Expression of juvenile hormone acid O‐methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone (JH), a sesquiterpenoid synthetized by the insect corpora allata (CA), plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O‐methyltransferase (JHAMT). Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica (L.) (BgJHAMT). BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3′‐UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi‐triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body vitellogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin (TOR). Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH‐specific branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.     

沙棘木蠹蛾(鳞翅目：木蠹蛾科)性信息素组分鉴定及其生物活性

报道了沙棘木蠹蛾性信息素的结构及组分,并报道了不同组分：顺-3-十四碳烯醇;反-3-十四碳烯醇;顺-3-十四碳乙酸酯;反-3-十四碳乙酸酯;顺-7-十四碳烯醇;顺-7-十四碳乙酸酯;顺-9-十四碳乙酸酯的室内触角电位反应和林间诱捕试验。林间诱蛾结果表明2组分和5组分的诱蛾活性高于其它组合。     

沙蒿木蠹蛾生物学特性研究

沙蒿木蠹蛾Holcocerus artemisiae Chou et Hua是危害油蒿Artemisia ordosica Krasch和籽蒿A. sphaerocephala Krasch的重要钻蛀性害虫, 以幼虫蛀食根部, 导致寄主植物长势衰弱甚至死亡。为有效控制其危害, 我们对该虫形态和生物学特征结合野外调查和室内饲养观察进行了研究。结果表明： 在宁夏, 该虫2年发生1代, 以幼虫在被害油蒿根部越冬; 老熟幼虫于5月中旬从受害根部钻出, 在周围的沙土里结茧化蛹, 蛹期平均19.5±3.5 d; 成虫始见于6月初, 终见于8月末, 期间经历3次羽化高峰, 分别为6, 7, 8月的上旬。成虫羽化主要在14:00-17:00, 整个过程持续30~45 min; 羽化当日即可交配, 交配时间为20:00-21:00, 高峰期为20:30左右; 雄虫有2次交尾现象。雄虫寿命2~3 d, 雌虫寿命1~3 d, 极少4 d。雌雄性比约为1∶2.16。卵初见于6月中旬, 初孵幼虫初见于6月下旬。幼虫常单独危害, 且具有转移危害的习性, 蛀食坑道不规则。5-9月, 油蒿受害率为11%~44%, 平均24%, 株虫口密度为0.13头/株。幼虫对油蒿的树龄和地径有较明显的选择性, 主要危害1~4年生、 地径16~34 mm的油蒿。沙蒿木蠹蛾生物学特征的系统研究为其控制措施的制定提供了科学依据。     

Spatial distribution of Holcocerus hippophaecolus (Lepidopetera: Cossidae) pupae in a seabuckthorn (Hippophae rhamnoides) stand.

The seabuckthorn carpenter moth,Holcocerus hippophaecolus,which has a generation time of four years,is recently becoming one of the major pests of the seabuckthorn (Hippophae rhamnoides) in Inner Mongolia,Liaoning,Shanxi,Ningxia and Shaanxi of China (Hua et al.,1990).The larvae of the H.hippophaecolus mainly damage the stems and roots of the seabuckthorn,and the mature larvae pupate in the soil.The spatial distribution of the pupae was analyzed by using biostatistics and geostatistics in order to effectively control the insect and further study the spatial distribution of the population.Results show that most of the pupae (90%) had an eclosion time span from early June to the end of July.The sex ratio of the pupae was nearly 1:1 in the woodland samples.In addition,24.3% of the 971 trees investigated had pupae and it ranged from 0 to 4 per tree within a distance of 1.3 m from the base of the stem.90% of the pupae were aggregated within a distance of 1 m from the base of the stem.The pupae show intense spatial aggregation in the sampled woodland which had an 11.1 m spatial dependence and a 90.7% intensity in the local spatial continuity.Moreover,the population presented an intensive spotted distribution and many aggregated spots were found in the woodlands.As for the relationship between grid size and variogram of the pupae,the variations in the range,the intensity of local spatial continuity and the sill were all very low or non-existent when the grid size was 5 m,6 m or 7 m.Whereas,the value of the decisive coefficient was the biggest when the grid size was 5 m making it the ideal grid size.     

