The authors wish to acknowledge support from the Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists [Grant Number 19J13123 and 21J00895].

1. Insect collection and mass rearing
<ol>
	<li>Collect tortricid insects from fields following previously published References8,12. 
	NOTE:&#160;H. magnanima and A. honmai larvae are collected from damaged tea leaves (Figure 1A); adults are attracted using 4 W portable UV light (365 nm wavelength, see Table of Materials, Figure 1B).</li>
	<li>Rear the collected larvae (Figure 1C,D) individually on a piece of artificial diet in a 1/2-oz cup for 2-3 weeks until adult eclosion (Figure 1E,F). Confirm the sex of pupae and adults by morphological traits (Figure 1G,I).</li>
	<li>Mate the males and females in a plastic box (30 cm x 20 cm x 5 cm) to oviposit egg masses on a paraffin paper (Figure 1J-L). Place a female and two males in a 120 mL plastic cup with a piece of paraffin paper to establish a matriline. 
	NOTE: It is important to make creases on the paraffin paper. For H. magnanima, paraffin paper needs to be on the bottom of the case. Meanwhile, for A. honmai, put a paper on the case ceiling to collect eggs (Figure 1L) since H. magnanima oviposit egg mass on the upper side of tea leaves, but A. honmai lays eggs on the underside of the leaves8. The procedures were also verified in other tortricid species, and it is better to place papers on both sides if the ecology and behavior of the target species are unclarified.</li>
	<li>Cut out the egg masses (approximately 100-200 eggs per egg mass12, Figure 1M) on the paraffin paper with scissors. Place the eggs in a 1/2 oz cup with dumped paper for 5-7 days. 
	NOTE: The matured embryo exhibits a blackhead capsule (Figure 1N). The periods of embryogenesis are diversely attributed to tortricid species, but generally 5 days post oviposition (dpo) for H. magnanima and 4 dpo for A. honmai at 25 &#176;C with 60% relative humidity, 16 h light/8 h dark cycles7.</li>
	<li>Store the egg masses showing black head capsules at 4-8 &#176;C for 7 days.</li>
	<li>Slice ~60 g artificial diets using a grater for mass rearing. Place the egg mass filled with matured embryos on the sliced artificial diet in a plastic container (23 cm x 16 cm x 8 cm). Place paraffin papers on the egg mass with the sliced diets (Figure 10). 
	NOTE: Below 30% relative humidity is considered over dry, while over 70% is too humid. Creating holes on the plastic container&#39;s lid is better to enable better ventilation. Fill the holes with cotton to prevent the escapes of small larvae.</li>
	<li>To eliminate surface contaminants from the eggs, soak the egg mass into 3% formalin and 0.2% Benzalkonium chloride solution for 5 min, respectively12. To eradicate gut or intracellular bacteria, utilize an artificial diet supplemented with 0.05% (w/w) Tetracycline hydrochloride or 0.06% (w/w) rifampicin instead of a normal artificial diet (see Table of Materials). 
	NOTE: This step is optional. It is important to knead Silk Mate 2S and 0.05% (w/w) Tetracycline hydrochloride or 0.06% (w/w) rifampicin equably for consistency.</li>
	<li>Collect the pupae from the plastic container and distinguish the sex based on morphological12 characteristics (Figure 1G-I). 
	NOTE: Generally, tortricids present 5-6 instars until pupation12. The H. magnanima and A. honmai larvae take 3 weeks and 2 weeks, respectively, after hatching until pupation.</li>
	<li>Place 15 males and 10 females in a plastic box (30 cm x 20 cm x 5 cm, Figure 1K) for mating with 25 &#176;C, 16 L/8 D. Collect eggs per 5-7 days and repeat steps 1.4-1.9. for each generation. 
	&#8203;NOTE: If collecting newly oviposited egg masses for subsequent analysis is needed, set the dark period&#39;s start and end, e.g., from 9 AM to 5 PM. In this condition, H. magnanima and A. honami usually oviposit eggs after 5 h (2 PM).</li>
</ol>
2. Separation of eggs and pharate larvae from egg masses for fixation, permeabilization, and staining
<ol>
	<li>Soak the egg mass into 1,000 &#181;L of 1.2% sodium hypochlorite aqueous solution for 10 min or into 1,000 &#181;L of 5 M potassium hydroxide aqueous solution for 30 min to separate the eggs.</li>
	<li>Wash the separated eggs in 1,000 &#181;L of PBSt (137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.05% of Polyoxyethylene (20) Sorbitan Monolaurate [Tween-20] pH 7.4, see Table of Materials) following the previously published report7.</li>
	<li>Soak eggs in a mixture of 500 &#181;L of 100% heptane and 500 &#181;L of 4% paraformaldehyde-PBSt solution (w/v). Mix for 10 min at 1,500 rpm using a vortex mixer.</li>
	<li>Soak the eggs into a mixture of 500 &#181;L of 100% heptane and 500 &#181;L of 100% methanol. Mix for 10 min at 1,500 rpm.</li>
	<li>Wash the eggs twice with 1,000 &#181;L of 100% methanol and store at 4 &#176;C in 100% methanol until further experiments (Figure 2A). 
	NOTE: The eggs can be stored for at least 1 year at 4 &#176;C.</li>
	<li>Soak the eggs sequentially in 99%, 70%, 50% ethanol, and PBSt for 5 min each for hydrophilization following the previously published report7.</li>
	<li>Wash the eggs with PBSt, immerse the eggs in 1 &#181;g/mL of DAPI solution for 5 min, and then wash the eggs with 1x PBS twice. Soak the eggs in 20 &#181;L of 1.25% (w/w) lactic-acetic orcein solution (see Table of Materials)11 to visualize heterochromatin until the nuclei exhibit a brilliant red color (this varies from 5-60 min) (Figure 2B,C). 
