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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 bi....

Protocol

1. Insect collection and mass rearing

  1. Collect tortricid insects from fields following previously published References8,12.
    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. Re.......

Representative 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.......

Acknowledgements

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].

....

Materials

NameCompanyCatalog NumberComments
1/2 ounce cupFP CHUPACP070009insect rearing; https://www.askul.co.jp/p/6010417/
1/2 ounce cup lidFP CHUPACP070011insect rearing; https://www.askul.co.jp/p/6010434/?int_id=recom_DtTogether
99.7% acetic acidFUJIFILM Wako Chemicals Co., Osaka, Japan36289fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01ALF036289.html
Agilent 2100 BioanalyzerAgilent Technologiesnot shownNucleic acids quantification; https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument
Agilent RNA6000 nano kitAgilent Technologies5067-1511Nucleic acids quantification; https://www.agilent.com/cs/library/usermanuals/Public/G2938-90034_RNA6000Nano_KG
.pdf
benzalkonium chloride solutionNihon Pharmaceutical Co., LtdNo.4987123116046Sterilization; https://www.nihon-pharm.co.jp/consumer/products/disinfection.html
CottonAOUME8-1611-02insect rearing; https://item.rakuten.co.jp/athlete-med/10006937/?scid=af_pc_etc&sc2id=af_113_0_1
DAPI solutionDojindo, Tokyo, Japan340-07971stainings; https://www.dojindo.co.jp/products/D523/
Disodium HydrogenphosphateFUJIFILM Wako Chemicals Co.4.98748E+12Na2HPO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0286.html
dsDNA HS quantification kitInvitrogenQ33231Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33230?SID=srch-srp-Q33230
Econospin RNA II columnEpoch Life Science Inc.EP-11201RNA extraction; http://www.epochlifescience.com/Product/SpinColumn/minispin.aspx
EthanolFUJIFILM Wako Chemicals Co., Osaka, Japan4.98748E+12fixation; 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+12Cell lysis buffer (EDTA); https://labchem-wako.fujifilm.com/jp/product/detail/W01T02N003.html
Glassine paperHEIKO2100010insect rearing; https://www.monotaro.com/p/8927/0964/?utm_id=g_pla&
utm_medium=cpc&utm_source=
Adw
heat block WSC-2620 PowerBLOCKATTO, Tokyo, Japan4002620incubation; https://www.attoeng.site/
heptaneFUJIFILM Wako Chemicals Co., Osaka, Japan4.98748E+12fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0108-0015.html
INSECTA LFNosan Co., Ltdnot shownArtificial diet; https://www.nosan.co.jp/business/fodder/ist.htm
ISOGENIINippon Gene311-07361RNA extraction; https://www.nippongene.com/siyaku/product/extraction/isogen2/isogen2.html
isopropanolFUJIFILM Wako Chemicals Co.4.98748E+12nucleic acids extraction; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0232-0004.html
Lactic acidFUJIFILM Wako Chemicals Co.4.98748E+12Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0112-0005.html
methanolFUJIFILM Wako Chemicals Co., Osaka, Japan4.98748E+12fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0113-0182.html
MSV-3500 vortexBiosanBS-010210-TAKVoltex mixer; https://biosan.lv/products/-msv-3500-multi-speed-vortex/
Nano Photometer NP 80Implennot shownNucleic acids quantification; https://www.implen.de/product-page/implen-nanophotometer-np80-microvolume-cuvette-spectrophotometer/tech-specs/
Natural pack wideInomata chemical1859insect 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 IlluminaNew England BioLabsE7770SLibrary preparation; https://www.nebj.jp/products/detail/2039
orceinFUJIFILM Wako Chemicals Co.4.98748E+12Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0115-0094.html
ParaformaldehydeFUJIFILM Wako Chemicals Co.160-16061fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-1606.html
Polyoxyethylene(20) Sorbitan MonolaurateFUJIFILM Wako Chemicals Co.4.98748E+12Tween-20; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-2121.html
Portable UV Black Light (4W, 365nm wavelength)Southwalker Co., Ltd., Kanagawa, Japannot shownInsect collection; http://www.southwalker.com/shopping/?pid=1364614057-467328
Potassium ChlorideFUJIFILM Wako Chemicals Co.4.98748E+12KCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0354.html
Potassium Dihydrogen PhosphateFUJIFILM Wako Chemicals Co.4.98748E+12KH2PO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0424.html
ProLong Diamond Antifade MountantInvitrogen, MA, USAP36965antifade; https://www.thermofisher.com/order/catalog/product/P36965
Proteinase K SolutionMerck71049-4CNDNA extraction; https://www.merckmillipore.com/JP/ja/product/Proteinase-K-Solution-600-mAU-ml,EMD_BIO-71049
protein precipitation solutionQiagen158912DNA 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 V4InvitrogenQ33238Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33238
rifampicinFUJIFILM Wako Chemicals Co., Osaka, Japan4.98748E+12Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0118-0100.html
RNA HS quantification kitInvitrogenQ32855Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q32852
RNase solutionNippon Gene313-01461RNA extraction; https://www.nippongene.com/siyaku/product/modifying-enzymes/rnase-a/rnase-s.html
Silk Mate 2SNosan Co., Ltdnot shownArtificial diet; https://www.nosan.co.jp/business/fodder/ist.htm
Sodium ChlorideFUJIFILM Wako Chemicals Co.4.98748E+12NaCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0166.html
Sodium Dodecyl SulfateFUJIFILM Wako Chemicals Co.4.98748E+12Cell lysis buffer (SDS); https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-1398.html
sodium hypochlorite aqueous solutionFUJIFILM Wako Chemicals Co., Osaka, Japan4.98748E+12egg separation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0220.html
Tetracycline HydrochlorideFUJIFILM Wako Chemicals Co., Osaka, Japan4.98748E+12Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1656.html
Tris-HClFUJIFILM Wako Chemicals Co.4.98748E+12Cell lysis buffer; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1536.html
ultra-pure distilled waterInvitrogen10977023RNA extraction; https://www.thermofisher.com/order/catalog/product/10977015

References

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TortricidaeMass RearingMolecular StudiesPest InsectsInsect EggsArtificial DietEgg MassSodium HypochloritePotassium HydroxideHeptaneParaformaldehydeMethanolEthanolDAPILactic AcidAcetic AcidLarval Dissection

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