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Morphological characteristics of Neural Tissue and Corazonin Neurons of Central Nervous System in Larval Stage of Scuttle Fly
Biomed Sci Letters 2022;28:290-297
Published online December 31, 2022;  https://doi.org/10.15616/BSL.2022.28.4.290
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Hohyun Park†,*

Department of Biomedical Laboratory Science, Mokpo Science University, Mokpo-si, Jeollanam-do 58644, Korea
Correspondence to: Hohyun Park. Department of Biomedical Laboratory Science, Mokpo Science University, Mokpo-si, Jeollanam-do 58644, Korea.
Tel: +82-61-270-2745, Fax: +82-61-270-2745, e-mail: phh7082@hanmail.net
*Professor.
Received November 14, 2022; Revised December 8, 2022; Accepted December 12, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Through previous studies, the central nervous system (CNS) was collected by dividing the scuttle fly into larval, pupa, and adult stages by developmental stage, and the morphological characteristics were observed. In situ hybridization (ISH) using the collected central nervous system, it was possible to confirm the location and extent of expression of the neurotransmitter corazonin (Crz) at each stage of development. In this study, paraffin specimens were prepared using central nervous system tissues of 3rd instar larval stage of scuttle fly, which had completed in situ hybridization, and general histochemical staining (hematoxylin-eosin, H-E) and special histochemical staining (luxol fast blue-cresyl violet) was performed to observe the histological and cytological morphology characteristics of corazonin neurons. As a result, a variety of nerve cell body existed between many myelin sheath. The corazonin neurons compose cortex of central nervous system with other neurons congregating in this tissue and show larger cell body relatively in neurohistochemical analysis.
Keywords : Scuttle fly, Corazonin, Central narve system, Larval stage, Hematoxylin-eosin, Luxol fast-cresyl violet
꽌 濡

理쒓렐뿉 媛옣 뿰援ш 留롮씠 吏꾪뻾릺怨 엳뒗 珥덊뙆由 醫낆쓽 븯굹濡 Drosophila melanogaster뒗 scuttle fly 쑀궗븳 援ъ“ 諛 듅吏 벑씠 留롮씠 愿李곕릺뿀怨, 留먭린 諛곗뿉꽌遺꽣 븷踰뚮젅 3졊 源뚯 24媛쒖쓽 꽭룷瑜 媛吏怨 엳뒗 3媛쒖쓽 떊寃 洹몃9뿉꽌 corazonin 돱윴씠 굹궃떎怨 蹂닿퀬븯떎(Shiga et al., 2003).

삉븳 蹂듭떊寃쎌깋(ventral nerve cord, VNC) 議곗쭅뿉꽌 1媛쒖쓽 떊寃 洹몃9, 뇤(brain) 議곗쭅뿉꽌 2媛쒖쓽 떊寃 洹몃9씠 愿李곕맂떎怨 븯떎(Choi et al., 2005; Lee et al., 2008; Choi, 2009). 뇤 議곗쭅뿉꽌 벑痢〓㈃ 븞履쎌쑝濡 諛쒗쁽릺뼱吏 corazonin 돱윴 쑀異⑺샇瑜대が(juvenile hormone)怨 吏吏덈룞썝샇瑜대が(adipokinetic hormone)쓣 遺꾨퉬븯뒗 썑몢 履쎌뿉꽌 愿李곕릺怨, 삤吏 벑痢〓㈃ 履쎌쓽 떊寃쎌썝 1媛쒖쓽 洹몃9 꽦異 떆湲곌퉴吏 吏냽릺뼱 諛쒗쁽맂떎怨 븯떎. Corazonin 돱윴 蹂듬 떊寃쎌젅뿉꽌 깉濡쒖슫 媛쒖옱 돱윴 4媛 洹몃9怨, 떆뿽 쇅痢 遺쐞쓽 1媛 뿽 떦 6~8媛 젙룄쓽 留롮 떊寃쎌꽭룷媛 吏묐떒쑝濡 諛쒗쁽릺怨 엳떎怨 蹂닿퀬릺뿀떎(Choi et al., 2005; Choi et al., 2006; Lee et al., 2008; Lee et al., 2011).

洹몃옒꽌 corazonin 돱윴씠 깮궛릺怨 엳뒗 怨듦컙쟻 遺꾪룷 援ъ뿭 鍮꾧탳쟻 以묒텛떊寃쎄퀎 議곗쭅 븞뿉 옒 蹂댁〈릺怨 엳怨(Veenstra and Davis, 1993; Cantera et al., 1994; Hansen et al., 2001; Choi et al., 2005; Roller et al., 2003; Roller et al., 2006; Verleyen et al., 2006), 떊寃 議곗쭅쓣 援ъ꽦븯뒗 떊寃쎌꽭룷 떊寃쎈텇鍮꾩꽭룷뱾쓽 옉 吏묓빀泥대줈 諛쒗쁽릺뼱 愿李곕릺뿀떎(Choi et al., 2005; Choi et al., 2008). 뇤 議곗쭅뿉꽌 빑떖 諛쒗쁽 遺쐞濡쒕뒗 쇅痢 遺遺(pars lateralis) 履쎌뿉 議댁옱븯怨 엳뒗 떊寃쎈궡遺꾨퉬꽭룷 洹몃9 遺遺꾩씠怨, 씠윭븳 遺쐞 肉먮쭔 븘땲씪 珥덊뙆由 醫(species)뿉 뵲씪꽌룄 醫 듅씠쟻쑝濡 諛쒗쁽맂 遺쐞媛 諛쒓껄릺怨 엳뿀떎. 듅吏뺤쟻쑝濡, 떆뿽 닔吏(optic lobe medulla) 履쎌뿉꽌 닔留롮 corazonin 돱윴씠 諛쒗쁽릺뼱 愿李곕릺怨 엳뒗 珥덊뙆由 醫낆쑝濡쒕뒗 Drosophila melanogaster濡 留롮씠 蹂닿퀬릺뿀떎(Choi et al., 2005; Lee et al., 2008).

