Revealing the Role of CYP346 Family Genes on Phosphine Resistance in Indian Populations of Tribolium castaneum.

BACKGROUND: Phosphine resistance in Tribolium castaneum challenges grain storage. This study investigates the impact of cytochrome P450 (CYP) enzymes and CYP346 family genes on phosphine resistance in Indian Tribolium castaneum populations. METHODS: Seven field populations of T. castaneum were compared with Lab- susceptible population for their resistance to phosphine. The levels of cytochrome P450 enzyme and expression of certain CYP346 family genes were tracked in these populations. RESULTS: The highly resistant Patiala population showed significantly increased CYP450 activity (11.26 ± 0.14 nmol/min/mg protein, 7.41-fold higher) compared to the lab-susceptible population (1.52 ± 0.09 nmol/min/mg protein) when assayed using 8 mM p-nitroanisole as the substrate. The mRNA expression was measured relative to the standard gene RPS18 and revealed significant upregulation of CYP346B1 and CYP346B3 in highly resistant populations Moga and Patiala (CYP346B1: 12.09 ± 2.19 to 21.74 ± 3.82; CYP346B3: 59.097 ± 10.265 to 50.148 ± 8.272). Patiala's CYP346B1 exhibited an impressive 685.76-fold change, and Moga's CYP346B3 showed a 361.893-fold change compared to lab-susceptible. Linear regression confirmed robust fits for each gene (R2: 0.693 to 0.756). Principal component analysis (PCA) demonstrated a strong positive correlation between CYP346 genes expression; and cytochrome P450 activity. Patiala, Moga, and Hapur populations showed conformity, associating higher resistance with increased P450 activity and CYP346 gene expression. Cluster analysis highlighted a potential correlation between CYP346B1, CYP346B2, and CYP346B3 and P450 activity, with Patiala and Moga clustering together. CONCLUSIONS: Variability in CYP346B1 and CYP346B3 in strong resistance populations may contribute to adaptation and resistance mechanisms. The study provides insights into specific CYP346 family genes associated with phosphine resistance, emphasizing the intricate interaction between CYP450 detoxifying enzymes, CYP346 family genes, and resistance mechanisms. The upregulation of CYP346 genes suggests a survival advantage for T. castaneum against phosphine, diminishing phosphine's efficacy as a pest control measure.

N(alpha)-acetyltransferase 40-mediated histone acetylation plays an important role in ecdysone regulation of metamorphosis in the red flour beetle, Tribolium castaneum.

Histone acetylation, a crucial epigenetic modification, is governed by histone acetyltransferases (HATs), that regulate many biological processes. Functions of HATs in insects are not well understood. We identified 27 HATs and determined their functions using RNA interference (RNAi) in the model insect, Tribolium castaneum. Among HATs studied, N-alpha-acetyltransferase 40 (NAA40) knockdown caused a severe phenotype of arrested larval development. The steroid hormone, ecdysone induced NAA40 expression through its receptor, EcR (ecdysone receptor). Interestingly, ecdysone-induced NAA40 regulates EcR expression. NAA40 acetylates histone H4 protein, associated with the promoters of ecdysone response genes: EcR, E74, E75, and HR3, and causes an increase in their expression. In the absence of ecdysone and NAA40, histone H4 methylation by arginine methyltransferase 1 (ART1) suppressed the above genes. However, elevated ecdysone levels at the end of the larval period induced NAA40, promoting histone H4 acetylation and increasing the expression of ecdysone response genes. NAA40 is also required for EcR, and steroid-receptor co-activator (SRC) mediated induction of E74, E75, and HR3. These findings highlight the key role of ecdysone-induced NAA40-mediated histone acetylation in the regulation of metamorphosis.

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.

A structural basis for the diverse linkage specificities within the ZUFSP deubiquitinase family.

Eukaryotic deubiquitinases are important regulators of ubiquitin signaling and can be subdivided into several structurally distinct classes. The ZUFSP family, with ZUP1 as its sole human member, has a modular architecture with a core catalytic domain highly active against the ubiquitin-derived peptide RLRGG, but not against ubiquitin itself. Ubiquitin recognition is conferred by additional non-catalytic domains, making full-length ZUP1 active against long K63-linked chains. However, non-mammalian ZUFSP family members contain different ubiquitin-binding domains in their N-terminal regions, despite their high conservation within the catalytic domain. Here, by working with representative ZUFSP family members from insects, fungi and plants, we show that different N-terminal domains are associated with different linkage preferences. Biochemical and structural studies suggest that the acquisition of two family-specific proximal domains have changed the default K48 preference of the ZUFSP family to the K63 preference observed in ZUP1 and its insect homolog. Additional N-terminal zinc finger domains promote chain cleavage without changing linkage-specificity.

