小地老虎性信息素通讯的分子和细胞机制
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摘要
取食、求爱及交配对昆虫来说是最基本和重要的生命活动，昆虫主要通过表达嗅觉蛋白的触角来检测环境中与生存和生殖相关的化学信号。性信息素分子和植物挥发物通过触角表皮上的极孔进入触角感器。一旦性信息素分子和植物挥发物进入到触角感器淋巴液，触角表达的结合蛋白(包括气味结合蛋白OBP和化学感受蛋白CSP)捕获外界的化学信号分子，然后将这些信号分子运输通过水溶性的触角感器淋巴液，最后将这些化学信号分子运送至嗅觉受体神经元ORN树突膜上与化学感受受体(包括ORs和IRs)结合，紧接着气味分子被气味降解酶迅速降解掉。昆虫对性信息素具有极高的敏感性和特异性，我们可以利用这个特点在害虫综合治理中利用性信息素来进行害虫种群的监测和大量诱捕。阐明昆虫嗅觉识别的机制可以帮助我们设计新的方法来进行害虫治理。小地老虎(鳞翅目:夜蛾科)是世界范围内多种作物上的重要害虫。研究表明小地老虎雄蛾对雌蛾释放的性信息素具有极高的趋性。但是雄蛾识别性信息素的分子和细胞机制目前还不清楚。小地老虎触角中嗅觉相关蛋白的鉴定以及功能分析将有利于我们阐明昆虫嗅觉识别的分子和细胞机制。更重要的是，可以为我们提供新的方法如通过干扰昆虫嗅觉识别，进而达到对靶标害虫进行有效防治的目的。在本论文中，通过二代转录组测序，我们从小地老虎的触角中鉴定了一系列嗅觉识别相关基因，在小地老虎雌蛾性腺中鉴定了数种参与小地老虎性信息素合成、运输和降解的基因。我们对小地老虎触角中的三类嗅觉蛋白进行了系统的功能研究，包括性信息素结合蛋白PBPs、性信息素受体PRs和感觉神经元膜蛋白SNMPs,最终在转录水平、蛋白水平和细胞水平证明小地老虎PBPs、PRs和SNMPs在小地老虎识别性信息素分子中的重要作用。主要研究结果如下：
1.小地老虎触角嗅觉感器类型和超微结构
扫描电镜和透射电镜结果显示:小地老虎的雌雄触角形态呈典型的性二态型:雄蛾触角呈双栉形，雌蛾触角呈丝状。雌雄触角平均长度为12mm。在小地老虎雌雄蛾触角上至少分布着6种不同类型的嗅觉感器，分别为:毛形感器、刺形感器、锥形感器、腔锥形感器、鳞形感器和B hm氏鬃毛。毛形感器的数量最多，并且每个感器有2-3个树突神经元；锥形感器细胞壁比较薄并且每个感器有12-25个树突神经元；腔锥形感器细胞壁有4-7个树突神经元；刺形感器没有神经元,细胞壁比较厚。
2.小地老虎触角转录组测序及嗅觉基因鉴定
采用Roche公司的454GS FLX测序平台，我们对小地老虎雌雄触角进行了转录组测序，通过生物信息学分析我们从小地老虎触角转录组中鉴别了一系列嗅觉识别相关基因，并用半定量RT-PCR和荧光定量qRT-PCR技术研究了这些嗅觉基因的组织表达图谱，主要结果如下:
(1)气味结合蛋白OBP基因33个。其中30个OBP基因具有完整的开放阅读框，长度为402-759bp不等。半定量RT-PCR和荧光定量qRT-PCR结果显示其中包括三个PBP在内的22个OBP基因(PBP1, PBP2, PBP3, GOBP1, GOBP2, OBP1, OBP2, OBP4, OBP5, OBP9, OBP11, OBP12,OBP13, OBP15, OBP16, OBP17, OBP19, OBP20, OBP21, OBP22, OBP24和OBP26)特异性或主要在雌雄触角中表达。表明在触角中特异性表达或高表达的OBP基因在小地老虎识别性信息素、定位寄主植物和寻找合适产卵位点过程中起重要作用。OBP6基因不在雌雄触角中表达，反而在性腺pheromone gland (PG)中特异性表达，与典型的触角表达的OBP基因不同，这个性腺高表达的OBP基因在识别性信息素和普通气味过程中可能起着完全不同的作用。
(2)化学感受蛋白CSP基因12个。半定量RT-PCR和荧光定量qRT-PCR结果显示3个CSP基因(CSP8, CSP9和CSP10)在雌雄触角中表达量最高，说明这三个触角高表达的CSP基因在小地老虎嗅觉识别过程中发挥关键作用。值得注意的是，一个CSP基因(CSP2)不在雌雄触角中表达，反而在性腺PG中特异性表达，暗示性腺中表达的CSP同样能够参与性信息素的结合与运输，并且昆虫能够利用性腺特异性或高表达的CSP蛋白检测并监测雌蛾自己释放的性信息素。
(3)气味受体OR基因42个，其中包括1个非典型嗅觉受体Orco,41个典型的嗅觉受体ORs。半定量RT-PCR和荧光定量qRT-PCR结果显示35个ORs基因特异性或主要在雌雄蛾触角中表达，其中4个ORs基因(OR1, OR3, OR4和OR14)特异性地在雄蛾触角中表达，表明这4个ORs极有可能为识别性信息素的受体PRs，在小地老虎识别性信息素过程中起关键作用。4个ORs基因(OR6, OR7, OR8和OR23)主要在雌蛾触角中表达，表明这几个基因在小地老虎雌蛾定位寄主植物和寻找产卵位点过程中发挥重要作用。
(4)离子型受体IR基因24个，其中包括两个保守的Coreceptors IR8a和IR25a.半定量RT-PCR和荧光定量qRT-PCR结果显示14个IR基因(IR8a, IR25a, IR21a, IR41a, IR75q.1, IR75q.2, IR76b,IR87a, IR1, IR3, IR4, IR8, IR12和IR13)主要在触角中表达，其中IR12基因特异性地在雄蛾触角中表达，表明IR12可能为识别性信息素受体。
3.小地老虎性信息结合蛋白的免疫定位及与性信息素的识别反应
通过同源克隆结合RACE PCR技术克隆了小地老虎性信息素结合蛋白PBP1-3基因。并对其功能进行了系统的研究，主要结果如下：
(1)三个小地老虎PBP1-3蛋白之间的序列相似性为40%。与法国小地老虎种群PBP1和PBP2序列相比，中国种群PBP1在其N端多了8个氨基酸序列，分别为MAPHPSVT.中国种群PBP2蛋白在其N端多了23个氨基酸序列，分别为MAASRWCIACLVCVLFAARSVMT.与此同时，法国种群和中国种群PBP1和PBP2蛋白在其他部位部分氨基酸也发生了变异，可能代表PBP1和PBP2蛋白在法国和中国种群小地老虎中的序列多态性。
(2)采用荧光定量qRT-PCR对三个小地老虎PBP基因在不同组织中的表达谱进行了分析，结果显示小地老虎AipsPBP1-3基因在触角中的表达量明显高于在其他组织中的表达量。同时AipsPBP1-3在味觉器官喙和下唇须中也有少量的表达。AipsPBP1-3在其他部位如头，胸，腹，足和翅中的表达量非常低。AipsPBP1-3在雄蛾触角中的表达量明显高于在雌蛾触角中的表达量，分别为雌蛾触角中表达量的2.2±0.3倍,4.5±0.4倍和2.6±0.3倍(p<0.01)。
(3)利用制备的AipsPBP1-3多克隆抗体，采用免疫组织化学技术系统研究了小地老虎AipsPBP1-3蛋白在不同触角感器中的表达分布情况，结果显示，在雄蛾触角中，胶体金颗粒主要标记在对性信息素敏感的毛形感受器的淋巴液中，有时也标记在对普通气味敏感的锥形感受器的淋巴液中。值得注意的是，有些毛形感器淋巴液并没有被胶体金颗粒标记上。在雌蛾触角中，仅有少数的毛形感器和锥形感器被标记上，说明AipsPBP1-3蛋白在雌蛾触角中的表达量极低。其余感器如刺形感器、腔锥形感器、鳞形感器和B hm氏鬃毛没有被标记上，说明AipsPBP1-3蛋白不在这些感受器中表达。
(4)荧光竞争结合实验结果表明小地老虎PBP1-3与荧光探针1-NPN的结合常数分别为:4.4±0.3μM,3.2±0.2μM和2.1±0.2μM. AipsPBP1蛋白同小地老虎的两种主要性信息素组分Z7-12:Ac和Z9-14:Ac有着很强的结合能力，IC50值分别为0.46±0.03μM和0.84±0.08μM. AipsPBP1同其余三种信息素Z11-16:Ac, Z5-10:Ac和Z8-12:Ac的结合能力较弱。AipsPBP2蛋白也同两种主要性信息素成分Z7-12:Ac和Z9-14:Ac有着很强的结合能力，IC50值分别为0.56±0.06μM和0.45±0.03μM，与此同时AipsPBP2同微量组分Z11-16:Ac也有着很强的结合能力，IC50值为0.79±0.05μM.值得注意的是, AipsPBP3只特异性的和微量组分Z11-16:Ac有很强的结合能力，IC50值为0.62±0.04μM。
(5)双相结合实验(two phase binding assays)得到的结果和荧光竞争结合实验的结果基本吻合。AipsPBP1蛋白结合主要性信息素成分Z7-12:Ac和Z9-14:Ac能力要明显高于其余三种微量组分Z11-16:Ac, Z5-10:Ac和Z8-12:Ac.每微克AipsPBP1能够结合Z7-12:Ac和Z9-14:Ac的量分别为2.2±0.5ng和2.0±0.5ng. AipsPBP2能够同时很好的结合两种主要性信息素组分Z7-12:Ac, Z9-14:Ac，同时也能够很好的结合微量性信息素组分Z11-16:Ac,每微克AipsPBP2蛋白能够结合这三种性信息素的量分别为1.9±0.4ng,2.1±0.5ng和2.0±0.4ng. AipsPBP3结合性信息素组分Z11-16:Ac的能力明显高于其余四种性信息素，每微克AipsPBP3能够结合Z11-16:Ac1.9±0.4ng.3个PBP蛋白中没有任何一个PBP蛋白特异性结合单一性信息素成分。
4.小地老虎性信息素受体的分子克隆及功能分析
通过同源克隆结合RACE PCR技术克隆了小地老虎非典型嗅觉受体Orco和性信息素受体PR基因3个，并利用非洲爪蟾卵母细胞表达系统结合双电极电压钳技术对其在识别性信息素过程中作用深入的研究。主要研究结论如下：
(1)荧光定量qRT-PCR结果显示非典型嗅觉受体AipsOrco和三个性信息素受体AipsOR1, AipsOR3和AipsOR4主要在触角中表达。AipsOrco在雌雄触角中的表达量相当(p>0.01)。AipsOrco同时在味觉器官喙和下唇须中也有少量的表达，在足和翅中也有微量的表达，在头、胸、腹中不表达。三个性信息素受体AipsOR1, AipsOR3和AipsOR4主要在雄蛾触角中表达，其在雄触角中的表达量是在雌触角中表达量的20倍以上(p<0.01)。三个性信息素受体同时在味觉器官喙和下唇须中也有少量表达。
(2)表达小地老虎性信息素受体的爪蟾卵母细胞对小地老虎5种性信息素呈现不同的反应图谱，性信息素受体OR3特异性识别小地老虎主要的性信息素组分Z7-12:Ac,在Z7-12:Ac浓度为10-4Mol/L的刺激下，表达OR3的卵母细胞的电流平均值能达到5100nA，性信息素受体OR1特异性识别小地老虎主要的第二主要性信息素组分Z9-14:Ac,在Z9-14:Ac浓度为10-4Mol/L的刺激下，表达OR1的卵母细胞的电流平均值为1335nA。性信息素受体OR4特异性识别小地老虎次要性信息素组分Z5-10:Ac,在Z59-10:Ac浓度为10-4Mol/L的刺激下，表达OR4的卵母细胞的电流平均值为362nA.