씠쟾 뿰援ъ뿉꽌 scuttle fly쓽 븷踰뚮젅 떆湲곗쓽 以묒텛떊寃쎄퀎뿉꽌 諛쒗쁽맂 corazonin 돱윴 2졊 븷踰뚮젅 떆湲곗뿉뒗 뇤 議곗쭅쓽 쟾뇤(procerebrum) 벑履 痢〓㈃(dorsolateral, DL) 媛옣옄由 履쎌뿉꽌 3 뙇쓽 corazonin 돱윴뱾씠 吏꾪븯寃 諛쒗쁽릺뿀怨, 뼇履쎌쓽 뇤 議곗쭅뿉꽌 벑以묒븰(dorsomedial, DM) 履쎌뿉 1뙇, 蹂듭떊寃쎌깋 議곗쭅긽뿉꽌룄 醫뚯슦濡 8뙇씠 슌졆븯寃 愿李곕릺뿀떎. 3졊 븷踰뚮젅 떆湲곗뿉뒗 븷踰뚮젅 2졊 떆湲곗 鍮꾩듂븳 뙣꽩씠吏留 쟾뇤쓽 벑履 痢〓㈃(DL) 遺遺 蹂대떎 쟾뇤 벑履 以묒븰(DM) 遺遺꾩씠 돱윴쓽 諛쒗쁽씠 빟빐졇 엳뿀怨 3졊 썑湲곗뿉뒗 돱윴씠 븯굹쓽 뙇쑝濡 愿李곕릺吏 븡쓣 븣룄 엳뿀떎. 蹂듭떊寃쎌깋 議곗쭅룄 醫뚯슦 移 8뙇쓽 corazonin 돱윴 以묒뿉꽌 留 븵 2뙇쓽 corazonin 돱윴 슌졆븯吏留 3踰덉㎏ corazonin 돱윴遺꽣뒗 젏젏 빟빐吏뒗 寃껋쓣 蹂 닔媛 엳뿀떎(Park, 2020).

踰덈뜲湲(pupae) 떆湲곕뒗 以묒텛떊寃쎄퀎뿉꽌 corazonin 돱윴쓽 2媛쒖쓽 洹몃9씠 궗씪吏怨 1媛쒖쓽 洹몃9뿉꽌留 諛쒗쁽븯떎. 븷踰뚮젅 떆湲곗뿉꽌쓽 corazonin 돱윴쓣 諛쒗쁽릺뿀뜕 쟾뇤 벑以묒븰(DM) 뼇履 1뙇, 蹂듭떊寃쎌깋 8뙇씠 踰덈뜲湲곕줈 蹂깭媛 씪뼱굹硫댁꽌 궗씪졇 踰꾨━뒗 寃껋쓣 愿李고븯怨, 떆뿽쓽 쟾뇤 벑痢〓㈃(DL) 履쎌뿉꽌 3뙇 븘吏곷룄 슌졆븯寃 諛쒗쁽릺怨 엳뿀떎. 3뙇쓽 corazonin 돱윴 踰덈뜲湲 떆湲 룞븞뿉 怨꾩냽 諛쒗쁽릺怨 엳뿀쑝硫 떆뿽 諛쒕떖濡 씤빐 以묒븰履쎌쑝濡 닔졃릺뿀怨 踰덈뜲湲 留먭린뿉뒗 吏냽쟻쑝濡 以묒븰 遺쐞뿉 쐞移섑븯떎.

꽦異(adult fly) 떆湲곗뿉뒗 以묒텛떊寃쎄퀎뿉꽌 corazonin 돱윴 3뙇쓽 벑痢〓㈃(DL) 履쎌쓽 젙以묒븰뿉 醫뚯슦濡 諛곗튂릺뿀怨, 뇤 議곗쭅쓽 벑履 痢〓㈃(DL) 遺遺꾩뿉꽌 諛쒗쁽맂 3뙇쓽 corazonin 돱윴씠 쟾뇤 븵履쎌쓽 以묒븰履 뇤媛 遺遺(pars intercerebralis)뿉꽌 吏묓빀릺뼱 議댁옱븯뒗 寃껋쓣 愿李고븷 닔 엳뿀떎(Park, 2021).

쐞 媛숈씠 씠쟾 뿰援щ 넻븯뿬 scuttle fly 븷踰뚮젅, 踰덈뜲湲, 꽦異 떆湲 諛쒖깮 떒怨꾨퀎濡 以묒텛떊寃쎄퀎뿉꽌쓽 corazonin 돱윴뿉 븳 諛쒗쁽 뼇긽 諛 삎깭븰쟻 蹂솕 벑쓣 궡렣蹂댁븯떎.

