意大利蝗卵发育过程保幼激素和蜕皮激素含量的变化及其代谢相关基因的表达谱

【目的】鉴于保幼激素(juvenile hormone, JH)与蜕皮激素(ecdysone, Ecd)在昆虫生长发育中的重要作用,本研究旨在明确这两种激素及其代谢相关基因对意大利蝗Calliptamus italicus卵发育过程的调控机制。【方法】采用ELISA技术检测意大利蝗卵发育过程中JH与Ecd含量的变化;采用qRT-PCR技术检测JH与Ecd代谢通路的重要基因(JHE,JHEH,DIB和EcR)在意大利蝗卵发育过程的表达模式。【结果】意大利蝗卵滞育阶段(阶段Ⅳ-Ⅵ)的JH含量显著高于早期发育阶段(阶段Ⅰ-Ⅲ)的,滞育后发育阶段(阶段Ⅶ-Ⅸ) JH含量显著下降;Ecd含量于滞育早期(阶段Ⅳ)显著上升,而后显著下降,滞育后发育阶段再次上升。JHE的表达量在意大利蝗卵早期发育阶段和滞育后发育阶段均呈先升后降的趋势,滞育阶段JHE的表达量较低;JHEH表达量在意大利蝗卵早期发育阶段也先升后降,滞育阶段变化不显著,滞育后发育阶段显著升高;DIB表达量在意大利蝗卵滞育阶段高于早期发育阶段的,在滞育后发育阶段下降;EcR表达量在意大利蝗卵早期发育阶段及滞育阶段均无显著变化,滞育后发育阶段...     

型变过程中亚洲小车蝗体内保幼激素滴度及其代谢相关基因表达量的变化

【目的】本研究旨在明确亚洲小车蝗Oedaleus asiaticus在不同发育阶段和型变过程中的保幼激素(juvenile hormone, JH) JHⅢ滴度及其代谢相关基因表达量的变化情况，为阐明保幼激素调控亚洲小车蝗型变中的生理功能奠定基础。【方法】采用高效液相色谱法测定散居型和群居型亚洲小车蝗不同发育阶段(4和5龄若虫及1, 4, 7, 10, 13和20日龄成虫)和3龄若虫群居化处理1, 3, 5和7 d时亚洲小车蝗血淋巴中的保幼激素JHⅢ的滴度变化；用qRT-PCR检测在群居化处理亚洲小车蝗3龄若虫1, 3, 5和7 d时保幼激素代谢相关的3种基因保幼激素酯酶(juvenile hormone esterase, JHE)基因、保幼激素甲基转移酶(juvenile hormone methyltransferase, JHAMT)基因和保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)基因的表达量；利用JHⅢ浸液处理散居型亚洲小车蝗3龄若虫后进行群居化处理，测定亚洲小车蝗型变率。【结果】JHⅢ在亚洲小车蝗血淋巴中的滴度甚微...     

麦红吸浆虫3-羟基-3-甲基戊二酰辅酶A还原酶基因SmHMGR的克隆及在滞育与发育变态过程中的表达动态

【目的】3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是保幼激素(JH)合成途径的限速酶。麦红吸浆虫Sitodiplosis mosellana是一种典型的专性幼虫滞育昆虫。本研究旨在探讨HMGR基因在麦红吸浆虫滞育和发育变态过程中的作用。【方法】通过RT-PCR和RACE技术克隆麦红吸浆虫滞育前幼虫HMGR基因全长cDNA序列;利用生物信息学软件分析HMGR基因核苷酸和其编码的蛋白氨基酸序列特性;采用qPCR技术测定其在麦红吸浆虫滞育不同时期3龄幼虫及不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹和后蛹以及雌雄成虫)中的mRNA表达水平。【结果】克隆获得一条麦红吸浆虫HMGR基因全长cDNA序列,命名为SmHMGR(GenBank登录号: MG876766)。该基因全长2 548 bp,其中开放阅读框长2 328 bp,编码775个氨基酸,预测的蛋白分子量为84.16 kD,理论等电点为8.29。序列分析发现该基因编码的蛋白具有HMGR蛋白家族典型的HMG-CoA-reductase-classⅠ催化功能域及其他保守功能基序;序列比对和系统发育分析表明,SmHMGR与达氏按蚊Anopheles darling等长角亚目(Nematocera)昆虫HMGR的相似性最高、亲缘关系最近。SmHMGR在麦红吸浆虫滞育前的3龄早期幼虫中表达量显著升高,进入滞育后一直维持较高水平,并在滞育后静息阶段的当年12月至翌年1月达到最高。SmHMGR在蛹期表达量低于幼虫期,预蛹期表达量最低;在雌成虫中表达量显著高于在蛹和雄成虫中的表达量。【结论】SmHMGR的表达与麦红吸浆虫发育密切相关,可能在滞育诱导、维持及滞育后静息状态的维持及生殖中发挥作用,其表达量的降低可能参与了幼虫到蛹的变态。     

