裴思慧　王蘭

摘 要：采用灼傷擠壓法摘除眼柄，通過常規(guī)石蠟切片、HE染色和實(shí)體顯微技術(shù)觀察河南華溪蟹（Sinopotamon Henanense）頭胸甲表皮組織細(xì)胞和第三顎足內(nèi)肢剛毛的形態(tài)學(xué)變化。實(shí)驗(yàn)發(fā)現(xiàn)，摘除眼柄后隨時(shí)間的延長，溪蟹頭胸甲表皮組織細(xì)胞出現(xiàn)染色較深的貯藏細(xì)胞，間質(zhì)增多，表明表皮中糖類或蛋白類物質(zhì)的合成活動(dòng)有所增強(qiáng)。溪蟹第三顎足內(nèi)肢在摘除眼柄后表皮厚度增加，邊緣輪廓逐漸模糊化，且邊緣厚度變薄，逐漸形成管狀樣的表皮結(jié)構(gòu)，剛毛腔增寬。通過研究，為預(yù)測河南華溪蟹的蛻皮周期、探究其蛻皮生理生化機(jī)制提供前期準(zhǔn)備。 關(guān)鍵詞：河南華溪蟹；摘除眼柄；體壁結(jié)構(gòu)；表皮組織

中圖分類號(hào)：Q95；S966.16 文獻(xiàn)標(biāo)識(shí)碼：A 文章編號(hào)：2095-6835（2014）04-0114-02

通過研究切除眼柄對(duì)甲殼動(dòng)物的影響，Zeleny等學(xué)者首次發(fā)現(xiàn)切除眼柄可以縮短蛻皮間期，從而加速蛻皮。隨后，許多學(xué)者也發(fā)現(xiàn)切除眼柄能誘導(dǎo)蛻皮并促進(jìn)生長的眼柄能明顯縮短稚蝦的蛻皮周期且加快蛻皮。在加速蛻皮的過程中，華溪蟹的形態(tài)、行為、結(jié)構(gòu)和生理都發(fā)生了哪些變化，目前還沒有明確的答案。探討河南華溪蟹被切除眼柄后表皮組織結(jié)構(gòu)和剛毛的變化，可以豐富甲殼動(dòng)物蛻皮機(jī)制內(nèi)容，為研究河南華溪蟹蛻皮的生理生化機(jī)制提供基礎(chǔ)，還可以為河南華溪蟹這一直接發(fā)育的物種的養(yǎng)殖提供理論指導(dǎo)。

1 材料與方法

1.1 材料與試劑

實(shí)驗(yàn)中研究的河南華溪蟹（下文簡稱“溪蟹”），于2013-05購自太原市五龍口東安水產(chǎn)批發(fā)市場，放置實(shí)驗(yàn)室水族缸（45 cm×30 cm×30 cm）中暫養(yǎng)2周。

選取頭胸甲寬為3～5 cm的健康溪蟹，養(yǎng)殖所用水采用曝氣48 h的自來水，水溫20±2 ℃，pH值為7.5，溶氧量8.0～8.3 mg/L。每日換水1次，并及時(shí)清理缸內(nèi)排泄物，每日投喂定量土豆1次。

準(zhǔn)備Bouin氏液、乙醇和甲醛等。

1.2 樣品處理

采用灼燒擠壓法切除眼柄，用體積分?jǐn)?shù)為75 %的乙醇擦拭鑷子、溪蟹眼柄，先用手術(shù)剪壓碎眼柄的基部并切除雙側(cè)眼柄，后用燒紅的鑷子灼燒使傷口愈合。在雙側(cè)眼柄摘除后將其放回缸中飼養(yǎng)，直到開始實(shí)驗(yàn)。

1.3 實(shí)驗(yàn)方法

分別在24 h、48 h、72 h、96 h、120 h、144 h和168 h隨機(jī)選取5只溪蟹進(jìn)行實(shí)驗(yàn)。每次實(shí)驗(yàn)隨機(jī)選取未切眼柄的5只溪蟹作為對(duì)照，重復(fù)3次。分別取正常組和切除眼柄組的溪蟹頭胸甲的表皮組織和第三顎足內(nèi)肢。表皮組織經(jīng)Bouin氏液固定48 h，流水沖洗，經(jīng)過系列酒精脫水，二甲苯透明，石蠟包埋，常規(guī)切片，Hematoxylin和Eosin染色。第三顎足內(nèi)肢經(jīng)體積分?jǐn)?shù)為4%的甲醛固定24 h，流水沖洗，并浸入體積分?jǐn)?shù)為75%的乙醇保存?zhèn)溆谩?/p>

