Juvenile hormone analog and injection effects on locust hemolymph protein synthesis

In adult female Locusta migratoria, at about day 8 after eclosion, when vitellogenin (Vg) is first produced as a result of induction by juvenile hormone (JH), the intensity of hemolymph protein electrophoretic bands at about 75 kDa and 20 kDa increases sharply, suggesting that JH may induce additional proteins. A major component of the elevated protein is persistent storage protein (PSP; subunit 74 kDa). Administration of the JH analog, methoprene, to precocene-treated adult locusts was followed by a rise in hemolymph levels of PSP but not in apolipophorin III (19 kDa), identified immunochemically and electrophoretically. The synthesis of PSP in adult fat body was confirmed by incorporation of [³H]leucine. At 48 h after treatment with methoprene, Vg synthesis was induced in females (as previously observed) and synthesis of PSP in both sexes was elevated above controls, while synthesis of apolipophorin III was not stimulated. We conclude that in adult locust fat body the synthesis of several proteins responds in different ways to the JH analog: Vg (and a 21 kDa protein described elsewhere) is induced de novo solely in females; PSP (and a 19 kDa protein described elsewhere) is stimulated in both sexes but is not fully JH-dependent; apolipophorin III is not stimulated. In these experiments, methoprene was administered both by injection in mineral oil and topically in acetone. After injection of mineral oil as a vector control, incorporation into secreted proteins was stimulated at 24 h, presumably due to a wound effect; topical application of acetone avoids this effect and is a preferred route for administration of JH analog. © 1992 Wiley-Liss, Inc.     

Growth of the male accessory gland in adult locusts: Roles of juvenile hormone,JH esterase,and JH binding proteins

The participation of juvenile hormone (JH) in the regulation of growth and protein synthesis in the accessory reproductive gland of male Locusta migratoria has been investigated. After elimination of endogenous JH with ethoxyprecocene, the accessory gland failed to grow, but growth was restored by a single application of the JH analog, pyriproxyfen. Pyriproxyfen appeared to stimulate total protein synthesis by 3 h, with a significant effect by 12 h, in contrast to 24 h observed in fat body. The dose curve for stimulation of protein synthesis 12 h after applying pyriproxyfen gave an ED₅₀ of 0.1 μg; the dose curve for gland growth at 72 h was biphasic, with steps at about 0.01 μg and 10 μg, suggesting two phases in JH action. SDS-PAGE analysis showed several components that were stimulated by pyriproxyfen, the effect being strongest in an 11 kDa band. A 5 kDa component was enhanced in the soluble and reduced in the particulate fraction after precocene treatment. The accessory gland contained JH esterase activity at levels about 100 times those in fat body or hemolymph, and was higher in precocene treated locusts. Binding activity for [³H]10R -JH III was high in cytosolic and nuclear fractions, and was identified immunologically as due to the previously described hemolymph JH binding protein. The results indicate that the mode of action of JH in the accessory gland may differ from that in the fat body. The presence of intracellular JH binding protein suggests a direct action of JH within the gland, that may be modulated by JH esterase. © 1995 Wiley-Liss, Inc.     

Protein synthesis and secretion by the locust fat body after ovariectomy

The synthesis and secretion of proteins, including vitellogenin, by the locust fat body were investigated in vivo and in vitro at various times after ovariectomy. The rate of overall protein synthesis and secretion by the fat body in vivo is significantly less in ovariectomized animals than in the controls two to three weeks after the operation, the difference in the rates of secretion of vitellin antibody-precipitable protein being even more pronounced and detectable earlier. The significantly greater amount of newly synthesized vitellogenin retained in the fat body of ovariectomized animals is insufficient to account for this difference. The secretion of newly synthesized protein by the fat bodies of ovariectomized locusts in vitro is significantly less than that of control fat bodies, the difference being particularly marked in the case of vitellogenin. The polysome populations of fat bodies of ovariectomized and control females are quantitatively similar and the amounts of total protein and vitellin antibody-precipitable protein synthesized by these polysomes in a cell-free system do not differ significantly.     

