Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle of Tribolium castaneum

The insect exoskeleton is composed of cuticle primarily formed from structural cuticular proteins (CPs) and the polysaccharide chitin. Two CPs, TcCPR27 and TcCPR18, are major proteins present in the elytron (highly sclerotized and pigmented modified forewing) as well as the pronotum (dorsal sclerite of the prothorax) and ventral abdominal cuticle of the red flour beetle, Tribolium castaneum. Both CPs belong to the CPR family, which includes proteins that have an amino acid sequence motif known as the Rebers & Riddiford (R&R) consensus sequence. Injection of double-stranded RNA (dsRNA) for TcCPR27 and TcCPR18 resulted in insects with shorter, wrinkled, warped and less rigid elytra than those from control insects. To gain a more comprehensive understanding of the roles of CPs in cuticle assembly, we analyzed for the precise localization of TcCPR27 and the ultrastructural architecture of cuticle in TcCPR27- and TcCPR18-deficient elytra. Transmission electron microscopic analysis combined with immunodetection using goldlabeled secondary antibody revealed that TcCPR27 is present in dorsal elytral procuticle both in the horizontal laminae and in vertical pore canals. dsRNA-mediated RNA interference (RNAi) of TcCPR27 resulted in abnormal electron-lucent laminae and pore canals in elytra except for the boundary between these two structures in which electron-dense molecule(s) apparently accumulated. Insects subjected to RNAi for TcCPR18 also had disorganized laminae and pore canals in the procuticle of elytra. Similar ultrastructural defects were also observed in other body wall regions with rigid cuticle such as the thorax and legs of adult T. castaneum. TcCPR27 and TcCPR18 are required for proper formation of the horizontal chitinous laminae and vertical pore canals that are critical for formation and stabilization of rigid adult cuticle

Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt

Citation: Mun, S., Noh, M. Y., Dittmer, N. T., Muthukrishnan, S., Kramer, K. J., Kanost, M. R., & Arakane, Y. (2015). Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt. Scientific Reports, 5, 11. doi:10.1038/srep10484In the insect cuticle, structural proteins (CPs) and the polysaccharide chitin are the major components. It has been hypothesized that CPs are cross-linked to other CPs and possibly to chitin by quinones or quinone methides produced by the laccase2-mediated oxidation of N-acylcatechols. In this study we investigated functions of TcCP30, the third most abundant CP in protein extracts of elytra (wing covers) from Tribolium castaneum adults. The mature TcCP30 protein has a low complexity and highly polar amino acid sequence. TcCP30 is localized with chitin in horizontal laminae and vertically oriented columnar structures in rigid cuticles, but not in soft and membranous cuticles. Immunoblot analysis revealed that TcCP30 undergoes laccase2-mediated cross-linking during cuticle maturation in vivo, a process confirmed in vitro using recombinant rTcCP30. We identified TcCPR27 and TcCPR18, the two most abundant proteins in the elytra, as putative crosslinking partners of TcCP30. RNAi for the TcCP30 gene had no effect on larval and pupal growth and development. However, during adult eclosion, similar to 70% of the adults were unable to shed their exuvium and died. These results support the hypothesis that TcCP30 plays an integral role as a cross-linked structural protein in the formation of lightweight rigid cuticle of the beetle

Comparative Genomic Analysis of Chitinase and Chitinase-Like Genes in the African Malaria Mosquito (Anopheles gambiae)

Chitinase is an important enzyme responsible for chitin metabolism in a wide range of organisms including bacteria, yeasts and other fungi, nematodes and arthropods. However, current knowledge on chitinolytic enzymes, especially their structures, functions and regulation is very limited. In this study we have identified 20 chitinase and chitinase-like genes in the African malaria mosquito, Anopheles gambiae, through genome-wide searching and transcript profiling. We assigned these genes into eight different chitinase groupings (groups I–VIII). Domain analysis of their predicted proteins showed that all contained at least one catalytic domain. However, only seven (AgCht4, AgCht5-1, AgCht6, AgCht7, AgCht8, AgCht10 and AgCht23) displayed one or more chitin-binding domains. Analyses of stage- and tissue-specific gene expression revealed that most of these genes were expressed in larval stages. However, AgCht8 was mainly expressed in the pupal and adult stages. AgCht2 and AgCht12 were specifically expressed in the foregut, whereas AgCht13 was only expressed in the midgut. The high diversity and complexity of An. gambiae chitinase and chitinase-like genes suggest their diverse functions during different developmental stages and in different tissues of the insect. A comparative genomic analysis of these genes along with those present in Drosophila melanogaster, Tribolium castaneum and several other insect species led to a uniform classification and nomenclature of these genes. Our investigation also provided important information for conducting future studies on the functions of chitinase and chitinase-like genes in this important malaria vector and other species of arthropods

