Chitin Research Revisited

Two centuries after the discovery of chitin, it is widely accepted that this biopolymer is an important biomaterial in many aspects. Numerous studies on chitin have focused on its biomedical applications. In this review, various aspects of chitin research including sources, structure, biosynthesis, chitinolytic enzyme, chitin binding protein, genetic engineering approach to produce chitin, chitin and evolution, and a wide range of applications in bio- and nanotechnology will be dealt with

Tribolium castaneum genes encoding proteins with the chitin-binding type II domain.

Doctor of PhilosophyDepartment of BiochemistrySubbarat MuthukrishnanThe extracellular matrices of cuticle and peritrophic matrix of insects are composed mainly of chitin complexed with proteins, some of which contain chitin-binding domains. This study is focused on the identification and functional characterization of genes encoding proteins that possess one or more copies of the six-cysteine-containing ChtBD2 domain (Peritrophin A motif =CBM_14 =Pfam 01607) in the red flour beetle, Tribolium castaneum. A bioinformatics search of T. castaneum genome yielded previously characterized chitin metabolic enzymes and several additional proteins. Using phylogenetic analyses, the exon-intron organization of the corresponding genes, domain organization of proteins, and temporal and tissue-specificity of expression patterns, these proteins were classified into three large families. The first family includes 11 proteins essentially made up of 1 to 14 repeats of the peritrophin A domain. Transcripts for these proteins are expressed only in the midgut and only during feeding stages of development. We therefore denote these proteins as “Peritrophic Matrix Proteins” or PMPs. The genes of the second and third families are expressed in cuticle-forming tissues throughout all stages of development but not in the midgut. These two families have been denoted as “Cuticular Proteins Analogous to Peritrophins 3” or CPAP3s and “Cuticular Proteins Analogous to Peritophins 1” or CPAP1s based on the number of ChtBD2 domains that they contain. Unlike other cuticular proteins studied so far, TcCPAP1-C protein is localized predominantly in the exocuticle and could contribute to the unique properties of this cuticular layer. RNA interference (RNAi), which down-regulates transcripts for any targeted gene, results in lethal and/or abnormal phenotypes for some, but not all, of these genes. Phenotypes are often unique and are manifested at different developmental stages, including embryonic, pupal and/or adult stages. The experiments presented in this dissertation reveal that while the vast majority of the CPAP3 genes serve distinct and essential functions affecting survival, molting or normal cuticle development. However, a minority of the CPAP1 and PMP family genes are indispensable for survival under laboratory conditions. Some of the non-essential genes may have functional redundancy or may be needed only under special circumstances such as exposure to stress or pathogens

Gene families of cuticular proteins analogous to peritrophins (CPAPs) in Tribolium castaneum have diverse functions

Citation: Jasrapuria S, Specht CA, Kramer KJ, Beeman RW, Muthukrishnan S (2012) Gene Families of Cuticular Proteins Analogous to Peritrophins (CPAPs) in Tribolium castaneum Have Diverse Functions. PLoS ONE 7(11): e49844. doi:10.1371/journal.pone.0049844The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal ‘stiff-jointed’ gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species

Summary of phenotypes after RNAi of <i>CPAP3</i> genes.

<p>(A) Insects injected with dsRNA at the penultimate and/or last instar larval stages for many of the <i>CPAP3</i> genes showed abnormal phenotypes (except for <i>TcCPAP3-A2</i> and <i>TcCPAP3-E).</i> RNAi for <i>TcCPAP3-A1</i> resulted in adult lethality (indicated by a red slash across the figure). dsRNA <i>TcCPAP3-A1+</i> dsRNA <i>TcCPAP3-A2:</i> the insects did not show additive mortality or additional abnormalities compared to animals injected with dsRNA <i>TcCPAP3-A1</i> alone; dsRNA <i>TcCPAP3-B:</i> adults show walking defects. Black arrows indicate dimpled pronotum. dsRNA <i>TcCPAP3C:</i> Insects showed pupal to adult molt arrest and 100% lethality. dsRNA <i>TcCPAP3-D1:</i> Insects had rough elytra, which did not completely cover the adult abdomen. dsRNA <i>TcCPAP3-D2</i>: The phenotype was similar to animals treated with dsRNA <i>TcCPAP3-D1,</i> but was less severe. dsRNA <i>TcVer</i>-injected insects (200 ng per insect; n = 40) were used as controls (white eye is shown in inset). (B) dsRNA <i>TcCPAP3-A1</i> treatment at the larval stages resulted in fat body depletion in one week-old adults compared to dsRNA <i>TcVer-</i>treated controls; shown by white arrows. Also the insects were unable to completely expel the fecal pellet; marked with blue arrow. (C) dsRNA <i>TcCPAP3-B</i> treated adults show a wobbly, stiff-jointed gait, resulting from abnormal articulation of the tibial-tarsal (shown in blue circle) and femoral-tibial joints for the metathoracic legs and of the femoral-tibial joint for the mesothoracic legs (shown as white arrows). (D) dsRNAs for <i>TcCPAP3-D1</i> and <i>TcCPAP3-D2</i> were co-injected into pharate pupae (n = 40). The resulting insects molted to otherwise normal adults with abnormal elytra. The elytra were wrinkled and alligator skin-like, and did not completely cover the abdomen, but none of the insects died. dsRNA <i>TcVer</i> was injected as a control and the elytra from these insects were normal (top panel). (E) Defects in a single elytron following RNAi for <i>TcCPAP3-D1</i> or <i>TcCPAP3-D2</i> in comparison to the <i>TcVer</i> dsRNA treatment. The differences are also shown in the form of the schematic line diagram on the right, where the lines are wavy in comparison to the control, <i>TcVer.</i></p

Observed RNAi phenotypes during embryonic development.

