Evidence for both humoral and neural regulation of sex pheromone biosynthesis in Spodoptera littoralis

Selected tissues presumably involved in the control of sex pheromone production were analyzed by ELISA for the presence of PBAN-like immunoreactivity (PBAN-IR) in Spodoptera littoralis. The temporal distribution pattern of PBAN-IR in the hemolymph is similar to that of pheromone production in the gland. On the other hand, analysis of the retrocerebral complex, brain-subesophageal ganglion complex, and terminal abdominal ganglion (TAG) revealed similar PBAN-IR levels in both photophase and scotophase periods. Pheromonotropic activity exhibited by both hemolymph and TAG, as determined by a modified in vitro bioassay, agrees with the results of the immunochemical analyses. Severing the ventral nerve cord anterior to the TAG impaired normal sex pheromone production by second-scotophase females. These results are discussed in the context of how sex pheromone biosynthesis is regulated by PBAN in S. littoralis. © 1996 Wiley-Liss, Inc.     

Effect of the pheromone biosynthesis activating neuropeptide on sex pheromone biosynthesis in Spodoptera littoralis isolated glands

The control of Spodoptera littoralis sex pheromone biosynthesis has been investigated with synthetic pheromone biosynthesis activating neuropeptide (PBAN) and different labeled tracers using an in vitro isolated gland system. Responsiveness of the glands to PBAN stimulation was impaired by careless tissue manipulation. The fact that PBAN is active in the isolated gland system suggests that this might be a target organ for this peptide in S. littoralis. As reported previously with Br-SOG extracts and intact females, label incorporation into the pheromone increased in glands treated with PBAN from all the precursors tested. However, the formation of labeled intermediates from d₅E11–14:Acid also occurred in glands incubated in the absence of the peptide, but the amounts of d₅Z9, E11–14:Acid were lower in PBAN treated glands than in controls. These results indicate that PBAN controls pheromone biosynthesis in S. littoralis by regulating the reduction of acyl moieties. © 1994 Wiley-Liss, Inc.     

Temporal distribution of PBAN‐like immunoreactivity in the hemolymph of Mamestra brassicae females in relation to sex pheromone production and calling behavior

A direct enzyme‐linked immunosorbent assay has been developed and applied to the analysis of PBAN immunoreactivity in female hemolymph of the cabbage armyworm, Mamestra brassicae. PBAN‐IR determinations have been carried out with third scotophase insects at different times of the photoperiod. The rhythm of calling and the pattern of pheromone production by third scotophase females at different times of the photoperiod have also been determined. PBAN‐IR and calling are well correlated. However, whereas pheromone titers decrease, both PBAN‐IR levels and percentage of calling females remain high in the last hours of the scotophase. These results are discussed in the context of the regulation of sex pheromone biosynthesis in M. brassicae. Arch. Insect Biochem. Physiol. 40:80–87, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of sex pheromone biosynthesis in two noctuid species,S. littoralis and M. brassicae,may involve both PBAN and the ventral nerve cord

In order to understand better the mechanism of regulation of pheromone production in moth species, we performed ELISA analyses to detect and follow pheromone biosynthesis activating neuropeptide-like immunoreactivity (PBAN-IR) in different tissues of the two noctuidae species, Spodoptera littoralis and Mamestra brassicae. Male S. littoralis and both male and female M. brassicae brain-subesophageal ganglion (Br-SEG), corpora cardiaca-corpora allata complex, and terminal abdominal ganglion extracts showed the presence of PBAN-IR during both the photophase and the scotophase. However, PBAN-IR was found only in scotophase in female hemolymph. Analysis of extracts of Br-SEG, terminal abdominal ganglion, and hemolymph after HPLC fractionation showed that the most immunoreactive fraction in all the extracts exhibited the same retention time as Hez-PBAN, suggesting that similar PBAN-like material is present in all these tissues. In vivo studies demonstrated that severing the ventral nerve cord in M. brassicae anterior to the terminal abdominal ganglion impaired normal sex pheromone production by third-scotophase females, as was previously shown in S. littoralis. Additionally, PBAN-IR levels were lower in hemolymph samples obtained at the peak of pheromone production in both S. littoralis and M. brassicae females that had the ventral nerve cord severed compared with sham operated animals. These results, along with earlier reported data, indicate that control of pheromone production in both species may involve both PBAN (or PBAN-like peptides) and the ventral nerve cord and support the hypothesis that a neural input from the ventral nerve cord triggers the release of the pheromonotropic peptide(s) into the hemolymph, which then acts directly on the pheromone gland to stimulate pheromone biosynthesis. Arch. Insect Biochem. Physiol. 37:295–304, 1998. © 1998 Wiley-Liss, Inc.