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.     

Mating in Heliothis virescens: Transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone

Studies were undertaken to determine whether adult males of Heliothis virescens transfer juvenile hormone (JH) to females during copulation, and an in vitro radiochemical assay was used to determine whether mating causes an allatotropic effect, i.e., stimulation of JH biosynthesis by corpora allata (CA). In vitro, CA from 3-day-old mated females synthesized and released approximately 2.5 times total JH as that of CA from comparably aged virgin females. Of the homologues, JH II exhibited significant increase in mated females; JH I also increased but not significantly. JH III remained similar to that of virgin females. This is the first demonstration of an allatotropic effect of mating in moths. In contrast to the female, CA of virgin males did not produce any JH, but accessory sex glands (ASG) in 3-day-old males synthesized small amounts of JH. Immediately after adult emergence, male ASG contained approximately 1.5 ng JH I and II, which increased by 12 h after emergence and remained at this high level up to 54 h after emergence. JH III was barely detected in ASG. JH in ASG of mated male immediately after uncoupling was depleted almost completely, and 24 h later recovered to levels comparable to that of 54-h-old virgin male. Virgin female bursa copulatrix did not contain any JH, but mated female bursa, immediately after uncoupling, had JH at levels comparable to that observed in virgin male ASG. By 6 h after uncoupling, JH levels decreased dramatically in mated female bursa. These data suggest the transfer of JH to females by the male. Arch. Insect Biochem. Physiol. 38:100–107, 1998. © 1998 Wiley-Liss, Inc.     

Spatial distribution of Holcocerus hippophaecolus (Lepidoptera: Cossidae) pupae in a seabuckthorn (Hippophae rhamnoides) stand

The seabuckthorn carpenter moth, Holcocerus hippophaecolus, which has a generation time of four years, is recently becoming one of the major pests of the seabuckthorn (Hippophae rhamnoides) in Inner Mongolia, Liaoning, Shanxi, Ningxia and Shaanxi of China (Hua et al., 1990). The larvae of the H. hippophaecolus mainly damage the stems and roots of the seabuckthorn, and the mature larvae pupate in the soil. The spatial distribution of the pupae was analyzed by using biostatistics and geostatistics in order to effectively control the insect and further study the spatial distribution of the population. Results show that most of the pupae (90%) had an eclosion time span from early June to the end of July. The sex ratio of the pupae was nearly 1:1 in the woodland samples. In addition, 24.3% of the 971 trees investigated had pupae and it ranged from 0 to 4 per tree within a distance of 1.3 m from the base of the stem. 90% of the pupae were aggregated within a distance of 1 m from the base of the stem. The pupae show intense spatial aggregation in the sampled woodland which had an 11.1 m spatial dependence and a 90.7% intensity in the local spatial continuity. Moreover, the population presented an intensive spotted distribution and many aggregated spots were found in the woodlands. As for the relationship between grid size and variogram of the pupae, the variations in the range, the intensity of local spatial continuity and the sill were all very low or non-existent when the grid size was 5 m, 6 m or 7 m. Whereas, the value of the decisive coefficient was the biggest when the grid size was 5 m making it the ideal grid size.     

cDNA CLONING AND SEQUENCE DETERMINATION OF THE PHEROMONE BIOSYNTHESIS ACTIVATING NEUROPEPTIDE FROM THE SEABUCKTHORN CARPENTERWORM,Holcocerus hippophaecolus (LEPIDOPTERA: COSSIDAE)

