Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum.

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus.

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.

Ovariole-specific Yellow-g and Yellow-g2 proteins are required for fecundity and egg chorion rigidity in the red flour beetle, Tribolium castaneum.

Most insects reproduce by laying eggs that have an eggshell/chorion secreted by follicle cells, which serves as a protective barrier for developing embryos. Thus, eggshell formation is vital for reproduction. Insect yellow family genes encode for secreted extracellular proteins that perform different, context-dependent functions in different tissues at various stages of development involving, for example, cuticle/eggshell coloration and morphology, molting, courtship behavior and embryo hatching. In this study we investigated the function of two of this family's genes, yellow-g (TcY-g) and yellow-g2 (TcY-g2), on the formation and morphology of the eggshell of the red flour beetle, Tribolium castaneum. Real-time PCR analysis revealed that both TcY-g and TcY-g2 were specifically expressed in the ovarioles of adult females. Loss of function produced by injection of double-stranded RNA (dsRNA) for either TcY-g or TcY-g2 gene resulted in failure of oviposition. There was no effect on maternal survival. Ovaries dissected from those dsRNA-treated females exhibited ovarioles containing not only developing oocytes but also mature eggs in their egg chambers. However, the ovulated eggs were collapsed and ruptured, resulting in swollen lateral oviducts and calyxes. TEM analysis showed that lateral oviducts were filled with electron-dense material, presumably from some cellular content leakage out of the collapsed eggs. In addition, morphological abnormalities in lateral oviduct epithelial cells and the tubular muscle sheath were evident. These results support the hypothesis that both TcY-g and TcY-g2 proteins are required for maintaining the rigidity and integrity of the chorion, which is critical for resistance to mechanical stress and/or rehydration during ovulation and egg activation in the oviducts of T. castaneum. Because Yellow-g and Yellow-g2 are highly conserved among insect species, both genes are potential targets for development of gene-based insect pest population control methods.

Temporal changes in the physical and mechanical properties of beetle elytra during maturation.

Changes in physical properties of Tenebrio molitor and Tribolium castaneum elytra (hardened forewings) were studied to understand how the development of microstructure and chemical interactions determine cuticle mechanical properties. Analysis of these properties supports a model in which cuticular material is continuously secreted from epidermal cells to produce an extracellular matrix so that the outermost layers mature first. It is hypothesized that enzymatic crosslinking and pigmentation reactions along with dehydration help to stabilize the protein-chitin network within the initial layers of cuticle shortly after eclosion. Mature layers are proposed to bear most of the mechanical loads. The frequency dependence of the storage modulus and the tan δ values decreased during the beginning of maturation, reaching constant values after 48 h post-eclosion. A decrease of tan δ indicates an increase in crosslinking of the material. The water content declined from 75% to 31%, with a significant portion lost from within the open spaces between the dorsal and ventral cuticular layers. Dehydration had a less significant influence than protein crosslinking on the mechanical properties of the elytron during maturation. When Tribolium cuticular protein TcCP30 expression was decreased by RNAi, the tan δ and frequency dependence of E' of the elytron did not change during maturation. This indicates that TcCP30 plays a role in the crosslinking process of the beetle's exoskeleton. This study was inspired by previous work on biomimetic multicomponent materials and helps inform future work on creating robust lightweight materials derived from natural sources. STATEMENT OF SIGNIFICANCE: Examination of changes in the physical properties of the elytra (hardened forewings) of two beetle species advanced understanding of how the molecular interactions influence the mechanical properties of the elytra. Physical characterization, including dynamic mechanical analysis, determined that the outer portion of the elytra matured first, while epidermal cells continued to secrete reactive components until the entire structure reached maturation. RNA interference was used to identify the role of a key protein in the elytra. Suppression of its expression reduced the formation of crosslinked polymeric components in the elytra. Identifying the molecular interactions in the matrix of proteins and polysaccharides in the elytra together with their hierarchical architecture provides important design concepts in the development of biomimetic materials.

Chitin deacetylases are necessary for insect femur muscle attachment and mobility.