	NOTE: The lactic-acetic orcein staining periods depend on temperature and humidity. It is better to check for proper staining using a microscope with 4x-10x magnification.</li>
	<li>Transfer the stained eggs using a pipette to a glass slide. Enclose the stained eggs with antifade reagent (see Table of Materials) and a cover glass7.</li>
	<li>Extract pharate H. magnanima and A. honmai larvae (4-5 days post oviposition (dpo)) from the egg masses using forceps (Figure 2D). Bisect larvae on a glass slide (Figure 2E).</li>
	<li>Fix any tissues (e.g., suboesophageal ganglion, thoracic ganglia, malpighian tubule, etc.) with a mixture of 1:3 (v/v) 99.7% acetic acid/100% methanol for 5 min. Stain those with 1.25% (w/w) lactic-acetic orcein solution11 until the nuclei are stained (this can take 5-60 min, depending on the temperature and humidity).</li>
	<li>Determine the sex of each specimen by observing the presence (female) or absence (male) of heterochromatin (the W chromosome, Figure 2F,G) under a microscope. 
	&#8203;NOTE: The sex is determined by visualizing the sex chromatin. Each cell represents the sex chromatin as a dot in females11,12, while the remaining portion of pharate larval tissues (not fixed) should be immediately immersed in cell lysis buffer12 (10 mM Tris-HCl, 100 mM EDTA, and 1% SDS, pH 8.0) or phenol-containing RNA extraction reagents for either DNA or RNA extraction. Before the dissections, dispense 20 &#956;L of the reagents into 0.2 mL PCR tubes in advance (Figure 3A). Immerse and store samples at -80 &#176;C until further extraction. The samples can be stored for at least 3 months, but it is better to proceed with the downstream experiments to prevent the degradation of nucleic acids.</li>
</ol>
3. DNA and RNA extractions from sex determined pharate larvae
<ol>
	<li>Pool 12 sex-determined male or female pharate larvae (5 dpo embryo) and add either cell lysis buffer or RNA extraction reagents (see step 2.11 and Table of Materials) into one 1.5 mL tube. 
	NOTE: Follow steps 3.2-3.7 for DNA extraction and steps 3.8-3.11 for RNA extraction.</li>
	<li>Homogenize tissues in 600 &#956;L of the cell lysis buffer (10 mM Tris-HCl, 100 mM EDTA, and 1% SDS, pH 8.0), and centrifuge the samples at 10,000 x g for 5 min at 4 &#176;C.</li>
	<li>Collect the supernatant (500 &#956;L) using a pipette and incubate at 50 &#176;C with 1.5 &#181;L of Proteinase K (20 mg/mL, see Table of Materials) for 5 h on a heat block.</li>
	<li>Treat the samples with 1.0 &#956;L of 10 mg/mL of RNase solution (see Table of Materials) at 37 &#176;C for 30 min.</li>
	<li>Add 200 &#956;L of protein precipitation solution (see Table of Materials) to the tubes7, followed by a centrifuge at 17,000 x g for 10 min at 4 &#176;C.</li>
	<li>Mix the supernatant (500 &#956;L) with 500 &#956;L of 100% isopropanol, and then centrifuge at 20,400 x g for 10 min at 4 &#176;C.</li>
	<li>Wash the pelleted DNA twice with 1,000 &#956;L of 70% ethanol. Then, air-dry (5-10 min at room temperature), dissolve the DNA in 30 &#956;L of 10 mM Tris-Cl buffer (pH 8.5).</li>
	<li>Homogenize the tissues in 600 &#956;L of RNA extraction reagents and add 240 &#956;L of ultra-pure distilled water to the tube. Centrifuge the tubes at 12,000 x g for 15 min at 4 &#176;C.</li>
	<li>Mix the supernatant (600 &#956;L) with 600 &#956;L of 100% isopropanol and transfer the mixture to a silica spin column (see Table of Materials). Centrifuge the tubes at 17,900 x g for 1 min at 4 &#176;C.</li>
	<li>Wash the column with 750 &#956;L of 70% ethanol, and centrifuge the column twice at 17,900 x g for 1 min each at 4 &#176;C.</li>
	<li>Load 15 &#956;L of Ultra-pure distilled water to the column. Centrifuge the tubes at 17,900 x g for 1 min at 4 &#176;C to elute the RNA.</li>
	<li>Calculate and verify the quality and quantity of DNA and RNA using a UV-based spectrophotometer. Assess the purity of nucleic acids using the A260/A280 (Nucleic acids/protein) and A260/A230 (Nucleic acids/salts and other contaminants) ratios13. 
	NOTE: For RNA extraction, the previously published procedure12 was followed, which resulted in phenol contamination (Table 1). The extracted DNA and RNA from a single embryo or pharate larvae yields low-amount and low-quality samples. Typically, DNA extraction from 12 pharate larvae yields 100-600 ng, while RNA extraction using the current method generates 900-1,500 ng, as shown Table 1. 
	NOTE: Steps 3.13-3.15 are optional for further assays of high throughput sequencings.</li>
	<li>Calculate the amounts of DNA and RNA using a fluorescence-based spectrophotometer. 
	NOTE: The ratio of RNA quantities (UV-based concentration (ng/&#956;L)/fluorescence-based concentration (ng/&#956;L)) was calculated to assess the quality for further experimental application. For example, when UV and fluorescence-based concentrations are 80 ng/uL and 60 ng/uL, respectively, the ratio will be 1.5 (80/60). Usually, ratios under 1.5 indicate sufficient purity for downstream applications using high throughput sequencing13.</li>
	<li>Analyze the quality of RNA by using microchip electrophoresis14.</li>
	<li>Utilize the qualified RNAs to prepare the RNA library using an RNA library preparation kit (see Table of Materials). Sequence the libraries using a platform suitable for the prepared libraries.</li>
</ol>

