Hormonal modulation of glycogen reserves In the fat body of castor semilooperAchaea janata Linn (Lepidoptera,Noctuidea)

Histochemical details of the fat body in the fifth instar larval stage, pupa and adult moth of the castor semilooperAchaea janata were elucidated in detail using light and electron microscopy in conjunction with glycogen storage patterns using polyacrylamide gel electrophoresis. The periodic-acid Schiff staining for glycogen in fat body was maximum in the spinning stage of the larva, when compared to the feeding stage and prepupal stages, and higher in the pupa than in the larva and the adult moth. In insulin injected and juvenile hormone treated fat body, glycogen deposition was more than in glucagon injected tissues. The periodic-acid Schiff stained bands in PAGE had electrophoretic mobility similar to the corresponding protein band numbers, indicating their glycoprotein nature.     

棉铃虫不同发育阶段微粒体P450酶系组成和活性的比较

比较了棉铃虫Helicoverpa armigera 6龄幼虫、蛹、成虫微粒体P450单加氧酶系组成及其活性。P450含量在6龄幼虫中肠>（脂肪体=蛹）>成虫,NADPH-细胞色素还原酶在幼虫中肠>幼虫脂肪体>蛹>成虫;6龄幼虫脂肪体微粒体与蛹脂肪体微粒体P450含量相近,但NADPH-细胞色素还原酶活性前者是后者的4.2倍;成虫微粒体的细胞色素P450和NADPH细胞色素P450还原酶含量很低,几乎未检测出。用对-硝基苯甲醚和艾氏剂为底物测定P450酶系活性表明,与6龄幼虫相比,蛹和成虫具有极低的单加氧酶活性,其O-脱甲基酶活性未检出,艾氏剂环氧化酶活性比幼虫低2～3个数量级。     

Haemolymph protein patterns in developing tobacco hornworm larvae

Quantitative changes in haemolymph proteins from each physiological phase of the last three larval instars of the tobacco hornworm, Manduca sexta, were studied by means of disk electrophoresis. Twelve anodical migrating protein bands were found, six of which occurred only sporadically. Total protein concentration increased from pharate third instar to late fifth instar larvae, then decreased slightly in the pharate pupal stages. Some individual bands showed cyclic patterns within each instar similar to the overall cyclic pattern of total protein, whereas other bands showed different patterns or no pattern.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

Prothoracicotropic hormone bioassay: Bombyx larva assay

Summary

An assay for the prothoracicotropic hormone (PTTH) has been established using in situ activation of the prothoracic glands (PG) of Bombyx mori in its larva-to-larva development. The timing of PTTH release was estimated by examining developmental response of 4th instar larvae to brain removal and neck ligation, and changes in the haemolymph ecdysteroid titer and ecdysone-releasing activity of PG in vitro during the development. Injection of Bombyx brain extracts into 4th instar larvae neck-ligated shortly before full activation of PG elicited larval moulting rather than precocious pupation in headless larvae. This developmental shift was regarded as due to the action of PTTH, and the PTTH unit has been defined from a linear log dose-response relationship. Materials chromatographically fractionated from Bombyx brain extracts were examined for the presence of stage- and species-specific PTTH molecules by using this Bombyx larva assay and Bombyx and Samia pupa assays previously developed. The same fractions were active when assayed by Bombyx larva and pupa assays.     

The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust

The presence of juvenile hormone in the haemolymph of larvae of Locusta has been detected by a modified Galleria bioassay and these results are compared with indirect methods of estimating corpus allatum activity. Juvenile hormone is present in the haemolymph during the fourth larval instar except on the last day of the instar, and is absent from the haemolymph of the fifth and final larval instar except on the last day of the instar. Changes in the volumes of the corpora allata simply reflect changes in the growth of the whole insect and are of no value in predicting endocrine activity. Changes in the size of the cells of the corpora allata can be correlated with the presence of juvenile hormone in the haemolymph in the fourth larval instar, but similar changes in cell size occur in the fifth larval instar when no juvenile hormone is present in the haemolymph. The effects of the implantation of corpora allata are unreliable as estimates of corpus allatum activity as isolated corpora allata from fifth instar larvae release juvenile hormone. Indirect methods of measuring corpus allatum activity are thus shown to be unreliable. The R_f value of Locusta juvenile hormone as determined by thin-layer chromatography differs from that of Roeller's juvenile hormone, suggesting that the two hormones might be chemically distinct.     