Muscle attachment sites (MASs, apodemes) in insects and other arthropods involve specialized epithelial cells, called tendon cells or tenocytes, that adhere to apical extracellular matrices containing chitin. Here, we have uncovered a function for chitin deacetylases (CDAs) in arthropod locomotion and muscle attachment using a double-stranded RNA-mediated gene-silencing approach targeted toward specific CDA isoforms in the red flour beetle, Tribolium castaneum (Tc). Depletion of TcCDA1 or the alternatively spliced TcCDA2 isoform, TcCDA2a, resulted in internal tendon cuticle breakage at the femur-tibia joint, muscle detachment from both internal and external tendon cells, and defective locomotion. TcCDA deficiency did not affect early muscle development and myofiber growth toward the cuticular MASs but instead resulted in aborted microtubule development, loss of hemiadherens junctions, and abnormal morphology of tendon cells, all features consistent with a loss of tension within and between cells. Moreover, simultaneous depletion of TcCDA1 or TcCDA2a and the zona pellucida domain protein, TcDumpy, prevented the internal tendon cuticle break, further supporting a role for force-dependent interactions between muscle and tendon cells. We propose that in T. castaneum, the absence of N-acetylglucosamine deacetylation within chitin leads to a loss of microtubule organization and reduced membrane contacts at MASs in the femur, which adversely affect musculoskeletal connectivity, force transmission, and physical mobility.

AA15 lytic polysaccharide monooxygenase is required for efficient chitinous cuticle turnover during insect molting.

Microbial lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of crystalline polysaccharides including chitin and cellulose. The discovery of a large assortment of LPMO-like proteins widely distributed in insect genomes suggests that they could be involved in assisting chitin degradation in the exoskeleton, tracheae and peritrophic matrix during development. However, the physiological functions of insect LPMO-like proteins are still undetermined. To investigate the functions of insect LPMO15 subgroup I-like proteins (LPMO15-1s), two evolutionarily distant species, Tribolium castaneum and Locusta migratoria, were chosen. Depletion by RNAi of T. castaneum TcLPMO15-1 caused molting arrest at all developmental stages, whereas depletion of the L. migratoria LmLPMO15-1, prevented only adult eclosion. In both species, LPMO15-1-deficient animals were unable to shed their exuviae and died. TEM analysis revealed failure of turnover of the chitinous cuticle, which is critical for completion of molting. Purified recombinant LPMO15-1-like protein from Ostrinia furnacalis (rOfLPMO15-1) exhibited oxidative cleavage activity and substrate preference for chitin. These results reveal the physiological importance of catalytically active LPMO15-1-like proteins from distant insect species and provide new insight into the enzymatic mechanism of cuticular chitin turnover during molting.

Yellow-y Functions in Egg Melanization and Chorion Morphology of the Asian Tiger Mosquito, Aedes albopictus.

The Asian tiger mosquito, Aedes albopictus, is one of the most serious public health pests, which can transmit various vector-borne diseases. Eggs from this mosquito species become dark black shortly after oviposition and exhibit high desiccation resistance. Some of the Yellow proteins that act as dopachrome conversion enzymes (DCEs) are involved in the tyrosine-mediated tanning (pigmentation and sclerotization) metabolic pathway that significantly accelerates melanization reactions in insects. In this research, we analyzed the function of one of the yellow genes, yellow-y (AalY-y), in eggshell/chorion melanization of Ae. albopictus eggs. Developmental and tissue-specific expression measured by real-time PCR showed that AalY-y transcripts were detected at all stages of development analyzed, with significantly higher levels in the ovaries from blood-fed adult females. Injection of double-stranded RNA for AalY-y (dsAalY-y) had no significant effect on fecundity. However, unlike dsEGFP-treated control eggs that become black by 2-3 h after oviposition (HAO), dsAalY-y eggs were yellow-brown at 2 HAO, and reddish-brown even at 48 HAO. dsEGFP eggs exhibited resistance to desiccation at 48 HAO, whereas approximately 50% of the dsAalY-y eggs collapsed when they were moved to a low humidity condition. In addition, TEM analysis revealed an abnormal morphology and ultrastructure of the outer-endochorion in the dsAalY-y eggs. These results support the hypothesis that AalY-y is involved in the tyrosine-induced melanin biosynthetic pathway, plays an important role in black melanization of the chorion and functions in conferring proper morphology of the outer-endochorion, a structure that is presumably required for egg desiccation resistance in Ae. albopictus.