1.4 實(shí)驗(yàn)觀察

溪蟹表皮組織HE染色后，經(jīng)過三目顯微鏡（Olympus -BX51TF）觀察；溪蟹第三顎足內(nèi)肢活體觀察采用體視顯微鏡（Olympus-SZX2-1LLK）觀察。

2 實(shí)驗(yàn)結(jié)果

2.1 對(duì)表皮組織的形態(tài)學(xué)變化

表皮是蛻皮激素調(diào)控蛻皮過程的關(guān)鍵靶組織。因此，我們研究了切除眼柄后，表皮組織的形態(tài)學(xué)變化。如圖1所示，圖1-a為正常組河南華溪蟹頭胸甲表皮組織，靠近外骨骼的一側(cè)是富含色素顆粒的色素細(xì)胞層，呈現(xiàn)黑褐色；內(nèi)側(cè)的上皮細(xì)胞排列較緊密，細(xì)胞密度大間質(zhì)少，包漿透亮，染色較均勻。圖1-b～1-h為切除眼柄24～168 h頭胸甲表皮組織。摘除眼柄72 h后，表皮組織細(xì)胞中開始出現(xiàn)染色較紅的顆粒物質(zhì)，隨著時(shí)間的延長，到96 h含顆粒的表皮組織細(xì)胞逐漸泡狀化（如1-e圖），120 h后形成貯藏細(xì)胞（如圖1-f、圖1-g、圖1-h），且表皮細(xì)胞數(shù)量減少，間質(zhì)增多，這表明表皮中糖類或蛋白類物質(zhì)的合成活動(dòng)有所增強(qiáng)。

2.2 對(duì)第三顎足內(nèi)肢剛毛的形態(tài)學(xué)變化

蟹在蛻去幾丁質(zhì)的舊殼時(shí)，蟹體上的剛毛會(huì)隨蟹舊殼一起蛻去，新生剛毛由新體長出，與舊剛毛無關(guān)。如圖2所示，圖2-a和圖2-b為正常組溪蟹第三顎足內(nèi)肢，正常組溪蟹處于蛻皮間期（stage C），外骨骼鈣化完全，輪廓清晰，表皮邊緣較均勻，剛毛腔（SL）狹窄，在剛毛基部形成剛毛圓錐（IC）（如圖2-b）；圖2-c～圖2-h為切除眼柄24～144 h，表皮厚度增加，邊緣輪廓逐漸模糊化，且邊緣厚度變薄，逐漸形成管狀樣的表皮結(jié)構(gòu)——?jiǎng)偯壽E，同時(shí)剛毛腔增寬（如圖2-f），此時(shí)溪蟹仍然處于蛻皮間期，但表皮與剛毛形態(tài)出現(xiàn)變化?，F(xiàn)擬定：圖2-c～圖2-d為C0期，圖2-e～圖2-f為 C1期，圖2-g～圖2-h為C2期。圖2-i中看出，表皮組織回縮，和外骨骼分離，產(chǎn)生一透明的空隙，回縮腔（RZ），這時(shí)溪蟹進(jìn)入蛻皮前早期D0 期，此時(shí)標(biāo)志著蛻皮的啟動(dòng)。

3 實(shí)驗(yàn)分析

參考文獻(xiàn)

[1]王芳，穆迎春，董雙林，等.去眼柄對(duì)凡納濱對(duì)蝦稚蝦蛻皮和生長的影響[J].中國海洋大學(xué)學(xué)報(bào)，2004，34（03）：371-376.

[2]劉繼濤.螃蟹的蛻殼與繁殖特點(diǎn)[J].水產(chǎn)養(yǎng)殖，2012（02）：46.

[3]徐在寬，何群益.河蟹無公害養(yǎng)殖重點(diǎn)、難點(diǎn)與實(shí)例[M].北京：科學(xué)技術(shù)文獻(xiàn)出版社，2005.

[4]趙紅霞.甲殼動(dòng)物蛻皮調(diào)控研究及蛻殼素的使用[J].廣東飼料，2006，15（01）：32-34.

[5]Watson R D，Lee K J，Qiu S，et al.Molecular cloning，expression，and tissue distribution of crustacean molt -inhibiting hormone[J].American Zoologist，2001，41：407-417.

[6]Zeleny C.Compensatory regulation[J].Journal of experimental biology，1905，2（01）.

[7]Chu K H，Chow W K.Effect of unilateral versus bilateral eyestalk ablation on moulting and growth of the shrimp Penaeus chinensis（Osbeck，1765）（Decapoda，Penaeidea）[J]. Crustaceana，1992，62（03）：225-233.

[8]Nan F H，Sheen S C，Liu P C，et al.The effect of eyestalk ablation on growth，haemolymph composition and gill Na+，K+- ATP activity of Penaeus monodon juveniles[J].Comparative Biochemistry and Physiology，1993，106A：621-626.