蹂 뿰援ъ뿉꽌뒗 珥덊뙆由 醫 以묒뿉꽌 Drosophila melanogaster媛 以묒텛떊寃쎄퀎 議곗쭅뿉 빐 留롮 뿰援ш 릺뼱 엳吏留 븘吏곴퉴吏뒗 떊寃 議곗쭅븰쟻 諛 꽭룷븰쟻 援ъ“쓽 삎깭뿉 빐꽌뒗 뿰援ш 씠猷⑥뼱吏怨 엳吏 븡븯떎. 洹몃옒꽌 Drosophila melanogaster뿉 鍮꾪빐 뿰援ш 留롮씠 릺뼱 엳吏 븡 씗洹 珥덊뙆由 醫낆씤 scuttle fly쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 梨꾩랬븯뿬 씠쟾 뿰援ъ쓽 젣옄由ы샎꽦솕踰(ISH)쓣 씠슜빐꽌 corazonin 돱윴쓣 諛쒗쁽떆궓 븷踰뚮젅 3졊쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 궗슜빐꽌 뙆씪 몴蹂몄쓣 留뚮뱾뿀怨, 씪諛 諛 듅닔議곗쭅븰쟻 뿼깋쓣 떎떆븯뿬 떊寃쎌쓽 議곗쭅 諛 꽭룷븰쟻 援ъ“ 삎깭쟻 듅吏, 諛쒗쁽맂 corazonin 돱윴怨 떎瑜 떊寃쎌꽭룷뱾怨쇱쓽 떊寃쎌꽭룷븰쟻 듅吏 벑쓣 궡렣蹂닿린濡 븯떎.

옱猷 諛 諛⑸쾿

궗쑁(feeding)

떎뿕뿉 씠슜븳 scuttle fly뒗 Megaselia scalaris씪뒗 븰紐낆쓣 媛吏 fly씠硫, 珥덊뙆由ъ 룞씪븳 癒뱀씠瑜 씠슜븯뿬 궗슜븯떎. 궗쑁쓣 쐞븯뿬 利앸쪟닔 250 mL뿉 dextrose 16 g, yeast flakes 6.5 g, cornmeal 20 g, agar 2.25 g쓣 援먮컲湲곕 씠슜븯뿬 샎빀븯怨 쟾옄젋吏뿉꽌 10遺 젙룄 걪뼱 꽆移섏 븡쓣 젙룄濡 媛뿴븯떎. 洹몃━怨 methylparaben 6.25 mL瑜 泥쒖쿇엳 뼥뼱쑉젮꽌 샎빀븯怨 諛곗 넻뿉 遺 썑 떇엺 떎쓬 꽦異⑹쓣 씠룞떆耳 궗쑁쓣 븯떎. 궗쑁 1二쇱씪 媛꾧꺽쑝濡 떎瑜 諛곗뿉 꽦異⑹쓣 삷寃⑥꽌 吏냽쟻쑝濡 떆뻾븯떎. 젣옉맂 궗쑁 諛곗뒗 4℃ 깋옣怨좎뿉 蹂닿븯쑝硫 궗슜 쟾 떎삩뿉 留욎텛湲 쐞빐 誘몃━ 爰쇰궡 넃 썑 궗슜븯떎.

以묒텛떊寃쎄퀎 議곗쭅쓽 梨꾩랬 諛 以鍮(collection and preparation of CNS tissue)

Scuttle fly쓽 븷踰뚮젅(larvae) 3졊 3~5씪 떆湲곗쓽 5媛 씠긽 媛쒖껜瑜 궗슜븯떎. 0.1% tween 20씠 룷븿릺뼱 엳뒗 PBS (phosphate buffer solution, pH 7.0)媛 梨꾩썙졇 엳뒗 솃씠 뙆씤 뒳씪씠뱶뿉 媛쒖껜瑜 떞洹 긽깭濡 떎泥댄쁽誘멸꼍(stereomicroscopy) 긽뿉꽌 媛뒛怨 젙諛븳 뀑쓣 씠슜븯뿬 踰덈뜲湲, 꽦異⑹쓽 猿띿쭏 遺遺꾩쓣 李뼱媛硫댁꽌 梨꾩랬븯怨, 以묒텛떊寃쎄퀎 議곗쭅씤 뇤 蹂듭떊寃쎌깋 議곗쭅쓣 쟻異쒗븯뿬 4% paraformaldehyde 슜븸뿉 꽔 썑 4℃ 깋옣怨좎뿉 1떆媛 씠긽 怨좎젙븯뿬 蹂닿븯떎. 洹몃━怨 씠쟾 뿰援щ 넻븯뿬 Probe 젣옉븯뿬 젣옄由ы샎꽦솕踰(in situ hybridization, ISH)쓣 씠誘 셿猷뚰븳 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅 寃泥대 洹몃濡 씠슜븯떎(Park, 2020) (Table 1).

Base sequences of primers used for PCR amplification in scuttle fly of corazonin gene & structure of Crz gene in scuttle fly

Name of primers Sequences (5' → 3')
Scut5' Crz_HindIII 5'-cgaagcttGAGTCGTATTCCGATATTC
Scut3' Crz_XbaI 5'-ggtctagaCTTCAAATGCGAAACCACA

A; map for the Crz coding region

B; sequences of the genomic Crz gene

The coding sequences are shown in capital letters and 5'-UT, intron, 3'-UT sequences are shown in lower case.