Nuclear development in locust fat body: the influence of juvenile hormone on inclusion bodies and the nuclear matrix

The hormonal induction of vitellogenesis in insects and in oviparous vertebrates are prime models of gene regulation in eukaryotes. In vertebrates the process is under estrogenic control and normally confined to females, although males can be artificially induced. In locust in contrast, juvenile hormone (JH) is central to fat body development in both males and females, yet the response is strongly sex limited not only for vitellogenin production but also in terms of total protein, DNA and RNA synthesis and nuclear ploidy levels. To differentiate further possible sex and/or JH related developmental aspects in locusts, large-scale nuclear events were examined during normal adult maturation and in animals treated with antiallatropins and JH analogs. Fat body nuclei undergo extensive restructuring during normal development in both sexes. This included progressive nuclear enlargement, accompanied by extensive proliferation of nuclear matrix components and elaboration of complex inclusion bodies (NB). The isolated protein matrix was unusually complex relative to similar structures from vertebrates and the NB were firmly anchored to it. Although matrix proteins were qualitatively similar to those from other sources, as assessed by SDS polyacrylamide gel electrophoresis, several major matrix polypeptides, including lamins A and B, and components greater than 150 kD, fluctuated quantitatively during development and in concert with nuclear enlargement. The number and morphology of the NB were unrelated to sex, but increased in direct proportion to absolute nuclear volumes. All changes were more pronounced in females, where higher ploidy levels, larger nuclei and correspondingly more internal matrix elements occurred. Suppression of JH production by precocene prevented all foregoing nuclear changes, but re-exposure to methoprene rapidly induced normal development. The results are compared to analogous nuclear changes in steroid responsive vertebrate tissues.     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body: Cytological development

Cytological development in the fat body of adult female Locusta migratoria, related to vitellogenin synthesis, has been studied by light and electron microscopy. In the newly-emerged adult, the cells are filled with lipid droplets, which indent the nucleus, and with fields of glycogen, while ribosomes and endoplasmic reticulum are scarce. Correlated with the onset of vitellogenin synthesis, about day 8 of adult life, the nucleus enlarges, lipid droplets and glycogen decrease, and rough endoplasmic reticulum and Golgi complexes become the most abundant organelles. These changes reflect a conversion of the principal role of the fat body from nutrient storage to the synthesis and secretion of protein. They are prevented by allatectomy, and restored by subsequent treatment with the juvenile hormone analogue, ZR-515. Late in the first gonotrophic cycle, about day 20, dense bodies, vesicle-containing bodies and lysosomes are seen, indicating recycling of cellular materials. Five days after ZR-515 treatment, when protein synthesis has declined, the rough endoplasmic reticulum appears in arrays adjacent to lipid droplets, possibly awaiting reactivation. By the use of ferritin-labelled antivitellin immunoglobulin, vitellogenin has been localized intracellularly in the RER saccules and Golgi vesicles, and extracellularly in channels between the folded plasma membranes, showing sites of accumulation and secretion of this protein.     

Evaluation of juvenile hormone analogues as rodent feed-through insecticides for control of immature phlebotomine sandflies

The juvenile hormone analogues methoprene and pyriproxyfen were evaluated as rodent feed-through insecticides for control of immature stages of the sandfly Phlebotomus papatasi Scopoli (Diptera: Psychodidae). The development and survival of P. papatasi second-instar larvae fed faeces from Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing methoprene (0, 9.788, 97.88 or 978.8 p.p.m.) or pyriproxyfen (0, 9.82, 98.2 or 982 p.p.m.) were evaluated. The faeces of methoprene-treated hamsters greatly reduced the percentage of larvae that pupated at all concentrations tested and prevented adult emergence at all but the lowest concentration (9.788 p.p.m.). Pyriproxyfen prevented both pupation and adult emergence at all concentrations tested. The results of this study suggest that a control strategy using rodent baits containing juvenile hormone analogues to control phlebotomine sandflies that live in rodent burrows and feed on rodent faeces may be possible. As rodent reservoirs and vectors of Leishmania major live in close association in many parts of the Middle East, control of the transmission of the agent of zoonotic cutaneous leishmaniasis may also be possible.     

