Progress of developmental programme during the last larval instar of Bombyx mori: Relationships with food intake,ecdysteroids and juvenile hormone

The progress of developmental programme in the epidermal cells of last instar larvae of Bombyx mori was determined by ecdysteroid injections in normal and in JH-treated larvae. To clarify the importance of food intake in the control of development, starved animals were also used.The instar begins with a period during which the larval programme is expressed: this occurs in the presence of 20-hydroxyecdysone. Epidermal cells can thereafter secrete pupal cuticle after ecdysteroid injection although the larval programme is normally still present. During the last period only pupal characters can be expressed either in normal or in 20-hydroxyecdysone-injected larvae.These different developmental phases are not correlated with obligatory and facultative feeding periods.Transition from the first to the second phases is correlated with the absence of JH effects on pupal genes. JH applications during the second period, however, prevent the expression of pupal characters after 20-hydroxyecdysone injection. Thus, during this period, the pupal programme is not stabilized. Cellular reprogramming itself occurs at the onset of the last developmental period and is probably under the control of ecdysteroids.     

Protein synthesis during salivary gland development,ageing, and cell death in Chironomus tentans larvae

Salivary gland protein synthesis in Chironomus tentans larvae was analyzed from the mid-third instar to larval pupation. Correcting for stage specific variations in the specific activity of the amino acid pool revealed a 30–40% reduction in the rate of protein synthesis during the larval moult. Except for a transient increase early in the fourth instar, this low rate of protein synthesis was maintained until the pharate pupal period when protein synthesis dramatically increased: maximum synthesis occurred in mid-pharate pupae with a subsequent decline correlating with gland autolysis and cell death at pupation. Each developmental period was characterized by a particular pattern of secretory protein synthesis: high and 35,000 daltons peptides were maximally synthesized only at particular larval stages, being reduced or absent in post-ecdysis, diapause and autolysing salivary glands.Except for the ecdysone puffs, and as otherwise previously noted (Clever, 1961, 1962), puffing activity during the peri-moult period remained relatively constant and did not decrease by the 30–40% predicted from the decreased rate of protein synthesis. The nearly complete loss in synthesis of the 35,000 daltons peptide was not accompanied by regression in any puff.     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

Repeated larval diapause and diapause-free development in geographic strains of the burnet moth Zygaena trifolii Esp. (Insecta,Lepidoptera)

Summary Zygaena trifolii is a long-day insect with temperature-dependent photoperiodic responses. All larval instars are sensitive to photoperiod; however, diapause may occur at the third larval stage or any subsequent larval instars. There were quantitative differences within populations in the threshold photoperiod for diapause induction. The diapause response was polymorphic, so that larvae might enter diapause at different instars under the same culture conditions. Furthermore, decreasing photoperiods below a critical daylength shifted the diapausing instar towards earlier stages. Geographic strains of Z. trifolii showed discontinuous clinal variation. Near the northern edge of the distribution [Cologne (Köln), FRG], there is first an obligatory diapause, mainly during early instars, and additional facultative (repeat) diapauses during later larval instars in subsequent years. In the southern part of its distribution, this burnet moth is partially bivoltine in the field with a facultative first developmental arrest and a decreased capacity for repeated diapause (Valencia, Spain; Marseille, France). Further experiments indicated that the photoperiodically controlled diapause reaction is also influenced by the number of photoperiodic cycles experienced during the period spent in each larval instar, which depends on temperature. The adaptive significance of obligatory and facultative repeated diapause, varying even among the offspring of a single female, may be to buffer the populations against the more extreme and, from year to year, unpredictable fluctuations in climatic conditions at the northern edge of the distribution.Abbreviations L₃ feeding 3rd larval instar - L₄D diapausing 4th larval instar - L₅D2 repeat-diapausing larval instar with second diapause at the 5th larval stage - LD light-dark cycle - KT shortday conditions (e.g. LD 8:16) - LT long-day conditions (e.g. LD 16:8)     

Control of activity and regression of the silk glands in the last-larval instar of Galleria mellonella

The weight increase of silk glands in isolated larval abdomens is enhanced by brain implants which stimulate RNA and protein synthesis. It is proposed that, in intact last-instar larvae, a blood-borne factor from the brain promotes silk gland activity during the facultative feeding period. In the post-feeding larvae, some starving larvae and in isolated larval abdomens the silk glands regress. The regression is accelerated and the glands degenerate under the action of 20-hydroxyecdysone. This effect is not associated with an increase in protease and RN-ase activities. In the normal larvae, the protease activity increases gradually during the last instar to a maximum at the time of regression and drops in the degenerating glands. RN-ase activity is maintained at a high level in the fully active, regressing and degenerating glands The results indicate that regression and degeneration of the silk glands are caused by structural cell reorganization rather than by an appearance of cytolytic enzymes.     

