劉微芬，廖懷建，石 雷，杜 婷，周成理，鄧 疆，馬云強(qiáng)

(中國林業(yè)科學(xué)研究院資源昆蟲研究所，國家林業(yè)局資源昆蟲培育與利用重點實驗室，云南 昆明 650224)

青斑蝶雌成蟲內(nèi)生殖器官形態(tài)與發(fā)育

劉微芬，廖懷建，石 雷*，杜 婷，周成理，鄧 疆，馬云強(qiáng)

(中國林業(yè)科學(xué)研究院資源昆蟲研究所，國家林業(yè)局資源昆蟲培育與利用重點實驗室，云南 昆明 650224)

青斑蝶；內(nèi)生殖器官；卵巢；發(fā)育分級

1 研究方法

1.1 蟲源準(zhǔn)備

1.2 內(nèi)生殖器官解剖方法

上午9:30將成蟲浸泡于40%乙醇中，使成蟲暈厥后，置于0 ℃蒸餾水中備用。在自制的圓形蠟盤(直徑：15 cm)中倒入0.7%的生理鹽水進(jìn)行蟲體的解剖。解剖時將蠟盤置于體視顯微鏡(Leica MZ16A, Leica有限公司，德國)的載物臺上，在10×目鏡和1×物鏡下進(jìn)行。

1.3 內(nèi)生殖器官圖片采集

將雌成蟲內(nèi)生殖器官置于超景深三維顯微鏡(VHX-1 000，KEYENCE，日本)載物臺下進(jìn)行拍照采集圖片。由于青斑蝶雌成蟲內(nèi)生殖器官尺寸較大，采集到的是內(nèi)生殖器官的不同組成結(jié)構(gòu)的局部圖片。利用顯微鏡的實時2D圖像合成功能，在放大20倍的情況下，將局部圖片合成為內(nèi)生殖器官的完整圖片。然后，利用顯微鏡自帶的XY測量系統(tǒng)，在合成的完整圖片上測量各結(jié)構(gòu)的物理參數(shù)(實驗測定的各生殖器官大小的樣本量與解剖的雌蝶樣本量不一致是由于解剖過程部分器官會出現(xiàn)破損的情況，而對于有破損的器官沒有進(jìn)行測定)。

1.4 內(nèi)生殖器官特征參數(shù)測定

表1 雌蝶內(nèi)生殖器官特征參數(shù)測定的成蟲樣本量Table 1 The sample capacity for measuring the characteristic parameters of female adultTirumala limniace

圖1 雌性青斑蝶卵巢內(nèi)成熟(A)與未成熟卵(B)
Fig.1 The mature and immature oocyte in the ovaries of the female adult Tirumala limniace

1.5 數(shù)據(jù)分析

不同日齡間雌成蟲卵巢小管長度與直徑、精巢縱軸長與橫軸長、以及側(cè)輸卵管直徑的差異采用One-Way ANOVA進(jìn)行檢驗，當(dāng)差異達(dá)到顯著時，則采用Duncan’s multiple-range test進(jìn)行檢驗。不同日齡間雌成蟲卵巢小管內(nèi)未成熟卵和成熟卵數(shù)量的差異采用One-Way ANOVA進(jìn)行檢驗，當(dāng)差異達(dá)到顯著時，則采用Duncan’s multiple-range test進(jìn)行檢驗。同日齡雌成蟲卵巢小管未成熟卵與成熟卵數(shù)量間差異，采用Student’s t test進(jìn)行檢驗。所有統(tǒng)計分析均在SAS 9.4中進(jìn)行。

2 結(jié)果分析

2.1 青斑蝶雌蝶內(nèi)生殖器官

圖2 雌性青斑蝶的內(nèi)生殖器官結(jié)構(gòu)
Fig.2 The female internal reproductive organs of the Tirumala limniace

2.2 雌性青斑蝶成蟲的卵巢管長度、直徑和側(cè)輸卵管直徑隨日齡的變化

注：不同小寫字母表示不同日齡間數(shù)據(jù)達(dá)到顯著差異(p<0.05)Note: Different letters indicate significant difference between ages. (p<0.05)圖3 青斑蝶雌蝶卵巢管的長度和直徑、側(cè)輸卵管的直徑隨日齡的變化Fig.3 Length and diameter of the ovariole, diameter of the oviduct of the female adult Tirumala limniace

圖4 雌性青斑蝶卵巢中成熟卵、未成熟卵的數(shù)量以及卵的總量隨日齡的變化Fig.4 Number of mature, immature, and total eggs in ovaries of one- to 12-day old female adult Tirumala limniace

2.4 青斑蝶雌蝶卵巢發(fā)育分級標(biāo)準(zhǔn)

注：圖左上角數(shù)字表示日齡，比例尺：2 mmNote:Numbers on top left corner of figure mean age,Scale bars:2.0 mm.圖5 不同日齡青斑蝶雌蝶的內(nèi)生殖器官結(jié)構(gòu)Fig.5 Developmental morphology of internal reproductive organs of 1- to 12-day old female adult Tirumala limniace