Juvenile hormone action on locust fat body

Production of vitellogenin (Vg) in fat body of adult female Locusta migratoria is abolished by removal or inactivation of the corpora allata and restored by administration of (RS)-methoprene. Juvenile hormone III injection alone has little effect, but when it is injected together with the JH esterase inhibitor OTFP, active Vg synthesis is induced. This supports the assumption that methoprene acts in place of the natural hormone in this system. When fat body from vitellogenic females is maintained in synthetic medium for 48 hr, the proportion of Vg in the secreted protein drops greatly, but when methoprene is present in the medium the proportion of Vg is sustained. When fat body from JH-withdrawn locusts, in which Vg synthesis has declined to zero, is cultured with methoprene, Vg synthesis is re-induced. These results show that the JH analog can act directly on locust fat body to bring about expression of the Vg genes. Experiments to optimize JH action on fat body in vitro are continuing.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Gene structure,cDNA sequence,and developmental regulation of a low molecular weight hemolymph protein from Locusta migratoria

From a Locusta migratoria genomic DNA library, a gene has been isolated that codes for a previously unrecognized hemolymph protein of M_r = 19,000, designated 19k protein. The gene has at least five exons, extending over about 9 kb of DNA. Its polypeptide product, obtained by cell-free translation of mRNA selected from adult fat body RNA by hybridization with the cloned DNA, is precipitated by antiserum against a low molecular weight hemolymph protein fraction. The mature protein product has been purified from locust hemolymph, and an N-terminal sequence of 20 amino acids has been determined. In polyacrylamide gel electrophoresis, this protein comigrates with apolipophorin III, from which it was previously not distinguished, but it is clearly distinct by amino acid composition and sequence. The genomic clone was used as a probe to isolate a fat body cDNA clone of the 19k protein mRNA. The 938-base pair cDNA clone contains a 516-base pair open reading frame. The deduced 172-amino acid polypeptide includes an apparent signal peptide, a sequence of four amino acids that may represent a prosegment, and a sequence identical (with a single exception, which may reflect polymorphism) with the N-terminal sequence of the hemolymph protein. Its mRNA occurs at a low level in late larval fat body, is abundant in the newly eclosed adult, then declines to a low level, and rises again at days 8–10; it is greatly reduced after destruction of the corpora allata with precocene and then is elevated after treatment with methoprene, suggesting stimulation by juvenile hormone. The biological role of 19k protein is unknown.     

Effects of juvenile hormone analogue treatment in adult females of the ovoviviparous cockroach,Blaptica dubia (Dictyoptera,Blaberidae)

Adult females of the ovoviviparous Argentinian cockroach, Blaptica dubia, were repeatedly treated with either 100?μg methoprene or 100?μg pyriproxyfen in 5?μL acetone either during the first vitellogenic cycle or during the period of gestation. Treatment during the first vitellogenic cycle (days 2–20 of adult life) did not inhibit vitellogenesis and oocyte growth, but prevented the formation of an ootheca. This was accompanied with a significant reduction of the titer of juvenile hormone (JH) III and an increased amount of ecdysone (E) and 20-hydroxyecdysone (20E) in the haemolymph of the animals. Treatment of adult females during the period of gestation (days 30–70) resulted in a complete degradation and resorption of the ootheca and induced another vitellogenic cycle. Again, this was associated with a decrease in haemolymph JH III titer, but an increase in the concentrations of free ecdysteroids.     