<ol>
	<li>Gaston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+magnitude+of+global+insect+species+richness.">The magnitude of global insect species richness.</a> Conservation Biology. 5 (3), 283-296 (1991).</li><li>Pogue, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+world+revision+of+the+genus+Spodoptera+Guen&#233;e+(Lepidoptera:+Noctuidae).">A world revision of the genus Spodoptera Guen&#233;e (Lepidoptera: Noctuidae).</a> Memoirs of the American Entomological Society. 43, 1 (2002).</li><li>Matsuura, H., Naito, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+cold-hardiness+and+overwintering+of+Spodoptera+litura+F.+(Lepidoptera:+Noctuidae):+VI.+Possible+overwintering+areas+predicted+from+meteorological+data+in+Japan.">Studies on the cold-hardiness and overwintering of Spodoptera litura F. (Lepidoptera: Noctuidae): VI. Possible overwintering areas predicted from meteorological data in Japan.</a> Applied Entomology and Zoology. 32 (1), 167-177 (1997).</li><li>Mita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+construction+of+an+EST+database+for+Bombyx+mori+and+its+application.">The construction of an EST database for Bombyx mori and its application.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (24), 14121-14126 (2003).</li><li>Kawamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+genome+assembly+of+the+silkworm,+Bombyx+mori.">High-quality genome assembly of the silkworm, Bombyx mori.</a> Insect Biochemistry and Molecular Biology. 107, 53-62 (2019).</li><li>Fukui, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+masculinizing+function+of+the+Ostrinia+furnacalis+Masculinizer+gene.">In vivo masculinizing function of the Ostrinia furnacalis Masculinizer gene.</a> Biochemical and Biophysical Research Communications. 503 (3), 1768-1772 (2018).</li><li>Arai, H., Ishitsubo, Y., Nakai, M., Inoue, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+disperse+eggs+from+lepidopteran+scale-like+egg+masses+and+to+observe+embryogenesis.">A simple method to disperse eggs from lepidopteran scale-like egg masses and to observe embryogenesis.</a> Entomological Science. 25 (1), 12497 (2022).</li><li>vander Geest, L. P., Evenhuis, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tortricid pests: their biology, natural enemies and control. , (1991).</li><li>Kiuchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+female-specific+piRNA+is+the+primary+determiner+of+sex+in+the+silkworm.">A single female-specific piRNA is the primary determiner of sex in the silkworm.</a> Nature. 509 (7502), 633-636 (2014).</li><li>Rand, M. D., Kearney, A. L., Dao, J., Clason, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+Drosophila+embryos+for+introduction+of+small+molecules.">Permeabilization of Drosophila embryos for introduction of small molecules.</a> Insect Biochemistry and Molecular Biology. 40 (11), 792-804 (2010).</li><li>Kageyama, D., Traut, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+sex-specific+effects+of+Wolbachia+and+interference+with+the+sex+determination+of+its+host+Ostrinia+scapulalis.">Opposite sex-specific effects of Wolbachia and interference with the sex determination of its host Ostrinia scapulalis.</a> Proceedings of the Royal Society B. 271 (1536), 251-258 (2004).</li><li>Arai, H., Lin, S. R., Nakai, M., Kunimi, Y., Inoue, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closely+related+male-killing+and+nonmale-killing+Wolbachia+strains+in+the+oriental+tea+tortrix+Homona+magnanima.">Closely related male-killing and nonmale-killing Wolbachia strains in the oriental tea tortrix Homona magnanima.</a> Microbial Ecology. 79 (4), 1011-1020 (2020).</li><li>Schalamun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+MinION:+An+example+of+how+to+establish+long-read+sequencing+in+a+laboratory+using+challenging+plant+tissue+from+Eucalyptus+pauciflora.">Harnessing the MinION: An example of how to establish long-read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora.</a> Molecular Ecology Resources. 19 (1), 77-89 (2019).</li><li>Winnebeck, E. C., Millar, C. D., Warman, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+does+insect+RNA+look+degraded.">Why does insect RNA look degraded.</a> Journal of Insect Science. 10 (1), 159 (2010).</li><li>Ivey, C. T., Carr, D. E., Eubanks, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+inbreeding+in+Mimulus+guttatus+on+tolerance+to+herbivory+in+natural+environments.">Effects of inbreeding in Mimulus guttatus on tolerance to herbivory in natural environments.</a> Ecology. 85 (2), 567-574 (2004).</li><li>Saccheri, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+and+extinction+in+a+butterfly+metapopulation.">Inbreeding and extinction in a butterfly metapopulation.</a> Nature. 392 (6675), 491-494 (1998).</li><li>Crnokrak, P., Roff, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+depression+in+the+wild.">Inbreeding depression in the wild.</a> Heredity. 83 (3), 260-270 (1999).</li><li>Keller, L. F., Waller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+effects+in+wild+populations.">Inbreeding effects in wild populations.</a> Trends in Ecology &amp; Evolution. 17 (5), 230-241 (2002).</li><li>Margaritis, L. H., Kafatos, F. C., Petri, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eggshell+of+Drosophila+melanogaster.+I.+Fine+structure+of+the+layers+and+regions+of+the+wild-type+eggshell.">The eggshell of Drosophila melanogaster. I. Fine structure of the layers and regions of the wild-type eggshell.</a> Journal of Cell Science. 43 (1), 1-35 (1980).</li><li>Sugimoto, T. N., Ishikawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+male-killing+Wolbachia+carries+a+feminizing+factor+and+is+associated+with+degradation+of+the+sex-determining+system+of+its+host.">A male-killing Wolbachia carries a feminizing factor and is associated with degradation of the sex-determining system of its host.</a> Biology Letters. 8 (3), 412-415 (2012).</li></ol>