1 We thank Germán Lázaro for insect rearing.

     

Inhibitory effect of 10,11-methyl-enetetradec-10-enoic acid on a Z9-desaturase in the sex pheromone biosynthesis of Spodoptera littoralis

The effect of 10,11-methylenetetradec-10-enoic acid on the sex pheromone biosynthetic pathway of Spodoptera littoralis is reported. This new cyclopropene fatty acid inhibited the biosynthesis of the main pheromone component from labeled myristicacid. The study of each Z desaturation step revealed that the Z9-desaturase of E11–14:Acid was inhibited, whereas the Z11-desaturase of 16:Acid was not affected. The results presented in this article agree with our hypothesis that the methylenehexadecenoic acids are beta-oxidized in the pheromone gland to the corresponding methylenetetradecenoic acids. © 1994 Wiley-Liss, Inc.     

Induction of cuticular melanization in Spodoptera littoralis larvae by PBAN/MRCH: Development of a quantitative bioassay and structure function analysis

PBAN (also termed melanization and reddish coloration hormone, MRCH) is a cerebral factor known to regulate sex pheromone biosynthesis and cuticular melanization in moths. In the present study we developed a quantitative method (based on computerized image analysis of cuticles) to determine the effect of Helicoverpa zea PBAN (Hez-PBAN) on cuticular melanization and to study the structure-activity relationship of the neuropeptide in Spodoptera littoralis larvae. The results indicate that Hez-PBAN stimulates cuticular melanization in an interspecific manner, and that the minimal dose evoking formation of melanins is between 3–10 pmol/larva. Higher doses of Hez-PBAN did not stimulate melanization any further. Examination of the structure-activity relationship of Hez-PBAN revealed that the first eight N-terminal amino acids are not essential for the melanotropic activity and that the activity resides in the C-terminal region. Within this region the C-terminal amide was found to play a very important role. © 1996 Wiley-Liss, Inc.     

Biosynthetic pathways of the pheromone of the Egyptian armyworm Spodoptera littoralis

Abstract Most insect pheromones comprise multicomponent blends of geometric or optical isomers, and one major question is how insects produce species‐specific ratios of components for successful reproductive isolation. Key enzymes suggested to be involved in pheromone biosynthesis are acetyl‐coenzyme A carboxylase and fatty acyl synthetase, chain‐shortening enzymes, desaturases, elongases, reductases, oxidases, and alcohol acetyl transferases. The female pheromone composition of the Egyptian armyworm Spodoptera littoralis (Boisd.) is highly dependent on the origin of the strain. In this review, we present a summary of the different reported pheromone compositions of the moth, including from our recent studies on this subject, as well as the biosynthetic routes to the different components and the molecular approaches involved. In addition, the key role played in the proposed biosynthetic pathways by a number of important biosynthetic enzymes, such as chain shortening enzymes, desaturases and alcohol acetyl transferases, is outlined, as well as the latest developments on the inhibition of these enzymes.     

双抗体夹心法检测3-硝基酪氨酸方法的建立及应用

目的：建立一种准确、快速的双抗体夹心酶免疫吸附的方法,以定量检测组织中过氧亚硝基阴离子的水平。方法：分别以小鼠源性抗3-硝基酪氨酸单克隆IgG抗体和兔源性抗3-硝基酪氨酸IgG抗体为包被抗体和检测抗体,采用正交设计方法摸索以上各抗体的浓度,建立定量检测3-硝基酪氨酸（3-NT）的双抗体夹心ELISA法。同时,测定心肌缺血再灌注大鼠心肌组织3-NT的含量。结果：本研究建立的双抗体夹心ELISA法检测3-NT的最低检测下限为0．10ng·ml^-1,具有良好线性关系的检测范围是（0．15-7．50）ng·ml^-1（r^2=0．995）;心肌缺血再灌注组大鼠的心肌组织中的3-NT水平为（1022．42±97．35）ng·mg pro^-1,明显高于假手术组（246．58±56．52ng·mg pro^-1,P〈0．01）。结论：本研究建立的定量检测3-NT的双抗体夹心ELISA法能够方便、准确、快速地检测组织中3-NT的含量,为定量检测组织中ONOO-的水平提供了新的方法。     