Yellow-g and Yellow-g2 proteins are required for egg desiccation resistance and temporal pigmentation in the Asian tiger mosquito, Aedes albopictus.

Eggs from Aedes mosquitoes exhibit desiccation resistance that helps them to survive and spread as human disease vectors throughout the world. Previous studies have suggested that eggshell/chorion melanization and/or serosal cuticle formation are important for desiccation resistance. In this study, using dsRNAs for target genes, we analyzed the functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the resistance to egg desiccation of the Asian tiger mosquito, Aedes albopictus, a species in which neither the timing of the melanization nor temporal development of the serosal cuticle is correlated with desiccation resistance. Injections of dsAalY-g, dsAalY-g2 or dsAalY-g/g2 (co-injection) into adult females have no effect on their fecundity. However, initial melanization is delayed by 1-2 h with the eggshells eventually becoming black similar to that observed in eggs from dsEGFP-injected control females. In addition, the shape of the eggs from dsAalY-g, -g2 and -g/g2-treated females is abnormally crescent-shaped and the outermost exochorion is more fragile and partially peeled off. dsEGFP control eggs, like those from the wild-type strain, acquire resistance to desiccation between 18 and 24 h after oviposition (HAO). In contrast, ~80% of the 24 HAO dsAalY-g and dsAalY-g2 eggs collapse when they are transferred to a low humidity environment. In addition, there is no electron-dense outer endochorion evident in either dsAalY-g or dsAalY-g2 eggs. These results support the hypothesis that AalY-g and AalY-g2 regulate the timing of eggshell darkening and are required for integrity of the exochorion as well as for rigidity, normal morphology and formation of the outer endochorion, a structure that apparently is critical for desiccation resistance of the Ae. albopictus egg.

Gene functions in adult cuticle pigmentation of the yellow mealworm, Tenebrio molitor.

In many arthropod species including insects, the cuticle tanning pathway for both pigmentation and sclerotization begins with tyrosine and is responsible for production of both melanin- and quinoid-type pigments, some of which are major pigments for body coloration. In this study we identified and cloned cDNAs of the yellow mealworm, Tenebrio molitor, encoding seven key enzymes involved in this pathway including tyrosine hydroxylase (TmTH), DOPA decarboxylase (TmDDC), laccase 2 (TmLac2), Yellow-y (TmY-y), arylalkylamine N-acetyltransferase (TmAANAT1), aspartate 1-decarboxylase (TmADC) and N-ß-alanyldopamine synthase (Tmebony). Expression profiles of these genes during development were analyzed by real-time PCR, revealing development-specific patterns of expression. Loss of function mediated by RNAi of either 1) TmTH or TmLac2, 2) TmDDC or TmY-y, and 3) TmAANAT1, TmADC or Tmebony resulted in pale/white, light yellow/brown and dark/black adult body coloration, respectively. In addition, there are three distinct layer/regional pigmentation differences in rigid types of adult cuticle, a brownish outer exocuticle (EX), a dark pigmented middle mesocuticle (ME) and a transparent inner endocuticle (EN). Decreases in pigmentation of the EX and/or ME layers were observed after RNAi of TmDDC or TmY-y. In TmADC- or Tmebony-deficient adults, a darker pigmented EX layer was observed. In TmAANAT1-deficient adults, trabeculae formed between the dorsal and ventral elytral cuticles as well as the transparent EN layer became highly pigmented. These results demonstrate that knocking down the level of gene expression of specific enzymes of this tyrosine metabolic pathway leads to abnormal pigmentation in individual layers and substructure of the rigid adult exoskeleton of T. molitor.

My Times with Professor Igor Kregar and some Hydrolytic Enzymes.

The late Professor Igor Kregar and this author had several overlapping biochemical research interests. One focus was the glycoside hydrolase, lysozyme, and the other was proteolytic enzymes and their inhibitors, in particular those present in tissues from insects and plants. Regarding lysozyme our results helped to understand its catalytic mechanism and the carboxylic acid functional groups involved. Another area was insect-plant interactions involving defensive responses of plants to insect feeding via proteolytic enzyme inhibitors. Those results can be utilized in transgenic plant and seed biotechnological applications, which would help to reduce damage to plants and seeds caused by coleopteran and other insect pests. Also described are some cultural and travel interactions that the author benefited from his friendship with Professor Kregar.