The authors have nothing to disclose.

Tortricid comprises several agricultural and forestry pests; the present study presented methods to rear tortrix over generations, observe embryogenesis and sex of the insects, and conduct molecular analysis using matured embryos.
One of the obstacles for pest insect study is to establish rearing methods. Especially, inbreeding sometimes affect the fitness of the species negatively. The fitness reduction by the inbred, called inbreeding depression, has widely been observed in various plants and animals, including insects15,16,17,18. As noted, both H. magnanima and A. honmai have been maintained for over 100 generations (more than 10 years) with no apparent fitness costs. Although it is not enough to assess whether other tortricids also have a high tolerance to inbreeding in general, the two species may have developed the characteristics by adapting monotonous environmental conditions (i.e., tea plantations) and against several endosymbiotic microorganisms (e.g., Wolbachia11,12). The eggs of the two species are easily obtained, but their oviposition behaviors are differently attributed to their ecology. Indeed, humidity and crease direction seem to directly affect the number of oviposited eggs, which might reflect the natural histories of the insects in fields. It is important to adjust rearing methods to species regarding their biological characteristics in nature.
The protocols established in this study enabled observation of embryogenesis and molecular studies using sex-determined matured embryos and larvae. The insect eggs are generally coated with several shell layers10,19. Moreover, the eggs of H. magnanima and A. honmai are covered with maternal secretions. To stain the eggs with such complexed structure, separation, fixation, and permeabilization seems to be all necessary. In Ostrinia furnacalis (Crambidae), which oviposits scale-like egg mass such as H. magnanima and A. honmai, PCR-based sex determination protocols using single embryos have been proposed, but the low quality and quantity of nucleic acids have been a problem for subsequent analysis6,20. In contrast, the present study suggested extracting nucleic acids from pooled sex-determined embryos after observing female-specific sex chromatin. RNAs are easily degraded, and the extraction procedure shown here did not affect the quality of RNA, which has also been confirmed by the transcriptome analysis (RNA-seq).The techniques shown here are applicable for studies&#160;of embryogenesis of various insect species. In addition, the protocols have the potentials to be used to assess the effects of chemical pesticides or intracellular microbes such as Wolbachia, which causes sex-specific defects during embryogenesis6,11,12.
The present study has several limitations. First, the sex of immature embryos (0-3 dpo) was difficult to determine using lactic-acetic orcein staining in H. magnanima and A. honmai. This is because the number and size of the nuclei are generally small during early embryogenesis, making it difficult to observe the W chromosome. To clarify the sex of the tortricids during early embryogenesis, detection and quantification of markers on sex chromosomes6,20 could be an alternative approach. Second, it was difficult to extract nucleic acids with high purity from a single individual after sex determination, possibly due to the small number of cells. However, the RNA or DNA extracted from a single embryo may apply to subsequent analyses such as PCR assays and single-cell sequencings.
In summary, the present protocol describes mass rearing, morphological observations, and genetic analyses of the eggs of two non-model lepidopteran pest insects, H. magnanima and A. honmai. These simple techniques are expected to be applicable for further research on tortricid, other lepidopterans insects, and other taxa.