棉铃虫保幼激素环氧水解酶基因的克隆及其原核表达

通过兼并引物PCR结合RACE技术,克隆了棉铃虫Helicoverpa armigera(Hubner)保幼激素环氧水解酶(juven-ile hormone epoxide hydrolase,JHEH)的基因,该基因的开放阅读框为1389 bp,编码463个氨基酸.预测蛋白分子量为52 kD,等电点为6.39.N末端存在由20个氨基酸组成的疏水性信号肽序列,存在组成JHEH催化三联体的氨基酸Asp(227)、Glu(403)和His(430)以及组成阴氧离子洞的氨基酸Tyr(298)、Tyr(373)和HGWP花样结构.其氨基酸序列与其它鳞翅目昆虫有很高的同源性.在大肠杆菌中表达JHEH基因的编码区,Western blot结果表明,棉铃虫JHEH已被成功表达.利用该基因序列可以在分子水平上研究棉铃虫JHEH基因的时空表达情况,进一步研究保幼激素(juvenile hormone,JH)的代谢途径及其功能.     

九香虫保幼激素环氧水解酶基因克隆、生物信息学及表达分析

[目的]保幼激素环氧水解酶(Juvenile hormone epoxide hydrolase,JHEH)是昆虫体内保幼激素(Juvenile hormone,JH)的主要降解酶.本文旨在分析九香虫Aspongopus chinensis Dallas保幼激素环氧水解酶基因序列(AcJHEH)信息,探索其在九香虫生长发育过程中的作用.[方法]以九香虫成虫的cDNA为模板,采用RT-PCR技术克隆获得AcJHEH基因序列,并利用生物信息学软件对其编码蛋白的理化特性、结构特征和系统进化进行分析;采用qRT-PCR技术检测并分析九香虫不同龄期和不同组织中AcJHEH的相对表达量.[结果]成功获得了AcJHEH的完整开放阅读框ORF序列,全长均为1 363 bp,共编码453个氨基酸,预测分子量为51.74 ku,等电点(pI)为7.66,分子蛋白式C2414H3683N581O653S14,含有N-端跨膜基序XWG、催化三联体、保守基序HGWP和2个酪氨酸,属于环氧水解酶家族.系统进化树分析表明,AcJHEH与茶翅蝽Halyomorpha halys的JHEH聚为一支,再与同为半翅目的臭虫Cimex lectulariu和绿盲蝽Apolygus lucorum的JHEH聚为一类.荧光定量PCR结果显示,AcJHEH在九香虫不同发育阶段和各组织中均有表达,且脂肪中的表达量极显著高于其它组织(P＜0.001);滞育九香虫成虫表达量最高,其次为4-5龄若虫都极显著高于其它发育阶段(P＜0.001).[结论]九香虫JHEH属于环氧水解酶家族,确定为保幼激素环氧水解酶.依据qPCR结果推测AcJHEH通过改变九香虫体内保幼激素浓度来影响九香虫4-5龄若虫蜕皮发育、成虫生殖系统发育和滞育等.     

