Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.     

Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro

One juvenile hormone was isolated from culture medium containing isolated corpora allata of the grasshopper $\text{Schistocerca vaga}$ (Orthoptera: Acrididae) and was shown by microchemical methods to be methyl (2$\text{E}$, 6$\text{E}$) - (10$\text{R}$) - 10, 11-epoxy-3, 7, 11-trimethyldodeca-2, 6-dienoate. This compound (JH III), which occurs in a sphingid moth $\text{Manduca sexta}$, is the first juvenile hormone identified in an insect order other than the Lepidoptera. Grasshopper organs incorporate both [2?¹⁴C] acetate and [methyl-¹⁴C] methionine into JH III showing $\text{de novo}$ biosynthesis, but no indication of the synthesis of JH I or JH II was seen.     

Partial morphological and functional characterization of the corpus allatum-corpus cardiacum complex from the two-spotted stinkbug, Perillus bioculatus (Hemiptera: Pentatomidae)

Summary Selected morphological and physiological properties of the corpus allatum (CA)-corpus cardiacum (CC) complex from the two-spotted stinkbug, Perillus bioculatus (Hemiptera: Pentatomidae), were studied. The CAs play an important role in insect physiology because of their production of the juvenile hormones (JHs), i.e., key hormones involved in development and reproduction. We found that the P. bioculatus CA-CC complex is present in two distinct morphological forms, the more frequently observed complex containing one “fused” CA between two CCs and the more rarely observed complex having one CA laterally attached to each CC. These complexes were tested for their ability to synthesize JH-like compounds. We found that the primary lipophilic compound synthesized by the CA-CCs migrated differently from JH III (a JH found in numerous insect species) when subjected to thin-layer chromatography. Furthermore, the synthesis of this compound is stimulated by 2E,6E-farnesol, a known precursor for JH III. These data indicate that the P. bioculatus CA-CC product has chemical properties similar to that of other (as of yet unidentified) hemipteran JHs. In addition, we found that the synthesis of this product is sensitive to pH and buffer type; minimally or not affected by the absence of the CC; expressed at similar levels in days 5–30 postemergent adults; and inhibited or decreased in adults reared under low temperature—short day conditions.     

Arbuscular mycorrhizal fungi associated with sedges on the Tibetan plateau

The arbuscular mycorrhizal (AM) status of nine dominant sedge species and the diversity of AM fungi in Tibetan grassland were surveyed in the autumn of 2003 and 2004. Most of the sedge species and ecotypes examined were mycorrhizal, but Carex moorcroftii and Kobresia pusilla were of doubtful AM status, and Kobresia humilis was facultatively mycorrhizal. This is the first report of the mycorrhizal status of eight of the nine sedge species examined. Intraradical vesicles and aseptate hyphae were the structures most frequently observed. Appressoria, coils, and arbuscules were found in the roots of a few sedge species. A strong negative correlation was found between soil organic matter content and the extent of mycorrhizal colonization. Using trap cultures, 26 species of AM fungi belonging to six genera, Glomus, Acaulospora, Paraglomus, Archaeospora, Pacispora, and Scutellospora, were isolated from the soil samples collected. The frequency of occurrence of different taxa of AM fungi varied greatly. Glomus and Acaulospora were the dominant genera, and Acaulospora scrobiculata was the most frequent and abundant species. The species richness of AM fungi was 2.73 in the study area. Species richness and diversity index differed among the sedge species but were not correlated with soil factors such as pH, available P, or organic matter content.     

Comparative metabolism of isoleucine by corpora allata of nonlepidopteran insects versus lepidopteran insects,in relation to juvenile hormone biosynthesis

We studied the metabolism of [U-¹⁴C]isoleucine by intact and homogenized corpora allata (CA) from various insect species to determine how this substrate is converted to precursors of juvenile hormone (JH). CA homogenates of the lepidopterans Manduca sexta, Hyalophora cecropia, and Samia cynthia metabolize [U-¹⁴C]isoleucine to several products including 2-keto-3-methyl-valerate, 2-methylbutyrate, CO₂, propionate, and acetate. Intact CA of male H. cecropia produce particularly high levels of 2-keto-3-methylvalerate, indicating a highly active branched-chain-amino acid transaminase. In contrast, CA homogenates from the nonlepidopterans Periplaneta americana, Schistocerca nitens, Tenebrio molitor, and Diploptera punctata barely metabolize [U-¹⁴C]isoleucine. However, P. americana CA homogenate metabolizes [U-¹⁴C]2-keto-3-methylvalerate, the transamination product of [U-¹⁴C]isoleucine, more rapidly than does a homogenate of M. sexta CA. Furthermore, intact CA from P. americana incubated with [U-¹⁴C]2-keto-3-methylvalerate incorporate low levels of ¹⁴C into JH III, but do not metabolize this substrate to JH II or JH I. Intact CA from female Diploptera punctata produce very high levels of JH III, but are also unable to incorporate radiolabel from [U-¹⁴C]isoleucine into JH III, which substantiates our findings with other nonlepidopteran CA. The results suggest that CA of nonlepidopteran insects lack an active branched-chain amino acid transaminase and, consequently, are unable to utilize these substrates for JH biosynthesis.     