<p>(A) Comparison of the numbers of eggs laid by adult females (n = 20) following dsRNA injection for each of the 10 <i>CPAP1</i> genes and then mated with 20 males. dsRNA <i>TcVer</i> was injected as a positive control. When compared to control insects, dsRNA <i>CPAP1</i>-injected animals did not exhibit significant reduction in the number of eggs. The X-axis shows the gene target for dsRNA-treatment. Y-axis: the number of eggs laid was plotted as the mean egg number per batch +/−SD during a 3-day period. (B) Comparison of the numbers of eggs laid by a pool of adult females (n = 20) following dsRNA injection for each of the seven <i>CPAP3</i> genes. When compared to dsRNA <i>TcVer</i>-treated control insects, dsRNA <i>TcCPAP3-D2</i>-injected animals exhibited a significant reduction in the number of eggs. X-axis shows the targeted gene. Y-axis is the number of eggs laid plotted as the mean egg number per batch with SD. The asterisks indicate insufficient egg numbers due to adult lethality. (C) Dissected ovaries of dsRNA <i>TcCPAP3-A1</i>-injected females lacked proper differentiation into ovarioles and did not contain eggs. In animals treated with dsRNA for <i>TcCPAP3-D2,</i> the ovaries had ovarioles but did not contain properly developed eggs. After <i>TcCPAP1-J</i> dsRNA-treatment, the adult females had normal ovaries with eggs, similar to dsRNA <i>TcVer-</i>injected animals. (D) dsRNA for <i>TcVer</i>-injected 2-weeks-old adult female controls laid normal number of eggs and the embryos had normal body segmentation and visible appendages just before hatching. dsRNA for <i>TcCPAP3-A1</i>: In rare cases where eggs could be collected, the body segmentation was not visible presumably due to failure of embryonic development. dsRNA <i>TcCPAP3-D2</i>-treated embryos also lacked body segment differentiation and the chorion was fragile and was detached upon Clorox treatment. dsRNA for <i>TcCPAP1-J</i> treatment resulted in embryos with visible appendages and the red eye spot, but the head was not bent down like in the <i>TcVer</i> control. Instead, they held up their heads. All of these embryos showed embryonic arrest and never hatched. A red slash line indicates lethal phenotypes.</p

Analysis of elytra for chitin levels following RNAi for <i>CPAP</i> genes.

<p>(A and B) Biochemical analysis of chitin content of 5 day-old pupae was carried out using a modified Morgan–Elson method (n = 5) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049844#pone.0049844-Arakane1" target="_blank">[11]</a>. Values of mean and standard errors are shown. dsRNA for <i>TcVer</i> was injected as a control that has a normal chitin content and dsRNA for <i>TcChs-A</i> served as a control that was depleted of chitin. When compared to control insects, the dsRNA <i>TcCPAP1-H</i>-treated pupae had a slightly reduced chitin content. GlcNAc = N-acetylglucosamine. (C) FITC-CBD staining of elytral cuticle after digestion with NaOH to remove cuticular proteins. Pharate pupae were treated with dsRNAs for <i>TcCPAP1-C</i>, <i>TcCPAP1-H</i>, <i>TcCPAP1-J</i> or <i>TcChs-A</i>. The elytra were dissected out from 5-day-old pupae. The elytra from insects injected with dsRNA for <i>TcCPAP1-H</i> or <i>TcChs-A</i> were fragile and became disintegrated by the alkali treatment (10 M NaOH at 95°C for 6 hours). Elytra from dsRNA <i>TcCPAP1-C-</i> and <i>TcCPAP1-J</i>-treated insects were also fragile and lost their original shape after NaOH treatment, but they remained intact and showed less staining after treatment with FITC-conjugated chitin-binding protein (FITC-CBD) when compared with elytra from dsRNA <i>TcVer</i>-treated control insects. Elytra from dsRNA <i>TcCPAP3-C</i> treated animals did not show any effect, compared to <i>Ver</i> controls even though these insects were arrested at the adult molt. (D) Confocal microscopic analysis of pharate adult cuticle from dsRNA <i>TcCPAP1-H</i> and dsRNA <i>TcVer</i>–treated (control) insects that were stained with a rhodamine-conjugated chitin-binding probe (red). An apparent decrease in chitin staining of elytral cuticle (green arrows) following RNAi for <i>TcCPAP1-H</i> compared to RNAi for <i>TcVer</i> (control) was observed. Scale bar = 10 µm. P, pupal cuticle; A, adult elytral cuticle. Plan Apochromat objective (40 X/1.4 oil).</p

RT-PCR analyses of target-specificity of RNAi mediated by dsRNAs for <i>CPAP1</i> and <i>CPAP3</i> genes.

<p>RT-PCR was carried out using RNA isolated four days after dsRNA injections as template to monitor the extent of depletion of the transcripts of the targeted <i>CPAP</i> gene as well as some of the closely related gene(s) of the same family. The RT-PCR (28 cycles) results indicated significant reduction in the transcripts of the targeted <i>CPAP</i> gene with no evidence of depletion of transcripts of other genes of the same family. (A) Specificity of transcript knock-down after injection of dsRNA for <i>CPAP1</i> genes. (B) Specificity of transcript knock-down after treatment with dsRNA for <i>CPAP3</i> genes.</p