Group I chitin deacetylases are essential for higher order organization of chitin fibers in beetle cuticle.

Roles in the organization of the cuticle (exoskeleton) of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi. Even though all TcCDAs are co-expressed in cuticle-forming cells from the hardened forewing (elytron) and ventral abdomen, as well as in the softer hindwing and dorsal abdomen, there are significant differences in the tissue specificity of expression of the alternatively spliced transcripts. Loss of either TcCDA1 or TcCDA2 protein by RNAi causes abnormalities in organization of chitinous horizontal laminae and vertical pore canals in all regions of the procuticle of both the hard and soft cuticles. Simultaneous RNAi for TcCDA1 and TcCDA2 produces the most serious abnormalities. RNAi of either TcCDA2a or TcCDA2b affects cuticle integrity to some extent. Following RNAi, there is accumulation of smaller disorganized fibers in both the horizontal laminae and pore canals, indicating that TcCDAs play a critical role in elongation/organization of smaller nanofibers into longer fibers, which is essential for structural integrity of both hard/thick and soft/thin cuticles. Immunolocalization of TcCDA1 and TcCDA2 proteins and effects of RNAi on their accumulation indicate that these two proteins function in concert exclusively in the assembly zone in a step involving the higher order organization of the procuticle.

A chitinase with two catalytic domains is required for organization of the cuticular extracellular matrix of a beetle.

Insect cuticle or exoskeleton is an extracellular matrix formed primarily from two different structural biopolymers, chitin and protein. During each molt cycle, a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the overlying old exoskeleton by molting fluid enzymes including epidermal chitinases. In this study we report a novel role for an epidermal endochitinase containing two catalytic domains, TcCHT7, from the red flour beetle, Tribolium castaneum, in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. Recombinant TcCHT7 expressed in insect cells is membrane-bound and capable of hydrolyzing an extracellular chitin substrate, whereas in vivo, this enzyme is also released from the plasma membrane and co-localizes with chitin in the entire procuticle. RNAi of TcCHT7 reveals that this enzyme is nonessential for any type of molt or degradation of the chitinous matrix in the old cuticle. In contrast, TcCHT7 is required for maintaining the integrity of the cuticle as a compact structure of alternating electron-dense and electron-lucent laminae. There is a reduction in thickness of elytral and leg cuticles after RNAi for TcCHT7. TcCHT7 is also required for formation of properly oriented long chitin fibers inside pore canals that are vertically oriented columnar structures, which contribute to the mechanical strength of a light-weight, yet rigid, adult cuticle. The conservation of CHT7-like proteins harboring such a unique domain configuration among many insect and other arthropod species indicates a critical role for the group III class of chitinases in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate exoskeletons.

Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle, Tribolium castaneum.

Insect exoskeletons are composed of the cuticle, a biomaterial primarily formed from the linear and relatively rigid polysaccharide, chitin, and structural proteins. This extracellular material serves both as a skin and skeleton, protecting insects from environmental stresses and mechanical damage. Despite its rather limited compositional palette, cuticles in different anatomical regions or developmental stages exhibit remarkably diverse physicochemical and mechanical properties because of differences in chemical composition, molecular interactions and morphological architecture of the various layers and sublayers throughout the cuticle including the envelope, epicuticle and procuticle (exocuticle and endocuticle). Even though the ultrastructure of the arthropod cuticle has been studied rather extensively, its temporal developmental pattern, in particular, the synchronous development of the functional layers in different cuticles during a molt, is not well understood. The beetle elytron, which is a highly modified and sclerotized forewing, offers excellent advantages for such a study because it can be easily isolated at precise time points during development. In this study, we describe the morphogenesis of the dorsal and ventral cuticles of the elytron of the red flour beetle, Tribolium castaneum, during the period from the 0 d-old pupa to the 9 d-old adult. The deposition of exocuticle and mesocuticle is substantially different in the two cuticles. The dorsal cuticle is four-fold thicker than the ventral. Unlike the ventral cuticle, the dorsal contains a thicker exocuticle consisting of a large number of horizontal laminae and vertical pore canals with pore canal fibers and rib-like veins and bristles as well as a mesocuticle, lying right above the enodcuticle. The degree of sclerotization appears to be much greater in the dorsal cuticle. All of these differences result in a relatively thick and tanned rigid dorsal cuticle and a much thinner and less pigmented membrane-like ventral cuticle.