Efficient separation of fatty acyl precursors of Spodoptera littoralis sex pheromone by reversed-phase high-performance liquid chromatography

Chromatographic conditions are reported for the efficient separation of fatty acyl precursors of Spodoptera littoralis sex pheromone by reversed-phase high-performance liquid chromatography. The procedure was optimized with a mixture of phenacyl derivative standards, using an octadecylsilane column, mixtures of acetonitrile-water, methanol-water, and methanol-isopropanol-water as mobile phases, and temperature control. This optimized method allowed the satisfactory separation of phenacyl esters obtained directly from S. littoralis sex pheromone gland extracts. © 1996 Wiley-Liss, Inc.     

The analysis of flight paths of male Egyptian cotton leafworm moths, Spodoptera littoralis, to a sex pheromone source in the field

ABSTRACT. The flight of male Spodoptera littoralis (Boisd.) (Noctuidae) towards a pheromone source was recorded during the early part of the night using a cine camera and an image intensifier. The cine films were analysed frame by frame to produce flight tracks from which it was possible to calculate the mean advance rate of moths towards the pheromone source and their projected ground speed, for a series of positions downwind of the source. As wind speed was measured the moth's air speed was also estimated. The moths compensated for changes in wind speed by varying their air speed, hence maintaining a ground speed independent of wind speed. The ground speed itself was found to decrease as moths flew closer to the pheromone source.     

The role of bacterial symbionts in suppressing the defence reaction in larval hemolymph of the cotton leafworm Spodoptera littoralis (Biosd.)

Abstract

Spodoptera littoralis hemolymph exhibited a decrease in phenoloxidase activity during the first hour of exposure to alive or dead Xenorhabdus nematophilus and Photorhabdus luminescens even in the presence or absence of laminarin and α-chymotrypsin. Also, as the bacterial numbers in the hemolymph of the larvae of S. littoralis increased, the suppression of the enzyme, phenoloxidase, activity in vivo increased. On the other hand, in the in vitro incubation of the infected hemolymph with X. nematophilus for 30 min with laminarin, the decrease in phenoloxidase activity reached 92% in the infection with the highest dose (1 × 10¹⁰ cells/ml) live bacteria, and reached 100% at the same dose in the infection with live bacteria in the absence of laminarin. A two-fold decrease in enzyme activity was recorded in the case of injection of (1 × 10⁶ cells/ml) dead X. nematophilus and the absence of laminarin compared with injection of the same dose in the presence of laminarin. The same trend was also observed by the end of incubation of X. nematophilus-treated hemolymph without α-chymotrypsin. The decrease in phenoloxidase activity was highly significantly different in injection of dead P. luminescens and the absence of laminarin during incubation. In the case of injection of (1 × 10⁸ cells/ml) live P. luminescens a higher degree in the reduction of enzyme activity was recorded in the absence of α-chymotrypsin, where it reached to nearly a two-fold decrease. The results of these studies indicated that both X. nematophilus and P. luminescens alive or dead suppress the phenoloxidase activity in the presence or absence of both laminarin and α-chymotrypsin but, the suppression in absence of both during the in vitro incubation was highly comparable.     

Identification of Arsenobetaine,a Water Soluble Organo-arsenic Compound in Muscle and Liver of a Shark,Prionace glaucus

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet for 12 days, 70 mg of streptozotocin/kg body weight (STZ group) or 70 mg of streptozotocin and 500 mg of nicotinamide/kg body weight (STZ-Nam group) was injected intraperitoneally into the rats. The rats were kept for 21 more days on the 20% casein diet and killed by decapitation. Urine was collected for the last 2 days. The level of blood glucose was 2-fold higher in the STZ group than in the STZ-Nam group. Urinary excretion of large amounts of glucose was observed only in the STZ group. Extremely reduction of urinary excretion of nicotinamide was observed in the STZ group, but, urinary excretion of N¹-methylnicotinamide (MNA) and N-¹-methyl-2-pyridone-5-carboxamide (2-py) was about the same in the two groups and that of N¹-methyl-4-pyridone-3-carboxamide (4-py) was higher in the STZ group than in the STZ-Nam group. The sum of urinary excretion of nicotinamide, MNA, 2-py, and 4-py was higher in the STZ group than in the STZ-Nam group. The levels of NAD in liver, pancreas, and blood in the STZ group tended to be higher, or rather not to decrease compared to the STZ-Nam group. For enzyme activities concerned with the tryptophan-NAD metabolism, a marked increase was observed in the activities of aminocarboxymuconate-semialdehyde decarboxylase, 3-hydroxyanthranilic acid oxygenase, and nicotinamide methyltransferase, on the other hand, the activity of NAD⁺ synthetase decreased in the STZ group compared to the STZ-Nam group. The activities of tryptophan oxygenase, kynureninase, NMN adenylyltransferase, and MNA oxidase were about the same in the two groups. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to NAD is lower in the streptozotocin diabetic rats than normal rats, but the tryptophan metabolites such as NAD and 4-py were higher in the STZ group than in the STZ-Nam group. This might be due to the higher food intake and the lower body weight gain in the STZ group than in the STZ-Nam group. Similar phenomena have reported in alloxan diabetic rats.     