議곗쭅몴蹂 젣옉(tissue specimen production)

씠쟾 뿰援щ 넻븯뿬 젣옄由ы샎꽦솕踰(ISH)쓣 셿猷뚯떆궓 썑 scuttle fly쓽 븷踰뚮젅(larvae) 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 諛붾줈 씠슜빐꽌 셿쟾엳 利앸쪟닔뿉 닔꽭븳 떎쓬 깉닔 怨쇱젙쓣 50%, 70%, 80%, 90%, 95%, 100% alcohol뿉꽌 媛곴컖 10遺꾧컙 떆뻾븯怨, 떎쓬쑝濡 닾紐 怨쇱젙쓣 xylene쓣 씠슜븯뿬 10遺꾧컙 몢 踰덉뿉 嫄몄퀜 떆뻾븯떎.

닾紐 怨쇱젙씠 걹궃 썑 60℃ 뿴뙋 쐞뿉 湲덉냽 슜湲곕 삱젮넃怨 뙆씪(Leica, Germany)쓣 끃씤 썑 닾紐 怨쇱젙씠 걹궃 議곗쭅 뱾쓣 삷寃⑤넃怨 1~2떆媛 젙룄 移⑦닾 옉뾽쓣 떎떆븯떎. 移⑦닾媛 걹궃 썑 룷留ㅼ옣移(Sandon, USA) 쐞뿉꽌 湲덉냽 룷留 쓣 삱젮넃怨 떎泥댄쁽誘멸꼍쓣 씠슜븯뿬 議곗쭅쓣 젙諛븯怨 삁由ы븳 뀑쑝濡 젙룉떆궓 썑 議곗쭅移댁꽭듃瑜 삱젮넃怨 끃븘엳뒗 뙆씪쓣 걹源뚯 梨꾩슫 썑 -5℃쓽 깋媛곹뙋뿉 쐞移섏떆耳 룷留 옉뾽쓣 셿猷뚰븯뿬 뙆씪 釉붾줉쓣 젣옉븯떎.

룷留ㅺ 셿猷뚮맂 뙆씪 釉붾줉쓣 쉶쟾삎諛뺤젅湲곗뿉 옣李⑺븯뿬 4 μm쓽 몢猿섏쓽 젅렪쓣 留뚮뱾怨 45℃쓽 遺쑀 삩닔議곗뿉 쓣슫 썑 쑀由ъ뒳씪씠뱶뿉 議곗쭅젅렪쓣 遺숈뿬꽌 60℃ 뒳씪씠뱶 떊쟾湲(slide warmer)뿉 30遺 젙룄 삱젮넃븘 議곗쭅怨 뒳씪씠뱶瑜 뿴뿉 쓽빐 遺李⑹떆궎硫댁꽌 議곗쭅몴蹂 젣옉 怨쇱젙쓣 셿猷뚰븯떎.

뿤留덊넚떎由-뿉삤吏 뿼깋(hematoxylin-eosin stain)

Scuttle fly뿉꽌 븷踰뚮젅쓽 以묒텛떊寃쎄퀎 議곗쭅쓽 삎깭瑜 愿李고븯湲 쐞븯뿬 議곗쭅몴蹂 젣옉 怨쇱젙씠 걹궃 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅몴蹂 뒳씪씠뱶瑜 xylene씠 뱾뼱엳뒗 슜湲곗뿉 10遺꾧컙 꽭 踰덉뿉 嫄몄퀜꽌 깉뙆씪 떒怨꾨 떆뻾븯怨, 떎쓬쑝濡 100%, 95%, 90%, 80%, 70%, 50% alcohol쓣 씠슜븯뿬 븿닔 怨쇱젙쓣 넻븯뿬 利앸쪟닔뿉 닔꽭瑜 븯떎.

뿤由ъ뒪 뿤留덊넚떎由(Harris hematoxylin)쓣 씠슜븯뿬 빑 뿼깋쓣 떆뻾븯怨, 빟媛꾩쓽 닔꽭瑜 븳 썑 遺꾨퀎븸(1% HCl in 70% alcohol)쓣 씠슜븯뿬 빑 씠쇅쓽 뿼깋븸쓣 젣嫄고븯쑝硫 利앸쪟닔뿉 異⑸텇븳 닔꽭瑜 븯뿬 以묓솕 怨쇱젙쓣 떆뻾븯떎(Bracegirdle et al., 2002; Gill, 2009; Carson and Hladik, 2009; Cardon, 2007; Titford, 2005).

떎쓬쑝濡 뿉삤吏(eosin) 뿼깋븸쓣 씠슜븯뿬 꽭룷吏 諛 寃고빀 議곗쭅 벑쓽 뿼깋쓣 떆뻾븳 떎쓬 50%, 70%, 80%, 90%, 95%, 100% alcohol쓣 씠슜븯뿬 媛곴컖 1遺꾧컙 깉닔 怨쇱젙쓣 떆뻾븯떎. 깉닔 怨쇱젙씠 걹궃 썑 xylene쑝濡 꽭 踰덉뿉 嫄몄퀜 1遺꾧컙 닾紐 怨쇱젙쓣 걹留덉튂怨 canada balsam뿉 닾紐낆젣씤 xylene쓣 냼웾 꽔뼱꽌 끃씤 썑 遊됱엯 옉뾽쓣 떆뻾븯怨, H&E (hematoxylin&eosin) 뿼깋 뒳씪씠뱶 젣옉쓣 셿꽦떆耳 愿묓븰쁽誘멸꼍쓣 씠슜븯뿬 以묒텛떊寃쎄퀎 議곗쭅쓣 愿李고븯떎.

