cDNA cloning and expression analysis of the juvenile hormone acid methyltransferase from seabuckthorn carpenterworm,Holcocerus hippophaecolus (Lepidoptera: Cossidae)

In this study, we report the cDNA cloning and sequence determination of Hh‐JHAMT from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full‐length cDNA of putative Hh‐JHAMT was 1659 bp and contained a highly conserved Motif I, SAM motif I, which showed that Hh‐JHAMT like enzyme was a member of SAM‐dependent MTases. Moreover, putative Hh‐JHAMT had high homology to the other members of the JHAMT peptide family: 59% with Spodoptera litura, 54% with Bombyx mori and 54% with Helicoverpa armigera. Multiple alignments and phylogenetic analysis revealed that Hh‐JHAMT was closely related to JHAMT from Lepidoptera. Real‐time quantitative PCR experiments showed that Hh‐JHAMT mRNA expression was highest in the corpora allata (CA) complex, and was also detected at high levels during earlier larval and adult stages. The JHAMT mRNA level gradually declined during larval development, and the lowest amount of expression was observed in the pupal stage, while it increased to a higher level during adult stages. The pattern of Hh‐JHAMT expression was similar to the mode of JH biosynthesis. These results provided information concerning molecular characteristics of Hh‐JHAMT, whose expression profile suggests that the Hh‐JHAMT gene might be changed with larval development, metamorphosis and adult reproduction of the H. hippophaecolus.     

Reproductive biology of the German cockroach,Blattella germanica: Juvenile hormone as a pleiotropic master regulator

Juvenile hormone (JH) exerts major pleiotropic effects on cockroach development and reproduction. The production of JH by the corpora allata (CA) in the adult female German cockroach, Blattella germanica, is dependent upon and modulated by both internal and environmental stimuli. Mating, intake of high-quality food, social interactions, and the presence of vitellogenic ovaries facilitate JH synthesis. Conversely, starvation, deficient diets, enforced virginity, isolation, and a pre- or post-vitellogenic ovary cause the CA to produce less JH. Sensory stimulation of the genital vestibulum by the ootheca also inhibits the CA via signals that ascend the ventral nerve cord. All these stimulatory and inhibitory signals are integrated by the brain, and a preponderance of favorable signals results in a graded lifting of brain inhibition, permitting the synthesis and release of JH. The effects of inhibitory signals on JH biosynthesis can be lifted experimentally by severing nervous connections between the brain and the CA. Such an operation accelerates activation of the CA. Besides controlling gonadal maturation in females, JH concurrently regulates the production of sexual signals, including both attractant- and courtship-eliciting pheromones, and the behavioral expression of calling (pheromone release) and sexual receptivity. Although JH is required for the expression of copulatory readiness in female B. germanica, it appears that signals associated with copulation (spermatophore, sperm, accessory secretions) can inhibit this behavioral state even when titers of JH are permissive for receptivity. These observations suggest that JH might regulate sexual receptivity in females indirectly through other directives. In males, JH accelerates not only the onset of sexual readiness but also synthesis of accessory reproductive products. Lastly, we present a novel cockroach control strategy that is based on the intimate association between food intake and rising JH titers in B. germanica females. JH analogs cause abortion of fertile oothecae in gravid females. In turn, rising JH titers and vitellogenic oocytes induce feeding in females. With strategic placement of insecticidal baits and JH analogs, gravid females, which normally feed little and are difficult to control, can thus be effectively targeted for elimination. Arch. Insect Biochem. Physiol. 35:405–426, 1997. © 1997 Wiley-Liss, Inc.     