5.小地老虎感觉神经元膜蛋白的表达和免疫定位分析
通过同源克隆结合RACE PCR技术克隆了小地老虎感觉神经元膜蛋白基因SNMP1和SNMP2，利用荧光定量qRT-PCR研究了小地老虎SNMP在成虫不同组织部位、不同发育阶段的触角以及交配前后在触角中的表达情况；利用免疫组织化学方法系统研究小地老虎SNMP在不同类型触角感受器中的表达情况。同时讨论了AipsSNMPs蛋白在昆虫嗅觉识别中的功能，主要研究结论如下：
(1)荧光定量qRT-PCR结果显示AipsSNMP1和AipsSNMP2基因在雌雄蛾触角中的表达量要显著高于其在其他组织中的表达量，是在腹部表达量的100倍以上(p<0.05)。另外，AipsSNMP1和AipsSNMP2在雄蛾触角中的表达量分别是在雌蛾触角中表达量的1.5倍和2.3倍(p<0.05)。AipsSNMP1和AipsSNMP2在喙、下唇须、头、足和翅中的表达量显著低于在触角中的表达量，但是却显著高于在胸、腹部、雄性附腺和雌雄性腺中的表达量。AipsSNMP1和AipsSNMP2在雌雄蛾触角的不同发育阶段呈现相似的表达模式：在羽化之前3天的雌雄蛹中开始表达；然后从羽化前一天表达量开始急剧上升，在羽化当天表达量为羽化前一天表达量的2倍；在羽化后1到4天在雌雄蛾触角中一直保持如此高的的表达水平；在羽化4天之后，AipsSNMP1和AipsSNMP2在雌雄触角中的表达量开始下降，羽化后5到7天的表达水平降至与羽化1天后相当的表达水平。AipsSNMP1和AipsSNMP2基因在4day-old交配前后雌雄蛾触角中的表达水平无显著的变化(p>0.05)。
(2)免疫组织化学结果显示AipsSNMP1和AipsSNMP2都在对性信息素敏感的毛形感受器中表达，但是表达模式却完全不同，AipsSNMP1在毛形感器嗅觉神经元ORNs膜上表达，而AipsSNMP2蛋白却在毛形感器中的淋巴液中表达。同时发现AipsSNMP1也在腔锥形感器神经元ORNs上表达。另外，AipsSNMP2也在锥形感器和腔锥形感器的淋巴液中表达。AipsSNMP1和AipsSNMP2在雄蛾触角标记的感器数量要显著高于在雌蛾触角中标记的感器数量。
6.小地老虎性腺转录组分析及性信息素合成、运输和降解基因的鉴定
利用二代测序技术，对小地老虎的性腺转录组进行了测序，利用生物信息学方法从中筛选鉴定了一系列参与小地老虎性信息素合成、运输、识别和降解的基因，并利用半定量RT-PCR和荧光定量qRT-PCR技术研究了这些基因在性腺和body中的表达情况。主要研究结果如下：
(1)信息素合成激活神经肽(pheromone biosynthesis activating neuropeptide, PBAN)受体基因。小地老虎性腺转录本Unigene_3821编码一个PBAN受体B亚型蛋白。但是其在性腺中的表达量非常的低，RPKM值只有31。
(2)乙酰辅酶A羧化酶。在小地老虎性腺转录组中我们鉴定了2个转录本(Unigene_2338和Unigene_6244)编码不同的乙酰辅酶A羧化酶基因。半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_2338和Unigene_6244在性腺中的表达量要明显高于其在body中的表达量。但是RPKM值分析显示这两个基因在小地老虎性腺中表达量很低，RPKM值分别为81和21.
(3)脂肪酸合成酶基因。从小地老虎性腺中鉴定了1个FAS基因(Unigene_18120)。开放阅读框ORF为7176bp.半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_18120主要在性腺中表达，其在性腺中的表达量为在body中表达量的40倍左右。RPKM值分析发现Unigene_18120在小地老虎性腺中的表达量也很高，RPKM值为343.
(4)去饱和酶基因。在小地老虎性腺转录组中我们鉴定了5个转录本编码去饱和酶基因。Unigene_65编码去饱和酶acyl-CoA9-desaturase，Unigene_741编码去饱和酶acyl-CoA11desaturase，这两个编码去饱和酶的转录本极有可能参与小地老虎性信息素合成过程。半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_741和Unigene_780在性腺中的表达量要显著高于在body中的表达量，分别是body中表达量的85和63倍。RPKM分析显示Unigene_780在性腺转录组中的表达量也很高, RPKM值为1206,推测Unigene_780在小地老虎性信息素生物合成中起关键作用。其余三个Unigene_65、Unigene_10494和Unigene_15401在性腺和body中的表达量相当。
(5)脂肪酰辅酶A还原酶基因。在小地老虎性腺转录组中我们一共鉴定了13个编码脂肪酰辅酶A还原酶的转录本。半定量RT-PCR和荧光定量qRT-PCR结果显示三个转录本(Unigene_163, Unigene_4302和Unigene_7344)主要在性腺中表达。其余10个转录本在性腺和body中的表达量相差不大或在主要在body中表达。RPKM分析显示两个转录本(Unigene_163和Unigene_2537)在性腺转录中的表达量相对较高，其余11个转录本在性腺转录组中的表达量很低，RPKM值在16到81之间。
(6)醛还原酶基因。在小地老虎性腺转录组中我们鉴定了11个转录本编码醛-酮还原酶aldo-ketoreductase。半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_3134和Unigene_4806主要在性腺中表达，其余9个编码醛还原酶的转录本在性腺和body中的表达量相差不大或主要在body中表达。RPKM分析显示鉴定的11个醛还原酶基因在小地老虎性腺转录组中的表达量非常低，RPKM值在10到67之间。
(7)乙酰基转移酶基因。在小地老虎性腺转录组中我们一共鉴定了5个编码乙酰基转移酶的转录本，半定量RT-PCR和荧光定量qRT-PCR结果显示三个转录本Unigene_407,Unigene_2015和Unigene_173在性腺中的表达量要显著高于在body中的表达量，RPKM分析显示这三个转录本在小地老虎性腺转录组中的相对表达量也比较高，RPKM值分别为195,155和71.