The PBAN (pheromone biosynthesis activating neuropeptide)/pyrokinin peptides comprise a major neuropeptide family characterized by a common FXPRL amide at the C‐terminus. These peptides are actively involved in many essential endocrine functions. For the first time, we reported the cDNA cloning and sequence determination of the PBAN from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full‐length cDNA of Hh‐DH‐PBAN contained five peptides: diapause hormone (DH) homolog, α‐neuropeptide (NP), β‐NP, PBAN, and γ‐NP. All of the peptides were amidated at their C‐terminus and shared a conserved motif, FXPR (or K) L. Moreover, Hh‐DH‐PBAN had high homology to the other members of the PBAN peptide family: 56% with Manduca sexta, 66% with Bombyx mori, 77% with Helicoverpa zea, and 47% with Plutella xylostella. Phylogenetic analysis revealed that Hh‐DH‐PBAN was closely related to PBANs from Noctuidae, demonstrated by the relatively higher similarity compared with H. zea. In addition, real‐time quantitative PCR (qRT‐PCR) analysis showed that Hh‐DH‐PBAN mRNA expression peaked in the brain–subesophageal ganglion (Br–SOG) complex, and was also detected at high levels during larval and adult stages. The expression decreased significantly after pupation. These results provided information concerning molecular structure characteristics of Hh‐DH‐PBAN, whose expression profile suggested that the Hh‐DH‐PBAN gene might be correlated with larval development and sex pheromone biosynthesis in females of the H. hippophaecolus.     

飞行对粘虫体内甘油酯积累与咽侧体活性的影响

研究了粘虫Mythimna separata (Walker)成虫飞行对甘油酯的恢复、保幼激素的滴度及飞行肌降解的影响, 结果表明,不同日龄粘虫的飞行活动对其能源物质的积累及保幼激素分泌产生不同的影响。1日龄蛾的飞行对粘虫这两方面产生的影响最大,其飞行个体能源物质的积累明显高于未飞行的对照个体; 3日龄飞行个体的能源物质积累与对照相当; 但5日龄飞行个体则很难达到对照水平。1日龄飞行个体咽侧体活性在36 h后明显高于对照,60 h后已是对照的10倍,108 h达到其峰值; 3日龄飞行个体咽侧体的活性稍高于对照,但差别不显著; 5日龄飞行个体的咽侧体活性则稍低于对照。不同日龄飞行对飞行肌的降解也产生不同的影响。1日龄飞行个体的飞行肌在飞行后6 天已经明显低于对照。3、5日龄的飞行活动对其飞行肌降解的影响不明显。因此推测,粘虫咽侧体活化的关键时期可能在羽化后1～3 天之间。     

Changes in the size of corpora allata,in the juvenile hormone III titer in the hemolymph,and in the protein content of terminal oocytes throughout the first gonadotropic cycle in Aiolopus thalassinus (Insecta: Orthoptera: Acrididae)

In Aiolopus thalassinus (Fabr.), the changes in the volume of the corpora allata (CA), in the concentration of juvenile hormone III (JH III) in the hemolymph, and in protein content in the terminal (t) oocytes were studied during the first gonadotropic cycle. These parameters could be better related to the volume of t-oocytes than to age after emergence. The JH III titer curve was maximum (2.9 pmol/10 μl) at an oocyte volume of 1.2 mm³. Before oviposition (days 10–16) the JH III titer decreased to 0.65 pmol/10 μl hemolyph. The increase in JH III titer reflected a period of high protein storage in the t-oocytes. The largest volume of the CA was reached at the beginning of yolk storage in the t-oocytes. The highest JH III titer did not correspond with the largest volume of CA, which occurred much earlier. © 1993 Wiley-Liss, Inc.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: Regulation at the terminal step in juvenile hormone biosynthesis

Changes in activity of the corpora allata (CA) during larval-pupal-adult development of the tobacco hornworm Manduca sexta were studied by transplantation assays, measurements of in vitro juvenile hormone (JH) and JH acid synthesis, and determination of JH acid methyltransferase (JHAMT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities. The data from these assays demonstrate that the CA cease to secrete JH by day 4 of the last larval instar (wandering stage). With regard to JH synthesis, they remain inactive throughout the prepupal, pupal, and most of the pharate adult periods. CA of females, but not of males, resume JH synthesis shortly before eclosion. The biochemical basis of the inactivation process is the loss of JHAMT activity. However, prepupal CA produce JH acids, as shown by enzyme and in vitro assays. Pupal and pharate adult CA do not synthesize JH acids although levels of HMG-CoA reductase activity seem to remain relatively high. Radiolabeled JH was recovered from hemolymph of allatectomized prepupae that had been injected with radiolabeled JH acid. These results provide further evidence that certain peripheral tissues (eg, imaginal discs) convert JH acid secreted by the prepupal CA to JH and, thus, that JH acid is a prohormone in the prepupal period. The CA change from hormone secretion to prohormone secretion during larval-prepupal transformation, a unique functional alteration in an endocrine gland.     