抑咽侧体素基因在大猿叶虫生殖滞育准备中的功能分析

【目的】本研究旨在明确大猿叶虫Colaphellus bowringi促咽侧体素(allatotropin, AT)和抑咽侧体素(allatostatin, AST)基因的分子特征,分析其在该虫生殖和滞育过程中的表达差异,并探究其在大猿叶虫生殖滞育准备中的功能。【方法】利用前期建立的大猿叶虫转录组数据库,鉴定大猿叶虫CbAT和CbASTs基因序列并克隆其开放阅读框(ORFs),并对其序列进行生物信息学分析;通过qRT-PCR技术检测CbAT和CbASTs在大猿叶虫注定滞育和注定非滞育雌蛹和雌成虫头部的表达模式;对注定滞育大猿叶虫2日龄雌蛹进行CbAST-B, CbAST-C以及CbAST-B+CbAST-C RNAi试验后,检测4日龄雌成虫保幼激素(juvenile hormone, JH)信号基因以及卵黄原蛋白基因的表达量变化。【结果】鉴定并克隆了大猿叶虫的一个促咽侧体素基因CbAT(GenBank登录号: MT977128)及两个抑咽侧体素基因CbAST-B(GenBank登录号: MT977126)和CbAST-C(GenBank登录号: MT977127),其开放阅读框分别长408, 600和303 bp。通过氨基酸序列比对和系统进化分析发现,CbAT, CbAST-B和CbAST-C均与鞘翅目昆虫的同源蛋白聚在一支,在进化上表现出较高保守性。qRT-PCR检测发现,CbAT基因在注定滞育与注定非滞育2日龄雌蛹到4日龄雌成虫头部中始终无表达差异;而CbAST-B和CbAST-C基因分别自雌成虫1和2日龄开始在注定滞育雌成虫头部显著高表达,且在注定滞育和注定非滞育个体中的差异表达状态一直持续到滞育准备期结束。在注定滞育2日龄雌蛹中沉默CbAST-B, CbAST-C以及同时沉默CbAST-B和CbAST-C后,检测4日龄雌成虫头部的干扰效率发现,CbAST-B和CbAST-C的表达均受到显著抑制,干扰效率达70%以上。在去除触角的头部中,JH合成途径基因AACT, FPPS和JHAMT以及脂肪体中JH应答基因Kr-h1和JHE1的表达在dsCbAST-B, dsCbAST-C和dsCbAST-B+dsCbAST-C处理组中均被显著上调,脂肪体中卵黄原蛋白基因Vg1和Vg2的表达也被显著上调。【结论】CbAT基因可能并非造成大猿叶虫产卵前期与滞育准备期保幼激素信号产生差异的主要原因,而在生殖滞育准备期CbAST-B和CbAST-C抑制了JH的合成,进而抑制Vgs的表达以促进滞育的发生。本研究进一步揭示了昆虫生殖滞育准备期保幼激素信号的上游调控机制,有助于进一步理解昆虫对环境的季节性适应策略,为挖掘害虫防治的新靶标提供了新的指导和借鉴。     

miR-252-5p通过靶向调控保幼激素酸甲基转移酶基因JHAMT的表达而影响果蝇变态发育

【目的】保幼激素酸甲基转移酶(juvenile hormone acid methyl transferase, JHAMT)是保幼激素(juvenile hormone, JH)合成通路中的关键限速酶。本研究旨在筛选并验证靶向调控黑腹果蝇Drosophila melanogaster JHAMT转录表达的miRNA,揭示miRNA在JH生物合成中的作用机理。【方法】首先通过miRanda, TargetScan和microT-CDS在线网站对靶向黑腹果蝇JHAMT的miRNA进行预测,取3个网站均能预测到的miRNA作为候选靶向JHAMT的miRNA;利用双荧光素酶系统对候选miRNA与JHAMT的靶向关系进行验证;qRT-PCR检测候选miRNA与JHAMT在黑腹果蝇生长发育中的表达模式;利用qRT-PCR和果蝇GAL4-UAS系统分别检测黑腹果蝇咽侧体中超表达miRNA对JHAMT的表达以及对黑腹果蝇变态发育的影响。【结果】 miRanda, TargetScan和microT-CDS分别预测到5, 18和16个靶向JHAMT的miRNA,共同预测到4个miRNA,分别是miR-252-5p, miR-277-3p, miR-1002-5p和miR-987-5p。双荧光素酶检测结果表明,miR-252-5p mimics可显著降低野生型JHAMT 3′UTR荧光素酶报告基因载体所表达的荧光素酶活性,而JHAMT 3′UTR区中miR-252-5p结合位点突变后,该抑制作用被解除。qRT-PCR检测结果表明,miR-252-5p与JHAMT在黑腹果蝇卵、幼虫及预蛹期的转录表达模式相反。咽侧体中超表达miR-252后,可显著降低JHAMT和JH初级反应基因Kr-h1的表达水平;且表现出类似JH缺失的表型,如化蛹时间推迟、体重变轻以及蛹期死亡增加。【结论】miR-252-5p可通过靶向作用于JHAMT参与JH生物合成调控,从而影响果蝇变态发育。     