Stage-dependent effects of 20-hydroxyecdysone on DNA synthesis of corpus allatum cells in the silkworm,Bombyx mori

DNA synthesis in cells of the corpus allata (CA) of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU); developmental changes during the 3rd, 4th, and last larval instars and effects of 20-hydroxyecdysone treatment were examined. During both the 3rd and 4th larval instars, the number of DNA-synthesizing cells fluctuated, and relatively low levels were observed during the middle stages. On day 0 of the last larval instar, the number of DNA-synthesizing cells per gland was 9.2, which then increased on day 1 and remained at levels ranging from 12.9 and 16.9 cells per gland. A major peak level (28 BrdU-labeled cells per gland) occurred on day 8, two days after larvae entered the wandering stage. When last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 0 or 1, the number of DNA-synthesizing cells dramatically decreased to very low levels and these low levels were maintained throughout the remainder of the instar. However, no effect was observed when last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 3, indicating the stage-specific action of 20-hydroxyecdysone. The mechanism by which 20-hydroxyecdysone treatment inhibits DNA synthesis of CA cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. It was found that the decrease in responsiveness of DNA synthesis of CA cells of 20-hydroxyecdysone-treated larvae to stimulation by growth factors from hemolymph may have been, at least in part, responsible for the indirect inhibitory effects of 20-hydroxyecdysone.     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.     

Synthesis of two storage proteins during larval development of the tobacco hornworm, Manduca sexta

Studies of synthesis and accumulation of the two storage proteins arylphorin and female-specific protein (FSP) during the final two larval instars of the tobacco hornworm showed both stage and temporal specificity. Arylphorin was present in both stages, but its synthesis ceased during the molt, during starvation, and at the wandering stage, and then resumed about 24 hr after the onset of feeding. During the larval molt about 25% of injected iodinated arylphorin was incorporated into the newly forming fifth instar cuticle. The cessation of arylphorin synthesis was mimicked by exposure of the fat body to 1 microgram/ml 20-hydroxyecdysone (20HE) in complete Grace's medium or to dilutions of Grace's medium greater than 50%. Lower concentrations of 20HE were ineffective, indicating that the cessation of synthesis in vivo was likely due to a combination of lack of excess nutrients and the hormonal milieu. The female-specific protein was not synthesized until the final larval instar, appearing first in females on Day 2 and later in males at the time of wandering, with synthesis continuing throughout the prepupal period. In vitro studies showed that this protein was synthesized as a 620-kDa protein, and then during secretion a 730-kDa immunoreactive form also appeared. Synthesis of FSP was inhibited by exposure of Day 2 fat body to 1 microgram/ml 20HE for 24 hr. Ligation followed by 20HE infusion showed that the disappearance of FSP from the hemolymph during the prepupal period was controlled by the rising ecdysteroid titer.     

Temporal changes in DNA synthesis of prothoracic gland cells during larval development and their correlation with ecdysteroidogenic activity in the silkworm, Bombyx mori

DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the 3rd, 4th, and last larval instars were examined. During the early stages of both the 3rd and 4th larval instars, a dramatic increase in the number of DNA-synthesizing cells of the prothoracic glands was detected. However, during the latter stages of each instar, the number of DNA-synthesizing cells greatly decreased. The determination of glandular protein content showed that dramatic increases occurred during the latter stages of each larval instar. Comparison of changes in prothoracic gland cell DNA synthesis with ecdysteroidogenic activity showed that the increase in DNA synthesis precedes ecdysteroidogenesis. The cellular mechanism underlying changes in prothoracic gland cell DNA synthesis during the last two larval instars was further analyzed by determining the in vitro DNA synthesis of the glands, their responsiveness to hemolymph growth factors, and changes in the growth-promoting activity of hemolymph during development. It was found that both growth factors and the responsiveness of the prothoracic gland cells to growth factors from hemolymph may play roles in regulating DNA synthesis of gland cells.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