Mechanism of PINK1 activation by autophosphorylation and insights into assembly on the TOM complex.

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

Assessment of the potential of non-thermal atmospheric pressure plasma discharge and microwave energy against Tribolium castaneum and Trogoderma granarium.

This study was carried out to investigate the efficacy of the non-thermal atmospheric pressure plasma produced with dielectric barrier discharge (APPD) using air as a processing gas and microwave energy to control Tribolium castaneum and Trogoderma granarium adults and larvae in wheat grains. Insects' mortality was found to be power and time-dependent. The results indicated that non-thermal APPD and the microwave have enough insecticidal effect on the target pests. From the bioassay, LT50's and LT90's levels were estimated, T. granarium larvae appeared more tolerant to non-thermal APPD and the microwave energy than adults 7 days post-exposure. The germination percentage of wheat grains increased as the time of exposure to the non-thermal APPD increased. On the contrary, the germination percentage of wheat grains decreased as the time of exposure to the microwave increased. In addition, changes in antioxidant enzyme activities, catalase (CAT), glutathione S-transferase (GST) and peroxidase, in adults and larvae were examined after 24 h post-treatment to non-thermal APPD at 15.9 W power level, which caused 50% mortality. The activity of CAT, GST and lipid peroxide in the treated larvae showed a significant increase post-exposure to the non-thermal APPD at 15.9 W power level. On the other hand, no significant change in GSH-Px activity was observed. Reductions in the level of glutathione (GSH) and protein content occurred in treated larvae in comparison with the control.

A structurally conserved human and Tetrahymena telomerase catalytic core.

Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates how domains of TER and TERT, including the TEN-TRAP complex, can interact in a conserved manner to regulate telomere synthesis.

Identification of catalytically distinct arylalkylamine N-acetyltransferase splicoforms from Tribolium castaneum.

The assumption that structural or sequential homology between enzymes implies functional homology is a common misconception. Through in-depth structural and kinetic analysis, we are now beginning to understand the minute differences in primary structure that can alter the function of an enzyme completely. Alternative splicing is one method for which the activity of an enzyme can be controlled, simply by altering its length. Arylalkylamine N-acetyltransferase A (AANATA) in D. melanogaster, which catalyzes the N-acetylation of biogenic amines, has multiple splicoforms - alternatively spliced enzyme isoforms - with differing tissue distribution. As demonstrated here, AANAT1 from Tribolium castaneum is another such enzyme with multiple splicoforms. A screening assay was developed and utilized to determine that, despite only a 35 amino acid truncation, the shortened form of TcAANAT1 is a more active form of the enzyme. This implies regulation of enzyme metabolic activity via alternative splicing.

Mechanisms of nucleotide selection by telomerase.

Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Characterization of cysteine sulfinic acid decarboxylase from Tribolium castaneum and its application in the production of ß-alanine.

ß-alanine is a precursor for the production of pharmaceuticals and food additives that is produced by chemical methods in industry. As concerns about the environment and energy are increasing, biocatalysis using L-aspartate-α-decarboxylase (ADC) to convert L-aspartate to ß-alanine has great potential. Many studies have focused on the catalytic activity of ADC, but these researches were limited to the prokaryotic enzymes. In this study, the gene encoding cysteine sulfinic acid decarboxylase from Tribolium castaneum (TcCSADC) was synthesized and overexpressed in Escherichia coli, and the enzyme was purified and characterized for the first time. It could use L-aspartate as its substrate, and the specific activity was 4.83 µmol/min/mg, which was much higher than that of ADCs from prokaryotes. A homology modeling assay indicated that TcCSADC had a dimer structure. Based on the evolutionary information from thermophilic bacteria, twenty-three variants were constructed to attempt to improve its abilities that transform L-aspartate to ß-alanine. One mutant, G369A, was screened that had improved thermal stability. An analysis of the suitability of the catalytic process showed that the up to 162 g/L ß-alanine could be produced using cells expressing the recombinant G369A variant, which is the highest yield to date. The CSADC from T. castaneum has important value for studies of the mechanism of ADCs and CSADCs from eukaryotes, and the engineered strain containing the G369A variant has great potential for the industrial production of ß-alanine.