[9]Spaziani E.Morphology，histology，and ultrastructure of the ecdysial gland（Y-organ）in Crustacea∥Gupta A P.Morp -hogenetic Hormones of Arthropods，vol[J].New Brunswick： Rutgers University.Press，1990，233-267.

[10]Lachaise F，Le Roux A，Huber M，et al.The molting gland of crustaceans：localization，activity，and endocrine controla[J].Crustacean Biology，1993，13：198-234.

[11]Chung J S，Webster S G.Moult cycle-related changes in biological activity of moult-inhibiting hormone（MIH）and crustacean hyperglycaemic hormone（CHH）in the crab，Carcinus maenas from target to transcript[J].European Journal of Biochemistry，2003，270（15）：3280-3288.

[12]Imayavaramban L，Dhayaparan D，Devaraj H.Molecular mechanism of molt-inhibiting hormone（MIH）induced suppression of ecdysteroidogenesis in the Y-organ of mud crab：Scylla serrata[J].FEBS Letters，2007，581（27）：5167-5172.

[13]Laverdure A M，Breuzet M，Soyez D.Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization[J].General and comparative endocrinology，1992，87（03）： 443-450.

[14]Tian Z H，Kang X J，Mu S M.The molt stages and the hepatopancreas contents of lipids，glycogen and selected inorganic elements during the molt cycle of the Chinese mitten crab Eriocheir sinensis[J].Fish science，2012，78：67-74.

[15]Promwikorn W，Kirirat P，Thaweethamsewee P.Index of molt staging in the black tiger shrimp（Penaeus monodon） [J].Songklanakarin Journal of Science and Technology，2004，26（05）：765 -772.

作者簡介：裴思慧（1988—），女，碩士，主要從事甲殼動(dòng)物蛻皮的生理生化調(diào)控研究。

〔編輯：李玨〕

Abstract： The removal of eye burns extrusion handle by conventional paraffin sections， HE staining and microscopy techniques to observe physical Sinopotamon Henanense morphological changes carapace epidermal cells and the third jaw foot limb setae. It was found that after the removal of the eye pattern over time， the Sinopotamon Henanense carapace epidermal cells appear darker stain storage cells， increased quality， indicating that the synthesis of carbohydrate or protein activity in the epidermis substances has been enhanced. Sinopotamon Henanense within the third limb jaw foot increase in epidermal thickness after removal of the eye stalk， gradually blurring the edge contours and edges thinner， gradually forming a tubular-like structure of the epidermis， bristle cavity widened. Through research， to predict Sinopotamon Henanense molting cycle， explore its physiological and biochemical mechanisms molt preparation.

Key words： Sinopotamon Henanense； removal of eye stalk； body wall structure； epidermal tissue

[6]Zeleny C.Compensatory regulation[J].Journal of experimental biology，1905，2（01）.

[7]Chu K H，Chow W K.Effect of unilateral versus bilateral eyestalk ablation on moulting and growth of the shrimp Penaeus chinensis（Osbeck，1765）（Decapoda，Penaeidea）[J]. Crustaceana，1992，62（03）：225-233.

[8]Nan F H，Sheen S C，Liu P C，et al.The effect of eyestalk ablation on growth，haemolymph composition and gill Na+，K+- ATP activity of Penaeus monodon juveniles[J].Comparative Biochemistry and Physiology，1993，106A：621-626.

[9]Spaziani E.Morphology，histology，and ultrastructure of the ecdysial gland（Y-organ）in Crustacea∥Gupta A P.Morp -hogenetic Hormones of Arthropods，vol[J].New Brunswick： Rutgers University.Press，1990，233-267.

[10]Lachaise F，Le Roux A，Huber M，et al.The molting gland of crustaceans：localization，activity，and endocrine controla[J].Crustacean Biology，1993，13：198-234.

[11]Chung J S，Webster S G.Moult cycle-related changes in biological activity of moult-inhibiting hormone（MIH）and crustacean hyperglycaemic hormone（CHH）in the crab，Carcinus maenas from target to transcript[J].European Journal of Biochemistry，2003，270（15）：3280-3288.

[12]Imayavaramban L，Dhayaparan D，Devaraj H.Molecular mechanism of molt-inhibiting hormone（MIH）induced suppression of ecdysteroidogenesis in the Y-organ of mud crab：Scylla serrata[J].FEBS Letters，2007，581（27）：5167-5172.

[13]Laverdure A M，Breuzet M，Soyez D.Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization[J].General and comparative endocrinology，1992，87（03）： 443-450.

[14]Tian Z H，Kang X J，Mu S M.The molt stages and the hepatopancreas contents of lipids，glycogen and selected inorganic elements during the molt cycle of the Chinese mitten crab Eriocheir sinensis[J].Fish science，2012，78：67-74.