(8)性信息素降解酶基因。在小地老虎性腺转录组我们一共鉴定了17个酯酶基因。荧光定量qRT-PCR结果显示AipsCXE3, AipsCXE7, AipsCXE8, AipsCXE9, AipsCXE11, AipsCXE14和AipsCXE207个酯酶基因在触角中表达量非常高,三个酯酶基因AipsCXE5, AipsCXE10和AipsCXE15同时在触角和性腺中高量表达，剩余的7个酯酶基因AipsCXE1, AipsCXE2,AipsCXE4, AipsCXE6, AipsCXE12, AipsCXE13和AipsCXE16在触角、body、性腺中的表达量差异不显著，说明这7个酯酶基因不是性信息素特异的。
(9)气味结合蛋白OBP和化学感受蛋白CSP基因。我们从小地老虎性腺转录组中鉴定了7个OBP基因和8个CSP基因。荧光定量qRT-PCR结果显示在7个OBP基因中，Unigene_15711编码的OBP基因主要在性腺中表达。三个OBP基因Unigene_520, Unigene_2120和Unigene_8860主要在触角中表达。在8个CSP基因中，Unigene_1704编码的CSP基因特异性地在性腺中表达，其表达量是触角和body中表达量的100倍以上。RPKM分析显示Unigene_1704在小地老虎性腺转录组中的表达量也非常高。另外一个编码CSP基因的Unigene_721也主要在性腺中表达，RPKM分析显示Unigene_721在小地老虎性腺转录组中的表达量非常高，RPKM值为1364.上述在性腺中特异性表达或者高表达的OBP和CSP基因可能参与性信息素的运输与释放过程。
Feeding, courtship and mating are fundamental for insects, and most insects rely on their sensitiveantennae that express specific olfactory proteins to detect survival-and reproductive-related chemicalcues from the environment. Pheromones and plant volatiles diffuse into the sensilla via the multiporesthat penetrate the cuticular surface. When pheromones and plant volatiles enter the sensillum lymph, theantennae-enriched binding proteins (odorant binding proteins (OBPs) and chemosensory proteins(CSPs)) capture these semiochemicals and transport them across the aqueous lumen to themembrane-bound chemosensory receptors (odorant receptors (ORs) and ionotropic receptors (IRs)).Subsequently, the odorant molecules are rapidly degraded by odorant-degrading enzymes (ODEs). Thehigh specificity and sensitivity of insect to sex pheromones make them as effective biological controlagents for population monitoring and mass trapping of noxious insects in integrated pest management(IPM) programs. Elucidating the mechanism of insect olfactory perception can further facilitate thedesign and implementation of novel intervention strategies against these pests. The black cutworm mothAgrotis ipsilon (Hufnagel)(Lepidoptera: Noctuidae) is a destructive pest affecting many cropsworldwide and the male A. ipsilon moth is high attracted by the sex pheromones released by the femalemoth. However, the molecular and cellular mechanisms of how sex pheromones are perceived by maleA. ipsilon moths are still rudimentary. The identification and functional characterization of antennalolfactory proteins in A. ipsilon will enhance our knowledge of the molecular and cellular basis of insectchemoreception. More importantly, can provide us new methods to control this pest through interringtheir olfaction perception. In this study, several kinds of olfactory genes putatively involved in theolfactory reception in A. ipsilon antennae, and several kinds of genes putatively involved in pheromoneproduction, transport and degradation in the A. ipsilon female moth pheromone gland were identifiedthrough next-generation transcriptome sequencing. We have taken systematic functional studies of threekind of key olfactory proteins in the A. ipsilon antennae, including pheromone binding proteins (PBPs),pheromone receptors (PRs) and sensory neuron membrane proteins (SNMPs), we have provideddetailed evidences of PBPs, PRs and SNMPs in A. ipsilon sex pheromone reception at transcriptionallevel, proteins level and cellular level. The main results are as follows:
1. Types and ultrastructures of olfactory sensilla in A. ipsilon antennae
The scanning and transmission electron microscopic examinations revealed that the grossmorphology between male and female A. ipsilon moth antennae was sexually dimorphic: bipectinate inmale antennae, threadlike in female antennae. The mean length of antennae was about12mm andsimilar between male and female moths. There were at least six types of olfactory sensilla in eachantenna: trichodea sensilla (str), chaetica sensilla (sch), basiconica sensilla (sba), coeloconica sensilla(sco), squamiformia sensilla (ssq) and B hm bristles (bbr). Trichoid sensilla (str) were most numerous on the antennae with2–3dendritic profiles in each sensillum. Basiconica sensilla (sba) have a thincuticular wall and12–25dendritic profiles. The coeloconica sensilla (sco) have4–7neurons in eachsensillum. The chaetica sensilla (sch) had no neurons but a thick cuticular wall.
2. Global transcriptional analysis and chemosensory genes in A. ipsilonantennae
Using Roche454GS-FLX sequencing platform and particular bioinformatics analysis, we haveidentified several olfactory gene families in A. ipsilon antennal transcriptome, the differential expressionprofiles of these olfactory genes are evaluated by RT-PCR and qRT-PCR. The main results aresummarized as follows:
(1) A total of33OBP genes were annotated from the A. ipsilon antennal transcriptome. Among them30have intact ORF with the length from402bp to759bp. The RT-PCR results indicated that22OBPgenes (PBP1, PBP2, PBP3, GOBP1, GOBP2, OBP1, OBP2, OBP4, OBP5, OBP9, OBP11, OBP12,OBP13, OBP15, OBP16, OBP17, OBP19, OBP20, OBP21, OBP22, OBP24and OBP26) areuniquely or mostly expressed in the male and female antennae, this suggested antennae-specific or-enriched expressed OBPs play important roles in sex pheromone detection, suitable host plantsearching and oviposition site location. Interestingly, one OBP (OBP6) was mostly expressed in thepheromone gland (PG), unlike the common antennae-enriched OBPs, this PG-expressed OBP mayplay completely different role in the odorant and pheromone detection and transportation.
(2)12novel CSP genes were identified in the A. ipsilon antennae. The RT-PCR and qRT-PCR resultsindicated that3CSP genes (CSP8, CSP9and CSP10) are highly expressed in the male and femaleantennae, suggesting these three antennae-enriched CSPs may play essential role in the chemicalcommunication process in A. ipsilon. Interestingly, one CSP gene (CSP2) was not expressed in theantennae but specially expressed in the female pheromone gland (PG), suggesting a possibleinvolvement of these proteins in carrying and releasing sex pheromones as demonstrated for theantennal OBPs and CSPs. The insect may use these female PG-enriched CSPs to auto-detect andmonitor the sex pheromones released by themselves.
(3)42OR genes (41typical ORs and the atypical coreceptor) from the A. ipsilon antennaltranscriptome were identified. The RT-PCR and qRT-PCR results indicated that35ORs exclusivelyor mainly expressed in the antennae, among them4ORs (OR1, OR3, OR4and OR14) are maleantennae-specific expression, suggesting they may play essential role in the detection of sexpheromones as pheromone receptors.4ORs (OR6, OR7, OR8and OR23) are femaleantennae-enriched expression, suggesting they may play important roles in the detection of generalodorants such as host plant volatiles.
(4)24IRs including two highly conserved coreceptors IR8a and IR25a were identified from the A.ipsilon antennal transcriptome. RT-PCR and qRT-PCR results indicated that14A. ipsilon IRs (IR8a, IR25a, IR21a, IR41a, IR75q.1, IR75q.2, IR76b, IR87a, IR1, IR3, IR4, IR8, IR12and IR13) arehighly expressed in the antennae, particularly, IR12was specially expressed in the male antennae,suggesting it may be a novel pheromone receptor gene and devoted to response to the female sexpheromone.
3. Sex pheromone recognition and immunolocalization of threepheromone binding proteins in A. ipsilon
The full-length of three pheromone binding protein (PBP) genes were obtained using homologouscloning combined with RACE PCR strategy. We have undertaken systematic studies of the A. ipsilonPBP proteins, the main results are summarized as follows:
(1) The amino acid identity among AipsPBP1–3was about40%. The AipsPBP1and AipsPBP2full-length sequences described here added the missing N-terminal sequences of those reportedpreviously with additional8(MAPHPSVT) and23amino acids(MAASRWCIACLVCVLFAARSVMT), respectively. There are also several amino acid differencescompared to previously reported sequences, which likely represent sequence diversity between theChinese and French strains of A. ipsilon moth.
(2) The expression patterns of AipsPBP1–3transcripts measured by qRT-PCR indicated thatAipsPBP1–3transcripts were highly expressed in antennae than in other tissues but not specific toantennae, low expressions were also detected in the taste organs proboscis and labial palp.AipsPBP1–3expression levels were very low in the heads, thoraxes, abdomens, legs and wings.Furthermore, the transcripts of AipsPBP1–3were about2.2±0.3,4.5±0.4and2.6±0.3times higherin the male antennae than in the female antennae (p<0.01), respectively.
(3) Polyclonal antisera made from each purified AipsPBPs were used for the cellular localization ofAipsPBPs in A. ipsilon antennal sensilla. Our immunocytochemistry results indicated that in maleantennae the trichoid sensilla were strongly labeled by all three anti-PBP antisera, sometimes thegeneral odorant-sensitive basiconic sensilla were also slightly labeled. It was interesting to note thatsome trichoid sensilla were not labeled by any of three anti-PBP antisera. In the female antennae,only a few trichoid and basiconic sensilla were strongly labeled. Other sensilla such as chaetica,coeloconica, squamiformia and B hm bristles were not labeled in both sexes.
(4) Fluorescence competitive binding assay results indicated that the dissociation constants of theAipsPBPs/1-NPN complex were4.4±0.3μM,3.2±0.2μM and2.1±0.2μM for AipsPBP1,AipsPBP2and AipsPBP3, respectively. AipsPBP1had high binding affinities with the two majorsex pheromone components Z7-12:Ac and Z9-14:Ac with the IC50values of0.46±0.03μM and0.84±0.08μM, respectively, but showed a weak binding abilities to the remaining sex pheromoneconponents Z11-16:Ac, Z5-10:Ac and Z8-12:Ac. AipsPBP2had high binding affinities withZ7-12:Ac, Z9-14:Ac as well as Z11-16:Ac with the IC50values of0.56±0.06μM,0.45±0.03μM and0.79±0.05μM, respectively. Interestingly, AipsPBP3only had a high binding affinity toZ11-16:Ac with the IC50values of0.62±0.04μM.