Arylalkylamine N-acetyltransferase 1 gene (TcAANAT1) is required for cuticle morphology and pigmentation of the adult red flour beetle, Tribolium castaneum.

In the insect cuticle tanning pathway (sclerotization and pigmentation), the enzyme arylalkylamine N-acetyltransferase (AANAT) catalyzes the acetylation of dopamine to form N-acetyldopamine (NADA), which is one of the major precursors for quinone-mediated tanning. In this study we characterized and investigated the function of TcAANAT1 in cuticle pigmentation of the red flour beetle, Tribolium castaneum. We isolated a full length TcAANAT1 cDNA that encodes a protein of 256 amino acid residues with a predicted GCN5-related acetyltransferase domain containing an acetyl-CoA binding motif. TcAANAT1 transcripts were detected at all stages of development with lowest expressions at the embryonic and pharate pupal stages. We expressed and purified the encoded recombinant TcAANAT1 protein (rTcAANAT1) that exhibited highest activity at slightly basic pH values (for example, pH 7.5 to 8.5 using dopamine as the substrate). In addition, rTcAANAT1 acts on a wide range of substrates including tryptamine, octopamine and norepinephrine with similar substrate affinities with Km values in the range of 0.05-0.11 mM except for tyramine (Km = 0.56 mM). Loss of function of TcAANAT1 caused by RNAi had no effect on larval and pupal development. The tanning of pupal setae, gin traps and urogomphi proceeded normally. However, the resulting adults (∼70%) exhibited a roughened exoskeletal surface, separated elytra and improperly folded hindwings. The body wall, elytra and veins of the hindwing of the mature adults were significantly darker than those of control insects probably due to the accumulation of dopamine melanin. A dark pigmentation surrounding the bristles located on the inter-veins of the elytron was evident primarily because of the underlying darkly pigmented trabeculae that partition the dorsal and ventral layers of the elytron. These results support the hypothesis that TcAANAT1 acetylates dopamine and plays a role in development of the morphology and pigmentation of T. castaneum adult cuticle.

Cuticle formation and pigmentation in beetles.

Adult beetles (Coleoptera) are covered primarily by a hard exoskeleton or cuticle. For example, the beetle elytron is a cuticle-rich highly modified forewing structure that shields the underlying hindwing and dorsal body surface from a variety of harmful environmental factors by acting as an armor plate. The elytron comes in a variety of colors and shapes depending on the coleopteran species. As in many other insect species, the cuticular tanning pathway begins with tyrosine and is responsible for production of a variety of melanin-like and other types of pigments. Tanning metabolism involves quinones and quinone methides, which also act as protein cross-linking agents for cuticle sclerotization. Electron microscopic analyses of rigid cuticles of the red flour beetle, Tribolium castaneum, have revealed not only numerous horizontal chitin-protein laminae but also vertically oriented columnar structures called pore canal fibers. This structural architecture together with tyrosine metabolism for cuticle tanning is likely to contribute to the rigidity and coloration of the beetle exoskeleton.

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

In 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 cross-linking partners of TcCP30. RNAi for the TcCP30 gene had no effect on larval and pupal growth and development. However, during adult eclosion, ~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.

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

Chitin, a homopolymer of ß-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.

Tribolium castaneum RR-1 cuticular protein TcCPR4 is required for formation of pore canals in rigid cuticle.