猷⑹넄 뙣뒪듃 釉붾(-겕젅떎 諛붿씠삤젢 뿼깋(Luxol fast blue-cresyl violet stain)

Scuttle fly 븷踰뚮젅쓽 以묒텛떊寃쎄퀎 議곗쭅뿉꽌쓽 꽭룷븰쟻 듅吏뺤쓣 愿李고븯湲 쐞븯뿬 씠쟾뿉 뿰援щ릺뼱吏 젣옄由ы샎꽦솕踰(ISH)쑝濡 corazonin 돱윴씠 諛쒗쁽맂 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 媛吏怨 議곗쭅몴蹂 젣옉 怨쇱젙쓣 떎떆븯뿬 뙆씪 議곗쭅몴蹂 뒳씪씠뱶瑜 젣옉븯뿬 떊寃쎌꽭룷 닔珥 뿼깋諛⑸쾿씤 luxol fast blue-cresyl violet 뿼깋쓣 떆뻾븯떎.

젣옉맂 議곗쭅몴蹂 뒳씪씠뱶瑜 xylene씠 뱾뼱엳뒗 슜湲곗뿉 媛곴컖 10遺꾧컙 꽭 踰덉뿉 嫄몄퀜꽌 뙆씪쓣 젣嫄고븯뒗 떒怨꾨 떆뻾븯떎. 100%, 95% alcohol쓣 씠슜븯뿬 媛곴컖 3遺꾧컙 xylene 젣嫄곕 븯떎. Luxol fast blue (Sigma, USA) 젣옉 슜븸쓣 뿼깋 슜湲곗뿉 誘몃━ 留뚮뱾뼱 37℃ 빆삩湲곗뿉 꽔뼱 삩룄瑜 留욎텣 썑 깉뙆씪씠 셿猷뚮맂 議곗쭅몴蹂 뒳씪씠뱶瑜 꽔뼱 16~24떆媛 룞븞 뿼깋쓣 븯떎. 뿼깋씠 걹궃 썑 95% alcohol쓣 씠슜븯뿬 怨쇰웾쓽 뿼깋떆빟쓣 젣嫄고븯怨 0.05% lithium carbonate 슜븸뿉꽌 10珥 젙룄 遺꾨퀎븳 떎쓬, 利됱떆 70% alcohol濡 삷寃 닔珥 씠쇅쓽 援먯썝꽟쑀 벑뿉 뿼깋맂 泥깋쓣 젣嫄고븳 썑 利앸쪟닔濡 닔꽭븯떎(Bancroft and Gamble, 2008; Carson and Hladik, 2009).

떎쓬쑝濡 cresyl violet (Sigma, USA) 젣옉 슜븸쓣 뿼깋 슜湲곗뿉 誘몃━ 37℃ 빆삩湲곗뿉 꽔뼱 삩룄瑜 삱由 썑 luxol fast blue 뿼깋씠 걹궃 뒳씪씠뱶瑜 20遺 룞븞 뿼깋쓣 븯떎. 뿼깋씠 걹궃 썑 利앸쪟닔瑜 씠슜븯뿬 닔꽭븯怨 씪젙떆媛 70% alcohol뿉 떞媛몢怨 1~2珥 룞븞 遺꾨퀎븸(95% alcohol 90 mL, chloroform 10 mL, glacial acetic acid 3 drops)뿉 꽔뼱꽌 諛붾줈 95% alcohol쓣 씠슜븯뿬 遺꾨퀎븸쓣 젣嫄고븯떎(Klüver and Barrera, 1953; Luna, 1992).

95%, 100%, 100% alcohol쓣 씠슜븯뿬 깉닔 怨쇱젙쓣 떆뻾븳 썑 xylene쑝濡 꽭 踰덉뿉 嫄몄퀜 3遺꾧컙 닾紐 怨쇱젙쓣 걹留덉튂怨 canada balsam뿉 닾紐낆젣씤 xylene쓣 냼웾 꽔뼱 끃씤 썑 遊됱엯 옉뾽쓣 떆뻾븯뿬 뿼깋 뒳씪씠뱶 젣옉쓣 셿꽦떆耳 愿묓븰쁽誘멸꼍쓣 씠슜븯뿬 corazonin 諛쒗쁽 遺쐞 二쇱쐞쓽 떎瑜 떊寃쎌꽭룷 뱾쓽 듅吏뺤쓣 愿李고븯떎.

寃 怨

떊寃쎌“吏곹븰쟻 듅吏

Scuttle fly뿉꽌 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 뙆씪 몴蹂몄쓣 젣옉븯뿬 씪諛 뿼깋(H&E stain)쓣 넻븯뿬 議곗쭅븰쟻 援ъ“瑜 궡렣 蹂댁븯떎.

뇤(brain) 議곗쭅쓽 寃쎌슦뒗 궗엺쓽 뇤 媛숈씠 寃됱쭏 (cortex) 履쎌뿉 떊寃쎌꽭룷泥(nerve cell body)뱾씠 援ъ꽦릺뼱 엳뿀怨, 냽吏(medulla) 履쎌 떊寃쎌꽟쑀 寃고빀 議곗쭅쑝濡 씠猷⑥뼱졇 엳뿀떎. 떊寃쎌꽭룷泥닿 諛吏묓븳 뵾吏 遺쐞뿉뒗 뿬윭 醫낅쪟쓽 떊寃쎌꽭룷媛 愿李곕릺怨 엳뿀怨, 二쇰줈 뵾씪誘몃뱶 꽭룷(pyramidal cell) 蹂 꽭룷(stellate cell) 벑씠 媛옣 留롮씠 議댁옱븯怨 엳뿀떎. 븘슱윭 떊寃쎌꽭룷쓽 留먯씠吏(myelin sheth)怨 떊寃쎌꽟쑀媛 二쇰뿉 留롮씠 愿李곗씠 릺吏留 슌졆븯吏뒗 븡븯떎. 븞履쎌쓽 냽吏 遺쐞뿉뒗 嫄곗쓽 빑쓣 媛吏怨 엳뒗 떊寃쎌꽭룷媛 議댁옱븯吏 븡븯怨 떊寃쎌꽟쑀 寃고빀 議곗쭅 벑쑝濡쒕쭔 梨꾩썙졇 엳뒗 援ъ“濡 愿李곗씠 릺뿀떎(Fig. 1).