(5) The results obtained by the two phase binding assays were similar to those of the fluorescencecompetitive binding assay. AipsPBP1bound significantly more with Z7-12:Ac and Z9-14:Ac thanother pheromone components. The amount of Z7-12:Ac and Z9-14:Ac bound with each μgAipsPBP1were2.2±0.5ng and2.0±0.5ng, respectively. AipsPBP2bound equally well withZ7-12:Ac, Z9-14:Ac and Z11-16:Ac, with the amount as1.9±0.4ng,2.1±0.5ng and2.0±0.4ng perμg protein, respectively. AipsPBP3bound significantly more Z11-16:Ac with the amount as1.9±0.4ng per μg AipsPBP3protein than any other pheromone components. However, none of AipsPBPswere specific to any one of the pheromone components.
4. Molecular cloning and functional characterization of pheromonereceptors in A. ipsilon
The full-length of atypical olfactory coreceptor Orco and three pheromone receptor PR genes wereobtained using homologous cloning combined with RACE PCR strategy. The differential responses ofpheromone receptors AipsOR1, AipsOR3and AipsOR4to A. ipsilon sex pheromone molecules arecharacterized using heterologous expression system in Xenopus oocytes in vitro and two-electrode,voltage-clamp physiological recordings. The main results are summarized as follows:
(1) The qRT-PCR results indicated that the atypical olfactory coreceptor AipsOrco and three pheromonereceptor gene AipsOR1, AipsOR3and AipsOR4were mainly expressed in the antennae. AipsOrcoshowed similar expression level between male and female antennae (p>0.01) and also showed lowexpression levels in the taste organs proboscis and Labial palp, and also can be detected in thewings and legs. The pheromone receptor gene AipsOR1, AipsOR3and AipsOR4mainly expressedin male antennae, with more than20times higher expressed in the male antennae than in the femaleantennae(p<0.01). The three pheromone receptor genes also showed low expression levels in thetaste organs proboscis and labial palp.
(2) The Xenopus oocytes which expressed different pheromone receptors showed completely differentresponse profiles to A. ipsilon five sex pheromone components. The AipsOR3/Orco specificallytuned to the major pheromone component Z7-12:Ac, the mean inward current responses of Xenopusoocytes with co-expresed AipsOR3/Orco in response to10-4Mol/L was5100nA. The Xenopusoocytes expressing AipsOR3/Orco responsed robustly to the second main sex pheromone comnentZ9-14:Ac, with a mean amplitude as1335nA when response to Z9-14:Ac at the concentration of10-4Mol/L. The Xenopus oocytes expressing AipsOR4/Orco specifically responsed the minor sexpheromone comnent Z5-10:Ac, with a mean amplitude as362nA when response to Z5-10:Ac at theconcentration of10-4Mol/L.
5. Differential expression and immunolocalization of sensory neuronmembrane proteins in the antennae of A. ipsilon moth
Two full-length SNMP transcripts in the A. ipsilon, AipsSNMP1and AipsSNMP2, were obtainedusing homologous cloning combined with RACE PCR strategy. We performed extensive expressionprofiling of AipsSNMP1and AipsSNMP2transcripts among different tissues of both sexes, in theantennae at different developmental stages and in the antennae of both sexes before and after mating byquantitative real-time PCR at the transcript level. The specific subcellular distribution of AipsSNMP1and AipsSNMP2proteins in the various olfactory sensilla was also investigated usingimmunocytochemistry techniques. The possible functions of the SNMPs in moth odorant detection arediscussed. The main results are summarized as follows:
(1) The qRT-PCR results revealed that both AipsSNMP1and AipsSNMP2genes were expressedsignificantly highly in the male and female antennae than in other tissues (more than100-foldhigher than in the abdomen, p<0.05) with insignificant differences between sexes and between twotranscripts in each tissue type. The relative expression levels of AipsSNMP1and AipsSNMP2transcripts in the male antennae were approximately1.5-fold and2.3-fold higher than in the femaleantennae, respectively (p<0.05). The expression levels in the proboscis, and labial palp, head, legsand wings were lower than in the antennae but higher than in the thorax, abdomen and accessoryglands, where no transcripts could be detected. Both AipsSNMP1and AipsSNMP2genes began inthe pupae stage about3days before adult emergence, increased dramatically from one day beforethe emergence, doubled at the day of the emergence and maintained at the high level for4daysduring adult life. On the4th day after the emergence, the expression levels began to decrease to alow level similar to that of one day before the emergence and remained at this low level from day5to day7after the emergence. There were no significant differences in the AipsSNMP1andAipsSNMP2transcript expression levels before and after mating in the4-day-old male or femaleantennae (p>0.05).
(2) The immunocytochemistry results at protein level demonstrated that both AipsSNMP1andAipsSNMP2were expressed in pheromone-sensitive sensilla trichodea but with a completelydifferent expression profile. AipsSNMP1is more uniformed and highly expressed along themembrane of the ORN dendrites, whereas AipsSNMP2is widely distributed at the bottom of thesensilla trichodea and highly localized in the sensillum lymph. Furthermore, the AipsSNMP2protein was also expressed in the general odorant-sensitive sensilla basiconica and sensillacoeloconica. The visual observations showed that a higher number of each type of olfactory sensillawas labeled with the SNMP antisera in male antennae compared with female antennae.
6. Transcriptome analysis of A. ipsilon pheromone gland revealedcandidate genes with putative functions in pheromone production,transport and degradation
Here we report next-generation sequencing of the A. ipsilon pheromone gland transcriptome,identification and expression profiling of genes putatively involved in pheromone production, transportand degradation. The main results are summarized as follows:
(1) Pheromone biosynthesis activating neuropeptide (PBAN) receptor gene. One transcriptUnigene_3821encoding protein highly homologous to PBAN receptor isoform B was identified. Ithad very low abundance in the A. ipsilon transcriptome with the RPKM value as31.
(2) Acetyl-CoA carboxylase (ACC). Two transcripts (Unigene_2338and Unigene_6244) encodingACCs were identified. The RT-PCR and qRT-PCR revealed that both Unigene_2338andUnigene_6244are highly expressed in the pheromone gland as compared to the body. However,they have very low abundance (81and21RPKM) in the transcriptome.
(3) Fatty acid synthase (FAS). We have identified one putative FAS transcript (Unigene_18120) in the A.ipsilon pheromone gland, containing an ORF of7176bp. The RT-PCR and qRT-PCR revealed thatUnigene_18120is highly expressed in the pheromone gland (40-fold higher than in bodies) and alsohas a high abundance (343RPKM) in the PG transcriptome.
(4) Desaturases (DES). In the A. ipsilon pheromone gland transctiptome5transcripts have highhomology to genes encoding desaturases. Unigene_65is encoding an acyl-CoA9-desaturase andUnigene_741encodes an acyl-CoA11desaturase, these two genes possibly involves in the A.ipsilon sex pheromone biosynthesis. RT-PCR and qRT-PCR results indicated that Unigene_741andUnigene_780are highly expressed in the A. ipsilon pheromone gland compared with the body (85and63fold higher, respectively). One of the transcripts (Unigene_780) is also highly abundant(1206RPKM) in the pheromone gland transcriptome, suggesting a possible role in A. ipsilon sexpheromone biosynthesis. The remaining three transcripts Unigene_65, Unigene_10494andUnigene_15401showed similar expressed intensity between the PG and the body part.
(5) Fatty acyl-CoA reductase (FAR). In the A. ipsilon pheromone gland transcriptome there are13transcripts homologous to putative FAR genes. RT-PCR and qRT-PCR results indicated that threetranscripts (Unigene_163, Unigene_4302and Unigene_7344) are highly expressed in thepheromone gland. The other ten transcripts seem equally expressed in the pheromone gland and thebody or highly expressed in the body. All FAR transcripts except two (Unigene_163andUnigene_2537) have low abundance (from81and16RPKM) in the pheromone glandtranscriptome.
(6) Aldehyde reductase (AR). In the A. ipsilon pheromone gland we identified11transcripts encodingaldo-ketoreductase. The RT-PCR and qRT-PCR results indicated that Unigene_3134andUnigene_4806are mainly expressed in the pheromone gland, while the other9putative aldehyde reductase transcripts have equal expression levels between the pheromone gland and the body or ahigher expression level in the body. All aldehyde reductase transcripts are present at low abundance(from67to10RPKM) in the pheromone gland transcriptome.
(7) Acetyltransferase (ATF). In the A. ipsilon pheromone gland transcriptome5acetyltransferasehomologous transcripts were identified. RT-PCR and qRT-PCR revealed that three transcripts(Unigene_407, Unigene_2015and Unigene_173) are mainly expressed in the pheromone gland andhave a relative high abundance of195,155and71RPKM, respectively in the pheromone glandtranscriptome.
(8) Pheromone degrading enzymes (PDEs). We have identified17transcripts predicted to encodeesterases in the A. ipsilon pheromone gland. Our qRT-PCR results revealed that7of the transcripts(AipsCXE3, AipsCXE7, AipsCXE8, AipsCXE9, AipsCXE11, AipsCXE14and AipsCXE20) areantennal-enriched, three (AipsCXE5, AipsCXE10and AipsCXE15) are both antennal-andpheromone gland-enriched and the remaining7(AipsCXE1, AipsCXE2, AipsCXE4, AipsCXE6,AipsCXE12, AipsCXE13and AipsCXE16) have similar expression levels in antennae, body andpheromone gland, suggesting they are not pheromone specific.