Endocrine regulation of female contact sex pheromone production in the German cockroach, Blattella germanica

ABSTRACT The amount of the major component of the cuticular sex pheromone, 3, 11-dimethyl-2-nonacosanone, on individual female German cockroaches, Blattella germanica (L.), as a function of age was determined by gas-liquid-chromatographic analysis. Accumulation of phermone increased with age in both virgin and mated females. During the first gono-trophic cycle, the pheromone accumulated most rapidly when oocyte growth rates were maximal (days 5–10), and least rapidly while the female carried an ootheca (days 11–32). Pheromone accumulation was similar in virgin and mated females when the same physiological stages (determined by oocyte maturation) were considered. Inhibition of Juvenile Hormone release, through allatectomy, chemicals (precocene or fluoromevalonate), or through mechanical egg case implants, suppressed or delayed pheromone production and oocyte growth. The Juvenile Hormone analogue ZR512 induced allatectomized or head-ligated females and females with chemically or mechanically inhibited corpora allata to produce pheromone and enlarge their basal oocytes. Finally, ZR512 applied to intact females stimulated pheromone production in a dose-dependent manner.     

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.     

Juvenile Hormone inhibition in corpora allata from ovariectomized Blattella germanica

Abstract. Ovariectomy has been used to study the role of the ovary in endocrine homeostasis. Our studies on young virgin adults of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) show that the cytological development of the corpora allata (CA) in ovariectomized females proceeds as in intact specimens, whereas the rates of Juvenile Hormone (JH) synthesis are lower. Stimulation of the CA from ovariectomized females in vitro by mevalonolactone suggests that enzymatic mechanisms which follow mevalonate formation in the biosynthetic pathway are functional. The synthetic capabilities of these CA are also illustrated by the kinetics of JH production in vitro , because hormonal release increases with time to reach 'normal' levels after 8h of incubation. Our data suggest that the absence of ovaries leads to effective inhibition of JH biosynthesis rather than to an impairment of the developmental process in the CA cells.     

Effects of ovariectomy and mating on the activity of the corpora allata in adult female Blattella germanica (L.) (Dictyoptera: Blattellidae)

In adult female cockroaches, the ovary greatly affects the synthesis of Juvenile Hormone (JH) by the corpora allata, and in females of some cockroach species, removal of the ovaries results in a permanent depression of JH synthesis. We report that the corpora allata in ovariectomised, adult virgins of the German cockroach, Blattella germanica (L.), increase and then decrease in activity, as they do in intact females. Moreover, the distal tubules in the left colleterial glands of ovariectomised females accumulate abundant protein, the production of which is regulated by JH. In both ovariectomised and sham‐operated females, the activity of the corpora allata more than tripled between days 1 and 4 of adulthood, during which the oöcytes of sham‐operated females grew considerably in length. The corpora allata of sham‐operated females produced even more JH on day 7, but very little on day 10, by which time all females had oviposited. The glands of ovariectomised females, by constrast, produced a similar amount of JH on day 7 as on day 4, but much less on day 10. Beginning on day 13, the activity of the corpora allata increased again in ovariectomised females, an increase that did not occur until day 22 in sham‐operated females. Mating of ovariectomised females on day 6 resulted in a significant increase in the activity of the corpora allata by day 10. We conclude that both the ovary and mating stimulate the synthesis of JH early in the reproductive cycle, but that neither is needed for the occurrence of a complete cycle of JH synthesis.     

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.     

德国小蠊抗药性机理

德国小蠊(Blattella germanica)是一种重要的室内卫生害虫,化学防治是目前主要的防治手段,但抗药性已逐渐成为制约德国小蠊防治的关键因素。德国小蠊抗药性的产生存在多种机理,本文从行为抗性、表皮穿透性降低、代谢抗性、靶标抗性及微生物降解等方面对德国小蠊抗药性产生机理进行了综述,重点阐述了近年来德国小蠊抗药性机理最新的研究进展。     

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。     

The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata

Following unilateral allatectomy in the viviparous cockroach, Diploptera punctata, the remaining corpus allatum (CA) can synthesize juvenile hormone (JH) at rates equal to that of a pair of control CA. A single CA undergoes changes in volume and cell number similar in magnitude to those occurring during the normal cycle of JH synthesis. There is no hyptertrophy of the CA associated with compensation in JH synthesis. Therefore, the rate of JH synthesis per cell or per unit volume of single CA following unilateral allatectomy is twice that of a single control CA.     