Developmental regulation of silk protein P25 in the silkwormBombyx mori

P₂₅ protein was extracted from cocoons of the silkwormBombyx mori by alkali solubilization and purified by gel elution. The purity and authenticity of the protein were confirmed by SDS-PAGE, 2-dimensional gel electrophoresis and peptide mapping. Polyclonal anti-P25 sera were raised in rabbit and mice. The relative abundance of P₂₅ protein present in the larva during different developmental stages was analysed by SDS-PAGE, and quantified by sandwich ELISA. The minimum level (0.2 μg/animal) of this protein was recorded at the beginning of the first instar and maximum (16.7mg/pair of silkgland) on the final day of the V instar. During each moult period, P₂₅ protein level was suppressed; the level increased with the initiation of feeding and reached maximum on the 3rd day of each instar except the final instar where the maximum was recorded prior to pupal moult. Western blot analysis also confirmed the developmental stage-specific accumulation of P₂₅ protein in the silkwormBombyx mori.     

桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用

【目的】明确桃蛀螟Conogethes punctiferalis幼虫取食Cry1Ab蛋白后体内CYP6AE76的过表达及对Cry1Ab蛋白有解毒作用。【方法】分析桃蛀螟CYP6AE76序列特征;利用RT-qPCR检测CYP6AE76在不同发育阶段(1-5龄幼虫)和4龄幼虫不同组织(头、中肠、血淋巴和脂肪体)以及4龄幼虫取食含有Cry1Ab蛋白(LC₅₀=1.08 ng/cm2)的人工饲料3 d存活的幼虫中肠和血淋巴中的表达量;利用RNAi饲喂法沉默桃蛀螟4 龄幼虫CYP6AE76后检测中肠中CYP6AE76的表达量,并统计120 h后幼虫体重并计算幼虫存活率;利用RNAi饲喂法沉默桃蛀螟初孵幼虫CYP6AE76后饲喂含1.08 ng/cm² Cry1Ab蛋白的饲料,7 d后统计幼虫体重并计算幼虫存活率。【结果】桃蛀螟CYP6AE76基因开放阅读框长1 572 bp,编码524个氨基酸,分子量约为60.34 kD,属于CYP6家族基因。发育表达谱结果表明, CYP6AE76基因在桃蛀螟整个幼虫阶段均有表达且在1龄幼虫期表达量最高,随着幼虫龄期增大而表达量降低;组织表达谱结果表明,CYP6AE76在4龄幼虫中肠中表达量最高。4龄幼虫取食含有Cry1Ab蛋白(1.08 ng/cm²)的人工饲料后,CYP6AE76在中肠和血淋巴中的表达量相比对照显著上调。通过RNAi沉默CYP6AE76后,桃蛀螟初孵幼虫再取食含有Cry1Ab蛋白的人工饲料后体重显著降低。【结论】CYP6AE76可能参与对桃蛀螟幼虫摄入的Cry1Ab蛋白的解毒。     

Expression of the major tyrosyl phosphoprotein of 54 KDa in the integument of the medflyCeratitis capitata

Expression of a 54 kDa tyrosyl phosphorylated protein in epidermal cells during the third instar larval stage was followed. It was demonstrated that the 54 kDa protein moiety and its phosphorylated counterpart follow the same developmental profile. The system seems to be regulated only at the onset of the second moult, by an initial signal which regulates both the synthesis and phosphorylation of a 54 kDa protein. The continuous presence this protein in epidermal cells during the third instar stage, as well as during apolysis and histolysis, suggests that it might participate in cell activities taking place during this developmental period. However, the 54 kDa protein could no be involved in specific epidermal cell activities such as histolysis, melanization and sclerotization, since these activities occur only at specific times during the third instar stage.     

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.     