Lepidopteran insects represent more than 10% of all described living species1, and certain taxa species cause severe damage to plants and serious agricultural losses2,3. Although molecular and genetic studies have been developed using model insects such as the silkworm Bombyx mori4,5, pest insects remain uninvestigated, partly because of the difficulties for rearing and handling6,7. Therefore, fundamental studies and techniques are necessary to understand the biology of such non-model pest insects.
The Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests8. Of the insect taxa, the oriental tea tortrix Homona magnanima Diakonoff and the summer fruit tortrix Adoxophyes honmai Yasuda are serious polyphagous pests known to damage tea trees in East Asia7. The two species lay flat and oval scale-like egg clusters (or egg masses) consisting of thin, soft, and fragile eggs covered by maternal secretions. Although embryogenesis stages are crucial for insect development and sex determinations9, structures of the eggs prevent further analysis from understanding the biology of the insects. It is important to overcome the difficulties for further study on pests ovipositing such complex egg mass.
Here, to understand the biology of tortricids, methods for mass rearing, observations, and molecular studies have been developed using A. honmai and H. magnanima. First, mass rearing methods maintain both tortricids over 100 generations by inbred. The separation of eggs from the concatenated scale-like egg mass facilitated embryogenesis observation of the tortricids using alkaline and organic solvents previously developed from techniques used in flies10. In addition, the present study established sex discrimination of small embryos by developing staining methods of the sex chromatin of lepidopteran females using lactic-acetic orcein11. By combining these methods, high quality and quantity nucleic acids were extracted from sex determined embryos, which was otherwise difficult to establish6. The extracted RNA was utilized for next-generation sequencing. Collectively, the methods presented here apply to other lepidopteran insects and other insect taxa.