Fig. 1. Histochemical staining of brain of central nerve system (CNS) in the scuttle fly larval period by hematoxylin-eosin (H-E) stain. A; whole sample (×100). B, C; partial enlargement of brain (×400). Black arrows, pyramidal cell; red arrows, stellate cell; yellow arrows, myelin sheath.

蹂듭떊寃쎌깋(ventral nerve cord, VNC) 議곗쭅쓽 寃쎌슦뒗 궗엺쓽 泥숈닔 떖由 뇤 議곗쭅怨 留덉갔媛吏濡 諛붽묑履쎌뿉 떊寃쎌꽭룷泥닿 諛吏묐릺怨, 븞履쎌뿉 꽟쑀議곗쭅쑝濡 굹늻뼱졇 엳뿀떎. 諛붽묑履 뵾吏 遺遺꾩뿉뒗 뵾씪誘몃뱶 꽭룷, 蹂 꽭룷, 떊寃쎌젅 꽭룷(ganglion cell) 벑씠 留롮씠 愿李곗씠 릺뿀怨, 二쇱쐞뿉 떊寃쎌꽟쑀, 留먯씠吏묒쑝濡 梨꾩썙졇 엳뒗 援ъ“瑜 븯怨 엳뿀吏留 슌졆븯寃 愿李곗씠 릺吏뒗 븡븯떎. 븞履쎌쓽 냽吏 遺遺꾩 떊寃쎌꽟쑀 寃고빀 議곗쭅쑝濡 援ъ꽦맂 援ъ“瑜 븯怨 엳뿀쑝硫 뇤 議곗쭅怨 떎瑜 젏 以묒븰 遺遺꾩뿉 'T' 옄 삎깭濡 빑쓣 媛吏怨 엳뒗 떊寃쎌꽭룷뱾씠 議댁옱븯뒗 寃껋쓣 愿李고븷 닔 엳뿀떎(Fig. 2).

Fig. 2. Histochemical staining of ventral nerve cord (VNC) of central nerve system (CNS) in the scuttle fly larval period by hematoxylin-eosin (H-E) stain. A; whole sample (×100). B, C; partial enlargement of VNC (×400). Black arrows, pyramidal cell; red arrows, stellate cell; yellow arrows, ganglion cell; blue arrows, myelin sheath; ring, 'T' shaped nucleus.

떊寃쎌꽭룷븰쟻 듅吏

Scuttle fly뿉꽌 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 젣옄由ы샎꽦솕踰(ISH)쓣 씠슜빐꽌 corazonin 돱윴쓣 뿼깋븳 떎쓬 씠寃껋쓣 떎떆 뙆씪 몴蹂몄쓣 留뚮뱾뼱꽌, 뿼깋맂 corazonin 돱윴쓣 鍮꾨’븯뿬 二쇱쐞쓽 떊寃쎌꽭룷 諛 땲뒳냼泥 (Nissle body) 삉뒗 留먯씠吏묒쓣 슌졆븯寃 蹂닿린 쐞빐 留먯씠吏-땲뒳냼泥 뿼깋(luxol fast blue-cresyl violet stain)쓣 떆뻾븯뿬 떊寃쎌꽭룷븰쟻씤 듅吏뺤쓣 愿李고븯떎.

븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 뇤 議곗쭅뿉꽌 꽭룷뱾쓣 궡렣蹂대㈃ 젣씪 늿뿉 쓣뒗 寃껋 留먯씠吏묒씠 留롮씠 議댁옱븯怨 엳뒗 寃껋쓣 愿李고븯떎. 留먯씠吏묒 異뺤궘(axon)쓣 媛먯떥怨 엳뒗 遺遺꾩쑝濡 異뺤궘 嫄곗쓽 遺遺 吏諛⑹쑝濡 릺뼱 엳뼱꽌 뿼깋씠 릺吏 븡怨, 留먯씠吏묐쭔 吏숈 뙆깋쑝濡 뿼깋씠 릺뼱 愿李곕릺뿀떎. 留롮 留먯씠吏 궗씠뿉 떎뼇븳 떊寃쎌꽭룷쓽 떊寃쎌꽭룷泥닿 議댁옱븯怨 엳뿀쑝硫 떊寃쎌꽭룷泥 븞뿉 빑 겕怨 뫁洹몃윴 紐⑥뒿쑝濡 븯뼏寃 蹂댁떎. 떊寃쎌꽭룷쓽 꽭룷泥 븞뿉뒗 땲뒳냼泥대 蹂 닔 엳뿀怨 怨쇰┰씠 뭾遺빐꽌 吏꾪븯寃 뿼깋씠 릺뼱 엳뒗 寃껋쓣 愿李고븷 닔 엳뿀떎. 씠윭븳 留먯씠吏묒 떊寃쎌꽭룷泥대뱾 궗씠뿉꽌 諛쒗쁽맂 corazonin 돱윴씠 쐞移섑빐 엳뒗 寃껋쓣 愿李 븯怨, corazonin 돱윴씠 떎瑜 떊寃쎌꽭룷뿉 鍮꾪빐 긽떦엳 겙 꽭룷씪뒗 寃껋쓣 愿李고븷 닔 엳뿀떎(Fig. 3).

