DNA replication events during larval silk gland development in the silkworm, Bombyx mori

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.     

Cell cycle events during the development of the silk glands in the mulberry silkworm<Emphasis Type="Italic"> Bombyx mori</Emphasis>

Silk glands of the mulberry silkworm Bombyx mori are long and paired structures originating from the labial region and are anatomically and physiologically divided into three major compartments, the anterior, middle and posterior silk glands. The silk gland morphogenesis is complete by 8 days post egg laying. Extensive growth of silk glands during the larval stages is due to increase in tissue mass and not cell number. The cells in a completely formed silk gland pursue an endoreplicative cell cycle, and the genome undergoes multiple rounds of replication without mitosis or nuclear division. The expression patterns of cyclin B (mitotic cyclin) and cyclin E (G1 cyclin, essential for G1/S transition in both mitotic and endoreplicative cell cycles) in the course of silk gland development revealed that mitotic cell divisions take place only in the apex of the growing silk gland. However, the persistence of another mitotic focus in the middle silk gland even when the growing apex has moved well past this zone suggested the continued operation of mitosis for a while in this restricted region. The lack of cyclin B expression and abundance of cyclin E in the rest of the areas confirmed an alternation of the G1 and S phases of the cell cycle without an intervening mitotic phase. No expression of cyclin B was noticed anywhere in the silk glands after stage 25 of embryogenesis, indicating a complete switch over to the endomitotic mode of the cell cycle. The onset of expression of various genes encoding different silk proteins correlated with the onset of endomitotic events.Edited by D. Tautz     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

Regulation of genes for ubiquitination and SUMO-specific protease involved in larval development of the silkworm,Bombyx mori

Protein modifications with highly conserved small proteins, such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO), regulate various cellular processes; however, the contribution of these protein modifications to larval development in insects has not yet been elucidated. We investigated the regulation of genes for these protein modifications in the posterior silk gland (PSG) during larval development of the silkworm Bombyx mori. We found that several genes encoding enzymes (E1, E2, and E3) for ubiquitination and SUMO-specific protease were upregulated by 20-hydroxyecdysone (20E), and, consistently, increases in ubiquitinated proteins were observed during the fourth molting stage. An injection of 20E into larvae at the fourth feeding stage induced higher expression levels of these E1, E2, and E3 genes and ecdysis approximately one day earlier than in mock-treated larvae. The expression of the fibroin heavy-chain gene (fibH) was simultaneously suppressed approximately one day earlier in 20E-injected larvae. The treatment of cultured PSG with 20E also induced these genes, which could be categorized into at least two types: those induced by a high dose of 20E, or by a pulse of 20E. In contrast to the 20E treatment, the administration of PR-619, an inhibitor of Ub- and SUMO-specific proteases in larvae, delayed ecdysis and prolonged the expression of fibH. These results suggest that the regulation of genes for ubiquitination and SUMO-specific protease is involved in the larval development of B. mori.     

Fenoxycarb modulates ecdysone receptor B1 and programmed cell death of the anterior silk gland of silkworm Bombyx mori

Fenoxycarb, O‐ethyl N‐(2‐(4‐phenoxyphenoxy)‐ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth‐instar larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior silk glands (ASG) and ecdysone receptor B1 (EcR‐B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20‐hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior silk glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR‐B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior silk glands and EcR‐B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR‐B1 expression in the anterior silk glands for programmed cell death process.     

Endomitosis and the effect of gibberellic acid in different Pisum sativum L. cultivars

The evolution of the total amount of DNA in epicotyls and of the amount of DNA per cell nucleus in epicotyl cortex cells during germination was followed in two closely related pea varieties, Pisum sativum cv. Finale and Pisum sativum cv. Rondo. Under etiolating conditions, growth of the cv. Rondo occurs only by cell elongation which is preceded by endomitotic DNA synthesis, while in the cv. Finale growth is the result of cell elongation accompanied by endomitotic DNA synthesis and cell division. The maximum C-level attained in both cultivars under etiolating conditions is 8 C (C=haploid amount of DNA in a gamete cell). Both the maximum C-level reached and the percentage of cells reaching this C-level seem to be under strict genetic control. In both cultivars, light inhibits the endomitotic DNA replication.Neither gibberellic acid (GA₃), nor AMO 1618 alter the maximum C-level or the percentage distribution of the C-classes. Both growth regulators are effective, although in an opposite way, only in tissues where cell division occurs or where endomitotic DNA synthesis is blocked, as in light-grown pea epicotyls.     

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori

Small non-protein coding RNAs （ncRNAs） play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs （four small nucleolar RNAs and five unclassified ncRNAs） that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm- 152, exhibited converse expression pattem with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.     