<table><tbody><tr><td>1/2 ounce cup</td><td>FP CHUPA</td><td>CP070009</td><td>insect rearing; https://www.askul.co.jp/p/6010417/</td></tr><tr><td>1/2 ounce cup lid</td><td>FP CHUPA</td><td>CP070011</td><td>insect rearing; https://www.askul.co.jp/p/6010434/?int_id=recom_DtTogether</td></tr><tr><td>99.7% acetic acid</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>36289</td><td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01ALF036289.html</td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>not shown</td><td>Nucleic acids quantification; https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument</td></tr><tr><td>Agilent RNA6000 nano kit</td><td>Agilent Technologies</td><td>5067-1511</td><td>Nucleic acids quantification; https://www.agilent.com/cs/library/usermanuals/Public/G2938-90034_RNA6000Nano_KG&lt;br/&gt; .pdf</td></tr><tr><td>benzalkonium chloride solution</td><td>Nihon Pharmaceutical Co., Ltd</td><td>No.4987123116046</td><td>Sterilization; https://www.nihon-pharm.co.jp/consumer/products/disinfection.html</td></tr><tr><td>Cotton</td><td>AOUME</td><td>8-1611-02</td><td>insect rearing; https://item.rakuten.co.jp/athlete-med/10006937/?scid=af_pc_etc&amp;amp;sc2id=af_113_0_1</td></tr><tr><td>DAPI solution</td><td>Dojindo, Tokyo, Japan</td><td>340-07971</td><td>stainings; https://www.dojindo.co.jp/products/D523/</td></tr><tr><td>Disodium Hydrogenphosphate</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Na2HPO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0286.html</td></tr><tr><td>dsDNA HS quantification kit</td><td>Invitrogen</td><td>Q33231</td><td>Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33230?SID=srch-srp-Q33230</td></tr><tr><td>Econospin RNA II column</td><td>Epoch Life Science Inc.</td><td>EP-11201</td><td>RNA extraction; http://www.epochlifescience.com/Product/SpinColumn/minispin.aspx</td></tr><tr><td>Ethanol</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>4.98748E+12</td><td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0105-0045.html</td></tr><tr><td>Ethylenediamine-N,N,N&#039;,N&#039;-tetraacetic Acid Tetrasodium Salt Tetrahydrate (4NA)</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Cell lysis buffer (EDTA); https://labchem-wako.fujifilm.com/jp/product/detail/W01T02N003.html</td></tr><tr><td>Glassine paper</td><td>HEIKO</td><td>2100010</td><td>insect rearing; https://www.monotaro.com/p/8927/0964/?utm_id=g_pla&amp;amp;&lt;br/&gt; utm_medium=cpc&amp;amp;utm_source=&lt;br/&gt; Adw</td></tr><tr><td>heat block WSC-2620 PowerBLOCK</td><td>ATTO, Tokyo, Japan</td><td>4002620</td><td>incubation; https://www.attoeng.site/</td></tr><tr><td>heptane</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>4.98748E+12</td><td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0108-0015.html</td></tr><tr><td>INSECTA LF</td><td>Nosan Co., Ltd</td><td>not shown</td><td>Artificial diet; https://www.nosan.co.jp/business/fodder/ist.htm</td></tr><tr><td>ISOGENII</td><td>Nippon Gene</td><td>311-07361</td><td>RNA extraction; https://www.nippongene.com/siyaku/product/extraction/isogen2/isogen2.html</td></tr><tr><td>isopropanol</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>nucleic acids extraction; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0232-0004.html</td></tr><tr><td>Lactic acid</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0112-0005.html</td></tr><tr><td>methanol</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>4.98748E+12</td><td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0113-0182.html</td></tr><tr><td>MSV-3500 vortex</td><td>Biosan</td><td>BS-010210-TAK</td><td>Voltex mixer; https://biosan.lv/products/-msv-3500-multi-speed-vortex/</td></tr><tr><td>Nano Photometer NP 80</td><td>Implen</td><td>not shown</td><td>Nucleic acids quantification; https://www.implen.de/product-page/implen-nanophotometer-np80-microvolume-cuvette-spectrophotometer/tech-specs/</td></tr><tr><td>Natural pack wide</td><td>Inomata chemical</td><td>1859</td><td>insect rearing; https://www.monotaro.com/g/03035766/?t.q=%E3%83%8A%E3%83%81%E3%83%A5%E3%&lt;br/&gt; 83%A9%E3%83%AB%E3%83%91%&lt;br/&gt; E3%83%83%E3%82%AF%E3%83%&lt;br/&gt; AF%E3%82%A4%E3%83%89</td></tr><tr><td>NEBNext Ultra II RNA Library Prep Kit for Illumina</td><td>New England BioLabs</td><td>E7770S</td><td>Library preparation; https://www.nebj.jp/products/detail/2039</td></tr><tr><td>orcein</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0115-0094.html</td></tr><tr><td>Paraformaldehyde</td><td>FUJIFILM Wako Chemicals Co.</td><td>160-16061</td><td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-1606.html</td></tr><tr><td>Polyoxyethylene(20) Sorbitan Monolaurate</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Tween-20; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-2121.html</td></tr><tr><td>Portable UV Black Light (4W, 365nm wavelength)</td><td>Southwalker Co., Ltd., Kanagawa, Japan</td><td>not shown</td><td>Insect collection; http://www.southwalker.com/shopping/?pid=1364614057-467328</td></tr><tr><td>Potassium Chloride</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>KCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0354.html</td></tr><tr><td>Potassium Dihydrogen Phosphate</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>KH2PO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0424.html</td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Invitrogen, MA, USA</td><td>P36965</td><td>antifade; https://www.thermofisher.com/order/catalog/product/P36965</td></tr><tr><td>Proteinase K Solution</td><td>Merck</td><td>71049-4CN</td><td>DNA extraction; https://www.merckmillipore.com/JP/ja/product/Proteinase-K-Solution-600-mAU-ml,EMD_BIO-71049</td></tr><tr><td>protein precipitation solution</td><td>Qiagen</td><td>158912</td><td>DNA extraction; https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/buffers-reagents/puregene-accessories/?cmpid=PC_DA_NON_&lt;br/&gt; BIOCOMPARE_ProductListing_&lt;br/&gt; 0121_RD_MarketPlace_ProductC</td></tr><tr><td>Qubit V4</td><td>Invitrogen</td><td>Q33238</td><td>Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33238</td></tr><tr><td>rifampicin</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>4.98748E+12</td><td>Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0118-0100.html</td></tr><tr><td>RNA HS quantification kit</td><td>Invitrogen</td><td>Q32855</td><td>Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q32852</td></tr><tr><td>RNase solution</td><td>Nippon Gene</td><td>313-01461</td><td>RNA extraction; https://www.nippongene.com/siyaku/product/modifying-enzymes/rnase-a/rnase-s.html</td></tr><tr><td>Silk Mate 2S</td><td>Nosan Co., Ltd</td><td>not shown</td><td>Artificial diet; https://www.nosan.co.jp/business/fodder/ist.htm</td></tr><tr><td>Sodium Chloride</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>NaCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0166.html</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Cell lysis buffer (SDS); https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-1398.html</td></tr><tr><td>sodium hypochlorite aqueous solution</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>4.98748E+12</td><td>egg separation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0220.html</td></tr><tr><td>Tetracycline Hydrochloride</td><td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td><td>4.98748E+12</td><td>Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1656.html</td></tr><tr><td>Tris-HCl</td><td>FUJIFILM Wako Chemicals Co.</td><td>4.98748E+12</td><td>Cell lysis buffer; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1536.html</td></tr><tr><td>ultra-pure distilled water</td><td>Invitrogen</td><td>10977023</td><td>RNA extraction; https://www.thermofisher.com/order/catalog/product/10977015</td></tr></tbody></table>