3 討論

昆蟲雌性的內(nèi)生殖器官通常由1對卵巢小管、附腺、一個或多個受精囊以及連接這些組織的導(dǎo)管組成[13]。青斑蝶雌性內(nèi)生殖器官組成與之相同，包括1對卵巢、2根側(cè)輸卵管、1根中輸卵管、交配囊腺、外生殖腔和產(chǎn)卵孔；不過，在青斑蝶雌性內(nèi)生殖器官中沒有發(fā)現(xiàn)附腺結(jié)構(gòu)，這與黃粉蝶和橙黃豆粉蝶雌性內(nèi)生殖器官結(jié)構(gòu)存在差異[5,14]。

本研究對雌性青斑蝶的內(nèi)生殖系統(tǒng)結(jié)構(gòu)與發(fā)育的研究，均在恒溫恒濕條件下進(jìn)行。研究指出，適宜的溫濕度及其昆蟲的取食對昆蟲的卵巢的發(fā)育起著至關(guān)重要的作用，溫度太高而濕度太低會導(dǎo)致卵巢滯育，發(fā)育停滯不前；而當(dāng)昆蟲可取食的寄主食物由不可獲取變?yōu)榭梢垣@取時，同等溫濕度條件下滯育卵巢會變成熟[20]。因此，在實際工作中需要根據(jù)不同溫濕度來判定具體的卵巢發(fā)育進(jìn)度。

4 結(jié)論

雌性青斑蝶的內(nèi)生殖器官由1對卵巢、1對側(cè)輸卵管、1根中輸卵管、交配囊腺和受精囊組成；對卵巢小管長度、卵巢小管和側(cè)輸卵管直徑測定發(fā)現(xiàn)隨著日齡增大，其大小顯著變大；隨著日齡的增長，卵巢中成熟卵的數(shù)量先增大后減小，未成熟卵數(shù)量始終緩慢增加；根據(jù)卵巢的發(fā)育特征，將卵巢發(fā)育分為乳白透明期、卵黃沉積期、成熟待產(chǎn)期、產(chǎn)卵盛期、產(chǎn)卵末期。本研究將在青斑蝶的人工繁殖中為人為調(diào)控雌蝶產(chǎn)卵行為和有效提高其產(chǎn)卵量提供理論依據(jù)。

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(責(zé)任編輯：張 玲)

Morphology and Development of Reproductive System of Female AdultTirumalalimniace(Cramer)

LIUWei-fen,LIAOHuai-jian,SHILei,DUTing,ZHOUCheng-li,DENGJiang,MAYun-qiang

(Research Institute of Resources Insects of the Chinese Academy of Forestry/ Key Laboratory of Cultivation and Utilization of Resource Insects,State Forestry Administration, Kunming 650224, Yunnan, China)

[Objective]Tirumalalimniaceis a kind of important ornamental insects. To understand the structure and developmental state of internal reproductive organs can lay a theoretical basis for artificial reproduction and utilization ofT.limniace.[Method]The internal reproductive system of femaleT.limniacewith ages from one- to twelve-day old were dissected and observed, the characteristic parameters were measured, and the ovarian development was graded according to the developmental characteristics of ovary.[Result](1) The internal reproductive organs of femaleT.limniacecontain a couple of ovaries, a couple of lateral oviducts, a common oviduct, bursa copulatrix gland and spermatheca. (2) The development of internal reproductive organ was influenced significantly by age. The length of ovariole increased significantly with the age increasing and reached the maximum at the10th day, but the diameter of ovariole and lateral oviduct increased slowly with the increase of age. (3) With the increase of age, the amount of mature eggs in ovary increased at initial and then deduced, but the amount of immature eggs increased slowly all the time. (4) Based on the developmental characteristics, the ovarian development can be divided into 5 grades, i.e. milky and translucent stage, yolk deposition stage, egg maturation stage, peak stage of oviposition and final stage of oviposition. [Conclusion]The structure of internal reproductive system of femaleT.limniaceis the same as other butterflies, the development of internal reproductive organs is influenced significantly by age, and the eggs mature gradually with the increase of age. Ovarian grading ofT.limniacecan theoretically guide the artificial control of egg laying amount.

Tirumalalimniace(Cramer); reproductive system; ovary; development grading

10.13275/j.cnki.lykxyj.2017.01.017

2016-06-15

林業(yè)公益性行業(yè)科研專項經(jīng)費(201504305); 云南省技術(shù)創(chuàng)新人才培養(yǎng)計劃(2012HB054)。

劉微芬(1990—)，女，碩士研究生，主要從事蝴蝶行為生理生態(tài)學(xué)研究.
* 通訊作者：石 雷, 研究員.

S899

A

1001-1498(2017)01-0125-06