Diversification and Independent Evolution of Troponin C Genes in Insects

Troponin C (TpnC), the calcium-binding subunit of the troponin regulatory complex in the muscle thin filament, is encoded by multiple genes in insects. To understand how TpnC genes have evolved, we characterized the gene number and structure in a number of insect species. The TpnC gene complement is five genes in Drosophilidae as previously reported for D. melanogaster. Gene structures are almost identical in D. pseudoobscura, D. suboboscura, and D. virilis. Developmental patterns of expression are also conserved in Drosophila subobscura and D. virilis. Similar, but not completely equivalent, TpnC gene repertoires have been identified in the Anopheles gambiae and Apis mellifera genomes. Insect TpnC sequences can be divided into three groups, allowing a systematic classification of newly identified genes. The pattern of expression of the Apis mellifera genes essentially agrees with the pattern in Drosophilidae, providing further functional support to the classification. A model for the evolution of the TpnC genes is proposed including the most likely pathway of insect TpnC diversification. Our results suggest that the rapid increase in number and sequence specialization of the adult Type III isoforms can be correlated with the evolution of the holometabolous mode of development and the acquisition of asynchronous indirect flight muscle function in insects. This evolutionarily specialization has probably been achieved independently in different insect orders.Reviewing Editor: Dr. Rüdiger Cerff     

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.     

Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: The search for a unifying arthropod endocrinology

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.     

Synthesis of the Thiirane Analogs of Juvenile Hormone III and Other Juvenile Hormone Mimics

Methyl (2E,6E)-10,11-epithio-3,7,11-trimethyl-2,6-dodecadienoate (the thiirane analog of JH III), 6,7-epithiogeranyl 4-methylphenyl ether and 6,7-epithiogeranyl 3,4-methylenedioxyphenyl ether were synthesized. An infrared absorption band at ~1090 cm^?1 was attributable to the thiirane group. The biological activity of these three sulfur-containing JH mimics was tested on Culex pipiens, Aedes aegypti and Spodoptera litura to reveal weak or no JH-like activity.     

Notes on New Zealand mammals 10. Effect of observer position on group size and level of aggression of mating brown hares

Abstract

The sex pheromones of two endemic New Zealand tortricid species were investigated. Females of the first species, Cnephasia jactatana, a pest of kiwifruit, were found to produce (Z)-11-tetradecenyl acetate (Z11-14:OAc) in their sex pheromone gland. When tested in the field, this compound on its own elicited significant catches of male C. jactatana. However, the addition of even very small amounts (0.3%) of the geometric isomer, (E)-11-tetradecenyl acetate (E11-14:OAc), to Z 11-14:OAc virtually suppressed trap catches. The sex pheromone of the second species, Merophyas leucaniana, was found to consist of a mixture of Z11-14:OAc, E11-14:OAc, tetradecyl acetate, and (Z)-11-tetradecenol. This species is morphologically very similar to the congeneric species, M. divulsana, the Australian lucerne leafroller. The sex pheromone of M. leucaniana is also very similar to that of the so-called “Z-type” of M. divulsana, which is now found in New Zealand. The pheromone components of C. jactatana and M. leucaniana are discussed in relation to those found in other tortricids.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

Erratum

Allatotropin (AT) is a 13 amino acid neuropeptide isolated from the tobacco hornworm, Manduca sexta, which stimulates the biosynthesis of juvenile hormone (JH) in adult females of this insect. In this paper we report that AT stimulates inositol phosphate formation in both female and male corpora allata (CA) of adult Manduca sexta. Furthermore, thapsigargin, a potent Ca²⁺-ATPase inhibitor, staurosporine, a potent inhibitor of protein kinases A and C, and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca²⁺ sequestration, all stimulate JH biosynthesis without changing the basal level of inositol phosphates. These data suggest that AT activates an inositol 1,4,5-trisphosphate (IP₃) pathway. Furthermore, increases in the free intracellular Ca²⁺ concentration in the CA stimulate JH biosynthesis. © 1992 Wiley-Liss, Inc.     