Effect of 20-Hydroxyecdysone, a Phytoecdysteroid, on Development, Digestive, and Detoxification Enzyme Activities of Tribolium castaneum (Coleoptera: Tenebrionidae).

Plants present a delimited reservoir of biologically active compounds. Many plants synthesize several compounds of secondary metabolism, such as alkaloids, terpenoids, phenolics, steroids, etc. Such compounds are generally thought to be involved in plant-insect interactions. Phytoecdysteroids are a class of chemicals that plants synthesize; these compounds are analogues of molting hormones produced by insects. In this work, the effect of the 20-hydroxyecdysone, which is a molecule that belongs to the family of phytoecdysteroids, was tested on an insect pest, Tribolium castaneum (Herbst). Firstly, the effect of this molecule on post-embryonic development parameters was tested after ingestion at 300, 600, 900, and 1,200 ppm. Secondly, the effect of the 20-hydroxyecdysone was also tested on the biological parameters (proteins, alpha-amylase, detoxification enzymes). The results of the post-embryonic parameters test showed an important induction of larval mortality and a significant reduction of pupation and adult emergence rates. On the other hand, the test on the biological parameters showed that the 20-hydroxyecdysone caused a significant decrease in the levels of soluble proteins in treated larvae. In addition, the alpha-amylase activity was significantly inhibited by the ingestion of the phytoecdysteroid. And there was also a disruption of detoxification enzymes. The whole of the disturbances recorded in this work prove that phytoecdysteroids are thought to have potential value on T. castaneum control.

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism.

Polyphosphates (polyPs) have been found in all cell types examined to date and play diverse roles, depending on the cell type. In eukaryotic organisms, polyPs have been mainly investigated in mammalian cells, with few studies on insects. In this study, we investigated mitochondrial polyphosphate metabolism in the red flour beetle, Tribolium castaneum. Substrate specificity for different chain lengths demonstrated the presence of two exopolyphosphatase isoforms in mitochondria. T. castaneum mitochondrial polyP levels decreased after injection with soluble pyrophosphatase (Tc-sPPase) dsRNA, while the membrane exopolyphosphate activity increased. Mitochondrial respiration modulated exopolyphosphatase activity only in wild-type beetles. Tripolyphosphate was able to increase the F-ATPase activity in wild-type and Tc-sPPase RNAi beetles. We suggest that inorganic pyrophosphatase modulates polyphosphate metabolism in mitochondria and affects the link between mitochondrial activity and polyphosphate metabolism in T. castaneum.

Target of rapamycin (TOR) determines appendage size during pupa formation of the red flour beetle Tribolium castaneum.

The adult body size is species-specific and controlled by complex interactions between hormones and the IIS/TOR pathway. To analyze the role of target of rapamycin (TOR) in the growth and development of the insect, expression levels of TOR were silenced in the model and pest insect red flour beetle, Tribolium castaneum. Injection of dsRNA into the last larval instar decreased pupal mass and size, while the amount of food intake by the larvae was not affected. These results place TcTOR downstream of nutrition as a transducer for nutritional signals to increase larval growth. In addition, TcTOR-silencing notably decreased the size of the adult appendages. Analysis of the wings and elytra revealed a decrease in cell size and number of these appendages in the TcTOR-silenced insects. This reduction in size was correlated with a decrease of transcriptional levels of marker genes controlling the cell cycle. Altogether, these results suggest a pivotal role for TcTOR in integrating nutritional signals and regulation of body and appendages growth.

Molecular characterization of class I histone deacetylases and their expression in response to thermal and oxidative stresses in the red flour beetle, Tribolium castaneum.

Reversible acetylation of core histones plays an important role in the epigenetic regulation of gene transcription, and is controlled by the action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). While HDACs have been well studied in Drosophila melanogaster, information from insect pests is still limited. In the current study, we cloned and characterized three class I enzymes, TcHDAC1, TcHDAC 3 and TcHDAC 8, in the red flour beetle, Tribolium castaneum. Expression profiling showed that T. castaneum HDAC genes are expressed in all developmental stages and tissues examined. A dramatic increase of mRNA expression level was observed from prepupae to 1-day-old pupae for all three T. castaneum HDAC genes. Both TcHDAC1 and TcHDAC3 exhibited the highest mRNA expression levels in thorax, whereas TcHDAC8 was highly expressed in fat body. Furthermore, T. castaneum HDAC genes were found to respond to heat, cold and oxidative stresses. While the heat-stress treatment decreased the mRNA expression levels of T. castaneum HDAC genes, their transcripts were induced by paraquat treatment. These results suggest a possible role for class I HDAC genes in the epigenetic regulation of T. castaneum development and stress responses.