(9) OBPs and CSPs. We have identified transcripts of7OBPs and8CSPs from the A. ipsilonpheromone gland. There is one OBP transcript (Unigene_15711) which is highly expressed in thepheromone gland, and3OBPs (Unigene_520, Unigene_2120and Unigene_8860) are highlyexpressed in the antennae. One CSP transcript Unigene_1704seems to be gland-specific and has anextremely high expression level (>100folds) in the pheromone glands compared with the antennaeand bodies and a relative high abundance in the pheromone gland transcriptome. Another CSPtranscript Unigene_721shows a higher expression level in the pheromone gland (10-fold higherthan in bodies) and is extremely abundant with1364RPKM in the pheromone gland transcriptome.The high expression levels of OBPs and CSPs in the pheromone gland is interesting because itsuggests a possible involvement in carrying and releasing sex pheromones.
1.小地老虎触角嗅觉感器类型和超微结构
扫描电镜和透射电镜结果显示:小地老虎的雌雄触角形态呈典型的性二态型:雄蛾触角呈双栉形，雌蛾触角呈丝状。雌雄触角平均长度为12mm。在小地老虎雌雄蛾触角上至少分布着6种不同类型的嗅觉感器，分别为:毛形感器、刺形感器、锥形感器、腔锥形感器、鳞形感器和B hm氏鬃毛。毛形感器的数量最多，并且每个感器有2-3个树突神经元；锥形感器细胞壁比较薄并且每个感器有12-25个树突神经元；腔锥形感器细胞壁有4-7个树突神经元；刺形感器没有神经元,细胞壁比较厚。
2.小地老虎触角转录组测序及嗅觉基因鉴定
采用Roche公司的454GS FLX测序平台，我们对小地老虎雌雄触角进行了转录组测序，通过生物信息学分析我们从小地老虎触角转录组中鉴别了一系列嗅觉识别相关基因，并用半定量RT-PCR和荧光定量qRT-PCR技术研究了这些嗅觉基因的组织表达图谱，主要结果如下:
(1)气味结合蛋白OBP基因33个。其中30个OBP基因具有完整的开放阅读框，长度为402-759bp不等。半定量RT-PCR和荧光定量qRT-PCR结果显示其中包括三个PBP在内的22个OBP基因(PBP1, PBP2, PBP3, GOBP1, GOBP2, OBP1, OBP2, OBP4, OBP5, OBP9, OBP11, OBP12,OBP13, OBP15, OBP16, OBP17, OBP19, OBP20, OBP21, OBP22, OBP24和OBP26)特异性或主要在雌雄触角中表达。表明在触角中特异性表达或高表达的OBP基因在小地老虎识别性信息素、定位寄主植物和寻找合适产卵位点过程中起重要作用。OBP6基因不在雌雄触角中表达，反而在性腺pheromone gland (PG)中特异性表达，与典型的触角表达的OBP基因不同，这个性腺高表达的OBP基因在识别性信息素和普通气味过程中可能起着完全不同的作用。
(2)化学感受蛋白CSP基因12个。半定量RT-PCR和荧光定量qRT-PCR结果显示3个CSP基因(CSP8, CSP9和CSP10)在雌雄触角中表达量最高，说明这三个触角高表达的CSP基因在小地老虎嗅觉识别过程中发挥关键作用。值得注意的是，一个CSP基因(CSP2)不在雌雄触角中表达，反而在性腺PG中特异性表达，暗示性腺中表达的CSP同样能够参与性信息素的结合与运输，并且昆虫能够利用性腺特异性或高表达的CSP蛋白检测并监测雌蛾自己释放的性信息素。
(3)气味受体OR基因42个，其中包括1个非典型嗅觉受体Orco,41个典型的嗅觉受体ORs。半定量RT-PCR和荧光定量qRT-PCR结果显示35个ORs基因特异性或主要在雌雄蛾触角中表达，其中4个ORs基因(OR1, OR3, OR4和OR14)特异性地在雄蛾触角中表达，表明这4个ORs极有可能为识别性信息素的受体PRs，在小地老虎识别性信息素过程中起关键作用。4个ORs基因(OR6, OR7, OR8和OR23)主要在雌蛾触角中表达，表明这几个基因在小地老虎雌蛾定位寄主植物和寻找产卵位点过程中发挥重要作用。
(4)离子型受体IR基因24个，其中包括两个保守的Coreceptors IR8a和IR25a.半定量RT-PCR和荧光定量qRT-PCR结果显示14个IR基因(IR8a, IR25a, IR21a, IR41a, IR75q.1, IR75q.2, IR76b,IR87a, IR1, IR3, IR4, IR8, IR12和IR13)主要在触角中表达，其中IR12基因特异性地在雄蛾触角中表达，表明IR12可能为识别性信息素受体。
3.小地老虎性信息结合蛋白的免疫定位及与性信息素的识别反应
通过同源克隆结合RACE PCR技术克隆了小地老虎性信息素结合蛋白PBP1-3基因。并对其功能进行了系统的研究，主要结果如下：
(1)三个小地老虎PBP1-3蛋白之间的序列相似性为40%。与法国小地老虎种群PBP1和PBP2序列相比，中国种群PBP1在其N端多了8个氨基酸序列，分别为MAPHPSVT.中国种群PBP2蛋白在其N端多了23个氨基酸序列，分别为MAASRWCIACLVCVLFAARSVMT.与此同时，法国种群和中国种群PBP1和PBP2蛋白在其他部位部分氨基酸也发生了变异，可能代表PBP1和PBP2蛋白在法国和中国种群小地老虎中的序列多态性。
(2)采用荧光定量qRT-PCR对三个小地老虎PBP基因在不同组织中的表达谱进行了分析，结果显示小地老虎AipsPBP1-3基因在触角中的表达量明显高于在其他组织中的表达量。同时AipsPBP1-3在味觉器官喙和下唇须中也有少量的表达。AipsPBP1-3在其他部位如头，胸，腹，足和翅中的表达量非常低。AipsPBP1-3在雄蛾触角中的表达量明显高于在雌蛾触角中的表达量，分别为雌蛾触角中表达量的2.2±0.3倍,4.5±0.4倍和2.6±0.3倍(p<0.01)。
(3)利用制备的AipsPBP1-3多克隆抗体，采用免疫组织化学技术系统研究了小地老虎AipsPBP1-3蛋白在不同触角感器中的表达分布情况，结果显示，在雄蛾触角中，胶体金颗粒主要标记在对性信息素敏感的毛形感受器的淋巴液中，有时也标记在对普通气味敏感的锥形感受器的淋巴液中。值得注意的是，有些毛形感器淋巴液并没有被胶体金颗粒标记上。在雌蛾触角中，仅有少数的毛形感器和锥形感器被标记上，说明AipsPBP1-3蛋白在雌蛾触角中的表达量极低。其余感器如刺形感器、腔锥形感器、鳞形感器和B hm氏鬃毛没有被标记上，说明AipsPBP1-3蛋白不在这些感受器中表达。
(4)荧光竞争结合实验结果表明小地老虎PBP1-3与荧光探针1-NPN的结合常数分别为:4.4±0.3μM,3.2±0.2μM和2.1±0.2μM. AipsPBP1蛋白同小地老虎的两种主要性信息素组分Z7-12:Ac和Z9-14:Ac有着很强的结合能力，IC50值分别为0.46±0.03μM和0.84±0.08μM. AipsPBP1同其余三种信息素Z11-16:Ac, Z5-10:Ac和Z8-12:Ac的结合能力较弱。AipsPBP2蛋白也同两种主要性信息素成分Z7-12:Ac和Z9-14:Ac有着很强的结合能力，IC50值分别为0.56±0.06μM和0.45±0.03μM，与此同时AipsPBP2同微量组分Z11-16:Ac也有着很强的结合能力，IC50值为0.79±0.05μM.值得注意的是, AipsPBP3只特异性的和微量组分Z11-16:Ac有很强的结合能力，IC50值为0.62±0.04μM。
(5)双相结合实验(two phase binding assays)得到的结果和荧光竞争结合实验的结果基本吻合。AipsPBP1蛋白结合主要性信息素成分Z7-12:Ac和Z9-14:Ac能力要明显高于其余三种微量组分Z11-16:Ac, Z5-10:Ac和Z8-12:Ac.每微克AipsPBP1能够结合Z7-12:Ac和Z9-14:Ac的量分别为2.2±0.5ng和2.0±0.5ng. AipsPBP2能够同时很好的结合两种主要性信息素组分Z7-12:Ac, Z9-14:Ac，同时也能够很好的结合微量性信息素组分Z11-16:Ac,每微克AipsPBP2蛋白能够结合这三种性信息素的量分别为1.9±0.4ng,2.1±0.5ng和2.0±0.4ng. AipsPBP3结合性信息素组分Z11-16:Ac的能力明显高于其余四种性信息素，每微克AipsPBP3能够结合Z11-16:Ac1.9±0.4ng.3个PBP蛋白中没有任何一个PBP蛋白特异性结合单一性信息素成分。
4.小地老虎性信息素受体的分子克隆及功能分析
通过同源克隆结合RACE PCR技术克隆了小地老虎非典型嗅觉受体Orco和性信息素受体PR基因3个，并利用非洲爪蟾卵母细胞表达系统结合双电极电压钳技术对其在识别性信息素过程中作用深入的研究。主要研究结论如下：
(1)荧光定量qRT-PCR结果显示非典型嗅觉受体AipsOrco和三个性信息素受体AipsOR1, AipsOR3和AipsOR4主要在触角中表达。AipsOrco在雌雄触角中的表达量相当(p>0.01)。AipsOrco同时在味觉器官喙和下唇须中也有少量的表达，在足和翅中也有微量的表达，在头、胸、腹中不表达。三个性信息素受体AipsOR1, AipsOR3和AipsOR4主要在雄蛾触角中表达，其在雄触角中的表达量是在雌触角中表达量的20倍以上(p<0.01)。三个性信息素受体同时在味觉器官喙和下唇须中也有少量表达。
(2)表达小地老虎性信息素受体的爪蟾卵母细胞对小地老虎5种性信息素呈现不同的反应图谱，性信息素受体OR3特异性识别小地老虎主要的性信息素组分Z7-12:Ac,在Z7-12:Ac浓度为10-4Mol/L的刺激下，表达OR3的卵母细胞的电流平均值能达到5100nA，性信息素受体OR1特异性识别小地老虎主要的第二主要性信息素组分Z9-14:Ac,在Z9-14:Ac浓度为10-4Mol/L的刺激下，表达OR1的卵母细胞的电流平均值为1335nA。性信息素受体OR4特异性识别小地老虎次要性信息素组分Z5-10:Ac,在Z59-10:Ac浓度为10-4Mol/L的刺激下，表达OR4的卵母细胞的电流平均值为362nA.
