Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Summary

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.

Abstract

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.

Introduction

Lepidopteran insects represent more than 10% of all described living species¹, and certain taxa species cause severe damage to plants and serious agricultural losses^2,3. Although molecular and genetic studies have been developed using model insects such as the silkworm Bombyx mori^4,5, pest insects remain uninvestigated, partly because of the difficulties for rearing and handling^6,7. 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.

Protocol

1. Insect collection and mass rearing

1. Collect tortricid insects from fields following previously published References^8,12.
   NOTE: 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).
2. 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).
3. 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.
4. 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 °C with 60% relative humidity, 16 h light/8 h dark cycles⁷.
5. Store the egg masses showing black head capsules at 4-8 °C for 7 days.
6. 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's lid is better to enable better ventilation. Fill the holes with cotton to prevent the escapes of small larvae.
7. 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.
8. 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.
9. Place 15 males and 10 females in a plastic box (30 cm x 20 cm x 5 cm, Figure 1K) for mating with 25 °C, 16 L/8 D. Collect eggs per 5-7 days and repeat steps 1.4-1.9. for each generation.
   ​NOTE: If collecting newly oviposited egg masses for subsequent analysis is needed, set the dark period'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).

2. Separation of eggs and pharate larvae from egg masses for fixation, permeabilization, and staining

1. Soak the egg mass into 1,000 µL of 1.2% sodium hypochlorite aqueous solution for 10 min or into 1,000 µL of 5 M potassium hydroxide aqueous solution for 30 min to separate the eggs.
2. Wash the separated eggs in 1,000 µL 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.
3. Soak eggs in a mixture of 500 µL of 100% heptane and 500 µL of 4% paraformaldehyde-PBSt solution (w/v). Mix for 10 min at 1,500 rpm using a vortex mixer.
4. Soak the eggs into a mixture of 500 µL of 100% heptane and 500 µL of 100% methanol. Mix for 10 min at 1,500 rpm.
5. Wash the eggs twice with 1,000 µL of 100% methanol and store at 4 °C in 100% methanol until further experiments (Figure 2A).
   NOTE: The eggs can be stored for at least 1 year at 4 °C.
6. Soak the eggs sequentially in 99%, 70%, 50% ethanol, and PBSt for 5 min each for hydrophilization following the previously published report⁷.
7. Wash the eggs with PBSt, immerse the eggs in 1 µg/mL of DAPI solution for 5 min, and then wash the eggs with 1x PBS twice. Soak the eggs in 20 µL 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.
8. 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.
9. 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).
10. 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).
11. 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.
    ​NOTE: The sex is determined by visualizing the sex chromatin. Each cell represents the sex chromatin as a dot in females^11,12, 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 μL of the reagents into 0.2 mL PCR tubes in advance (Figure 3A). Immerse and store samples at -80 °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.

3. DNA and RNA extractions from sex determined pharate larvae

1. 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.
2. Homogenize tissues in 600 μL 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 °C.
3. Collect the supernatant (500 μL) using a pipette and incubate at 50 °C with 1.5 µL of Proteinase K (20 mg/mL, see Table of Materials) for 5 h on a heat block.
4. Treat the samples with 1.0 μL of 10 mg/mL of RNase solution (see Table of Materials) at 37 °C for 30 min.
5. Add 200 μL 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 °C.
6. Mix the supernatant (500 μL) with 500 μL of 100% isopropanol, and then centrifuge at 20,400 x g for 10 min at 4 °C.
7. Wash the pelleted DNA twice with 1,000 μL of 70% ethanol. Then, air-dry (5-10 min at room temperature), dissolve the DNA in 30 μL of 10 mM Tris-Cl buffer (pH 8.5).
8. Homogenize the tissues in 600 μL of RNA extraction reagents and add 240 μL of ultra-pure distilled water to the tube. Centrifuge the tubes at 12,000 x g for 15 min at 4 °C.
9. Mix the supernatant (600 μL) with 600 μL 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 °C.
10. Wash the column with 750 μL of 70% ethanol, and centrifuge the column twice at 17,900 x g for 1 min each at 4 °C.
11. Load 15 μL of Ultra-pure distilled water to the column. Centrifuge the tubes at 17,900 x g for 1 min at 4 °C to elute the RNA.
12. 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.
13. Calculate the amounts of DNA and RNA using a fluorescence-based spectrophotometer.
    NOTE: The ratio of RNA quantities (UV-based concentration (ng/μL)/fluorescence-based concentration (ng/μL)) 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.
14. Analyze the quality of RNA by using microchip electrophoresis¹⁴.
15. 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.

Results

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.

Morphol...

Discussion

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 an...

Materials

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