mass-rearing and molecular studies in tortricidae pest insects

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

Establishment of host lines and their maintenance 
The viability of field-collected larvae is differently attributed to field location, seasons, and rearing conditions (e.g., 90% of viability in Taiwan, Taoyuan, as shown in Arai et al.12). Approximately 30%-50% of pairs will generate the next generation as usual. For H. magnanima and A. honmai, matrilines have been maintained by inbreeding for over 100 generations.
Morphological observations and sex determinations 
The treatment with either potassium hydroxide (3 or 5 M KOH) or sodium hypochlorite (1.2% Cl2 or NaClO) separates eggs from their concatenated egg masses (Figure 2A). The lower concentration of the reagents could not achieve separation. All the fixation, permeabilization, and staining steps enabled the visualization of nuclei with DAPI solution. The eggs treated with only KOH or NaClO (separated) without the fixation and permeabilization steps were not stained with these dyes7. Non-treated H. magnanima egg masses were not stained. The lactic-acetic orcein stains heterochromatin (W chromosome) with a dark red color and nuclei with a bright red color as usual (Figure 3A). This staining enables easy and fast sex determination from 4 dpo embryo to sixth instar larvae. Although nuclei were visualized in 0-3 dpo embryos, it was hard to observe the W chromosome with 400x magnification.
High-quality DNA and RNA were obtained from sex-determined and pooled embryos 
From 12 pharate larvae, high quality DNA (A260/A280 = 1.7-2.0; A260/A230 = 1.7-2.4) were extracted with 100-600 ng in total amount (Table 1). Although the RNA extracted following the previously published method12 yielded 500-1,000 ng product with low quality: A260/A280 = 1.7-2.1; A260/A230 = 0.1-0.5 (Table 1), the modified protocols using a spin column improved the quality of the RNA, producing 900-1,500 ng product with A260/A280 = 1.9-2.1 and A260/A230 = 1.9-2.3 (Table 1). Furthermore, the RNA concentration ratio (UV-based/Qbit fluorescence-based values) for the modified protocol was below 1.2, meeting the quality requirements for next-generation sequencing13. Conversely, the quality and quantity of nucleic acids extracted from a single embryo were too low to be calculated. The intactness of the RNA extracted from sex-determined pharate larvae using the modified protocol was also confirmed using microchip electrophoresis (Figure 3B). The prepared libraries were confirmed to yield high Q30 score reads (Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63737/63737fig01.jpg" /> 
Figure 1: Overview of insect collection and mass rearing of H. magnanima and A. honmai. (A) Tea leaves and nests damaged by a larva. (B) Light trap with UV light. (C,D) A female (C) and a male (D) 6th instar larva of H. magnanima. The Orange triangle indicates the testis. (E,F) Collected larvae were reared individually in a 1/2 oz cup with a piece of artificial diet. (G,H) Pupae of H. magnanima (G) and A. honmai (H). The female is shown on the left, while the male is shown on the right. (I) Adults of H. magnanima (above) and A. honmai (below). Both males and females are shown on the right and left, respectively. (J,K) A plastic bag (J) or plastic case (K) for egg mass collection. (L) A. honmai oviposit eggs on paraffin paper placed on the lid, and H. magnanima lay eggs on paraffin paper placed on the bottom of the case. (M,N) Egg mass of H. magnanima. The matured embryos exhibit black head capsules indicated with white arrows (N). (O) Sliced artificial diets with a grater for mass rearing. The larvae hatched from eggs, placed on the sliced diet, will form pupae 3-4 weeks after hatching at 25 &#176;C (under 16 h light/8 h dark cycle). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63737/63737fig01large.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/63737/63737fig02.jpg" /> 
Figure 2: Observations of embryos. (A) The separated, fixed, and permeabilized eggs of H. magnanima using 5 N KOH, 4% PFA / heptane, methanol/heptane, and methanol. A separated egg is highlighted with broken lines. (B) The 3 dpo embryo was stained with lactic-acetic orcein. The black arrowhead indicates the embryo. (C) The 4 dpo embryo was stained with DAPI. The white arrowhead indicates the embryo. (D,E) Dissection of pharate larvae (5 dpo) using forceps. The pharate larvae are extracted from egg-mass (D), bisected on a glass slide (E). (F,G) Lactic-acetic orcein staining using the dissected pharate larvae. Females exhibit heterochromatin as a dot (indicated with black arrowhead, (F), but males lack the heterochromatin (G). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63737/63737fig02large.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/63737/63737fig03.jpg" /> 
Figure 3: Graphical summary of nucleic acid extraction using sex-determined pharate larvae and RNA qualities of H. magnanima and A. honmai. (A) Tissues of dissected pharate larvae on glass slides were stained with lactic-acetic orcein to determine their sex. The remaining tissues were soaked in reagents, pooled into one tube sorted by the sex of the individuals, and then subjected to DNA/RNA extraction. (B) The quality of RNA extracted from 12 male H. magnanima or A. honmai was assessed using a microchip electrophoresis system. Abbreviations: [nt], nucleotide size; [FU], fluorescence unit; M, molecular marker; Hm, H. magnanima; Ad, A. honmai. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63737/63737fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Concentration and quality of nucleic acids extracted from sex determined pharate larvae of H. magnanima. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63737/63737_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Qualities of RNA-sequencing raw data. 1Q20 (%) indicates the probability of reads with 99% read accuracy. 2Q30 (%) indicates the probability of reads with 99.9% read accuracy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63737/63737_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Mass-Rearing and Molecular Studies in Tortricidae Pest Insects at JoVE.com