[15]Promwikorn W，Kirirat P，Thaweethamsewee P.Index of molt staging in the black tiger shrimp（Penaeus monodon） [J].Songklanakarin Journal of Science and Technology，2004，26（05）：765 -772.

作者簡介：裴思慧（1988—），女，碩士，主要從事甲殼動(dòng)物蛻皮的生理生化調(diào)控研究。

〔編輯：李玨〕

Abstract： The removal of eye burns extrusion handle by conventional paraffin sections， HE staining and microscopy techniques to observe physical Sinopotamon Henanense morphological changes carapace epidermal cells and the third jaw foot limb setae. It was found that after the removal of the eye pattern over time， the Sinopotamon Henanense carapace epidermal cells appear darker stain storage cells， increased quality， indicating that the synthesis of carbohydrate or protein activity in the epidermis substances has been enhanced. Sinopotamon Henanense within the third limb jaw foot increase in epidermal thickness after removal of the eye stalk， gradually blurring the edge contours and edges thinner， gradually forming a tubular-like structure of the epidermis， bristle cavity widened. Through research， to predict Sinopotamon Henanense molting cycle， explore its physiological and biochemical mechanisms molt preparation.

Key words： Sinopotamon Henanense； removal of eye stalk； body wall structure； epidermal tissue

[6]Zeleny C.Compensatory regulation[J].Journal of experimental biology，1905，2（01）.

[7]Chu K H，Chow W K.Effect of unilateral versus bilateral eyestalk ablation on moulting and growth of the shrimp Penaeus chinensis（Osbeck，1765）（Decapoda，Penaeidea）[J]. Crustaceana，1992，62（03）：225-233.

[8]Nan F H，Sheen S C，Liu P C，et al.The effect of eyestalk ablation on growth，haemolymph composition and gill Na+，K+- ATP activity of Penaeus monodon juveniles[J].Comparative Biochemistry and Physiology，1993，106A：621-626.

[9]Spaziani E.Morphology，histology，and ultrastructure of the ecdysial gland（Y-organ）in Crustacea∥Gupta A P.Morp -hogenetic Hormones of Arthropods，vol[J].New Brunswick： Rutgers University.Press，1990，233-267.

[10]Lachaise F，Le Roux A，Huber M，et al.The molting gland of crustaceans：localization，activity，and endocrine controla[J].Crustacean Biology，1993，13：198-234.

[11]Chung J S，Webster S G.Moult cycle-related changes in biological activity of moult-inhibiting hormone（MIH）and crustacean hyperglycaemic hormone（CHH）in the crab，Carcinus maenas from target to transcript[J].European Journal of Biochemistry，2003，270（15）：3280-3288.

[12]Imayavaramban L，Dhayaparan D，Devaraj H.Molecular mechanism of molt-inhibiting hormone（MIH）induced suppression of ecdysteroidogenesis in the Y-organ of mud crab：Scylla serrata[J].FEBS Letters，2007，581（27）：5167-5172.

[13]Laverdure A M，Breuzet M，Soyez D.Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization[J].General and comparative endocrinology，1992，87（03）： 443-450.

[14]Tian Z H，Kang X J，Mu S M.The molt stages and the hepatopancreas contents of lipids，glycogen and selected inorganic elements during the molt cycle of the Chinese mitten crab Eriocheir sinensis[J].Fish science，2012，78：67-74.

[15]Promwikorn W，Kirirat P，Thaweethamsewee P.Index of molt staging in the black tiger shrimp（Penaeus monodon） [J].Songklanakarin Journal of Science and Technology，2004，26（05）：765 -772.

作者簡介：裴思慧（1988—），女，碩士，主要從事甲殼動(dòng)物蛻皮的生理生化調(diào)控研究。

〔編輯：李玨〕

Abstract： The removal of eye burns extrusion handle by conventional paraffin sections， HE staining and microscopy techniques to observe physical Sinopotamon Henanense morphological changes carapace epidermal cells and the third jaw foot limb setae. It was found that after the removal of the eye pattern over time， the Sinopotamon Henanense carapace epidermal cells appear darker stain storage cells， increased quality， indicating that the synthesis of carbohydrate or protein activity in the epidermis substances has been enhanced. Sinopotamon Henanense within the third limb jaw foot increase in epidermal thickness after removal of the eye stalk， gradually blurring the edge contours and edges thinner， gradually forming a tubular-like structure of the epidermis， bristle cavity widened. Through research， to predict Sinopotamon Henanense molting cycle， explore its physiological and biochemical mechanisms molt preparation.

Key words： Sinopotamon Henanense； removal of eye stalk； body wall structure； epidermal tissue