Distribution and metabolism of exogenous ecdysteroids in the egyptian cotton leafworm Spodoptera littoralis (lepidoptera: noctuidae)

After ingestion of various amounts of either [³H]ecdysone or [³H]20-hydroxyecdysone (0.8 ng to 10 μg) by sixth instar larvae of the Egyptian cotton leafworm Spodoptera littoralis, apolar metabolites are rapidly detected in the gut and frass. Hydrolysis of the apolar products with Helix hydrolases releases solely [³H]ecdysone or [³H]20-hydroxyecdysone, respectively. This, coupled with the formation of chemical derivatives (acetonide and acetate) which cochromatograph with authentic reference compounds on hptlc and hplc demonstrates that these apolar metabolites consist of ecdysone or 20-hydroxyecdysone esterified at C-22 with common long-chain fatty acids. The major fatty acids have been identified by RP-hplc and their contribution to the mixture determined. In contrast, [³H]ecdysone injected into the haemolymph of S. littoralis is metabolized to yield 20-hydroxyecdysone, ecdysonoic acid, and 20-hydroxyecdysonoic acid. Thus, two different pathways exist for the metabolism of ecdysteroids in this species. In addition to an essentially polar pathway operating on injected and endogenous ecdysteroids, exogenous ecdysteroids entering the gut of S. littoralis are detoxified, yielding apolar ecdysteroid 22-fatty acyl esters which are rapidly excreted. The significance of these results in relation to the effects of ingested ecdysteroids on S. littoralis is discussed. Arch. Insect Biochem. Physiol. 34:329–346, 1997. © 1997 Wiley-Liss, Inc.     

Ferritin acts as a target site for the snowdrop lectin (GNA) in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA (Galanthus nivalis agglutinin) has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor(s) responsible for binding of GNA in the larval midgut of a major lepidopteran pest (the cotton leafworm, Spodoptera littoralis) to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA‐binding proteins and BLAST analyses revealed that the N‐terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta (76% sequence identity), Calpodes ethlius (80% sequence identity) and Bombyx mori (61% sequence identity). Furthermore, the N‐terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta (88% sequence identity).     

Sensitive enzyme-linked immunosorbent assays for the detection of bacterial superantigens and antibodies against them in human plasma

Enzyme-linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic-shock syndrome toxin-1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3 pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150 min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme-linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.     

Molecular cloning,developmental expression and tissue distribution of diapause hormone and pheromone biosynthesis activating neuropeptide in the bamboo borer Omphisa fuscidentalis

Diapause, an arrested period of post‐embryonic development in insects, is under the control of hormonal interactions. In the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae), larvae remain in diapause for as long as 9 months during the dry season, from September to the following June, although the factors that regulate larval diapause are poorly understood. The present study describes the cloning and expression analysis of the diapause hormone and pheromone biosynthesis activating neuropeptide (DH‐PBAN) precursor of O. fuscidentalis (Ompfu‐DH‐PBAN cDNA), aiming to reveal how it may be involved regulating larval diapause in this species in combination with environmental factors. The open reading frame (ORF) of the cDNA encodes a 199‐amino acid precursor protein that contains DH, PBAN and three other neuropeptides, all of which share a conservative C‐terminal pentapeptide motif FXPR/KL (X = G, T or S). The Ompfu‐DH‐PBAN is highly similar (74%) to the DH‐PBAN of the legume pod borer (Maruca vitrata). A quantitative real‐time polymerase chain reaction reveals that Ompfu‐DH‐PBAN mRNA is expressed only in neural tissues and that expression is highest in the suboesophageal ganglion. In addition, the expression level of Ompfu‐DH‐PBAN mRNA in the suboesophageal ganglion is consistently high during the fifth larval instar, increasing moderately in early diapause before reaching a peak during late diapause. After pupation, expression of the Ompfu‐DH‐PBAN precursor decreases to a low level. In addition to endocrine factors, the results demonstrate that photoperiod increases the expression level of Ompfu‐DH‐PBAN mRNA in larval diapause. These results also suggest that the expression of the Ompfu‐DH‐PBAN gene correlates with larval diapause development and may be activated by photoperiod in O. fuscidentalis.     