5.小地老虎感觉神经元膜蛋白的表达和免疫定位分析
通过同源克隆结合RACE PCR技术克隆了小地老虎感觉神经元膜蛋白基因SNMP1和SNMP2，利用荧光定量qRT-PCR研究了小地老虎SNMP在成虫不同组织部位、不同发育阶段的触角以及交配前后在触角中的表达情况；利用免疫组织化学方法系统研究小地老虎SNMP在不同类型触角感受器中的表达情况。同时讨论了AipsSNMPs蛋白在昆虫嗅觉识别中的功能，主要研究结论如下：
(1)荧光定量qRT-PCR结果显示AipsSNMP1和AipsSNMP2基因在雌雄蛾触角中的表达量要显著高于其在其他组织中的表达量，是在腹部表达量的100倍以上(p<0.05)。另外，AipsSNMP1和AipsSNMP2在雄蛾触角中的表达量分别是在雌蛾触角中表达量的1.5倍和2.3倍(p<0.05)。AipsSNMP1和AipsSNMP2在喙、下唇须、头、足和翅中的表达量显著低于在触角中的表达量，但是却显著高于在胸、腹部、雄性附腺和雌雄性腺中的表达量。AipsSNMP1和AipsSNMP2在雌雄蛾触角的不同发育阶段呈现相似的表达模式：在羽化之前3天的雌雄蛹中开始表达；然后从羽化前一天表达量开始急剧上升，在羽化当天表达量为羽化前一天表达量的2倍；在羽化后1到4天在雌雄蛾触角中一直保持如此高的的表达水平；在羽化4天之后，AipsSNMP1和AipsSNMP2在雌雄触角中的表达量开始下降，羽化后5到7天的表达水平降至与羽化1天后相当的表达水平。AipsSNMP1和AipsSNMP2基因在4day-old交配前后雌雄蛾触角中的表达水平无显著的变化(p>0.05)。
(2)免疫组织化学结果显示AipsSNMP1和AipsSNMP2都在对性信息素敏感的毛形感受器中表达，但是表达模式却完全不同，AipsSNMP1在毛形感器嗅觉神经元ORNs膜上表达，而AipsSNMP2蛋白却在毛形感器中的淋巴液中表达。同时发现AipsSNMP1也在腔锥形感器神经元ORNs上表达。另外，AipsSNMP2也在锥形感器和腔锥形感器的淋巴液中表达。AipsSNMP1和AipsSNMP2在雄蛾触角标记的感器数量要显著高于在雌蛾触角中标记的感器数量。
6.小地老虎性腺转录组分析及性信息素合成、运输和降解基因的鉴定
利用二代测序技术，对小地老虎的性腺转录组进行了测序，利用生物信息学方法从中筛选鉴定了一系列参与小地老虎性信息素合成、运输、识别和降解的基因，并利用半定量RT-PCR和荧光定量qRT-PCR技术研究了这些基因在性腺和body中的表达情况。主要研究结果如下：
(1)信息素合成激活神经肽(pheromone biosynthesis activating neuropeptide, PBAN)受体基因。小地老虎性腺转录本Unigene_3821编码一个PBAN受体B亚型蛋白。但是其在性腺中的表达量非常的低，RPKM值只有31。
(2)乙酰辅酶A羧化酶。在小地老虎性腺转录组中我们鉴定了2个转录本(Unigene_2338和Unigene_6244)编码不同的乙酰辅酶A羧化酶基因。半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_2338和Unigene_6244在性腺中的表达量要明显高于其在body中的表达量。但是RPKM值分析显示这两个基因在小地老虎性腺中表达量很低，RPKM值分别为81和21.
(3)脂肪酸合成酶基因。从小地老虎性腺中鉴定了1个FAS基因(Unigene_18120)。开放阅读框ORF为7176bp.半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_18120主要在性腺中表达，其在性腺中的表达量为在body中表达量的40倍左右。RPKM值分析发现Unigene_18120在小地老虎性腺中的表达量也很高，RPKM值为343.
(4)去饱和酶基因。在小地老虎性腺转录组中我们鉴定了5个转录本编码去饱和酶基因。Unigene_65编码去饱和酶acyl-CoA9-desaturase，Unigene_741编码去饱和酶acyl-CoA11desaturase，这两个编码去饱和酶的转录本极有可能参与小地老虎性信息素合成过程。半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_741和Unigene_780在性腺中的表达量要显著高于在body中的表达量，分别是body中表达量的85和63倍。RPKM分析显示Unigene_780在性腺转录组中的表达量也很高, RPKM值为1206,推测Unigene_780在小地老虎性信息素生物合成中起关键作用。其余三个Unigene_65、Unigene_10494和Unigene_15401在性腺和body中的表达量相当。
(5)脂肪酰辅酶A还原酶基因。在小地老虎性腺转录组中我们一共鉴定了13个编码脂肪酰辅酶A还原酶的转录本。半定量RT-PCR和荧光定量qRT-PCR结果显示三个转录本(Unigene_163, Unigene_4302和Unigene_7344)主要在性腺中表达。其余10个转录本在性腺和body中的表达量相差不大或在主要在body中表达。RPKM分析显示两个转录本(Unigene_163和Unigene_2537)在性腺转录中的表达量相对较高，其余11个转录本在性腺转录组中的表达量很低，RPKM值在16到81之间。
(6)醛还原酶基因。在小地老虎性腺转录组中我们鉴定了11个转录本编码醛-酮还原酶aldo-ketoreductase。半定量RT-PCR和荧光定量qRT-PCR结果显示Unigene_3134和Unigene_4806主要在性腺中表达，其余9个编码醛还原酶的转录本在性腺和body中的表达量相差不大或主要在body中表达。RPKM分析显示鉴定的11个醛还原酶基因在小地老虎性腺转录组中的表达量非常低，RPKM值在10到67之间。
(7)乙酰基转移酶基因。在小地老虎性腺转录组中我们一共鉴定了5个编码乙酰基转移酶的转录本，半定量RT-PCR和荧光定量qRT-PCR结果显示三个转录本Unigene_407,Unigene_2015和Unigene_173在性腺中的表达量要显著高于在body中的表达量，RPKM分析显示这三个转录本在小地老虎性腺转录组中的相对表达量也比较高，RPKM值分别为195,155和71.