七星瓢虫雌成虫卵巢发育不同阶段体内保幼激素的含量

本文以大蜡螟Galleria mellonella(L.)为生测昆虫,应用蜡测定法(Wax test)测定了取食天然食物及人工饲料的七星瓢虫雌成虫在不同卵巢发育阶段体内保幼激素(JH)的含量。取食蚜虫者羽化后8日左右(卵巢管内出现第三卵母细胞,卵黄大量沉积即将排卵),雅虫JH含量达到高峰(593.3G.U.),但一经开始排卵(羽化后约10日左右)保幼激素的含量猛然下降。直至持续产卵达10次的雌虫(羽化后约25日)JH含量仍保持在比较平稳的状态。取食人工饲料者卵巢发育缓慢,羽化30日后不产卵个体的JH水平仍很低,仅及取食蚜虫组高峰的1/6。这一结果证实了JH对七星瓢虫雌成虫卵巢发育及排卵的调控,也指出了取食人工饲料的雌虫产卵率及产卵量明显偏低的重要原因。 在应用蜡测法的过程中肯定了JH浓度与大蜡螟蛾中胸背板保持的部分蛹皮面积之部的关系。     

外源保幼激素对中华通草蛉滞育解除及滞育后发育的影响

【目的】本研究旨在明确外源保幼激素(juvenile hormone, JH)在中华通草蛉Chrysoperla sinica滞育快速解除过程中的使用剂量及最佳使用时期。【方法】测定点滴不同剂量(0, 5, 15, 25和35 μg/成虫)外源JH后中华通草蛉滞育成虫的产卵前期、产卵历期、雌虫寿命及单雌产卵量,以及15 μg/成虫外源JH处理对不同日龄(0, 5, 10, 20, 30和40日龄)滞育成虫处理后这4个指标的变化。【结果】在不同点滴剂量处理中,15和25 μg/成虫外源JH处理的中华通草蛉滞育成虫的产卵前期分别为6.82和6.29 d,显著短于点滴丙酮的对照组(10.55 d),且15 μg/成虫外源JH处理组产卵历期、雌虫寿命及单雌产卵量均最大,均显著大于对照组。不同日龄的中华通草蛉滞育成虫点滴15 μg/成虫外源JH,0, 5, 10和20日龄处理的成虫产卵前期均显著短于未经外源JH处理的对照组。10, 20和30日龄处理的成虫产卵历期和雌虫寿命均与未经外源JH处理的对照组无显著差异,但10, 20和30日龄处理的成虫生殖力显著降低,而20日龄处理时对照组和外源JH处理组均有较高的生殖力。【结论】综合考虑产卵前期、产卵历期、产卵量和雌虫寿命4个指标,15 μg/成虫的外源JH剂量是中华通草蛉快速解除滞育的最佳剂量;最佳使用时期为滞育20 d的成虫,此时对中华通草蛉成虫滞育解除后的生殖能力影响最小。本研究解决了中华通草蛉滞育解除耗费时间长的瓶颈问题,为成虫滞育天敌的高效存储利用与滞育的快速解除提供了新思路。     

保幼激素类似物ZR-515对杜仲梦尼夜蛾幼虫生长发育的影响

【目的】为了解保幼激素对杜仲梦尼夜蛾Orthosia songi Chen et Zhang幼虫生长发育的影响。【方法】本研究采用不同浓度的保幼激素类似物ZR-515处理其4龄、5龄和6龄幼虫,测定了幼虫的发育历期、取食量、排粪量以及后期的化蛹率和羽化率。【结果】(1)保幼激素类似物ZR-515处理可延长杜仲梦尼夜蛾幼虫的发育历期,增加体重,提高取食量和排粪量;但是对后期的化蛹率和成虫羽化率影响较大,导致幼虫和蛹的死亡率较高,且处理浓度越大,影响越显著。(2)处理4龄或5龄幼虫,对其后的各龄期的生长发育产生很大的影响。(3)综合考虑激素处理对幼虫生长产生的有利影响及后期的不利影响,认为1 000 ng/μL为最适处理浓度,5龄为最佳处理龄期。【结论】研究结果可为后续杜仲梦尼夜蛾的规模化人工饲养和开发利用提供理论依据。     

