Organic and inorganic compound variations in haemolymph, epidermal tissue and cuticle over the molt cycle in Scylla serrata (Decapoda)

Analysis of organic and inorganic compounds in haemolymph, epidermal tissue and cuticle, allowed the correlation of their content variation during the molt cycle in Scylla serrata (Decapoda). The extrusion of carbohydrate, chitin, H(2)O- and NaOH-soluble proteins of the epidermal tissue from D2 stage on suggested an early formation of the organic fraction in the new cuticle. The additional secretion of HCl-soluble protein, glycosaminoglycans, calcium, phosphorus and sulfur in D2-D3 stages suggests mineral nucleation shortly before or at ecdysis. This is consistent with the dominant content of proteins and chitin associated with peaks of carbohydrates and GAG-sulfur groups detected in the cuticle immediately after ecdysis. Furthermore, the maximal phosphorus content at this moment suggests calcium, magnesium and manganese phosphate deposits. After an intense accumulation of calcium, chitin and sharp decrease of protein from A to B stages, a constant rate of their deposition in intermolt and dissolution in premolt occurred. The variations concerning proteins, carbohydrates, glycosaminoglycans, calcium, magnesium and sulfur in the haemolymph, before and after ecdysis, suggest a transitory phenomenon for calcium binding and releasing. Other major elements such as, sodium, potassium and chloride may function as osmotic regulators in the haemolymph and in epidermal compartments. The copper profile presented an indicator role for variations of osmolality over the molt cycle.     

Chitinase activity during Drosophila development

Before both larval moults in Drosophila melanogaster, the chitin in the cuticle is digested to a significant degree by the moulting fluid. A spurt of chitinase activity appears just before each ecdysis, drops sharply after the first ecdysis, and begins to rise again just about the time that chitin degradation becomes evident. The level of enzyme activity/mg of soluble protein reached just before the second ecdysis is about twice that reached before the first, and this declines gradually after the ecdysis until puparium formation. Chitinase activity is measured with a viscometric assay on a chitosan substrate.The enzyme activity is stable, with no loosely bound cofactor. Data also exist supporting the presence of more than one enzyme fraction in Drosophila with chitinase activity.     

The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.     

Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

Femur cuticle from fifth instar larvae of the desert locust, Schistocerca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted to the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotization continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned.     

Cuticular strength and pigmentation of rust-red and black strains of Tribolium castaneum: Correlation with catecholamine and β-alanine content

Rust-red wild and black mutant strains of the red flour beetle, Tribolium castaneum, were used to investigate temporal patterns of catecholamine and β-alanine content during sclerotization and pigmentation of adult cuticle and to relate these patterns to corresponding changes in cuticle resistance to puncture. Rust-red elytral cuticle sclerotized more rapidly than black cuticle until 6 days after adult eclosion when both became equal in puncture resistance. The cuticular concentrations of $\text{N-β-}\text{alanyldopamine}$ (NBAD), β-alanine and 3,4-dihydroxyphenylacetic acid (DOPAC) increased more rapidly in the rust-red strain than in the black strain during the first 7 days following adult eclosion. Conversely, cuticular dopamine increased more rapidly in black than in the red strain. Thus the rust-red pigmentation and rapid sclerotization appear to be related to the availability of β-alanine, $\text{N-β-}\text{alanyldopamine}$ and DOPAC. Melanization was prevented and rust-red pigmentation induced by injections of β-alanine or NBAD into newly ecdysed black mutant beetles. Crosses of the two strains generally had intermediate levels of cuticular dopamine and β-alanine, but the NBAD levels were similar to those of the rust-red strain. Dopamine, NBAD and DOPAC levels became similar in both black and rust-red strains about 6 days after adult ecdysis as did resistance to puncture. Therefore, dopamine appears to be directed initially into the melanin pathway in black adults due to a temporary lack of $\text{N-}\text{acylation}$ with β-alanine. After the melanization phase, dopamine is metabolized to sclerotization precursors eventually resulting in normal physical properties of the exoskeleton.     

Quantitative studies on the activity of the corpora allata in adult male Locusta and Schistocerca

Juvenile hormone has been detected in the haemolymph and corpora allata of adult male Locusta and the haemolymph of adult male Schistocera by a modified Galleria bioassay. The hormone was readily detected in the haemolymph of insects immediately after the final ecdysis, but then became difficult to detect until 2 days prior to the onset of sexual maturation. In sexually mature insects the titre of juvenile hormone was maintained at a constant level. The corpora allata of adult male Locusta increased in size throughout adult life. The juvenile hormone content of the corpora allata was low during the period of somatic growth, but increased at the onset of sexual maturation. Sectioning of the nervi corporis allati I in insects immediately after the final ecdysis prevented the normal increase in size of the corpora allata, but did not render them inactive since juvenile hormone was detected in the haemolymph after the operation. The half life of juvenile hormone in the haemolymph of allatectomized adult male Locusta was 1 to 2 hr.     

Possible involvement of cockroach haemocytes in the storage and synthesis of cuticle proteins

The injection of UL¹⁴C-leucine into newly ecdysed immature cockroaches resulted in the labelling of both haemocyte and serum proteins. Serum proteins were purified by gel filtration, concentrated and reinjected into other freshly ecdysed animals. After incubations of one hour, radioactivity was detected in serum, haemocyte, and cuticle proteins. Similar experiments using labelled soluble blood cell proteins also produced radioactivity in the serum, cells and cuticle. The possible rôle of haemocytes in cuticle protein synthesis is denoted and its significance in regard to cuticular tanning is discussed.     

Cuticle sclerotization by the American cockroach: Differential incorporation of leucine and tyrosine during post-ecdysis

The incorporation of U-¹⁴C-leucine into the cuticle occurs within the first 2 hr after ecdysis whereas U-¹⁴C-tyrosine is incorporated at a steady rate for approximately 8 hr. These data suggest that most of the cuticle protein is synthesized and laid down within a short time after ecdysis. On the other hand, tyrosine and/or metabolites (such as N-acetyl-dopamine) are translocated into the cuticle for several hours. This indicates that the sclerotization process may take place over an extended period.     

Cuticle laccase of the silkworm,Bombyx mori: Purification,gene identification and presence of its inactive precursor in the cuticle.

Laccase is a multi-copper enzyme found in variety of organisms including plants, fungi and bacteria. In insects, laccase is thought to play an important role in cuticle sclerotization with its ability to catalyze the oxidation of phenolic compounds to their corresponding quinones. From the newly ecdysed pupae of the silkworm, Bombyx mori, we purified a dimer form of cuticular laccase with 70-kDa polypeptides. Mass spectrometric analysis of the tryptic fragments and cDNA sequence analysis revealed that the gene for the purified laccase (BmLaccase2) is an ortholog of laccase2, one of the multiple laccase genes found in insect genomes. BmLaccase2 is highly expressed in the epidermis prior to ecdysis, suggesting that the BmLaccase2 protein accumulates before ecdysis. However, the cuticle of newly ecdysed pupa does not have laccase activity, and the activity only becomes detectable several hours after ecdysis. These data suggest that cuticle laccase is synthesized as an inactive precursor, which is later activated after ecdysis. We also found that urea-solubilized cuticle protein extract contains an inactive form of laccase that can be activated by trypsin treatment.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper,Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1-¹⁴C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1-¹⁴C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.     

Identification of a cuticle protein with unique repeated motifs in the silkworm,Bombyx mori

The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at the N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.     

Catecholamines in the cuticles of four strains of the german cockroach Blattella germanica (L.) during sclerotization and melanization

Catecholamines were extracted from the cuticles of four strains of the cockroach Blattella germanica at different times 48 h after adult ecdysis and analyzed by reverse phase HPLC with electrochemical detection. The wild (VPl), black (Bl), orange (or), and yellow (y) phenotypes differ in cuticular pigmentation, particularly in the extent of melanization. N-β-Alanyldopamine (NBAD) and N-β-alanylnorepinephrine (NBANE) were major o-diphenolic compounds in extracts from cuticle of all strains during the main period of sclerotization. N-Acetyldopamine (NADA) and N-acetylnorepinephrine (NANE) were minor the first day after ecdysis, but accumulated to higher levels thereafter. Dopamine (DA) concentrations were higher in the darker pigmented cuticles of strains Bl and or than in the lighter-colored cuticles of strains VPl and y. Extractable DA rapidly increased in VPl, Bl, and or cuticles shortly after ecdysis, reached peak levels 6–24 h later, and then decreased after melanization. Only small amounts of DA were detected in strain y cuticle, whereas NBANE concentrations were very high. Therefore, high DA levels in cuticle are correlated with melanization that occurs during the first few hours after adult ecdysis, whereas sclerotization is correlated with high levels of the N-β-alanylcatecholamines. Sclerotization appears to be delayed in strain Bl, since only low concentrations of the N-acylated catecholamines accumulate until after melanization is completed.     

The control of chitin deposition by ecdysterone in larvae of Bombyx mori

The effect of ecdysterone on the deposition of chitin in Bombyx larvae was examined. The chitin content in the abdomen decreased following hormone treatment to a value half that of the controls. Studies with ¹⁴C-glucose revealed that whereas controls exhibited a gradual decrease in the rate of ¹⁴C-glucose incorporation into chitin, the administration of ecdysterone resulted in a rapid fall in the rate of incorporation followed by a rise until ecdysis occurred. Chitin catabolism was estimated at 0·11 mg chitin/hr, based on the chitin content and incorporation rates. A dual rôle is indicated for ecdysterone in chitin metabolism, namely the activation of both synthetic and lytic systems.     

闹羊花素类化合物对斜纹夜蛾幼虫表皮成分的影响及构效关系

从我国特有的高效杀虫植物闹羊花 Rhododendron molle G. Don中分离提纯了14个活性化合物,并研究了这些化合物对斜纹夜蛾Spodoptera litura幼虫表皮成分的影响及构效关系,为揭示闹羊花素类化合物的作用机制、优化结构及确定先导化合物提供了参考。通过IR,UV, NMR,MS等光谱鉴定了3个新化合物黄杜鹃素A, B, C (rhodomolin A, B,C)。结果表明: 闹羊花素Ⅲ（rhodojaponin Ⅲ）,黄杜鹃素A,羊踯躅素Ⅰ（rhodomollein Ⅰ）,黄杜鹃素B ,黄杜鹃素C,羊踯躅素ⅩⅧ (rhodomollein ⅩⅧ),木藜芦素Ⅲ (grayant oxin Ⅲ)和对照药剂印楝素（azadirachtin）的生长发育抑制活性明显高于其他化合物,以50 mg/L浓度浸渍叶碟饲喂斜纹夜蛾4龄幼虫后,虫重、蛹重以及羽化率均显著低于空白对照。处理后120 h,各化合物处理和空白对照试虫表皮几丁质相对含量为28.15%～35.18%,差异不显著。闹羊花素Ⅲ,黄杜鹃素A, B, C,羊踯躅素ⅩⅧ和印楝素处理显著降低试虫表皮总蛋白的相对含量,提高了表皮总脂肪的相对含量。进一步研究发现,闹羊花素 Ⅲ和印楝素处理后,试虫表皮水溶性蛋白、氢键结合蛋白和共价键结合蛋白相对含量显著降低,弱键结合蛋白相对含量显著提高,而对电价键结合蛋白相对含量无明显影响。闹羊花素 Ⅲ处理试虫表皮水溶性蛋白相对含量为对照的50%。闹羊花素类化合物对昆虫生长发育抑制作用不属于“几丁质合成抑制型”,而属于“内分泌干扰型 ”,显著降低表皮水溶性蛋白是其重要机制之一。构效关系定性分析表明,木藜芦烷类闹羊花素化合物基本结构中的C-2,3环氧基、C-6、C-10和C-14取代基结构对化合物的生长发育抑制活性具有重要意义。还讨论比较了闹羊花素类化合物与印楝素对昆虫生长发育抑制作用机 制的差异。     

A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin

The physical properties of cuticle are determined by the structure of its two major components, cuticular proteins (CPs) and chitin, and, also, by their interactions.A common consensus region (extended R&R Consensus) found in the majority of cuticular proteins, the CPRs, binds to chitin. Previous work established that β-pleated sheet predominates in the Consensus region and we proposed that it is responsible for the formation of helicoidal cuticle. Remote sequence similarity between CPRs and a lipocalin, bovine plasma retinol binding protein (RBP), led us to suggest an antiparallel β-sheet half-barrel structure as the basic folding motif of the R&R Consensus. There are several other families of cuticular proteins. One of the best defined is CPF. Its four members in Anopheles gambiae are expressed during the early stages of either pharate pupal or pharate adult development, suggesting that the proteins contribute to the outer regions of the cuticle, the epi- and/or exo-cuticle. These proteins did not bind to chitin in the same assay used successfully for CPRs. Although CPFs are distinct in sequence from CPRs, the same lipocalin could also be used to derive homology models for one A. gambiae and one Drosophila melanogaster CPF. For the CPFs, the basic folding motif predicted is an eight-stranded, antiparallel β-sheet, full-barrel structure. Possible implications of this structure are discussed and docking experiments were carried out with one possible Drosophila ligand, 7(Z),11(Z)-heptacosadiene.     

