Use of chitinases as a Biopesticides

(Subbaratnam Muthukrishnan) 한국키틴키토산학회 2000 한국키틴키토산학회지 Vol.5 No.3

<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>

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>

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

Noh, Mi Young,Muthukrishnan, Subbaratnam,Kramer, Karl J.,Arakane, Yasuyuki Public Library of Science 2018 PLoS genetics Vol.14 No.3

<▼1><P>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, <I>Tribolium castaneum</I>, 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 <I>in vivo</I>, this enzyme is also released from the plasma membrane and co-localizes with chitin in the entire procuticle. RNAi of <I>TcCHT7</I> 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 <I>TcCHT7</I>. 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.</P></▼1><▼2><P><B>Author summary</B></P><P>Insect cuticle or exoskeleton is an extracellular matrix consisting of three major morphologically distinct layers, the water-proofing envelope, the protein-rich epicuticle and the chitin/protein-rich procuticle. To accommodate growth, insects must periodically replace their cuticles in a process called “molting or ecdysis”. During each molt cycle a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the old one by molting fluid enzymes including epidermal chitinases. We show that a chitinase, CHT7, from the red flour beetle, <I>Tribolium castaneum</I>, belonging to a subfamily (group III) of chitinases that have two catalytic domains, is necessary for organization of chitin-containing structures in nascent cuticle, which contributes to the rigidity of the extracellular matrix. This unexpected function is distinct from that of other groups of epidermal chitinases that catalyze the turnover of chitin in old cuticle during the molting process. Because group III chitinases are highly conserved among insect and other arthropod species, we propose that these enzymes have a novel function in processing nascent chitin chains during cuticle assembly and organization into higher order structures that include horizontally stacked laminae and vertically oriented pore canals of many invertebrate cuticular extracellular matrices.</P></▼2>

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>

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

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).

Groups I and II chitinases, TcCHT5 and TcCHT10, function in turnover of chitinous cuticle during embryo hatch and post-embryonic molts in the red flour beetle, Tribolium castaneum

Myeongjin Kim,Mi Young Noh,Seulgi Mun,Subbaratnam Muthukrishnan,Karl J. Kramer,Yasuyuki Arakane 한국응용곤충학회 2023 한국응용곤충학회 학술대회논문집 Vol.2023 No.10

Insect eggshell and cuticle/exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous cuticular extracellular matrices must be degraded at least in part during embryo hatching and molting/ecdysis periods to accommodate continuous growth all the way to the adult stage. In this study, we investigated the functional importance of groups I and II chitinases, TcCHT5 and TcCHT10, in the turnover of chitinous cuticle during both embryonic and post-embryonic development in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. TcCHT10 appears to be able to substitute for TcCHT5 in all these vital physiological events except for the pupal-adult molting in which TcCHT5 is indispensable for complete digestion of chitin in the old pupal cuticle.

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 2022 한국응용곤충학회 학술대회논문집 Vol.2022 No.04

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