麦红吸浆虫滞育发生和解除过程中总脂和甘油三酯含量变化

【目的】脂类与昆虫滞育密切相关。本研究旨在明确麦红吸浆虫Sitodiplosis mosellana (Géhin)滞育过程中脂类物质含量的变化规律,探讨麦红吸浆虫滞育与脂类物质变化的关系。【方法】采用香兰素硫酸显色法测定了2008年5月－2010年1月不同时间陕西杨凌养虫圃内麦红吸浆虫滞育前、滞育期及滞育解除后幼虫和蛹体内总脂和甘油三酯含量。【结果】 滞育前幼虫总脂和甘油三酯含量分别为378.12和291.67 μg/mg,显著高于整个滞育期（P<0.05）。滞育年周期中,冬季总脂和甘油三酯含量最高,翌年1月结茧幼虫二者含量分别为335.29 和275.72 μg/mg,显著高于其他季节（P<0.05）;整个滞育期间,裸露幼虫和结茧幼虫总脂和甘油三酯随季节变化趋势相同,但同期结茧幼虫含量高于裸露幼虫;滞育当年与第2年同期幼虫总脂和甘油三酯含量差异不显著（P>0.05）。滞育解除后,总脂和甘油三酯含量随着幼虫的发育和变态逐渐降低,其中中蛹和后蛹显著低于活动幼虫（P<0.05）。【结论】麦红吸浆虫滞育不同时期幼虫及蛹总脂和甘油三酯含量存在明显差异,其滞育与脂类物质的含量密切相关。     

Juvenile hormone esterase activity in the pupating and diapausing larvae of Sesamia nonagrioides

The development of the Mediterranean corn borer, Sesamia nonagrioides, under long-day (LD) photoperiod is associated with juvenile hormone (JH) decline and pupation in the 5th or 6th larval instar. The larvae grown under short-day (SD) conditions maintain a moderate JH titer and enter diapause during which they undergo several extra larval molts. Both types of larvae exhibit similar levels of juvenile hormone esterase (JHE) activity that increases in each instar during the period of low ecdysteroid titer and drops when the titer rises to a molt-inducing peak. A suppression of JHE activity within 24h after application of an ecdysteroid agonist suggests that the drop of activity is a rapid and possibly direct response to ecdysteroids or their agonist. Esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) suppressed more than 98% of the JHE activity without affecting pupation timing and adult development. The data indicate that JHE is not crucial for the switch between larval development, diapause, and metamorphosis in S. nonagrioides.     

Cloning,partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.     

Expression pattern of enzymes related to juvenile hormone metabolism in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis> L.

The physiological balance of juvenile hormone (JH) in insects depends on its biosynthesis and degradation pathway. Three key enzymes namely, juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK) are required for degradation in insects. Our present results showed that JHE and JHEH exhibited expression in almost all the tissues. This indicated that JHE and JHEH might degrade JH simultaneously. In addition, the highest levels of JHDK were observed in the midgut, with trace level being found in the malpighian tubule and haemocytes. Since the midgut is a digestive organ and not a JH target, it was hypothesized that both JHE and JHEH hydrolyzed JH to JH diol (JHd) which was then transported to midgut and hydrolyzed further by JHDK, to be finally excreted out of the body. Also the expression studies on JH degradation enzymes in different tissues and stages indicated that the activities of the three enzymes are specific and coincident with the JH functions in silkworm, Bombyx mori L.     

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.     

The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Trichoplusia ni

Brain (median or lateral regions) or suboesophageal ganglion (SOG) homogenates of Day 1 fifth instar larvae of Trichoplusia ni induced the appearance of haemolymph juvenile hormone esterase (JHE) when injected into Day 1, Day 2 or early Day 4 fifth instar ligated hosts. Brain and SOG homogenates of late fourth instars also induced JHE when injected into Day 1 hosts, whole late fifth instar and pupal tissue did not. The pattern of JHE induction by early fourth through Day 3 fifth instar brain and SOG homogenates correlated with natural haemolymph JHE activity occurring at these times. Implantation of late fourth and Day 1 fifth instar brains and/or SOG into similar age hosts similarly induced JHE activity while prothoracic and abdominal ganglia did not. The relative levels of induction following implantation were SOG<brain<brain+SOG. JHE activity which appears in the haemolymph following injection of brain homogenates appears to be largely due to a single enzyme which has an isoelectric point indistinguishable from that of the natural haemolymph enzyme. Evidence is presented which suggests that inhibitory as well as stimulatory brain factors are involved in JHE regulation.