Prenyltransferase of larval and adult M. sexta corpora allata

Prenyltransferase activity derived from the corpora allata (CA) of the lepidopteran insect, Manduca sexta, has been characterized. The coupling of allylic substrates DMAPP and GPP with the non-allylic substrate IPP was evaluated using CA homogenates of both the larval and adult stages of development. The effect of additives and inhibitors, assay conditions, and metal preference were examined. The cellular location of prenyltransferase activity was also investigated. We found subtle differences between larval and adult preparations, including metal and detergent preference, and while larval prenyltransferase activity was strictly cytosolic, prenyltransferase derived from adult CA was found in both the cytosolic and pellet fractions. Differences in kinetics as a function of development were also noted. When GPP was utilized as allylic substrate, adult prenyltransferase displayed cooperative behavior; while with DMAPP, biphasic kinetics were observed. In fifth instar larvae, prenyltransferase activity was highest on days 1-2 and reaction end products changed as a result of insect age. Taken together, these results suggest that larval and adult prenyltransferase of M. sexta have distinct enzymological properties and that the adult CA possess more than one prenyltransferase.     

麦红吸浆虫3-羟基-3-甲基戊二酰辅酶A还原酶基因SmHMGR的克隆及在滞育与发育变态过程中的表达动态

【目的】3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是保幼激素(JH)合成途径的限速酶。麦红吸浆虫Sitodiplosis mosellana是一种典型的专性幼虫滞育昆虫。本研究旨在探讨HMGR基因在麦红吸浆虫滞育和发育变态过程中的作用。【方法】通过RT-PCR和RACE技术克隆麦红吸浆虫滞育前幼虫HMGR基因全长cDNA序列;利用生物信息学软件分析HMGR基因核苷酸和其编码的蛋白氨基酸序列特性;采用qPCR技术测定其在麦红吸浆虫滞育不同时期3龄幼虫及不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹和后蛹以及雌雄成虫)中的mRNA表达水平。【结果】克隆获得一条麦红吸浆虫HMGR基因全长cDNA序列,命名为SmHMGR(GenBank登录号: MG876766)。该基因全长2 548 bp,其中开放阅读框长2 328 bp,编码775个氨基酸,预测的蛋白分子量为84.16 kD,理论等电点为8.29。序列分析发现该基因编码的蛋白具有HMGR蛋白家族典型的HMG-CoA-reductase-classⅠ催化功能域及其他保守功能基序;序列比对和系统发育分析表明,SmHMGR与达氏按蚊Anopheles darling等长角亚目(Nematocera)昆虫HMGR的相似性最高、亲缘关系最近。SmHMGR在麦红吸浆虫滞育前的3龄早期幼虫中表达量显著升高,进入滞育后一直维持较高水平,并在滞育后静息阶段的当年12月至翌年1月达到最高。SmHMGR在蛹期表达量低于幼虫期,预蛹期表达量最低;在雌成虫中表达量显著高于在蛹和雄成虫中的表达量。【结论】SmHMGR的表达与麦红吸浆虫发育密切相关,可能在滞育诱导、维持及滞育后静息状态的维持及生殖中发挥作用,其表达量的降低可能参与了幼虫到蛹的变态。     

Ecdysteroid-dependent protein synthesis in caste-specific development of the larval honey bee ovary

In the honey bee, Apis mellifera, the fifth larval instar is a critical period for caste differentiation. During this premetamorphic phase the hormonal milieu shows pronounced caste differences and several organs, particularly the ovaries, enter different developmental pathways leading to highly fertile queens and nearly sterile workers. Developmental profiles of total protein synthesis in larval ovaries showed marked caste differences starting with the early fifth instar. By two-dimensional electrophoresis, caste-specific patterns could be detected in the synthesis of a 29 kDa/pI 4.6 and two 24 kDa/pI 5.2–5.5. proteins (pI=isoelectric point). A marked decrease in the expression of these proteins was found to coincide with caste-specific differences in the haemolymph ecdysteroid titer. In vitro exposure of larval worker ovaries to physiological (10^–7 m) concentrations of synthetic makisterone A elicited an identical response. Juvenile hormone did not affect protein synthesis patterns in larval ovaries, and also did not inhibit or reverse the ecdysteroid-induced effects. Heat shock experiments revealed that the 29 kDa/pI 4.6 ecdysteroid-regulated protein belongs to the class of small heat shock proteins.     