Genome organization and polyploid evolution in the genus Eleusine (Poaceae)

 Eleusine (Poaceae) includes six diploid and three polyploid species and has three basic chromosome numbers, x=8, 9 and 10. The species are annual as well as perennial and all are wild except E. coracana, which is cultivated for grain and fodder in Africa and the Indian subcontinent. Eleusine coracana and E. africana have the same genome and chromosome number (2n=36). Eleusine indica and E. floccifolia are identified as two genome donors to these polyploid species. Eleusine kigeziensis is the third polyploid species of the genus with 2n=38. Its genome may have come from E. jaegeri and from one of the species with x=9, most probably from E. indica. Eleusine indica, E. tristachya, E. floccifolia and E. intermedia with x=9 and two polyploid species, E. coracana and E. africana, are closely related and there is free genetic flow between them. Eleusine multiflora with x=8 is significantly different in morphology and at genomic level from other species. Eleusine jaegeri with x=10 is morphologically similar to E. indica, however, more information is needed to ascertain its position in the genus. Eleusine coracana, which is commonly called finger millet, is a potential and nutritious crop for the increasing population of the world, particularly in arid and semi-arid regions. It can also serve as a gene pool for various important characters and disease resistant genes. Received February 11, 2002; accepted May 27, 2002 Published online: October 14, 2002 Addresses of the authors: Madho Singh Bisht and Yasuhiko Mukai (e-mail: ymukai@cc.osaka-kyoiku.ac.jp), Laboratory of Plant Molecular Genetics, Division of Natural Science, Osaka Kyoiku University, 4-698-1 Asahigaoka, Kashiwara, Osaka 582-8582, Japan.     

Plant defense against leaf herbivory based on metal accumulation: examples from a tropical high altitude ecosystem

Species that evolved in high‐altitude grasslands, where soils are dystrophic and metal rich, developed adaptations for these extreme conditions, such as metal accumulation and sclerophyllous leaves, and these adaptations may secondarily affect insect herbivory activity. The present study investigates the hypothesis that costs related to accumulation of certain metals may be evolutionarily compensated for by decreasing leaf herbivory in some plant species from rupestrian fields. Studies were conducted in different locations at the Ferriferous Quadrangle, a metal‐rich region in south‐east Brazil, with four species adapted to high‐altitude grasslands: Eremanthus erythropappus, Eremanthus incanus, Lychnophora ericoides and Byrsonima variabilis. Sample design varied according to population sizes and spatial distribution of individuals. We found that concentrations of manganese (Mn) and iron (Fe) significantly reduced the herbivory in the leaves of E. erythroppapus and E. incanus, whereas aluminum (Al) reduced herbivory in L. ericoides, and Mn affected negatively the herbivory in B. variabilis. These results support the hypothesis that metal‐accumulating plants, as a response to the harsh environment in which they evolved, are protected against foliar damage caused by insect herbivores in rupestrian fields.     

Detection of insect Juvenile hormone III and Its precursors from<Emphasis Type="Italic">in Vitro</Emphasis> plantlets of<Emphasis Type="Italic">Cyperus aromaticus</Emphasis>

Juvenile hormone III (JH III) is one of the five closely related sesquiterpenoid compounds important in the regulation of an insect physiological processes in both the developing and the mature insect. JH III and its biosynthetic intermediates, farnesol and methyl farnesoate were detected in thein vitro Cyperus aromaticus plantlets and the mature plants growing in the field. A higher amount of methyl farnesoate (52.7%) and farnesol (31.4%) were found to be present in thein vitro plantlets as compared to the field grown mother plants which contained lower methyl farnesoate (19.5%) and farnesol (14.8%). On the other hand, the mature mother plant (4 months old) contained a higher percentage of JH III than thein vitro plantlets. The estimated content of JHIII analyzed by gas chromatography indicated that the amount of JHIII in the 10 weeks oldin vitro plantlets and the four months old mature plants of C.aromaticus was 0.076 μg/g and 0.085 μg/g respectively. This study indicated thatin vitro culture system could be a potential tool for the production of this natural derived bio-insecticide and could provide a material source for further investigations of the biosynthesis of JH III inC. aromaticus.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.