Immersion and TOFD

Time of flight diffraction (TOFD) is a well-developed ultrasonic non-destructive testing (NDT) technique which has been applied successfully for accurate sizing of defects in thick sections. Codes of practice such as ASME now permit TOFD for routine examination as an alternative to radiography for thick weldments. However, examination of thinner sections by TOFD has its limitations. The main limitation is that as the thickness of the specimen reduces, the lateral wave, diffracted wave and the back wall echo merge together and it is very difficult to identify and size the discontinuity. Also, the size of conventional transducers limits the required probe separation. Limited success has been obtained internationally through the application of miniature probes and software for extending TOFD to lower thicknesses. In these cases, the minimum thickness that has been examined is 7 mm. A new methodology based on a simple and novel combination of TOFD and the immersion technique has been proposed by the authors that successfully extends the application of TOFD to thinner sections down to 3 mm. Immersion coupling provides a delay line, the necessary angles and probe separation making it possible to examine the thin components successfully. This paper highlights the results of detailed experimental investigations on immersion TOFD and its successful application for the evaluation of welds in hexcan used for encapsulating nuclear fuel pins

Mechanism of Oxidation of p-Phenylenediamine by Manganese(III) Acetate in Aqueous Sulphuric Acid Medium

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Time of flight diffraction testing of austenitic stainless steel weldments at elevated temperatures

Ultrasonic testing is a mandatory requirement during inservice inspection of the welds in the main and safety vessels of a prototype fast breeder reactor. Presently, conventional ultrasonic techniques have been proposed for use. Two of the main challenges likely to be encountered during ultrasonic testing are the high temperature of the vessels (around 423-473 K during shutdown) and the background radiation. Ultrasonic time of flight diffraction is now well established as a technique for NDT of thick (greater than 12.5 mm) weldments. The main advantage of using time of flight diffraction is its increased accuracy for discontinuity detection and fast scanning times. Conventionally, time of flight diffraction has been applied only for carbon steel weldments and at ambient temperatures. This paper highlights the successful application of time of flight diffraction for testing of stainless steel weldments at high temperature (423 K). Experimental studies reveal that the diffracted signal amplitude decreases and, hence, the time of flight diffraction image contrast decreases with increasing temperature. Additional gain was necessary to compensate for the decreased signal amplitude. Analysis of the experimental data indicated that the additional gain necessary to compensate for the lower signal amplitude varied nonlinearly (quadratically) with temperature. The errors in the discontinuity dimensional measurement were observed to be less than 5% compared to measurements at ambient temperature

Time of flight diffraction and synthetic aperture focusing technique for NDT of weldments

The limits of anomaly detection by use of nondestructive testing (NDT) methods are based on the interaction of the material with the applied probing medium. Various methods are employed for detection of anomalies in weldments. Investigations using the radiographic, ultrasonic pulse/echo, time of flight diffraction, and synthetic aperture focusing techniques were carried out on a carbon steel weld pad (thickness 18 mm) with natural anomalies introduced at predetermined locations. The anomalies were first imaged by radiography. The pulse/echo technique, using different parameters, could detect almost all the planar anomalies except a cluster of porosity. Although time of flight diffraction could recognize most of the anomalies, it was not possible to clearly resolve and characterize the cluster of pores and group of cracks. Cracks with cleft edges were almost impossible to identify. The experimental results revealed that time of flight diffraction at a minimum meets code requirements for detection of anomalies. Sizing of length and through-wall extent can be carried out using the initial time of flight diffraction data. Using the synthetic aperture focusing technique, it was possible to resolve and characterize all the anomalies on par with the radiographic information. In addition, two clusters of porosity were distinctly seen, separated by 7 mm. Hence, the reporting criteria of the "ASME Boiler and Pressure Vessel Code" (ASME, 2004) can be satisfied using ultrasonic imaging techniques such as time of flight diffraction and synthetic aperture focusing for detection, sizing and characterization of anomalies in weldments in lieu of radiography. Destructive tests were also employed at a later stage to corroborate and validate the findings of anomalies by NDT methods