Factors influencing the developmental capacity of Galleria larval epidermis

The nature of the cuticle secreted by integument from a day-1 penultimate instar larval Galleria when cultured in vivo in the abdomen of a last instar larva varied with the age of the host. When placed in a day-5 last instar larva, the implanted integument secreted a pupal cuticle at the time the host metamorphosed and became a pupa. However, when placed in a day-7 last instar larva the implant, from the same stage donor, secreted a larval cuticle at the time the host pupated. Experimental studies involving implantation of the integument for a 24 hr period, into various developmental stages of normal and ligated last instar larvae, pupae, and pharate adults, prior to placing it in a day-7 last instar larva suggest that a non-hormonal factor present in day-4 and -5 last instar larvae is important to initiate pupal syntheses.     

Caste-specific modulation of juvenile hormone III content and ecdysteroid titer in postembryonic development of the stingless bee,Scaptotrigona postica depilis

Summary Juvenile hormone III content and ecdysteroid titer were analyzed for larval and pupal development of the stingless bee,Scaptotrigona postica depilis. Castespecific differences in juvenile hormone III content were detected at three developmental phases: at the transition from the fourth to the fifth larval stadium, in the spinning phase of the fifth larval stadium, and shortly after the imaginal moult. During the fifth larval stadium, juvenile hormone content closely reflects corpora allata activity. Juvenile hormone synthesis may thus be responsible for the elevated hormone titer in spinning-phase queen larvae, a phase of known sensitivity for induction of queen characters by exogenous juvenile hormone. For ecdysteroids, two phases of caste-specific differences were found: in the pre-pupal phase, and shortly after the imaginal moult. In both periods the titer in queens is distinctly higher compared to workers.Abbreviations Im imago 1 day after eclosion - L3, L4, L5 larval instars 3, 4, and 5 - L5F1, L5F2 substages of feeding phase in fifth larval instar - L5S1, L5S2, L5S3 substages of spinning phase in fifth larval instar - PP1, PP2 substages of prepupal phase - Pw white eyed pupa - Pp pink eyed pupa - Pr red eyed pupa - Pd dark eyed pupa - Pdl, Pdm, Pdd dark eyed pupa with progressive tanning of cuticle - RIA radioimmunoassay     

The development of the sensory organs of the legs in the blowfly,Phormia regina

Summary The development of the sensory neurons of the legs of the blowfly,Phormia regina has been described from the third instar larva to the late pupa using immunohistochemical staining. The leg discs of the third instar larva contain 8 neurons of which 5 come to lie in the fifth tarsomere of the developing leg. Whereas 2 neurons persist at least to the late pupa, the other cells degenerate. The first neurons of gustatory sensilla arise in the fifth tarsomere at about 1.5 h after formation of the puparium. Most of these sensilla, however, appear within a short time period beginning at about 18 h. The femoral chordotonal sensory neurons first appear at the time of formation of the puparium, as a mass of cells situated in the distal femur. During later pupal development 2 groups of these cells come to lie at the femur-trochanter border, where they become the proximal femoral chordotonal organ of the adult; the remaining cells become the distal femoral chordotonal organ. Other scolopidial neurons appear later in development. The nerve pathways of the late pupal leg are established either by the axons of the cells that are present in the larval leg disc or by new outgrowing processes of sensory neurons. In the tibia, the initial direction of new outgrowth differs in different regions of the segment: proximal tibial neurons grow distally, while distal tibial neurons grow initially proximally.     

Metabolism of U14C leucine and U14C valine by adult female Glossina morsitans during pregnancy

After U¹⁴C leucine or U¹⁴C valine injections into haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the post-parturient female and its larval offspring in the injected material, lipids, and a range of non-essential amino acids. The level of radioactivity recorded from the third instar larva was higher than that remaining in the injected adult, and the activity was higher in amino acids than in the lipid fraction. Radiometric analysis of oöcyte and intra-uterine progeny 24 hr after haemocoelic administration to females of labelled leucine or valine revealed a pattern of radioactivity coincident with growth characteristics of these young stages. Rate of leucine uptake by the in utero third instar larva was slightly higher than that of valine, and this instar continues feeding even only a few hours before parturition. For both labelled materials, expired carbon dioxide and excreta from remales in early pregnancy showed significantly higher radioactivity than those in late pregnancy. Uric acid is the main nigrogenous waste of leucine and valine metabolism, though small amounts of these amino acids are also lost during excretion, with valine elimination being higher than leucine.     