家蚕蛹变态期丝腺组织的退化与细胞凋亡特征

利用形态学观察方法、分子生物学检测方法以及20-羟基蜕皮酮(20-hydroxyecdysone)和放线菌酮(cycloheximide)体外培养方法, 研究了家蚕Bombyx mori 蛹变态期丝腺组织的退化与细胞凋亡特征。显微镜的观察显示家蚕丝腺的逐渐退化发生在吐丝期间。DNA梯度电泳的分析表明程序性细胞死亡(programmed cell death)可能伴随发生在丝腺的退化过程中。在离体培养条件下, 用20-羟基蜕皮酮处理5龄第6天幼虫的丝腺, 导致的细胞凋亡提前于对照, 提示在进入蛹变态期前, 20-羟基蜕皮酮提早激发了介导家蚕丝腺细胞凋亡与水解机制的遗传调控级联系统。上述结果表明, 20-羟基蜕皮酮能够诱导家蚕丝腺组织在蛹变态期发生程序性细胞死亡。     

Effect of starvation and re‐feeding on growth performance and content of plasma lipids,glucose and insulin in cultured juvenile Persian sturgeon (Acipenser persicus Borodin, 1897)

The effect of starvation and subsequent re‐feeding to satiation on compensatory growth performance, insulin and blood serum values were investigated in juvenile Persian sturgeon (Acipencer persicus) with an average weight 108.04 ± 0.28 g (mean ± SEM) and in the same rearing condition over an 8‐week period. Sturgeons were allocated to one of five feeding treatments: controls (C, continuous feeding), W1 (1 week starvation), W2 (2 weeks starvation), W3 (3 weeks starvation) and W4 (4 weeks starvation), followed by a single 4 weeks of re‐feeding to satiation. Changes in growth performance and blood serum indices were examined at the end of weeks 4 and 8. Body weight, specific growth rate (SGR), condition factor (CF) and weight gain were determined to have significantly decreased during starvation. Fish starved for 1 week reached the same weight as the control fish after re‐feeding for 4 weeks, indicating that complete compensatory growth occurred. Although the specific growth rate in W2, W3 and W4 fish was greater than that in the control fish after re‐feeding, W2, W3 and W4 fish did not reach the same body weight as control fish at the end of re‐feeding period, and showed partial compensation only. Blood plasma, glucose and insulin concentrations did not change significantly during starvation and re‐feeding (P > 0.05). This suggests that sturgeon are able to maintain glycaemia during starvation, probably due to their non‐carbohydrate dietary source. Plasma total lipid and triglyceride levels increased in starvation treatments, whereas the increases were significant only in W3 treatment (P < 0.05). After a 4‐week re‐feeding period, their levels decreased in comparison to the starvation periods. Increases in plasma total lipid and triglyceride levels appear to be due to their roles as preferred nutrients for mobilization in Persian sturgeon and the magnitude and duration of compensatory growth depended on the length of food deprivation.     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm,Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearences are common for both cell death types. Autophagy features are also exhibited during the prepupal period. One of the lysosomal marker enzymes, acid phosphatase, levels are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that only apoptotic cell death not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.

Addendum to: Goncu E, Parlak O. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem 2008; In press.     

Developmental changes in the spinning apparatus over the life cycle of wolf spiders (Araneae: Lycosidae)

Spiders are characterized by their spinning activity. Much of the current knowledge of the spinning apparatus comes from studies on orb web spiders and their relatives, whereas wolf spiders have been more or less neglected in this respect. Therefore, we studied developmental changes in the spinning apparatus of four wolf spiders (Tricca lutetiana, Arctosa alpigena lamperti, Pardosa amentata, and Xerolycosa nemoralis) throughout their life cycles. Each of these lycosids has a stenochronous life cycle, but of varied length (from 1 to 3 years) and number of instars (from seven to ten). Use of the spinning apparatus begins in the first instar, after leaving the egg sac. Secondary ampullate, all piriform, and all but four aciniform glands are tartipore‐accommodated. The tartipores, collared openings through which silk gland ducts pass during proecdysis, appear on the spinning field starting with the second instar. Tartipore‐accommodated glands can function during proecdysis and their evolution corresponds with the way spiders secure themselves when molting. We suggest that the function of aciniform silk in juvenile wolf spiders is to serve as an ancillary “scaffold” supporting the spider's body during ecdysis.     

Examining growth rate and starvation endurance in pit‐building antlions from Mediterranean and desert regions

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Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori

Class B scavenger receptors (SR‐Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR‐Bs; it is necessary to study these SR‐Bs for revealing their function. In this study, we cloned the full‐length coding sequence of BmSCRBQ4, a SR‐B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.     

Control of ecdysteroidogenesis: Activation and inhibition of prothoracic gland activity

The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insectManduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca²⁺ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a β tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the gland's potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.