Treatment of larvae with repeated doses of precocene II induces precocious metamorphic changes in Spodoptera mauritia (Lepidoptera: Noctuidae)

In Spodoptera mauritia repeated daily treatments of larvae with 40 μg of precocene II throughout the fifth and sixth instar larval period had no effect on the larval-larval period but prolonged larval-pupal ecdysis. The resulting pupae showed precocious adult differentiation of mouth parts, wings, eyes, legs, and fat body.     

榆木蠹蛾幼虫龄数的确定

为弄清榆木蠹蛾Holcocerus vicarius Walker幼虫的发育情况及预测其发生时间, 通过测量榆木蠹蛾幼虫的头壳宽、 体长、 体宽、 前胸背板宽、 上颚长和上颚宽, 运用Crosby生长法则和线性回归方法分析来找出判定幼虫龄数的最佳形态指标, 推断其幼虫的龄数。结果表明： 各龄幼虫头壳宽平均值的变异系数和Crosby指数最小, 其他5项指标的变异系数和Crosby指数较大, 头壳宽为最佳分龄指标。根据头壳宽将榆木蠹蛾幼虫分为20龄, 不同龄幼虫头壳宽值符合Dyar定律提出的幼虫头壳宽增长规律, 头壳宽和龄数的回归方程为y=0.233+1.686x+0.127x²-0.005x³ （R²=0.996）。榆木蠹蛾幼虫龄数的确定为研究其发生规律、 生物学习性及进行综合防治提供依据。     

沙柳木蠹蛾性行为及其性信息素滴度的动态节律

沙柳木蠹蛾Holcocerus arenicola是为害沙柳Salix psammophila的主要钻蛀性害虫之一。在进入暗期后的6 h内,对其求偶行为和交配行为进行观察和记录,调查该虫求偶和交配的活动规律。通过触角电位技术（EAG）和毛细管气相色谱（GC）对性腺体内信息素的滴度进行了分析,以揭示性信息素产生的昼夜节律和雌虫日龄对性信息素合成的影响。结果表明：该虫在暗期0.5~1 h内即开始有求偶行为,最大求偶高峰出现在羽化后的第2晚。交配行为主要发生在暗期的1~2 h内。在各日龄的成虫中,1日龄成虫的交配百分率最高。交配平均时间为24.16±2.64 min,随着日龄的增加,交配时间前移。在实验中,未观察到5~6日龄成虫的交配行为。沙柳木蠹蛾性信息素的体内合成早于求偶行为1~2 h,并在暗期的前2 h内达到峰值。性信息素的滴度随着雌虫日龄的增减而减少,最高值为当日羽化雌虫的腺体提取物。林间诱蛾实验中,处女雌蛾和性腺体提取物对雄蛾均有较好的诱捕效果。本研究表明,在沙柳木蠹蛾的性信息素滴度和性活动之间具有同步关系,同时为进一步利用长距离性信息素防治该虫提供了参考。     

Rates of juvenile hormone synthesis control caste differentiation in the stingless bee Scaptotrigona postica depilis

Summary In social insects the expression of caste-specific characters is controlled by juvenile hormone (JH) during definite sensitive periods in preimaginal development. For a number of stingless bee species the existence of such a JH-sensitive period has already been demonstrated. Queen development can be induced by topical JH applications during the cocoon spinning phase of the last larval instar. Neither JH titers nor rates of JH synthesis were known so far for this subfamily of eusocial bees distinguished by a pronounced caste dimorphism. As the pantropically distributed stingless bees with approximately 400 recent species are the largest group of social bees, JH synthesis was studied in one of the species that can be kept under laboratory conditions. An in vitro radiochemical assay was used to measure stage- and caste-specific activities of the corpora allata (CA). For the first time in a eusocial hymenopteran species it was demonstrated how the endocrine system is reacting to trophogenic stimuli capable to induce caste differentiation during larval development. Generally JH synthesis in queen CA was found to be 30–80% higher than in workers during the penultimate and last larval instar, but a strong and distinct caste-specific modulation of JH synthesis was only observed right before the onset of a JH-sensitive period in the cocoon spinning phase of the fifth instar.     