(8)性信息素降解酶基因。在小地老虎性腺转录组我们一共鉴定了17个酯酶基因。荧光定量qRT-PCR结果显示AipsCXE3, AipsCXE7, AipsCXE8, AipsCXE9, AipsCXE11, AipsCXE14和AipsCXE207个酯酶基因在触角中表达量非常高,三个酯酶基因AipsCXE5, AipsCXE10和AipsCXE15同时在触角和性腺中高量表达，剩余的7个酯酶基因AipsCXE1, AipsCXE2,AipsCXE4, AipsCXE6, AipsCXE12, AipsCXE13和AipsCXE16在触角、body、性腺中的表达量差异不显著，说明这7个酯酶基因不是性信息素特异的。
(9)气味结合蛋白OBP和化学感受蛋白CSP基因。我们从小地老虎性腺转录组中鉴定了7个OBP基因和8个CSP基因。荧光定量qRT-PCR结果显示在7个OBP基因中，Unigene_15711编码的OBP基因主要在性腺中表达。三个OBP基因Unigene_520, Unigene_2120和Unigene_8860主要在触角中表达。在8个CSP基因中，Unigene_1704编码的CSP基因特异性地在性腺中表达，其表达量是触角和body中表达量的100倍以上。RPKM分析显示Unigene_1704在小地老虎性腺转录组中的表达量也非常高。另外一个编码CSP基因的Unigene_721也主要在性腺中表达，RPKM分析显示Unigene_721在小地老虎性腺转录组中的表达量非常高，RPKM值为1364.上述在性腺中特异性表达或者高表达的OBP和CSP基因可能参与性信息素的运输与释放过程。
Feeding, courtship and mating are fundamental for insects, and most insects rely on their sensitiveantennae that express specific olfactory proteins to detect survival-and reproductive-related chemicalcues from the environment. Pheromones and plant volatiles diffuse into the sensilla via the multiporesthat penetrate the cuticular surface. When pheromones and plant volatiles enter the sensillum lymph, theantennae-enriched binding proteins (odorant binding proteins (OBPs) and chemosensory proteins(CSPs)) capture these semiochemicals and transport them across the aqueous lumen to themembrane-bound chemosensory receptors (odorant receptors (ORs) and ionotropic receptors (IRs)).Subsequently, the odorant molecules are rapidly degraded by odorant-degrading enzymes (ODEs). Thehigh specificity and sensitivity of insect to sex pheromones make them as effective biological controlagents for population monitoring and mass trapping of noxious insects in integrated pest management(IPM) programs. Elucidating the mechanism of insect olfactory perception can further facilitate thedesign and implementation of novel intervention strategies against these pests. The black cutworm mothAgrotis ipsilon (Hufnagel)(Lepidoptera: Noctuidae) is a destructive pest affecting many cropsworldwide and the male A. ipsilon moth is high attracted by the sex pheromones released by the femalemoth. However, the molecular and cellular mechanisms of how sex pheromones are perceived by maleA. ipsilon moths are still rudimentary. The identification and functional characterization of antennalolfactory proteins in A. ipsilon will enhance our knowledge of the molecular and cellular basis of insectchemoreception. More importantly, can provide us new methods to control this pest through interringtheir olfaction perception. In this study, several kinds of olfactory genes putatively involved in theolfactory reception in A. ipsilon antennae, and several kinds of genes putatively involved in pheromoneproduction, transport and degradation in the A. ipsilon female moth pheromone gland were identifiedthrough next-generation transcriptome sequencing. We have taken systematic functional studies of threekind of key olfactory proteins in the A. ipsilon antennae, including pheromone binding proteins (PBPs),pheromone receptors (PRs) and sensory neuron membrane proteins (SNMPs), we have provideddetailed evidences of PBPs, PRs and SNMPs in A. ipsilon sex pheromone reception at transcriptionallevel, proteins level and cellular level. The main results are as follows:
1. Types and ultrastructures of olfactory sensilla in A. ipsilon antennae
The scanning and transmission electron microscopic examinations revealed that the grossmorphology between male and female A. ipsilon moth antennae was sexually dimorphic: bipectinate inmale antennae, threadlike in female antennae. The mean length of antennae was about12mm andsimilar between male and female moths. There were at least six types of olfactory sensilla in eachantenna: trichodea sensilla (str), chaetica sensilla (sch), basiconica sensilla (sba), coeloconica sensilla(sco), squamiformia sensilla (ssq) and B hm bristles (bbr). Trichoid sensilla (str) were most numerous on the antennae with2–3dendritic profiles in each sensillum. Basiconica sensilla (sba) have a thincuticular wall and12–25dendritic profiles. The coeloconica sensilla (sco) have4–7neurons in eachsensillum. The chaetica sensilla (sch) had no neurons but a thick cuticular wall.
2. Global transcriptional analysis and chemosensory genes in A. ipsilonantennae
Using Roche454GS-FLX sequencing platform and particular bioinformatics analysis, we haveidentified several olfactory gene families in A. ipsilon antennal transcriptome, the differential expressionprofiles of these olfactory genes are evaluated by RT-PCR and qRT-PCR. The main results aresummarized as follows:
(1) A total of33OBP genes were annotated from the A. ipsilon antennal transcriptome. Among them30have intact ORF with the length from402bp to759bp. The RT-PCR results indicated that22OBPgenes (PBP1, PBP2, PBP3, GOBP1, GOBP2, OBP1, OBP2, OBP4, OBP5, OBP9, OBP11, OBP12,OBP13, OBP15, OBP16, OBP17, OBP19, OBP20, OBP21, OBP22, OBP24and OBP26) areuniquely or mostly expressed in the male and female antennae, this suggested antennae-specific or-enriched expressed OBPs play important roles in sex pheromone detection, suitable host plantsearching and oviposition site location. Interestingly, one OBP (OBP6) was mostly expressed in thepheromone gland (PG), unlike the common antennae-enriched OBPs, this PG-expressed OBP mayplay completely different role in the odorant and pheromone detection and transportation.
(2)12novel CSP genes were identified in the A. ipsilon antennae. The RT-PCR and qRT-PCR resultsindicated that3CSP genes (CSP8, CSP9and CSP10) are highly expressed in the male and femaleantennae, suggesting these three antennae-enriched CSPs may play essential role in the chemicalcommunication process in A. ipsilon. Interestingly, one CSP gene (CSP2) was not expressed in theantennae but specially expressed in the female pheromone gland (PG), suggesting a possibleinvolvement of these proteins in carrying and releasing sex pheromones as demonstrated for theantennal OBPs and CSPs. The insect may use these female PG-enriched CSPs to auto-detect andmonitor the sex pheromones released by themselves.
(3)42OR genes (41typical ORs and the atypical coreceptor) from the A. ipsilon antennaltranscriptome were identified. The RT-PCR and qRT-PCR results indicated that35ORs exclusivelyor mainly expressed in the antennae, among them4ORs (OR1, OR3, OR4and OR14) are maleantennae-specific expression, suggesting they may play essential role in the detection of sexpheromones as pheromone receptors.4ORs (OR6, OR7, OR8and OR23) are femaleantennae-enriched expression, suggesting they may play important roles in the detection of generalodorants such as host plant volatiles.
(4)24IRs including two highly conserved coreceptors IR8a and IR25a were identified from the A.ipsilon antennal transcriptome. RT-PCR and qRT-PCR results indicated that14A. ipsilon IRs (IR8a, IR25a, IR21a, IR41a, IR75q.1, IR75q.2, IR76b, IR87a, IR1, IR3, IR4, IR8, IR12and IR13) arehighly expressed in the antennae, particularly, IR12was specially expressed in the male antennae,suggesting it may be a novel pheromone receptor gene and devoted to response to the female sexpheromone.
3. Sex pheromone recognition and immunolocalization of threepheromone binding proteins in A. ipsilon
The full-length of three pheromone binding protein (PBP) genes were obtained using homologouscloning combined with RACE PCR strategy. We have undertaken systematic studies of the A. ipsilonPBP proteins, the main results are summarized as follows:
(1) The amino acid identity among AipsPBP1–3was about40%. The AipsPBP1and AipsPBP2full-length sequences described here added the missing N-terminal sequences of those reportedpreviously with additional8(MAPHPSVT) and23amino acids(MAASRWCIACLVCVLFAARSVMT), respectively. There are also several amino acid differencescompared to previously reported sequences, which likely represent sequence diversity between theChinese and French strains of A. ipsilon moth.
(2) The expression patterns of AipsPBP1–3transcripts measured by qRT-PCR indicated thatAipsPBP1–3transcripts were highly expressed in antennae than in other tissues but not specific toantennae, low expressions were also detected in the taste organs proboscis and labial palp.AipsPBP1–3expression levels were very low in the heads, thoraxes, abdomens, legs and wings.Furthermore, the transcripts of AipsPBP1–3were about2.2±0.3,4.5±0.4and2.6±0.3times higherin the male antennae than in the female antennae (p<0.01), respectively.
(3) Polyclonal antisera made from each purified AipsPBPs were used for the cellular localization ofAipsPBPs in A. ipsilon antennal sensilla. Our immunocytochemistry results indicated that in maleantennae the trichoid sensilla were strongly labeled by all three anti-PBP antisera, sometimes thegeneral odorant-sensitive basiconic sensilla were also slightly labeled. It was interesting to note thatsome trichoid sensilla were not labeled by any of three anti-PBP antisera. In the female antennae,only a few trichoid and basiconic sensilla were strongly labeled. Other sensilla such as chaetica,coeloconica, squamiformia and B hm bristles were not labeled in both sexes.
(4) Fluorescence competitive binding assay results indicated that the dissociation constants of theAipsPBPs/1-NPN complex were4.4±0.3μM,3.2±0.2μM and2.1±0.2μM for AipsPBP1,AipsPBP2and AipsPBP3, respectively. AipsPBP1had high binding affinities with the two majorsex pheromone components Z7-12:Ac and Z9-14:Ac with the IC50values of0.46±0.03μM and0.84±0.08μM, respectively, but showed a weak binding abilities to the remaining sex pheromoneconponents Z11-16:Ac, Z5-10:Ac and Z8-12:Ac. AipsPBP2had high binding affinities withZ7-12:Ac, Z9-14:Ac as well as Z11-16:Ac with the IC50values of0.56±0.06μM,0.45±0.03μM and0.79±0.05μM, respectively. Interestingly, AipsPBP3only had a high binding affinity toZ11-16:Ac with the IC50values of0.62±0.04μM.
(5) The results obtained by the two phase binding assays were similar to those of the fluorescencecompetitive binding assay. AipsPBP1bound significantly more with Z7-12:Ac and Z9-14:Ac thanother pheromone components. The amount of Z7-12:Ac and Z9-14:Ac bound with each μgAipsPBP1were2.2±0.5ng and2.0±0.5ng, respectively. AipsPBP2bound equally well withZ7-12:Ac, Z9-14:Ac and Z11-16:Ac, with the amount as1.9±0.4ng,2.1±0.5ng and2.0±0.4ng perμg protein, respectively. AipsPBP3bound significantly more Z11-16:Ac with the amount as1.9±0.4ng per μg AipsPBP3protein than any other pheromone components. However, none of AipsPBPswere specific to any one of the pheromone components.