Fig. 3. Cytological staining of brain of central nerve system (CNS) in the scuttle fly larval period by luxol fast blue-cresyl violet stain. A, B; whole sample (×100). C, D; partial enlargement of brain (×400). Black arrows, corazonin-expressing neuron; red arrows, Nissl body; elliptical rings, myelin sheath.

以묒텛떊寃쎄퀎 蹂듭떊寃쎌깋 議곗쭅뿉꽌룄 留덉갔媛吏濡 뇤 議곗쭅뿉 鍮꾪빐꽌뒗 留먯씠吏묒씠 留롮씠 議댁옱븯吏뒗 븡븯怨 떎뼇븳 떊寃쎌꽭룷媛 留롮씠 議댁옱븯怨 엳뿀떎. 뇤 議곗쭅怨 留덉갔媛吏濡 corazonin쓣 諛쒗쁽븯뒗 꽭룷뒗 二쇱쐞쓽 떎뼇븳 떊寃쎌꽭룷뿉 鍮꾪빐 겕湲곌 留ㅼ슦 겙 寃껋쓣 蹂 닔 엳뿀떎. 臾쇰줎 젣옄由ы샎꽦솕踰 뿼깋씠 吏꾪븯寃 뿼깋씠 릺뼱꽌 corazonin 돱윴쓣 젙솗엳 솗씤쓣 븷 닔뒗 뾾뿀吏留 1媛쒖쓽 遺꾨퉬愿쓣 씠猷⑤뒗 돱윴泥섎읆 愿李 릺뒗 듅吏뺤씠 엳뿀떎(Fig. 4).

Fig. 4. Cytological staining of ventral nerve cord (VNC) of central nerve system (CNS) in the scuttle fly larval period by luxol fast blue-cresyl violet stain. A, B; whole sample (×100). C, D; partial enlargement of VNC (×400). Black arrows, corazonin-expressing neuron; blue arrows, myelin sheath.
怨 李

룞臾쇱뿉꽌쓽 떊寃쎌쟾떖臾쇱쭏쓣 遺꾨퉬븯뒗 꽭룷뒗 떊寃쎄퀎쓽 援ъ“뿉 룷븿릺뼱 룞臾쇱씠 뻾룞븯뒗뜲 媛옣 쁺뼢쓣 二쇨퀬 엳떎. 洹몃옒꽌 怨ㅼ땐쓽 뻾룞뿉 吏곸젒쟻쑝濡 쁺뼢쓣 誘몄튂怨 엳뒗 corazonin 돱윴뿉 愿떖쓣 媛議뚮떎. Corazonin 돱윴 떎뼇븳 뒪듃젅뒪뿉 뵲瑜 怨ㅼ땐쓽 援곗쭛 뻾룞 벑쓣 議곗젅븷 닔 엳쑝硫(Tawfix et al., 1999), 뵾遺 議곗쭅쓽 깋냼 벑뿉 愿뿬(Veenstra, 1991; Tawfik et al., 1999; Tanaka, 2001; Tanaka et al., 2002a; Porras et al., 2003), 깮떇 뒫젰 議곗젅(Tayler et al., 2012), 쓬떇 꽠痍⑥뿉 븳 꽠떇 뻾룞(Veenstra, 2009), 떖옣諛뺣룞 쑀吏(Veenstra, 1989; Tawfik et al., 1999; Tanaka, 2001; Yerushalmi et al., 2002) 벑쓣 깮由ъ쟻쑝濡 議곗젅븯뒗 寃껋쑝濡 뿰援щ릺뿀떎. 洹몃옒꽌 씠쟾뿉 뿰援щ릺뼱吏 젣옄由ы샎꽦솕踰뺤쓣 씠슜븳 寃泥대 씠슜븯뿬 corazonin 돱윴뿉 빐 떊寃 議곗쭅 諛 꽭룷븰쟻 援ъ“쟻씤 삎깭瑜 븣湲 쐞빐 뿰援щ 븯寃 릺뿀떎.

Scuttle fly뿉꽌 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 梨꾩쭛븯뿬 젣옄由ы샎꽦솕踰뺤쓣 셿猷뚰븳 썑, 뙆씪 몴蹂몄쓣 留뚮뱾뼱꽌 H&E (hematoxylin & eosin) 뿼깋쓣 떆뻾븯뿬 以묒텛떊寃쎄퀎쓽 쟾泥댁쟻씤 議곗쭅븰쟻씤 援ъ“瑜 궡렣蹂댁븯떎. 븷踰뚮젅 以묒텛떊寃쎄퀎 議곗쭅뿉꽌 뇤 議곗쭅 궗엺쓽 뇤 媛숈씠 寃됱쭏(cortex) 履쎌 쉶깋吏(gray matter), 냽吏(medulla) 履쎌 諛깆깋吏(white matter)쓽 鍮꾩듂븳 援ъ“濡 릺뼱 엳뿀떎.