条背萤的形态和生物学研究

首次在中国大陆发现水栖萤火虫条背萤Luciola substriata。形态观察发现,条背萤成虫橙黄色, 鞘翅末端灰黑色;发光器均为白色,雄虫发光器位于第5、6腹节,位于第5腹节的发光器呈带状,第6腹节的发光器呈“V”字形;雌虫的发光器呈带状,位于第5腹节;卵椭圆形,橙黄色。幼虫有两种形态,1～2龄具有7对呼吸鳃,3～6龄幼虫无呼吸鳃。幼虫具有一对发光器,位于第7腹节腹面;初蛹期仍保留幼虫形态的发光器,后呈现成虫的发光器,两种形态的发光器并存直至羽化。对条背萤生活史及习性调查发现,条背萤生活在水草较多的池塘、湖泊和流速缓慢的河流中。该虫1年发生1代,以幼虫在水中越冬,5月初老熟幼虫开始上岸化蛹。在25℃下,条背萤预蛹期平均为6.17天,蛹期平均为4.43天。成虫5月上旬至9月中旬发生。日落后的1 h内是条背萤成虫闪光求偶的最盛期。卵期平均12.5天。幼虫的猎物为静水椎实螺Lymnaea stagnalis,凸旋螺Gyraulus conwexiusculus等,天敌为克氏原螯虾Procambarus clarkii、中华绒毛蟹Eriocheir sinensis、草鱼Ctenopharyngodonidellus等。利用光谱仪对条背萤的发光光谱进行测定发现,条背萤的萤光光谱为425～603 nm,峰值为504 nm,颜色为黄绿混合色。     

Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10°C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.     

Quantitative determination of the juvenile hormones in the haemolymph of Locusta migratoria during normal development and after implantation of corpora allata

Simultaneous quantitative determination of the three naturally occurring juvenile hormones in insects (JH-I, JH-II and JH-III) was performed on haemolymph samples of both normally developing locusts and locusts implanted with active corpora allata, using capillary gas chromatography with electron capture detection.In fourth instar female larvae, 24–48 hr after the third ecdysis, as well as in adult females, 18 days after the imaginal ecdysis, only JH-III was detected. In fifth instar female larvae JH-III was present in very low concentrations, if at all.After implantation of four pairs of corpora allata taken from young fourth instar female larvae or one pair or corpora allata taken from adult females into fifth instar female larvae 0–24 hr after ecdysis, an elevation of the JH-III titre was observed. Neither JH-I nor JH-II could be detected. The amount of JH-III, already elevated 2 hr after implantation, remained high for several days in comparison to that of control insects. On the third day after the subsequent moult the JH-III level was comparable to that of normally developing fifth instar larvae. Factors involved in the achievement of the haemolymph JH-titre are discussed.     

Influence of larval stage and virus inoculum on virus yield in insect host Neodiprion abietis (Hymenoptera: Diprionidae)

Virus yield produced by dead larvae of balsam fir sawfly, Neodiprion abietis (Harris) (Hymenoptera: Diprionidae), that had been infected at four different larval stages (second, third, fourth, or fifth instar) with two virus concentrations (10(5) polyhedral inclusion bodies (PIB) /ml or 10(7) PIB/ml), were analyzed and compared to determine the effects of instar and amount of virus inoculum on virus production. The results indicate that both larval stage and inoculation dosage significantly affect virus yield. On average, each dead larva produced 1.36-12.21 x 10(7) PIB, depending upon larval age and virus concentration of inoculation. Although each dead larva produced more PIB when it was inoculated in the fourth or fifth stage, inoculation of these larvae did not result in the highest virus yield because of low larval mortality. In terms of net virus return, third instars would maximize virus yield when they are inoculated with a virus concentration that can cause 95-100% larval mortality.     

Proteomic analysis of the silkworm (Bombyx mori L.) hemolymph during developmental stage

We utilized the proteomic approach to investigate the proteome of the fifth instar hemolymph during growth and development, and to improve the understanding of this important bioprocess and gene expression situation. A total of 25 microL of hemolymph was used for 2D analysis, and the separated proteins were visualized by silver staining and analyzed using the ImageMaster 2D software. The report showed as many as 241 of protein spots were expressed in the beginning of the fifth instar. Among them, most were concentrated in pI 3.5-6.5, which reached 76% of the total protein spots. As for the protein molecular sizes, 182 protein spots concentrated between 35 and 90 kDa, which comes to 75% of the total spots. When the larvae grow to the seventh day (total fifth instar duration was 9 days), 298 protein spots were visualized through 2D electrophoresis. Fifty-seven spots were newly expressed compared to the image of the first day in fifth instar. The results implied that these proteins are related to biosynthesis of silk protein and metamorphosis preparation from larva to pupa. In total, 19 protein spots including 6 special spots expressed in seventh day were analyzed through MALDI-TOF-MS. The relations between proteins and growth and development of silkworm were discussed.