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.     

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.     

Putative‐farnesoic acid O‐methyltransferase (FAMeT) in medfly reproduction

A gene potentially involved in juvenile hormone (JH) biosynthesis was previously identified in Ceratitis capitata as the putative‐farnesoic acid O‐methyltransferase (FAMeT). Since JH is involved in insect reproduction, we silenced the putative‐FAMeT expression by RNA interference in Ceratitis capitata to evaluate its implication in egg production. FAMeT gene expression was knocked down in females and males after eclosion and in 1‐ and 2‐day‐old females. Treated specimens were left to mate with each other or with untreated partners to evaluate the extent of each sex influencing egg production. Gene silencing was investigated by Real‐Time PCR. Results unambiguously showed that FAMeT has a measurable role on the fertility of both medfly sexes. © 2010 Wiley Periodicals, Inc.     

德国小蠊泛素基因的克隆及序列分析

设计一对简并引物,从德国小蠊Blattellager manica细胞中克隆了泛素基因的编码区,GenBank登录号为AY501003。序列分析表明,该编码区的长度为228 bp,编码的多肽由76个氨基酸残基组成,相对分子质量为8.47 Kd ,其等电点为5.73。同源性比较发现,德国小蠊泛素基因与其他真核生物泛素基因在氨基酸水平上具有94%以上的相似性。     

Synergism of chlorpyrifos against the German cockroach, Blattella germanica

Of fifteen compounds tested as synergists for chlorpyrifos against susceptible and resistant strains of Blattella germanica, the German cockroach, eleven were active against the resistant strain but only seven were synergistic against the susceptible strain. Overall, the most effective synergist was S,S,S-tributyl phosphorotrithioate (DEF) followed by phenyl saligenin cyclic phosphonate (PSCP) and two substituted N,N-dimethylcarbamates: SK-102 and SK-37. The most effective synergist for overcoming chlorpyrifos resistance was SK-37 which reduced the resistance ratio by 3.2-fold. Four other synergists which reduced chlorpyrifos resistance, in order of their effectiveness, were: SK-102 > PSCP > DEF > tridiphane. The potential usefulness of these synergists for German cockroach control is discussed.     

S6 protein kinase activates Juvenile Hormone and vitellogenin production in the cockroach Blattella germanica

Nutritional status regulates different processes, such as growth and development, through TOR (target of rapamycin) and insulin receptor signalling pathways. The ribosomal S6 protein kinase (S6K) is a downstream element of both pathways. Using cDNA from the German cockroach Blattella germanica (L.), two S6K isoform sequences (BgS6K) are cloned. The long isoform differs from the short one by the insertion of a 22‐amino acid duplication, involving the key phosphorylation position Thr³⁹⁰. As a result of this, the long isoform presents a new, potentially regulatory phosphorylation site. RNA interference knockdown of both BgS6K isoforms induces an increase in the length of the last nymphal instar, together with a reduction in the mRNA levels of a number of enzymes of the Juvenile Hormone biosynthetic pathway in the corpora allata, vitellogenin mRNA levels in the fat body and basal oocyte length. Thus, BgS6K is important for nymphal development and is necessary for the full induction of Juvenile Hormone synthesis and vitellogenin production in adult females.     