The effect of the juvenile hormone analog, fenoxycarb, on ecdysone receptor B1 expression in the midgut of Bombyx mori during larval-pupal metamorphosis

The Bombyx mori (Lepidoptera: Bombycidae) midgut undergoes remodeling during the larval-pupal metamorphosis. All metamorphic events in insects are controlled by mainly two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate, has been shown to be one of the most potent juvenile hormone analogs against a variety of insect species. In this study, the effect of fenoxycarb on EcR-B1 protein expression in the midgut of Bombyx mori during the remodeling processwas investigated. Fenoxycarb was topically treated to the beginning of the fifth instar Bombyx larvae. Its application prolonged the last instar and prevented metamorphic events. Analyses were performed from day 6 of the fifth instar to 24 hr after pupation in controls and to day 14 of the fifth instar in the fenoxycarb treated group. According to our results, the presence of EcR-B1 in the midguts of the fenoxycarb treated group during the feeding period suggested that EcR-B1 was involved in the functioning of larval cells and during this period fenoxycarb did not affect EcR-B1 status. Immediately after termination of the feeding stage, the amount of EcR-B1 protein increased, which indicated that it may strengthen the ecdysone signal for commitment of remodeling process. In the fenoxycarb treated group, its upregulation was delayed, which may be related to the inhibition of ecdysone secretion from the prothoracic gland.     

A developmental study of the cuticular hydrocarbons of Sarcophaga bullata.

The accumulation of cuticular hydrocarbon was measured throughout the life of Sarcophaga bullata. Less than 5 μg hydrocarbon per insect are present until the third larval instar when synthesis increases the quantity present to 10 to 20 μg by pupariation. The rate of synthesis increases at this time to 5 to 8 μg/day and continues until 40 to 45 μg are present per insect. This amount remains constant until several days before the pupal-adult ecdysis when synthesis again occurs. The rate of synthesis by these pharate adults is >20 μg/day. When the adult emerges it contains between 90 and 100 μg which increases slightly during the adult stadium. Two periods of rapid accumulation of cuticular hydrocarbon are observed: (1) during pupariation and the 3 day period following pupariation, and (2) during the 4 day period preceding the pupal-adult ecdysis. When pupariation is inhibited by contact with water, the rate of hydrocarbon biosynthesis also fails to increase.     

Transition from larva to pupa: morphogenesis,cell proliferation and protein synthesis in Bombyx wing disc

Summary

In the present study, an attempt was made to clarify the timing of the transition from proliferation stage to pupal preparation stage of Bombyx wing discs and the correlation of its timing and ecdysteroid release. An histological study was carried out during the period of ecdysteroid increase in hemolymph in the last larval instar. The number of mitoses in wing discs from the feeding stage to the spinning stage was counted. During the feeding stage, the number of mitoses was about 200/disc, with the number increasing up to about 850/disc on the day of the beginning of spinning (S0); it then decreased to about 250/disc at the S1 stage, and finally mitotic figures were absent at S2. Comparing the mitotic number with hemolymph ecdysteroid titer, ecdysteroid during the high mitotic period is close to 100ng/ml, while ecdysteroid during the low mitotic period is about 10ng/ml at day 5 and 500ng/ml at S1, respectively. Two-dimensional gel electrophoresis was carried out to identify the ecdysone-induced polypeptides or the polypeptides synthesized during the spinning stage. Five ecdysteroid-inducible polypeptides were observed during the spinning stage or after 20-hydroxyecdysone (20E) treatment. From these findings, low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation and induce new protein synthesis.     

Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans

The concentration of free amino acids, total soluble protein, and haemoglobin in the haemolymph of fourth instar Chironomus tentans was investigated.The concentration of the free amino acid pool increases between the early (15.7 mM/l) and mid-(33.9 mM/l) fourth larval stages followed by a decline during the late (16.9 mM/l) fourth larval period. Alanine, serine, and the amides of aspartic acid and glutamic acid are the predominant free amino acids at all stages. Physiological fluid analysis of late fourth instar haemolymph detected 32 ninhydrin positive components including 18 common amino acids plus homoarginine, ornithine, citrulline, β-alanine, α-aminoadipic acid, α-aminoisobutyric acid, and sarcosine.The concentration of total soluble protein steadily increases during fourth instar larval development to a maximum of $\text{9.3}\text{g}\text{100}\text{ml}$ followed by a decline during the pharate pupal period. A similar pattern of variation occurs in haemoglobin content which comprises from 51 to 66% of Chironomus tentans haemolymph protein.The mM percentage of individual amino acids of total haemolymph protein varies little during the fourth instar. At all stages alanine and aspartic acid are the predominant amino acids.