Termination of sex pheromone production in mated females of the silkworm moth

A mating duration of more than 6 h was necessary to permanently terminate the production of the sex pheromone (bombykol) in the silkworm moth, Bombyx mori L. (Lepidoptera: Bombycidae), although the female formed a bursa copulatrix including a spermatophore and laid fertilized eggs even after mating for only 0.5 h. The 6-h mated female again produced bombykol if given an injection of synthetic pheromonotropic neuropeptide (PBAN), which is known to activate pheromone biosynthesis in a virgin female. Extracts of brain-suboesophageal ganglion (SG) complexes, which were removed from 6- and 24-h mated females, showed strong pheromonotropic activities. These results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it could not produce pheromone due to a suppression of PBAN secretion from the SG. Furthermore, bombykol titers did not decrease after mating in females with a transected ventral nerve cord, even after the injection of a spermatophore extract, suggesting that the suppression of PBAN secretion was mediated by a neural signal and not by a substance in the spermatophore. The mated females accumulated (10E, 12Z)-10,12-hexadecadienoic acid, a precursor of bombykol biosynthesis, in their pheromone glands as did decapitated females. © 1996 Wiley-Liss, Inc.     

烟实夜蛾性信息素合成激活肽基因的分子克隆

根据家蚕Bombyx mori和玉米夜蛾Helicoverpa zea的性信息素合成激活肽基因序列,设计若干套引物, 以烟实夜蛾Heliothis assulta基因组DNA为模板进行PCR扩增, 得到0.63 kb的特异性DNA片段。该片段克隆进适当载体,序列测定和同源比较, 查明烟实夜蛾的基因组中存在性信息素合成激活肽基因。烟实夜蛾的性信息素合成激活肽由33个氨基酸组成, C末端是FXPRL结构,是目前发现的第4种昆虫性信息素合成激活肽。在该神经肽第14和第15个氨基酸之间, 插入一个0.42 kb的内含子。 进一步的分析证明了烟实夜蛾的性信息素合成激活肽基因在潜成虫期的食道下神经节中表达。     

甜菜夜蛾性信息素的研究与应用

甜菜夜蛾Spodoptera exigua(Hbner)已成为我国的重要农业害虫,其高抗药性和危害具有隐蔽性,致使防治十分困难。文章介绍甜菜夜蛾性信息素的主要成分、活性、配比、诱芯剂型、诱捕器类型和利用性信息素监测、防治甜菜夜蛾技术的研究概况。认为改进现有技术后并应用于实际,将会更有效地监测和防治甜菜夜蛾的发生与危害。     

甜菜夜蛾对性信息素的行为反应及其田间诱捕效果

人工合成的甜菜夜蛾性信息素两组分（顺,反）-9,12-十四碳二烯醇醋酸酯（Z9,E12-14：Ac）和（顺）-9-十四碳烯醇（Z9-14：OH）按不同配比制成诱芯,在风洞中均能引起雄蛾搜索气迹、定向飞行及接近诱芯的行为反应.不同配比和剂量的诱芯诱虫效果差异显著（p＜0.05）.Z9,E12-14：Ac和Z9-14：OH按7：3配比,诱虫活性最高,表现在风洞中,搜索气迹、定向飞行和接近诱芯的蛾数最高,与处女蛾的引诱效果相当.以此配比制成的性诱剂在田间诱捕的虫量最多,与风洞的试验结果基本一致.诱芯剂量在0.05～0.5mg范围内均有显著的诱虫效果,但剂量过低或过高均导致诱捕率下降.同一配比诱芯在风洞中引起雄蛾接近诱芯的蛾量与田间诱蛾量呈显著相关（R1=0.968,R2=0.961）.并对这些结果在甜菜夜蛾田间性诱剂防治应用的前景进行了讨论.