Precocenes as pro-allatocidins in adult female Diploptera punctata: A functional and ultrastructural study

Adult mated females of the viviparous cockroach Diploptera punctata are moderately sensitive to precocenes. Oöcyte growth is inhibited and oviposition is delayed in insects topically treated with precocene II or precocene III. C₁₆ juvenile hormone release by corpora allata of precocene-treated insects is markedly inhibited when compared to corpora allata of acetone-treated controls. Electron microscopy of the corpora allata reveals that precocene treatment results in a disorganisation of the intracellular organelles. Topically applied precocene II reaches a high concentration in the haemolymph (0.5 mM 2 hr after topical application of 250 μg). C₁₆ juvenile hormone release by isolated corpora allata is inhibited by precocenes in vitro; half-maximal inhibition over a 3 hr period is obtained at 0.4 mM precocene II. In vitro inhibition of corpora allata by precocene II concentrations higher than 1 mM rapidly destroys the glands as evidenced by electron microscopy (total disintegration of cellular organelles) and by the virtual cessation of C₁₆ juvenile hormone synthesis by the corpora allata. Inhibition of C₁₆ juvenile hormone release by precocene is time-dependent and is not reversible over the short-term incubation in vitro. This inhibition does not appear to be related to the spontaneous activity of the glands in vitro, and it can be reduced by two epoxidase inhibitors. Precocenes are pro-allatocidins in this species: they are bioactivated within the corpora allata to cytotoxic epoxides.     

饵料蛋白质水平对德国小蠊营养效应及氮代谢的影响

【目的】本研究旨在探究饵料蛋白质水平对德国小蠊Blattella germanica营养利用及氮代谢的影响,为蟑螂毒饵的研发提供新思路。【方法】采用标准重量分析法评估了取食4种不同蛋白质水平(5%, 25%, 45%和65%)饵料的德国小蠊雄成虫营养效率指数和氮利用率;利用分光光度法测定了取食不同蛋白水平饵料的德国小蠊雄成虫体内黄嘌呤氧化酶(xanthine oxidase, XOD)、谷草转氨酶(glutamic oxaloacetic transaminase, GOT)、谷丙转氨酶(glutamic pyruvic transaminase, GPT)活性及尿酸含量。【结果】取食65%蛋白质饵料组的德国小蠊雄成虫的相对取食量最高,而取食45%蛋白质饵料组的雄成虫食物利用率、食物转化率及相对生长率均显著高于取食5%和65%蛋白质饵料组的雄成虫。且德国小蠊雄成虫体内氮、粪便氮、氮消耗速率、氮排泄率、氮生成率、氮同化效率、黄嘌呤氧化酶活性和尿酸含量均随饵料蛋白质水平的提高而提高,但65%蛋白质饵料组氮利用率最低,45%蛋白质饵料组谷草转氨酶活性最高,25%和45%蛋白质饵料组谷丙转氨酶活性显著高于5%和65%蛋白质饵料组。【结论】适量蛋白质饵料有利于德国小蠊对食物及氮的利用,而高蛋白质含量条件下德国小蠊谷草转氨酶和谷丙转氨酶活性下降,说明高蛋白不利于德国小蠊利用食物,且增加其代谢负担。     

Insecticide Susceptibility of Field‐collected Blattella germanica (Blattaria: Blattalidae) in Busan,Republic of Korea during 2014

The susceptibility values for field‐collected (BG‐BG) and laboratory (KS‐BG) strains of German cockroaches (BS‐BG), Blattella germanica (L.) to five synthetic pyrethroid and three organophosphate insecticides were assessed by topical application. Both KS‐BG and BS‐BG strains demonstrated the highest levels of susceptibility to deltamethrin with LD₅₀ values of 0.0035 μg/female and 1.5744 μg/female, respectively. The KS‐BG strain demonstrated the lowest susceptibility level to fenthion with an LD₅₀ value of 0.0698 μg/female, while the BS‐BG field population showed the lowest susceptibility to bifenthrin with 4.6811 μg/female. The BS‐BG field population showed resistance levels of 50 to 640 fold higher against the eight pesticides evaluated compared to the KS‐BG strain. The resistance ratios (RR) of the eight pesticides were compared using populations of B. germanica that were collected from the same locality during 2007 and 2014. The field population of B. germanica collected during 2014 demonstrated significantly high resistance to an organophosphates and all pyrethroids when compared with those collected during 2007, but RR values were <10 except for chlorpyrifos‐methyl and chlorpyrifos with RR values of 74 and 104, respectively.