, and place them back into the cage. Over a piece of black construction paper, pinch the cotton ball gently, and tear it slowly into thin filament, so that any eggs present fall onto the black paper. Take a standard Petri dish lid, and use a piece of black filter paper to cover the inside.Stick a piece of double-sided tape along the edge of a standard microscope slide. Place the microscope slide onto the black filter paper in the Petri dish lid. Taking the paper containing the collected embryos, gently tap to transfer the embryos to the Petri dish lid.Using a paintbrush, align the embryos on the tape perpendicular to the slide, with the anterior or posterior end towards the edge. To begin RNAi, first take up 2-4 microliters of dsRNA into a 20 microliter micro loader pipette tip. Insert the tip into a pre-pulled glass capillary, referred to as the needle, and gently pipette the solution into the needle.Fix the needle into a glass capillary holder and then assemble the capillary holder into the micromanipulator. Next, carefully transfer the slide with embryos attached onto the stage of the dissecting microscope. Move the slide to bring one embryo into the center of the field and focus the microscope.Whilst looking into the dissecting scope, position the tip of the capillary with the micromanipulator. Bring the tip close to the end of the first embryo in the row. Switch on the nitrogen or air supply, and then set the solenoid input selector switch to vacuum.Adjust the eject pressure regulator to 10-15 psi, depending on the apparatus. Open the capillary, and the colored dsRNA solution will fill the tip, ready for injection. Move the capillary tip forward, and gently puncture the embryo.Under ideal pressure settings, no embryonic fluid will flow into the capillary. Step on the foot switch to eject dsRNA into the embryo, until the appropriate amount is dispensed. Leave the tip inside the embryo for around two seconds, and then carefully remove it.Once all of the embryos have been injected, place the slide into a Petri dish. Add a wet cotton ball to the dish, taking care not to let it come into contact with the slide. Cover the Petri dish and wrap it with sealing film.Label it with the dsRNA type, concentration, date, time and number of injected embryos. Finally, place the dish into an incubator at 30 degrees Celsius until the embryos hatch. To perform pRNAi, first collect pupae from the colony.Sort male and female pupae into two separate dishes, and place these into a 30 degree Celcius incubator. Check the plates daily for eclosed beetles, and transfer these individuals to two separate Petri dishes for male and female adults. Feed these adults and maintain the plates every other day, until enough adults have eclosed to begin injection.Once the adult pool is complete, anesthetize a female beetle, and hold the individual ventral side up with one hand, holding the syringe in the other. Gently penetrate the segmacoria membrane between sternites 2 and 3. Check the syringe scale and slowly dispense around two microliters per female.After injection, hold the needle steady for at least 5 seconds and then carefully withdraw it. Transfer injected females to a Petri dish with cat food. Label the dish appropriately.Place the dish into a 30 degrees Celcius incubator. After 24 hours, transfer the injected females to a mini cage, and add an equal number of the uninjected young males. Label the cage, and add cat food.Place the cage into a 30 degree Celsius incubator, to allow mating. The resultant offspring can then be examined to determine the effects of the pRNAi. This image shows an offspring of a pRNAi individual injected with DS GFP, green fluorescent protein as a control.Here, as with wild-type individuals, the offspring hatched with one pigmented stripe per segment. Neighboring pigmented stripes were separated by a non-pigmented gap. Following paired pRNAi, affected offspring hatched with fused neighboring pigmented stripes, indicating defective segmental boundaries.Phenotypic severity was variable, but fusions consistently appeared at certain boundary regions, indicating pair rule like defects. Similarly, cuticle phenotypes of unhatched embryos after paired knockdown showed loss of abdominal segments, as well as shortened body length. Staining of early embryos with engrailed antibody to examine molecular defects showed stripes of expression of equal intensity in each segment in control individuals.In contrast, reduced expression was detected in alternate stripes in offspring of Dmac paired dsRNA injected females. This pattern of reduced expression is consistent with the defective cuticle pattern observed in the affected hatched larvae. Here, for the first time, we present our protocol for knocking down a gene of interest, during the early embryonic development in Dermestes maculatus, using RNA interference.While attempting these procedures, it is important to remember to use embryos or female adults at the appropriate stage. Once mastered, injecting about 100 embryos or 10 female adults can be done within half an hour. Following this procedure, other methods, like embryo staining or quantitative PCR can be performed in order to answer additional questions about gene regulatory mechanisms.After watching this video, you should have a good understanding of how to set up a Dermestes lab colony, and perform both embryonic and parental RNA interference in this beetle. Currently, only a small number of insect models are available for functional studies. The techniques presented here pave the way for researchers to use Dermestes as a model system, expanding the scope of insects amenable to molecular genetic analysis.

rearing-double-stranded-rna-mediated-gene-knockdown-hide-beetle

Research

JoVE Journal

Genetics

10.4K 조회수.  University of Maryland. The overall goal of this procedure is to explore gene function in Dermestes maculatus, a new insect model system, which represents the diverse clade of beetles, and facilitates the study of basic and applied research questions. This method can help answer key questions in the fields of developmental biology, evolutionary biology and beyond, by making it possible to test gene function in Dermestes embryos and adults. The main advantage of this technique is that RNA interference can be used to ...