4. Molecular cloning and functional characterization of pheromonereceptors in A. ipsilon
The full-length of atypical olfactory coreceptor Orco and three pheromone receptor PR genes wereobtained using homologous cloning combined with RACE PCR strategy. The differential responses ofpheromone receptors AipsOR1, AipsOR3and AipsOR4to A. ipsilon sex pheromone molecules arecharacterized using heterologous expression system in Xenopus oocytes in vitro and two-electrode,voltage-clamp physiological recordings. The main results are summarized as follows:
(1) The qRT-PCR results indicated that the atypical olfactory coreceptor AipsOrco and three pheromonereceptor gene AipsOR1, AipsOR3and AipsOR4were mainly expressed in the antennae. AipsOrcoshowed similar expression level between male and female antennae (p>0.01) and also showed lowexpression levels in the taste organs proboscis and Labial palp, and also can be detected in thewings and legs. The pheromone receptor gene AipsOR1, AipsOR3and AipsOR4mainly expressedin male antennae, with more than20times higher expressed in the male antennae than in the femaleantennae(p<0.01). The three pheromone receptor genes also showed low expression levels in thetaste organs proboscis and labial palp.
(2) The Xenopus oocytes which expressed different pheromone receptors showed completely differentresponse profiles to A. ipsilon five sex pheromone components. The AipsOR3/Orco specificallytuned to the major pheromone component Z7-12:Ac, the mean inward current responses of Xenopusoocytes with co-expresed AipsOR3/Orco in response to10-4Mol/L was5100nA. The Xenopusoocytes expressing AipsOR3/Orco responsed robustly to the second main sex pheromone comnentZ9-14:Ac, with a mean amplitude as1335nA when response to Z9-14:Ac at the concentration of10-4Mol/L. The Xenopus oocytes expressing AipsOR4/Orco specifically responsed the minor sexpheromone comnent Z5-10:Ac, with a mean amplitude as362nA when response to Z5-10:Ac at theconcentration of10-4Mol/L.
5. Differential expression and immunolocalization of sensory neuronmembrane proteins in the antennae of A. ipsilon moth
Two full-length SNMP transcripts in the A. ipsilon, AipsSNMP1and AipsSNMP2, were obtainedusing homologous cloning combined with RACE PCR strategy. We performed extensive expressionprofiling of AipsSNMP1and AipsSNMP2transcripts among different tissues of both sexes, in theantennae at different developmental stages and in the antennae of both sexes before and after mating byquantitative real-time PCR at the transcript level. The specific subcellular distribution of AipsSNMP1and AipsSNMP2proteins in the various olfactory sensilla was also investigated usingimmunocytochemistry techniques. The possible functions of the SNMPs in moth odorant detection arediscussed. The main results are summarized as follows:
(1) The qRT-PCR results revealed that both AipsSNMP1and AipsSNMP2genes were expressedsignificantly highly in the male and female antennae than in other tissues (more than100-foldhigher than in the abdomen, p<0.05) with insignificant differences between sexes and between twotranscripts in each tissue type. The relative expression levels of AipsSNMP1and AipsSNMP2transcripts in the male antennae were approximately1.5-fold and2.3-fold higher than in the femaleantennae, respectively (p<0.05). The expression levels in the proboscis, and labial palp, head, legsand wings were lower than in the antennae but higher than in the thorax, abdomen and accessoryglands, where no transcripts could be detected. Both AipsSNMP1and AipsSNMP2genes began inthe pupae stage about3days before adult emergence, increased dramatically from one day beforethe emergence, doubled at the day of the emergence and maintained at the high level for4daysduring adult life. On the4th day after the emergence, the expression levels began to decrease to alow level similar to that of one day before the emergence and remained at this low level from day5to day7after the emergence. There were no significant differences in the AipsSNMP1andAipsSNMP2transcript expression levels before and after mating in the4-day-old male or femaleantennae (p>0.05).
(2) The immunocytochemistry results at protein level demonstrated that both AipsSNMP1andAipsSNMP2were expressed in pheromone-sensitive sensilla trichodea but with a completelydifferent expression profile. AipsSNMP1is more uniformed and highly expressed along themembrane of the ORN dendrites, whereas AipsSNMP2is widely distributed at the bottom of thesensilla trichodea and highly localized in the sensillum lymph. Furthermore, the AipsSNMP2protein was also expressed in the general odorant-sensitive sensilla basiconica and sensillacoeloconica. The visual observations showed that a higher number of each type of olfactory sensillawas labeled with the SNMP antisera in male antennae compared with female antennae.
6. Transcriptome analysis of A. ipsilon pheromone gland revealedcandidate genes with putative functions in pheromone production,transport and degradation
Here we report next-generation sequencing of the A. ipsilon pheromone gland transcriptome,identification and expression profiling of genes putatively involved in pheromone production, transportand degradation. The main results are summarized as follows:
(1) Pheromone biosynthesis activating neuropeptide (PBAN) receptor gene. One transcriptUnigene_3821encoding protein highly homologous to PBAN receptor isoform B was identified. Ithad very low abundance in the A. ipsilon transcriptome with the RPKM value as31.
(2) Acetyl-CoA carboxylase (ACC). Two transcripts (Unigene_2338and Unigene_6244) encodingACCs were identified. The RT-PCR and qRT-PCR revealed that both Unigene_2338andUnigene_6244are highly expressed in the pheromone gland as compared to the body. However,they have very low abundance (81and21RPKM) in the transcriptome.
(3) Fatty acid synthase (FAS). We have identified one putative FAS transcript (Unigene_18120) in the A.ipsilon pheromone gland, containing an ORF of7176bp. The RT-PCR and qRT-PCR revealed thatUnigene_18120is highly expressed in the pheromone gland (40-fold higher than in bodies) and alsohas a high abundance (343RPKM) in the PG transcriptome.
(4) Desaturases (DES). In the A. ipsilon pheromone gland transctiptome5transcripts have highhomology to genes encoding desaturases. Unigene_65is encoding an acyl-CoA9-desaturase andUnigene_741encodes an acyl-CoA11desaturase, these two genes possibly involves in the A.ipsilon sex pheromone biosynthesis. RT-PCR and qRT-PCR results indicated that Unigene_741andUnigene_780are highly expressed in the A. ipsilon pheromone gland compared with the body (85and63fold higher, respectively). One of the transcripts (Unigene_780) is also highly abundant(1206RPKM) in the pheromone gland transcriptome, suggesting a possible role in A. ipsilon sexpheromone biosynthesis. The remaining three transcripts Unigene_65, Unigene_10494andUnigene_15401showed similar expressed intensity between the PG and the body part.
(5) Fatty acyl-CoA reductase (FAR). In the A. ipsilon pheromone gland transcriptome there are13transcripts homologous to putative FAR genes. RT-PCR and qRT-PCR results indicated that threetranscripts (Unigene_163, Unigene_4302and Unigene_7344) are highly expressed in thepheromone gland. The other ten transcripts seem equally expressed in the pheromone gland and thebody or highly expressed in the body. All FAR transcripts except two (Unigene_163andUnigene_2537) have low abundance (from81and16RPKM) in the pheromone glandtranscriptome.
(6) Aldehyde reductase (AR). In the A. ipsilon pheromone gland we identified11transcripts encodingaldo-ketoreductase. The RT-PCR and qRT-PCR results indicated that Unigene_3134andUnigene_4806are mainly expressed in the pheromone gland, while the other9putative aldehyde reductase transcripts have equal expression levels between the pheromone gland and the body or ahigher expression level in the body. All aldehyde reductase transcripts are present at low abundance(from67to10RPKM) in the pheromone gland transcriptome.
(7) Acetyltransferase (ATF). In the A. ipsilon pheromone gland transcriptome5acetyltransferasehomologous transcripts were identified. RT-PCR and qRT-PCR revealed that three transcripts(Unigene_407, Unigene_2015and Unigene_173) are mainly expressed in the pheromone gland andhave a relative high abundance of195,155and71RPKM, respectively in the pheromone glandtranscriptome.
(8) Pheromone degrading enzymes (PDEs). We have identified17transcripts predicted to encodeesterases in the A. ipsilon pheromone gland. Our qRT-PCR results revealed that7of the transcripts(AipsCXE3, AipsCXE7, AipsCXE8, AipsCXE9, AipsCXE11, AipsCXE14and AipsCXE20) areantennal-enriched, three (AipsCXE5, AipsCXE10and AipsCXE15) are both antennal-andpheromone gland-enriched and the remaining7(AipsCXE1, AipsCXE2, AipsCXE4, AipsCXE6,AipsCXE12, AipsCXE13and AipsCXE16) have similar expression levels in antennae, body andpheromone gland, suggesting they are not pheromone specific.
(9) OBPs and CSPs. We have identified transcripts of7OBPs and8CSPs from the A. ipsilonpheromone gland. There is one OBP transcript (Unigene_15711) which is highly expressed in thepheromone gland, and3OBPs (Unigene_520, Unigene_2120and Unigene_8860) are highlyexpressed in the antennae. One CSP transcript Unigene_1704seems to be gland-specific and has anextremely high expression level (>100folds) in the pheromone glands compared with the antennaeand bodies and a relative high abundance in the pheromone gland transcriptome. Another CSPtranscript Unigene_721shows a higher expression level in the pheromone gland (10-fold higherthan in bodies) and is extremely abundant with1364RPKM in the pheromone gland transcriptome.The high expression levels of OBPs and CSPs in the pheromone gland is interesting because itsuggests a possible involvement in carrying and releasing sex pheromones.
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