Unfolding and refolding of juvenile hormone binding protein

Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements, and spectroscopic methods. Molecules of JHBP change their conformation from a native state via two unstable intermediates to a denatured state. The first intermediate appears in a compact state, because it slightly changes its molecular size and preserves most of the JHBP secondary structure of the native state. Although the second intermediate also preserves a substantial part of the secondary structure, it undergoes a change into a noncompact state changing its Stokes radius from approximately 30 to 39 A. Refolding experiments showed that JHBP molecules recover their full protein structure, as judged from the CD spectrum, fluorescence experiments, and JH binding activity measurements. The free energy of unfolding in the absence of the denaturant, DeltaG(D-N), is calculated to be 4.1 kcal mol(-1).     

家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

家蚕头部是一个神经中枢和感受的器官,其头部含有触角和感觉毛,感受外界的信号,并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的,而保幼激素结合蛋白是保幼激素转运和发挥功能的载体,在昆虫体内具有极其重要的功能。文中通过Silk DB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白Bm TOL,其编码基因编号为BGIBMGA003404(Gen Bank登录号:KY681053)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm TOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm TOL在头部都是高量表达,且Bmtol基因在起蚕时表达量较高,在5龄和蛹期表达量较低,而在化蛾后表达量又开始上调。免疫组化结果显示Bm TOL蛋白定位在头部的皮层、触角和脑中,推测其可能与头部信息传递有关,为家蚕的生长发育和行为调控提供重要的信息来源。     

Yellowing and YPT gene expression in the desert locust,Schistocerca gregaria: Effects of developmental stages and fasting

The yellow protein of the takeout family (YPT) controls the development of yellow body color in the desert locust. This study focused on two aspects related to YPT in the locust. We first examined the expression pattern of YPT during nymphal stages because yellowing was not obvious during the early instars. YPT expression levels were extremely low in the second and third instars compared with the last two nymphal instars. Warm rearing temperature and juvenile hormone (JH) injection, which stimulated YPT expression in the late instars, had little effect in the second instar, suggesting that YPT expression during the early instars was suppressed and could not be stimulated by either of these factors. We also investigated delayed yellowing in fasting male adults, under the hypothesis that fasting decreased the JH titers and delayed the onset of YPT expression. Yellowing was delayed in fasting adults compared with well‐fed adults and YPT expression was stimulated by JH injections at Day 15. However, we failed to obtain evidence that fasting significantly influenced the expression levels of YPT and the JH early‐inducible gene Krüppel homolog 1 at Days 15 and 20 post‐adult emergence. Results suggest that a YPT‐independent mechanism possibly induces delayed yellowing in fasting males.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: Evidence for a fat body stimulatory factor

Juvenile hormone or ZR512 applied topically to day-5, fifth-instar, neck-ligated Manduca sexta larvae results in the acceleration of pharate pupal development when compared to neck-ligated, untreated larvae. This occurs as a result of an increase in the haemolymph ecdysteroid titre. Juvenile hormone, therefore, appears to stimulate ecdysone synthesis by the prothoracic glands of these animals, but not directly as shown by in vitro analysis. When ecdysone synthesis by the prothoracic glands of these ZR512- or juvenile hormone-treated animals was analyzed in vitro, increased gland activity was demonstrated but this did not occur until at least 2 days after treatment. This time lag in response supports the concept of an indirect stimulation of the prothoracic glands. Incubation of fat body from these ZR512- or juvenile hormone-treated, neck-ligated, larvae in 19AB culture medium revealed that the resulting pre-conditioned medium was capable of stimulating prothoracic glands in vitro up to 9-fold in a dose-dependent manner. A developmental profile was generated of the amount of this stimulatory factor released into the medium by fat body of untreated larvae representing each day of the last instar, and revealed that maximal release occurred with fat body from day-9 animals. The alterations in the amount of factor release by the fat body during larval-pupal development roughly correlated with the juvenile hormone titre and suggested a possible role for this factor in the regulation of the ecdysteroid titre. In contrast to the prothoracicotropic hormone, the fat body stimulatory factor is heat labile and has an apparent mol. wt in the 30,000 Dalton range. These data, particularly the kinetics of prothoracic gland stimulation, suggest that the factor may be a protein transporting a substrate for ecdysone biosynthesis to the prothoracic glands.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.