븯吏留 븷踰뚮젅쓽 蹂듭떊寃쎌깋 議곗쭅 궗엺쓽 泥숈닔媛 諛붽묑履쎌 諛깆깋吏, 븞履쎌 쉶깋吏덉씤 諛섎㈃뿉 寃됱쭏怨 냽吏 履쎌씠 媛곴컖 洹몃濡 븷踰뚮젅 뇤 議곗쭅怨 룞씪븳 援ъ“瑜 븯怨 엳뒗 李⑥씠젏쓣 솗씤븯떎. 븷踰뚮젅쓽 뇤 蹂듭떊寃쎌깋 議곗쭅 寃됱쭏 遺遺꾩뿉뒗 留롮 뿬윭 醫낅쪟쓽 떊寃쎌꽭룷媛 諛吏묐릺뼱 愿李곗씠 릺뿀怨, 냽吏 遺遺꾩뿉뒗 떊寃쎌꽭룷媛 議댁옱븯吏 븡怨 떊寃쎌꽟쑀 寃고빀 議곗쭅쑝濡쒕쭔 梨꾩썙졇 엳뒗 援ъ“瑜 븯怨 엳뿀떎. 洹몃━怨 蹂듭떊寃쎌깋 議곗쭅쓽 븳媛슫뜲뿉뒗 "T" 옄삎쓽 以꾩쓣 吏뼱 떊寃쎌꽭룷뱾씠 議댁옱븯怨 엳뒗 議곗쭅븰쟻 援ъ“쓽 듅吏 벑쓣 븣 닔 엳뿀떎(William et al., 2013).

以묒텛떊寃쎄퀎 議곗쭅뿉꽌 H&E 뿼깋쓣 넻빐꽌 떊寃 議곗쭅븰쟻 쟾泥 援ъ“뒗 솗씤븷 닔 엳뿀吏留 corazonin 諛쒗쁽 돱윴怨 湲고 떊寃쎌꽭룷쓽 援ъ“뒗 젙솗엳 솗씤쓣 븷 닔媛 뾾뿀떎. 洹몃옒꽌 scuttle fly 븷踰뚮젅 3졊 떆湲곗쓽 以묒텛떊寃쎄퀎 議곗쭅쓣 젣옄由ы샎꽦솕踰뺤쓣 떆뻾븯뿬 corazonin 돱윴쓣 諛쒗쁽떆궓 썑 뙆씪 몴蹂몄쓣 留뚮뱾뼱꽌 諛쒗쁽맂 corazonin 돱윴怨 떎瑜 떊寃쎌꽭룷 벑쓣 슌졆븯寃 愿李고븯湲 쐞븯뿬 留먯씠吏-땲뒳냼泥 뿼깋(luxol fast blue-cresyl violet stain)쓣 떆뻾븯뿬 떊寃쎌꽭룷븰쟻쓽 듅吏뺤쓣 愿李고븯떎.

븷踰뚮젅쓽 以묒텛떊寃쎄퀎 議곗쭅뿉 議댁옱븯뒗 꽭룷뱾쓣 궡렣蹂댁븯쓣 븣 뇤 蹂듭떊寃쎌깋 議곗쭅뿉꽌 떎뼇븯怨 留롮 떊寃쎌꽭룷瑜 鍮꾨’븯뿬 留먯씠吏, 땲뒳냼泥 벑쓣 솗씤븷 닔 엳뿀떎. 留롮 留먯씠吏묎낵 떊寃쎌꽭룷뱾 궗씠뿉꽌 corazonin 돱윴씠 諛쒗쁽릺뼱 엳뿀吏留 corazonin 돱윴씠 젣옄由ы샎꽦솕踰(ISH)뿉 쓽빐 뿼깋씠 吏꾪븯뿬 젙솗븳 援ъ“뒗 솗씤븷 닔 뾾뿀떎. 븯吏留 씪諛섏쟻씤 떊寃쎌꽭룷쓽 꽭룷泥댁 鍮꾧탳뻽쓣 븣 뵾吏 遺遺꾩쓽 留롮 떊寃쎌꽭룷 궗씠뿉 議댁옱븯怨 엳뒗 corazonin 돱윴 긽떦엳 겙 떊寃쎈궡遺꾨퉬꽭룷씪뒗 寃껋쓣 솗씤븯떎(William et al., 2013).

쐞쓽 寃곌낵瑜 넻븯뿬 scuttle fly쓽 以묒텛떊寃쎄퀎뿉꽌 젣옄由ы샎꽦솕踰(ISH)쓣 쓽빐 諛쒗쁽릺뒗 corazonin 돱윴씠 떎뼇븳 떊寃쎌꽭룷 궗씠뿉꽌 鍮꾧탳쟻 겙 떊寃쎌꽭룷濡 옄由ъ옟怨 엳뒗 寃껋쓣 삎깭쟻쑝濡 愿李고븷 닔 엳뿀떎. 븵쑝濡쒖쓽 뿰援щ뒗 scuttle fly쓽 以묒텛떊寃쎄퀎뿉꽌 떎뼇븳 떊寃쎌꽭룷 궗씠뿉 鍮꾧탳쟻 겙 떊寃쎌꽭룷濡 쐞移섑빐 엳뒗 corazonin 諛쒗쁽 돱윴뿉 빐 諛쒖깮 떒怨꾨퀎濡 corazonin 쑀쟾옄쓽 諛쒗쁽 뼇긽쓣 蹂닿린 쐞빐꽌 RNA瑜 異붿텧븯뿬 쟾湲곗쁺룞 긽뿉꽌 솗씤븷 삁젙씠떎.

ACKNOWLEDGEMENT

This study was supported by reserch fund Mokpo Science University, 2022.

CONFLICT OF INTEREST

The authors affirm that they have no academic, financial or rights interests.

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