The conservation and functionality of the oxygen-sensing enzyme Factor Inhibiting HIF (FIH) in non-vertebrates.

The asparaginyl hydroxylase, Factor Inhibiting HIF (FIH), is a cellular dioxygenase. Originally identified as oxygen sensor in the cellular response to hypoxia, where FIH acts as a repressor of the hypoxia inducible transcription factor alpha (HIF-α) proteins through asparaginyl hydroxylation, FIH also hydroxylates many proteins that contain ankyrin repeat domains (ARDs). Given FIH's promiscuity and the unclear functional effects of ARD hydroxylation, the biological relevance of HIF-α and ARD hydroxylation remains uncertain. Here, we have employed evolutionary and enzymatic analyses of FIH, and both HIF-α and ARD-containing substrates, in a broad range of metazoa to better understand their conservation and functional importance. Utilising Tribolium castaneum and Acropora millepora, we provide evidence that FIH from both species are able to hydroxylate HIF-α proteins, supporting conservation of this function beyond vertebrates. We further demonstrate that T. castaneum and A. millepora FIH homologs can also hydroxylate specific ARD proteins. Significantly, FIH is also conserved in several species with inefficiently-targeted or absent HIF, supporting the hypothesis of important HIF-independent functions for FIH. Overall, these data show that while oxygen-dependent HIF-α hydroxylation by FIH is highly conserved in many species, HIF-independent roles for FIH have evolved in others.

Development of a new method for collecting hemolymph and measuring phenoloxidase activity in Tribolium castaneum.

OBJECTIVE: Hemolymph plays many important roles in the physiology of an insect throughout its lifetime; however, for small-bodied insects, studies are lacking because of the difficulties encountered while collecting hemolymph. The objective of our study was to develop a method to collect hemolymph plasma from various stages of Tribolium castaneum and to evaluate phenoloxidase activity in the plasma samples. We first designed a procedure for easily and quickly collecting clear hemolymph plasma from T. castaneum. RESULTS: By using this method, we collected approximately 5 µl plasma from 30 individuals at the larval, pupal or adult stages. And then, we studied the expression of phenoloxidase by performing western blot analysis of the plasma samples and found that phenoloxidase is present in hemolymph in each developmental stage. We also measured phenoloxidase activity in control plasma and plasma treated with Gram-positive bacteria, Micrococcus luteus. Phenoloxidase activity was greater in some of the M. luteus-treated plasma samples compared with control samples. Thus, we developed a method to collect hemolymph plasma that is suitable for studies of phenoloxidase activity.

Telomerase Inhibition by a New Synthetic Derivative of the Aporphine Alkaloid Boldine.

Telomerase, the enzyme responsible for cell immortality, is an important target in anti-cancer drug discovery. Boldine, an abundant aporphine alkaloid of Peumus boldus, is known to inhibit telomerase at non-toxic concentrations. Cytotoxicity of N-benzylsecoboldine hydrochloride (BSB), a synthetic derivative of boldine, was determined using the MTT method in MCF7 and MDA-MB231 cells. Aliquots of cell lysates were incubated with various concentrations of BSB in qTRAP (quantitative telomere repeat amplification protocol)-ligand experiments before substrate elongation by telomerase or amplification by hot-start Taq polymerase. The crystal structure of TERT, the catalytic subunit of telomerase from Tribolium castaneum, was used for docking and molecular dynamics analysis. The qTRAP-ligand data gave an IC50 value of about 0.17 ± 0.1 µM for BSB, roughly 400 times stronger than boldine, while the LD50 in the cytotoxicity assays were 12.5 and 21.88 µM, respectively, in cells treated for 48 h. Although both compounds interacted well with the active site, MD analysis suggests a second binding site with which BSB interacts via two hydrogen bonds, much more strongly than boldine. Theoretical analyses also evaluated the IC50 for BSB as submicromolar. BSB, with greater hydrophobicity and flexibility than boldine, represents a promising structure to inhibit telomerase at non-toxic concentrations.

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.