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

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

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

mass-rearing-and-molecular-studies-in-tortricidae-pest-insects

Nanyang Technological University

Research

JoVE Journal

Environment

2.7K Views.  Tokyo University of Agriculture and Technology. The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Video: Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

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

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Developmental Biology

Quantifying Corticolous Arthropods Using Sticky Traps

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density requires a method with high detection probability and a consistent sampling area. We used manufactured sticky traps to compare abundance, total length (a surrogate for biomass), richness, and Shannon diversity of corticolous arthropods among the boles of 5 tree species. Efficacy of this method was adequate to detect variation in corticolous arthropods among tree species and provide a standard error of the mean that was &#60;20% of the mean for all estimates with sample sizes from 7 to 15 individual trees of each species. Our results indicate, even with these moderate sample sizes, the level of precision of arthropod community metrics produced with this approach is adequate to address most ecological questions regarding temporal and spatial variation in corticolous arthropods. Results from this method differ from other quantitative approaches such as chemical knockdown, visual inspection, and funnel traps in that they provide an indication of corticolous arthropod activity over a relatively long-term, better including temporary bole residents, flying arthropods that temporarily land on the tree bole and crawling arthropods that use the tree bole as a travel route from the ground to higher forest foliage. Furthermore, we believe that commercially manufactured sticky traps provide more precise estimates and are logistically simpler than the previously described method of directly applying a sticky material to tree bark or applying a sticky material to tape or other type of backing and applying that to the tree bark.

We describe a semi-quantitative approach of measuring characteristics of corticolous (bark-dwelling) arthropod communities. We placed commercially manufactured sticky traps on tree boles to estimate abundance, total length (a surrogate to biomass), richness, and Shannon diversity for comparison among tree species.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density ...

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern United States. Some populations have developed resistance to management strategies including transgenic corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Knowledge of western corn rootworm dispersal is of critical importance for models of resistance evolution, spread, and mitigation. Flight behavior of an insect, especially over a long distance, is inherently difficult to observe and characterize. Flight mills provide a means to directly test developmental and physiological impacts and consequences of flight in the laboratory that cannot be obtained in field studies. In this study, flight mills were used to measure the timing of flight activity, total number of flights, and the distance, duration, and speed of flights taken by female rootworms during a 22-h test period. Sixteen flight mills were housed in an environmental chamber with programmable lighting, temperature, and humidity control. The flight mill described is of a typical design, where a flight arm is free to rotate about a central pivot. Rotation is caused by flight of an insect tethered to one end of the flight arm, and each rotation is recorded by a sensor with a time-stamp. Raw data are compiled by software, which are subsequently processed to provide summary statistics for flight parameters of interest. The most difficult task for any flight mill study is attachment of the tether to the insect with an adhesive, and the method used must be tailored to each species. The attachment must be strong enough to hold the insect in a rigid orientation and to prevent detachment during movement, while not interfering with natural wing motion during flight. The attachment process requires dexterity, finesse, and speed, making video footage of the process for rootworms of value.

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect&#8217;s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern ...

Behavior

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Dissection for Identifying Migratory Insects via Ovarian Reproductive Analysis

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using searchlight traps. One of the most important techniques for migratory pest forecasting is to identify the migratory species. However, in most cases, it is difficult to get the information just by appearance. Therefore, using knowledge acquired by systematic analysis of the female reproductive system can help to understand the combined anatomic morphology of the ovarian mating sac and ovary developmental grading of wild-type migratory insects captured with searchlight traps. To demonstrate the applicability of this method, ovarian development status and egg grain development stages were directly assessed in Helicoverpa armigera, Mythimna separata, Spodoptera litura, and Spodoptera exigua for the ovarian anatomy, and the ovarian mating sacs were studied in Agrotis ipsilon, Spaelotis valida, Helicoverpa armigera, Athetis lepigone, Mythimna separata, Spodoptera litura, Mamestra brassicae, and Spodoptera exigua, to explore their relationships. This work shows the specific dissection method to predict wild-type migratory insects, comparing the unique reproductive system of different migratory insects. Then, both tissues, namely, the ovary and mating sacs, were further investigated. This method helps to predict the dynamics and the structural development of reproductive systems in wild-type female migratory insects.

Author Spotlight: Analysis of Ovarian Anatomy in Migratory Insects to Overcome Experimental Challenges 

The protocol demonstrates a simple and easy dissection method, suitable for wild-type migratory female insects captured with searchlight traps. This technique can significantly clarify the same species by comparing both reproductive tissues, namely the mating sac and ovarian development of wild-type female insects.

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

Neuroscience

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Summary

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Chapters in this video

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

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Quantifying Corticolous Arthropods Using Sticky Traps

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

A Simple Flight Mill for the Study of Tethered Flight in Insects

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology