Tyrosine metabolism for insect cuticle tanning

Insects have become one of the most successful animal groups in diversity and numbers through the development of a multifunctional exoskeleton and skin, which must be shed periodically in order for them to grow and develop into adults. The evolutionary choice of certain structural materials for the assembly and stabilization of a cuticle with remarkable mechanical and chemical properties has allowed insects to invade terrestrial environments and to evolve flight mechanics for dispersion relatively early in geological history. Diphenolic compounds derived from tyrosine play a central role in sclerotization or tanning of the new cuticle. The phenolic amino acid is stored during larval feeding, and it is mobilized for the production of both structural proteins and diphenolic tanning precursors that are transported into the cuticle. The latter compounds permeate the cuticle and serve as precursors for quinonoid derivatives that both sclerotize and pigment the exoskeleton. This report focuses on how tyrosine and derived diphenolic structures are stored as inactive molecules in preecdysial stages, and how they are released and metabolized to tanning chemicals that stabilize the new cuticle.     

Ecdysteroid production by a continuous insect cell line

The spent medium from ten established cell lines was extracted and tested for ecdysteroids by radioimmunoassay. Of the seven lepidopteran lines tested, only IAL-TNDI and MRRL-CH showed evidence of ecdysteroid production. However, the results were erratic and difficult to evaluate and these lines were dropped from further consideration. However, of the three cockroach cell lines tested, one, UMBGE 4, produces ecdysteroid and consistently releases virtually all of it into the medium. The main ecdysteroid was identified as ecdysone and the increase was logarithmic during the first 11 days of the subculture, with a decrease from day 11 to day 14. UMBGE 4 is a vesicle cell line which also tested positive for chitin synthesis. When the pH of the medium was lowered from pH 7.4 to pH 6.3, both the chitin synthesis and the ecdysone synthesis dropped by roughly 50%.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro

Summary This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0μg/ml); (b) continuous treatment with 0.2μg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0μg/mlβ-ecdysone in medium conditioned with larval fat body. Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change. i.e., cell aggregation, in response to treatment with 1 microM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20-30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.     

Chitinous cuticle and systematic position of tardigrada

The chitinous nature of the cuticle, jaws and stomodaeum of three species of Tardigrada has been definitely proven using a specific micromethod involving a preparation of purified chitinase. These chemical characteristics are in favour of the phylogenetic closeness between Tardigrada and the Arthropoda.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

A new form of insect cuticle

A hitherto unnoticed, harder form of cuticle, which occurs on the mandibles of the Australian plague locust, Chortoicetes terminifera , is described     

昆虫的变态发育研究

昆虫变态发育使得昆虫成为地球陆地上种类最多、数量最大、分布最广、生活环境最多样化的一群生物。变态使昆虫在其生命周期中的不同发育时期表现出完全不同的形态、结构、功能和生活习性的变化,有利于昆虫迁飞转移,扩大其求偶交配、生活和生存环境空间。昆虫变态发育的变化是长期自然环境适应、协同进化的结果,受激素、营养和基因的精确调控。本文简要介绍了昆虫变态的类型、激素调控、营养调控和基因调控方面的研究进展,以及研究昆虫变态发育的科学和应用意义。     

Genomic inference accurately predicts the timing and severity of a recent bottleneck in a nonmodel insect population

The analysis of molecular data from natural populations has allowed researchers to answer diverse ecological questions that were previously intractable. In particular, ecologists are often interested in the demographic history of populations, information that is rarely available from historical records. Methods have been developed to infer demographic parameters from genomic data, but it is not well understood how inferred parameters compare to true population history or depend on aspects of experimental design. Here, we present and evaluate a method of SNP discovery using RNA sequencing and demographic inference using the program δaδi, which uses a diffusion approximation to the allele frequency spectrum to fit demographic models. We test these methods in a population of the checkerspot butterfly Euphydryas gillettii. This population was intentionally introduced to Gothic, Colorado in 1977 and has as experienced extreme fluctuations including bottlenecks of fewer than 25 adults, as documented by nearly annual field surveys. Using RNA sequencing of eight individuals from Colorado and eight individuals from a native population in Wyoming, we generate the first genomic resources for this system. While demographic inference is commonly used to examine ancient demography, our study demonstrates that our inexpensive, all‐in‐one approach to marker discovery and genotyping provides sufficient data to accurately infer the timing of a recent bottleneck. This demographic scenario is relevant for many species of conservation concern, few of which have sequenced genomes. Our results are remarkably insensitive to sample size or number of genomic markers, which has important implications for applying this method to other nonmodel systems.     

Fine structure of the chitin-protein system in the crab cuticle

The fine structure of the organic matrix of the shore crab cuticle (Carcinus maenas L.), observed in transmission electron microscopy, reveals three different levels of organization of the chitin—protein complex. The highest level corresponds to the ‘twisted plywood’ organization described by Bouligand (1972). Horizontal microfibrils, parallel to the cuticle plane, rotate progressively from one level to another. When viewed in oblique section this structure gives superimposed series of nested arcs, visible in light microscopy or at the lowest magnifications of the electron microscope, in all the chitin-protein layers. At the highest magnifications of the electron microscope and with the best resolution, when the ultrathin sections are exactly transverse to the microfibril, a constant pattern can be observed which consists of rods transparent to electrons, which are embedded in an electron-opaque matrix. In cross-section, these rods often form more or less hexagonal arrays. We call a microfibril one rod and the adjacent opaque material, and question the usual interpretation of the microfibril molecular structure. Between these two levels of organization, there is an intermediate level, which corresponds to the grouping of microfibrils. Microfibrils form a dense structure, with few free spaces in the membranous layer, the deepest and non-calcified layer of the cuticle. In other parts of the cuticle, microfibrils are grouped into fibrils of various diameters or form a reticulate structure, the free spaces of the organic matrix being occupied by the mineral.     

An assembly zone antigen of the insect cuticle

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.     

Cell culture techniques in the search for cancer viruses of man

Summary A combination of biochemical and electron microscopic methods has been utilized to identify viruses present in cell cultures derived from human cancer patients. Two independently established cell lines from breast carcinoma patients have been shown to contian a virus that morphologically appears closely related to the murine mammary tumor virus. Presented at the 20th Annual Tissue Culture Association Meeting, Washington, D. C., June 1970.     

EPR techniques for studying radical enzymes

EPR studies on radical enzymes are reviewed under the aspects of the information that they can provide and of the techniques that are used. An overview of organic radicals derived from amino acids, modified amino acids, and cofactors is given and g tensor data are compiled. The information accessible from a spectroscopic point of view is contrasted with the information required to understand enzyme structure and function, and some precautions are discussed that must be taken to derive the latter kind of information from the former. Structural dynamics is identified as an aspect that has rarely been addressed in the past although it is highly relevant for enzyme function. It is proposed that techniques introduced recently on other classes of proteins could help to close this gap.     

EPR techniques for studying radical enzymes

EPR studies on radical enzymes are reviewed under the aspects of the information that they can provide and of the techniques that are used. An overview of organic radicals derived from amino acids, modified amino acids, and cofactors is given and g tensor data are compiled. The information accessible from a spectroscopic point of view is contrasted with the information required to understand enzyme structure and function, and some precautions are discussed that must be taken to derive the latter kind of information from the former. Structural dynamics is identified as an aspect that has rarely been addressed in the past although it is highly relevant for enzyme function. It is proposed that techniques introduced recently on other classes of proteins could help to close this gap.     

Protein cross-linking by peroxidase: Possible mechanism for sclerotization of insect cuticle

Incubation of test proteins with horseradish peroxidase in the presence of hydrogen peroxide and a catechol resulted in polymerization and precipitation of test proteins. SDS-PAGE readily revealed the generation of dimers, trimers, and higher oligomers in the reaction mixture. With the exception of 3,4-dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three catechols to accomplish polymerization is attributed to their high rate of intramolecular cyclization, which results in melanin formation. Radioactive studies with [³H]N-acetyldopamine clearly reveal both intermolecular and intramolecular cross-linking of test proteins by peroxidase. Based on these studies a possible mechanism for sclerotization and the biological significance of peroxidase in cuticle is discussed.     

Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation

Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration.     

Future questions in insect chitin biology: A microreview

This microreview stems from the Second Symposium on Insect Molecular Toxicology and Chitin Metabolism held at Shanxi University in Taiyuan, China (June 27 to 30, 2017) at the institute for Applied Biology headed by Professor Enbo Ma and Professor Jianzhen Zhang.