Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

Different Functions of the Insect Soluble and Membrane-Bound Trehalase Genes in Chitin Biosynthesis Revealed by RNA Interference

Background

Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported.

Principal Findings

The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%.

Conclusions

SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut.     

家蚕几丁质去乙酰化酶BmCDA2的基因表达和免疫定位

几丁质的去乙酰化修饰与昆虫的发育变态密切相关,几丁质去乙酰化酶(chitin deacetylase,CDA)是这个过程中的关键酶。家蚕(Bombyxmori)是鳞翅目昆虫的代表性昆虫,目前对家蚕CDAs的研究较少。为了更好地揭示BmCDAs对家蚕变态发育的作用,本研究采用生物信息学分析、蛋白表达纯化以及免疫荧光定位等方法对表皮中高量表达的BmCDA2进行了研究。结果发现,BmCDA2有两种mRNA拼接形式BmCDA2a和BmCDA2b,分别在幼虫眠期和化蛹期表皮高量表达,两个基因均有几丁质去乙酰化酶催化结构域(catalyticdomain)、几丁质结合结构域(chitinbinding domain)和低密度脂蛋白受体结构域(low density lipoprotein receptor domain);Western blotting结果显示,该蛋白在表皮存在,荧光免疫定位发现BmCDA2蛋白随着幼虫新表皮的生成而逐渐增多,推测BmCDA2可能参与了幼虫新表皮的形成。该结果丰富了家蚕CDAs的生物学功能信息,也为其他昆虫CDA的研究提供一些有价值的参考。     

The zygomycetous fungus,Benjaminiella poitrasii contains a large family of differentially regulated chitin synthase genes

Benjaminiella poitrasii is a zygomycetous, non-pathogenic dimorphic fungus. Chitin synthases are the membrane bound enzymes involved in the synthesis of chitin and are key enzymes in the cell wall metabolism. Multiplicity of these enzymes is a common occurrence. Here, we identify eight distinct CHS genes in B. poitrasii as confirmed through DNA sequence and Southern analysis. These genes are related to other fungal CHS genes. BpCHS1-4 are class I-III chitin synthases while BpCHS5-8 are class IV-V chitin synthases. These eight genes are differentially expressed during morphogenesis and under different growth conditions. Two of these genes viz. BpCHS2 and BpCHS3 appear to be specific to the mycelial growth form. These are the first B. poitrasii sequences to be reported. Based on CHS gene sequences, B. poitrasii chitin synthase genes place it with other zygomycetes on a fungal phylogenetic tree.     

The class A chitin synthase gene of Spodoptera exigua: molecular cloning and expression patterns

Chitin synthase (CHS) is an important enzymatic component required for chitin formation in the cuticles and cuticular linings of other tissues. In the present study, a new CHS gene was characterized from the beet army worm Spodoptera exigua (Hübner) (Se). Homologous alignment and phylogenetic analysis of S. exigua CHS (SeCHS) with other related proteins suggest that SeCHS belongs to the class A CHS family (SeCHSA). Northern blot analysis revealed that SeCHSA is transcribed preferentially in the cuticle and tracheae. Further investigation indicated that SeCHSA mRNA is highly expressed in the early and late stages of each larval instar, and consistently expressed in high level during the pupal stage. Using antibody specific for CHS, SeCHS was further localized in the underlying epidermal cells of the integument and tracheal cells, but not in the fat body or Malpighian tubules. These data suggest that SeCHS plays an important role in cuticle formation and development of S. exigua.     

甜菜夜蛾几丁质合成酶基因A和B启动子的克隆

几丁质不仅是昆虫的表皮和围食膜的主要成分,也是一个非常关键的害虫控制靶标,主要通过几丁质合成酶（chitin synthase,CHS）基因合成。本文在克隆甜菜夜蛾Spodoptera exigua的两个几丁质合成酶基因（SeCHSA和SeCHSB）cDNA和基因组序列的基础上,从基因的5′末端设计特异性引物和构建特定的基因组文库, 采用PCR的方法获得了5′端侧翼序列。通过5′RACE的方法确定SeCHSA和SeCHSB基因的转录起始位点后,获到了启动子序列。这为研究昆虫几丁质合成和转录调控奠定了基础。     

Chitin synthesis in larval and pupal epidermis of the Indian meal moth,Plodia interpunctella (Hübner), and the greater wax moth,Galleria mellonella (L.)

The level of chitin synthesis was determined in whole last-instar larvae and in pupae of Plodia interpunctella, and in epidermal tissue from similar stages of Galleria mellonella. The incorporation of radiolabelled N-acetyl-d-glucosamine into chitin was used to measure synthesis. Chitin production was similar in both species with peaks of synthesis occurring at the beginning of the last larval instar, in prepupae, in white pupae and prior to adult emergence. P. interpunctella also exhibited an additional small increase at mid-instar. Exposure of larval epidermis of P. interpunctella to 20-hydroxyecdysone in vitro stimulated chitin synthesis, but only after a 24 hr lag period subsequent to exposure to the hormone. This hormonal stimulation of chitin synthesis was inhibited by actinomycin-D and cycloheximide which suggested that 20-hydroxyecdysone-stimulated production of chitin depended on synthesis of RNA and protein. Comparison of the synthesis of chitin in epidermis of G. mellonella with previously published ecdysone titres, indicated that chitin production in vivo is preceded by an elevated ecdysone titre.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.