Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle, <i>Tribolium castaneum</i>

Noh, Mi Young,Muthukrishnan, Subbaratnam,Kramer, Karl J.,Arakane, Yasuyuki Elsevier 2017 Insect biochemistry and molecular biology Vol.91 No.-

<P><B>Abstract</B></P> <P>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, <I>Tribolium castaneum</I>, 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Development of <I>Tribolium</I> elytral dorsal and ventral cuticles is asynchronous except at certain phases of deposition. </LI> <LI> Exocuticle of the dorsal cuticle contains numerous horizontal laminae and vertical pore canals with chitin fibers. </LI> <LI> Mesocuticle is present between the exocuticle and the endocuticle in the dorsal cuticle but not in the ventral cuticle. </LI> <LI> Both dorsal and ventral cuticles contain an endocuticle consisting of a thick brick (macrofiber/Balken)-type of laminae. </LI> <LI> The dorsal cuticle is four-fold thicker and more sclerotized than the ventral. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

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

Noh, Mi Young,Muthukrishnan, Subbaratnam,Kramer, Karl J.,Arakane, Yasuyuki American Society for Biochemistry and Molecular Bi 2018 The Journal of biological chemistry Vol.293 No.18

<P>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.</P>

Gene functions in adult cuticle pigmentation of the yellow mealworm, <i>Tenebrio molitor</i>

Mun, Seulgi,Noh, Mi Young,Kramer, Karl J.,Muthukrishnan, Subbaratnam,Arakane, Yasuyuki Elsevier 2020 Insect biochemistry and molecular biology Vol.117 No.-

<P><B>Abstract</B></P> <P>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, <I>Tenebrio molitor</I>, encoding seven key enzymes involved in this pathway including tyrosine hydroxylase (TmTH), DOPA decarboxylase (TmDDC), laccase 2 (TmLac2), Yellow-y (TmY-y), arylalkylamine <I>N</I>-acetyltransferase (TmAANAT1), aspartate 1-decarboxylase (TmADC) and <I>N</I>-β-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 <I>TmDDC</I> or <I>TmY-y</I>. 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 <I>T. molitor</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CDNAs for seven key enzymes involved in <I>Tenebrio</I> cuticle tanning were cloned. </LI> <LI> Rigid cuticle has brownish exo-, dark meso- and transparent endocuticular layers. </LI> <LI> RNAi of target genes causes abnormal regional and temporal pigmentation. </LI> <LI> NBAD and NADA are associated with brownish exocuticle and less pigmented endocuticle. </LI> <LI> NADA-tanning produces a dark pigmented mesocuticle layer and overall adult body color. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

<i>Tribolium castaneum</i> RR-1 Cuticular Protein TcCPR4 Is Required for Formation of Pore Canals in Rigid Cuticle

Noh, Mi Young,Muthukrishnan, Subbaratnam,Kramer, Karl J.,Arakane, Yasuyuki Public Library of Science 2015 PLoS genetics Vol.11 No.2

<▼1><P>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, <I>Tribolium castaneum</I>. 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 <I>TcCPR4</I> 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 <I>T. castaneum</I>. 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 <I>TcCPR4</I> 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.</P></▼1><▼2><P><B>Author Summary</B></P><P>The insect cuticle is a remarkable biomaterial primarily formed from two different types of structural biopolymers, cuticular proteins and chitin. Despite a rather limited composition, insects produce diverse cuticles with the proper combination of mechanical properties such as strength, hardness and flexibility. Adult beetles are covered mostly by a hard cuticle, but they can fly because their cuticle is lightweight. The rigid cuticle is comprised of three major functional layers, namely the outermost envelope, the protein-rich epicuticle and the innermost chitin-protein rich procuticle. In addition, there are a large number of vertically oriented columnar structures denoted as pore canals that contain chitinous fibers (pore canal fibers) that are absent in soft and flexible cuticles. We have identified a cuticular structural protein, TcCPR4, which is predominantly localized in the pore canals of rigid cuticles of the red flour beetle. Loss of function of <I>TcCPR4</I> by RNA interference causes abnormal and amorphous pore canal fibers resulting in less organized pore canals that do not traverse the procuticle vertically. TcCPR4 plays a major role in determining the morphology of the vertical pore canals and pore canal fibers that contribute to the formation of a lightweight and rigid beetle cuticle.</P></▼2>

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

Seulgi Mun,Mi Young Noh,Karl J. Kramer,Subbaratnam Muthukrishnan,Yasuyuki Arakane 한국응용곤충학회 2020 한국응용곤충학회 학술대회논문집 Vol.2020 No.05

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

Seulgi Mun,Mi Young Noh,Karl J. Kramer,Subbaratnam Muthukrishnan,Yasuyuki Arakane 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.10

Functional importance of group I chitin deacetylases in cuticle morphology of Tribolium castaneum adult

Seulgi Mun,Mi Young Noh,Subbaratnam Muthukrishnan,Karl J. Kramer,Yasuyuki Arakane 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.10

Chitin deacetylases (CDAs) are extracellular-modifying enzymes that deacetylate chitin to produce chitosan. In insects, this modification may contribute to the affinity and/or cross-linking of chitin/chitosan-like polysaccharides for a variety of structural proteins, which may lead to diverse mechanical properties of the cuticle. We previously reported the functional importance of Group I CDAs, TcCDA1 and TcCDA2, as well as the two alternative spliced isoforms of the latter, TcCDA2a and TcCDA2b from the red flour beetle, Tribolium castaneum in molting, morphology of cuticle and movement of legs. In this study, we further analyzed protein localization, ultrastructural defects of the cuticles and leg joints after RNAi of those genes. Both proteins are mainly present in the innermost procuticle region called the “assembly zone”. Loss of function of either TcCDA1 or TcCDA2 caused disorganized chitinous horizontal laminae and vertical pore canals in both the rigid and soft cuticles. RNAi of TcCDA2b affects cuticle integrity similar to that seen in RNAi of the two alternatively spliced forms of TcCDA2. In contrast, TcCDA2a-deficient adult, like that seen in the hypomorphic phenotype produced by RNAi of TcCDA1, exhibited ruptured tendons between femur and tibia, resulting in loss of locomotion ability. These results suggest that Group I CDAs play critical roles in molting, morphology, ultrastructure and mobility in T. castaneum. This work was supported by NRFs (NRF-2015R1A6A3A04060323 and NRF-2018R1A2B6005106).

Functional importance of group I chitin deacetylases in cuticle morphology of Tribolium castaneum adult

Seulgi Mun,Mi Young Noh,Subbaratnam Muthukrishnan,Karl J. Kramer,Yasuyuki Arakane 한국농약과학회 2018 한국농약과학회 학술발표대회 논문집 Vol.2018 No.10

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

Myeongjin Kim,Seulgi Mun,Mi Young Noh,Karl J. Kramer,Subbaratnam Muthukrishnan,Yasuyuki Arakane 한국응용곤충학회 2023 한국응용곤충학회 학술대회논문집 Vol.2023 No.04

Lytic polysaccharide monooxygenase is required for chitinous cuticle turnover during insect molting

Myeongjin Kim,Seulgi Mun,Mi Young Noh,Karl J. Kramer,Subbaratnam Muthukrishnan,Yasuyuki Arakane 한국응용곤충학회 2022 한국응용곤충학회 학술대회논문집 Vol.2022 No.10