Insect cuticle is composed mainly of structural proteins and the polysaccharide chitin. The CPR family is the largest family of cuticle proteins (CPs), which can be further divided into three subgroups based on the presence of one of the three presumptive chitin-binding sequence motifs denoted as Rebers-Riddiford (R&R) consensus sequence motifs RR-1, RR-2 and RR-3. The TcCPR27 protein containing the RR-2 motif is one of the most abundant CPs present both in the horizontal laminae and in vertical pore canals in the procuticle of rigid cuticle found in the elytron of the red flour beetle, Tribolium castaneum. Depletion of TcCPR27 by RNA interference (RNAi) causes both unorganized laminae and pore canals, resulting in malformation and weakening of the elytron. In this study, we investigated the function(s) of another CP, TcCPR4, which contains the RR-1 motif and is easily extractable from elytra after RNAi to deplete the level of TcCPR27. Transcript levels of the TcCPR4 gene are dramatically increased in 3 d-old pupae when adult cuticle synthesis begins. Immunohistochemical studies revealed that TcCPR4 protein is present in the rigid cuticles of the dorsal elytron, ventral abdomen and leg but not in the flexible cuticles of the hindwing and dorsal abdomen of adult T. castaneum. Immunogold labeling and transmission electron microscopic analyses revealed that TcCPR4 is predominantly localized in pore canals and regions around the apical plasma membrane protrusions into the procuticle of rigid adult cuticles. RNAi for TcCPR4 resulted in an abnormal shape of the pore canals with amorphous pore canal fibers (PCFs) in their lumen. These results support the hypothesis that TcCPR4 is required for achieving proper morphology of the vertical pore canals and PCFs that contribute to the assembly of a cuticle that is both lightweight and rigid.

Loss of function of the yellow-e gene causes dehydration-induced mortality of adult Tribolium castaneum.

Yellow protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway that significantly accelerates pigmentation reactions in insects. Recent studies have suggested that the insect yellow genes represent a rapidly evolving gene family generating functionally diverse paralogs, but the exact physiological functions of several yellow genes are still not understood. To study the function(s) of one of the yellow genes, yellow-e (TcY-e), in the red flour beetle, Tribolium castaneum, we performed real-time PCR to analyze its developmental and tissue-specific expression, and utilized immunohistochemistry to identify the localization of the TcY-e protein in adult cuticle. Injection of double-stranded RNA for TcY-e (dsTcY-e) into late instar larvae had no effect on larval-pupal molting or pupal development. The pupal cuticle, including that lining the setae, gin traps and urogomphi, underwent normal tanning. Adult cuticle tanning including that of the head, mandibles and legs viewed through the translucent pupal cuticle was initiated on schedule (pupal days 4-5), indicating that TcY-e is not required for pupal or pharate adult cuticle pigmentation in T. castaneum. The subsequent pupal-adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (~25%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became highly desiccated. Interestingly, both the failure of the pupal-adult molt and desiccation-induced mortality were prevented by maintaining the dsTcY-e-treated insects at 100% relative humidity (rh). However, when the high humidity-rescued adults were removed from 100% rh and transferred to 50% rh, they rapidly dehydrated and died, whereas untreated beetles thrived throughout development at 50% rh. We also observed that the body color of the high humidity-rescued dsTcY-e-adults was slightly darker than that of control animals. These results support the hypothesis that TcY-e has a role not only in normal body pigmentation in T. castaneum adults but also has a vital waterproofing function.

Functional specialization among members of Knickkopf family of proteins in insect cuticle organization.

Our recent study on the functional analysis of the Knickkopf protein from T. castaneum (TcKnk), indicated a novel role for this protein in protection of chitin from degradation by chitinases. Knk is also required for the laminar organization of chitin in the procuticle. During a bioinformatics search using this protein sequence as the query, we discovered the existence of a small family of three Knk-like genes (including the prototypical TcKnk) in the T. castaneum genome as well as in all insects with completed genome assemblies. The two additional Knk-like genes have been named TcKnk2 and TcKnk3. Further complexity arises as a result of alternative splicing and alternative polyadenylation of transcripts of TcKnk3, leading to the production of three transcripts (and by inference, three proteins) from this gene. These transcripts are named TcKnk3-Full Length (TcKnk3-FL), TcKnk3-5' and TcKnk3-3'. All three Knk-family genes appear to have essential and non-redundant functions. RNAi for TcKnk led to developmental arrest at every molt, while down-regulation of either TcKnk2 or one of the three TcKnk3 transcripts (TcKnk3-3') resulted in specific molting arrest only at the pharate adult stage. All three Knk genes appear to influence the total chitin content at the pharate adult stage, but to variable extents. While TcKnk contributes mostly to the stability and laminar organization of chitin in the elytral and body wall procuticles, proteins encoded by TcKnk2 and TcKnk3-3' transcripts appear to be required for the integrity of the body wall denticles and tracheal taenidia, but not the elytral and body wall procuticles. Thus, the three members of the Knk-family of proteins perform different essential functions in cuticle formation at different developmental stages and in different parts of the insect anatomy.