The essential role of bursicon during <Emphasis Type="Italic">Drosophila</Emphasis>development

Background  

The protective external cuticle of insects does not accommodate growth during development. To compensate for this, the insect life cycle is punctuated by a series of molts. During the molt, a new and larger cuticle is produced underneath the old cuticle. Replacement of the smaller, old cuticle culminates with ecdysis, a stereotyped sequence of shedding behaviors. Following each ecdysis, the new cuticle must expand and harden. Studies from a variety of insect species indicate that this cuticle hardening is regulated by the neuropeptide bursicon. However, genetic evidence from Drosophila melanogaster only supports such a role for bursicon after the final ecdysis, when the adult fly emerges. The research presented here investigates the role that bursicon has at stages of Drosophila development which precede adult ecdysis.     

Catecholamines and related o-diphenols in the hemolymph and cuticle of the cockroach Leucophaea maderae (F.) during sclerotization and pigmentation

Developmental profiles of catecholamines and related o-diphenols in the hemolymph and cuticle of Leucophaea maderae were determined during sclerotization and pigmentation of last instar nymphs and adults. N-Acetyldopamine (NADA) and dopamine (DA) were the major o-diphenols in hemolymph, whereas 3,4-dihydroxyphenylketoethanol (DOPKET), N-β-alanyldopamine (NBAD), norepinephrine, 3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxyphenylalanine were detected at lower concentrations. The o-diphenols occurred primarily as acid-labile conjugates in hemolymph. Dopamine, conjugated as the 3-O-sulfate ester, and a NADA conjugate(s) were equal in concentration (0.06 mM) in nymphs shortly before adult apolysis. However, NADA increased after adult ecdysis to a peak at 6 h (0.18 mM), while its precursor DA decreased, suggesting N-acetylation of the latter or its metabolism to melanin pigments in the cuticle. In cuticle, NADA, N-acetylnorepinephrine (NANE), DOPKET, and N-β-alanylnorepinephrine (NBANE) accumulated during the early period of adult cuticle sclerotization. DOPKET and NADA (0.4 μmol g⁻¹ each), and NANE (0.2 μmole g⁻¹) occurred at the highest concentrations in tanned adult cuticle. Large amounts of DOPKET conjugates extracted by cold 1.2 M HCl from tanned cuticle which released DOPKET upon hydrolysis at 100°C for 10 min. DA and NBANE (0.2 μmole g⁻¹ each) predominated in tanned nymphal cuticle. Therefore, sclerotization of nymphal cuticle may require more of the N-β-alanyl catecholamines, whereas the adult cuticle contains larger quantities of the N-acetyl derivatives and ketocatechol (DOPKET) metabolites. Black pigmentation of nymphal and adult cuticle occurs during the first few hours after ecdysis, which correlates with relatively high levels of dopamine.     

Cuticular protein LmTwdl1 is involved in molt development of the migratory locust

The cuticle, an essential structure for insects, is produced from cuticular proteins and chitin via a series of biochemical reactions. Tweedle genes are important members of the cuticular protein family and have four conserved motifs binding to chitin. Tweedle family genes have been found to play a profound effect on cuticle development. Here, we report that the cuticular protein gene LmTwdl1 of Locusta migratoria belongs to the Tweedle family. In situ hybridization showed that LmTwdl1 is localized to epidermal cells of the cuticle. The expression patterns of LmTwdl1 showed low expression in the cuticle during the early and middle stages of the fifth‐instar nymphs; in contrast, its expression rapidly increased in the late stages of fifth‐instar nymphs. We performed RNA interference to examine the function of LmTwdl1 in locusts. Silencing of LmTwdl1 resulted in high mortality during the molting process before the next stage. Also, the epicuticle of nymphs failed to molt, tended to be thinner and the arrangement of chitin in the procuticle appeared to be disordered compare to the control group. These results demonstrate that LmTwdl1 plays a critical role in molting, which contributes to a better understanding of the distinct functions of the Tweedle family in locusts.