榆木蠹蛾性信息素通讯系统的超微结构观察

为了解榆木蠹蛾Holcocerus vicarius （Walker）雌蛾性信息素分泌腺（性信息素释放系统）位置、 表面形态和超微结构及雄蛾触角感受器（性信息素接收系统）的种类、 形态、 分布及功能, 利用扫描电镜和透射电镜对榆木蠹蛾雌蛾性信息素分泌腺和雄蛾的触角进行观察。结果表明： 榆木蠹蛾雌蛾性信息素分泌腺位于腹尖末端第8～9节节间膜上的背面中央区域, 腺体表面分布着许多饱满的锥状突起, 2日龄处女雌蛾腺体细胞间有明显的胞连接, 细胞基底膜基褶较多, 质膜上分布着微绒毛, 并与内表皮连接, 内表皮上含有多层几丁质, 胞质中含有脂质粒、 大量空泡、 光面内质网、 粗面内质网及线粒体; 雄蛾触角鞭节上有5种感受器, 为毛形感器、 刺形感器、 锥形感器、 腔锥形感器和曲毛形感器, 其中毛形感器数量最多, 曲毛形感器最少。柄节和梗节被大量鳞片覆盖, 未观察到感器。榆木蠹蛾性信息素通讯系统的研究为榆木蠹蛾性信息素的生物合成、 性信息素的提取、 鉴定及成虫生殖交配生物学行为提供了依据。     

Development of physogastry in the queen of the fungus-growing termite Macrotermes michaelseni (Isoptera: Macrotermitinae)

Female reproductives of the fungus-growing termite Macrotermes michaelseni never showed signs of physogastry if they were kept in petri dishes to establish an incipient colony. During the 200 days of observation, their corpora allata volume, juvenile hormone titre and number of active ovarioles remained on a more or less constant level, and their gut was filled with soil. In contrast, these parameters are enhanced in partially and especially in fully physogastric queens, and these females had only a transparent liquid in the gut.Application of the juvenile hormone mimic ZR 515 to young females accelerated yolk incorporation, increased the number of active ovarioles and brought about the release of the soil from the gut. The same changes were induced by adding workers to increase the population of incipient colonies from about 45 to 400. This also resulted in an increase of the corpus allatum volume and of the juvenile hormone titre in young females. Their intestine was in this case filled with transparent liquid associated with the increased population of workers. It was, therefore, concluded that the development of physogastry depends on a positive feedback: the more workers are with the young female, the more nutrients are available and the more juvenile hormone the female produces, the more eggs are laid and the more workers can again develop to nurture the queen.     

Allatotropic and nervous control of corpora allata in the adult male loreyi leafworm, Mythimna loreyi (Lepidoptera: Noctuidae)

Allatotropic activity is found in methanolic extracts of the brain–suboesophageal ganglion (SOG)–corpora cardiaca (CC) complex from virgin males of Mythimna loreyi Duponchel (Lepidoptera, Noctuidae). Corpora allata (CA) from 6‐day‐old virgin males exhibit low rates of release of Juvenile Hormone (JH) acid (JHA) in vitro. Release of JHA can be activated by the addition of an extract of brain–SOG–CC complex in a dose‐dependent manner, and this allatotropic activation can be sustained consistently in the continuous presence of such extracts. Based on its trypsin sensitivity and heat stability, the allatotropic factor is most likely a peptide. The allatotropic activity is dependent on the concentration of calcium ions in the medium, with the highest activation achieved beyond 2 m m . The results of nerve transection experiments suggest that both nervi corporis allati I (NCA I) and NCA II are involved in mediating the allatotropic control of CA in vitro. Isolated CA alone show significantly higher rates of release of JHA than the intact brain–SOG–CC–CA complex during the first 3 h of incubation, but the release of JHA reaches almost the same range in both groups by the end of the fourth hour of incubation.