Richard W. Beeman

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1‐specific RNA interference (RNAi) disrupted all three types of moult (larval–larval, larval–pupal and pupal–adult) and greatly reduced whole‐body chitin content. Exon‐specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval‐pupal and pupal‐adult moults, whereas splice variant 8b was required only for the latter. TcCHS2‐specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi‐mediated down‐regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

Functional specialization among insect chitinase family genes revealed by RNA interference

The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal–adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval–larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.

Genetic analysis of the homeotic gene complex (HOM-C) in the beetle Tribolium castaneum.

Our laboratories have undertaken both genetic and molecular studies of the homeotic gene complex (HOM-C) of the beetle Tribolium castaneum, and this paper discusses results from our genetic analyses. We describe here the adult phenotypes and complementation behavior of over 50 new mutations. Many of these homeotic phenotypes resemble those of Drosophila melanogaster, but few precisely parallel the segmental transformations seen in this fly. Analysis of putative loss-of-function mutations affecting the head and thorax suggests that the maxillopedia and Cephalothorax genes most closely resemble proboscipedia and Sex combs reduced of Drosophila. In the abdomen, putative loss-of-function alleles of Abdominal affect a domain corresponding to those of the combined abdominal-A and Abdominal-B genes of Drosophila. In contrast to the situation in flies, Abdominal loss-of-function variants in Tribolium cause anteriorward transformations in A3-A5a, but posteriorward transformations in A5p-A7. The implications of the differences in developmental strategies evolved in Tribolium vs Drosophila are discussed.

piggyBac-mediated germline transformation in the beetle Tribolium castaneum

The lepidopteran transposable element piggyBac can mediate germline insertions in at least four insect orders. It therefore shows promise as a broad‐spectrum transformation vector, but applications such as enhancer trapping and transposon‐tag mutagenesis are still lacking. We created, cloned, sequenced and genetically mapped a set of piggyBac insertions in the red flour beetle, Tribolium castaneum. Transpositions were precise, and specifically targeted the canonical TTAA recognition sequence. We detected several novel reporter‐expression domains, indicating that piggyBac could be used to identify enhancer regions. We also demonstrated that a primary insertion of a non‐autonomous element can be efficiently remobilized to non‐homologous chromosomes by injection of an immobile helper element into embryos harbouring the primary insertion. These developments suggest potential for more sophisticated methods of piggyBac‐mediated genome manipulation.

BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum

BeetleBase (http://www.beetlebase.org) has been updated to provide more comprehensive genomic information for the red flour beetle Tribolium castaneum. The database contains genomic sequence scaffolds mapped to 10 linkage groups (genome assembly release Tcas_3.0), genetic linkage maps, the official gene set, Reference Sequences from NCBI (RefSeq), predicted gene models, ESTs and whole-genome tiling array data representing several developmental stages. The database was reconstructed using the upgraded Generic Model Organism Database (GMOD) modules. The genomic data is stored in a PostgreSQL relatational database using the Chado schema and visualized as tracks in GBrowse. The updated genetic map is visualized using the comparative genetic map viewer CMAP. To enhance the database search capabilities, the BLAST and BLAT search tools have been integrated with the GMOD tools. BeetleBase serves as a long-term repository for Tribolium genomic data, and is compatible with other model organism databases.

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum

Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, we performed systemic RNA interference (RNAi) experiments utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods.

Molecular and Functional Analyses of Amino Acid Decarboxylases Involved in Cuticle Tanning in Tribolium castaneum

Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide β-alanine and dopamine used in insect cuticle tanning. β-Alanine is conjugated with dopamine to yield N-β-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of β-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA.

Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum

The expression profiles of nine genes encoding chitin deacetylase (CDA)-like proteins were studied during development and in various tissues of the red flour beetle, Tribolium castaneum, by RT-PCR. TcCDA1, TcCDA2 and TcCDA5 were expressed throughout all stages of development, while TcCDA6-9 were expressed predominantly during larval feeding stages. In situ hybridization experiments revealed that both TcCDA1 and TcCDA2 were expressed in epidermal cells. Polyclonal antibody to TcCDA1 detected an immunoreactive protein in larval tracheae. TcCDA6 through TcCDA9, which belong to a distinct subgroup of gut-specific CDAs, were transcribed in the cells lining the midgut, including epithelial cells. TcCDA3 was expressed in the thoracic muscles, whereas TcCDA4 was expressed in early imaginal appendages. To study the function(s) of individual TcCDA genes, double-stranded RNAs (dsRNA) specific for each gene were injected into insects at different developmental stages and the phenotypes were monitored. No visible phenotypic changes were observed after injection of dsRNAs for TcCDA3 to 9, whereas injection of dsRNAs for TcCDA1 or TcCDA2 affected all types of molts, including larval-larval, larval-pupal and pupal-adult. Insects treated with these dsRNAs could not shed the old cuticle and were trapped in their exuviae. Interestingly, unique and very dissimilar adult phenotypes were observed after injection of dsRNAs that specifically down-regulated either of the two alternatively spliced transcripts of TcCDA2, namely TcCDA2a or TcCDA2b. These results reveal functional specialization among T. castaneum CDA genes and splice variants.

Chitin synthases are required for survival, fecundity and egg hatch in the red flour beetle, Tribolium castaneum

The synthesis of chitin, the beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, TcCHS-A and TcCHS-B, during embryonic and adult development in the red flour beetle, Tribolium castaneum, RNA interference (RNAi) experiments were carried out. When dsRNA for TcCHS-A was injected into male or female pharate adults, all insects died 5-7 d after the adult molt, and the females failed to oviposit prior to death. When dsTcCHS-A was injected into young adults 1-2 d post-eclosion, a similar lethal phenotype was obtained after 5 d and no oviposition occurred. When dsTcCHS-A injections were delayed until after adult maturation (7-10 d post-eclosion), the treated females did oviposit and the resulting embryos appeared to develop normally. However, the chitin content of the eggs was dramatically reduced, the embryos became twisted and enlarged, and the eggs did not hatch. Adults treated with dsRNA for TcCHS-B exhibited little or no chitin in their PM and died about 2 wk after injection. None of the TcCHS-B-treated females oviposited, which was probably a secondary effect caused by starvation. These results extend our previous findings that CHS genes are required for all types of molt. The present study also demonstrates that these genes have additional roles in embryonic and adult development.

Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects

A bioinformatics-based investigation of three insect species with completed genome sequences has revealed that insect chitinase-like proteins (glycosylhydrolase family 18) are encoded by a rather large and diverse group of genes. We identified 16, 16 and 13 putative chitinase-like genes in the genomic databases of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. Chitinase-like proteins encoded by this gene family were classified into five groups based on phylogenetic analyses. Group I chitinases are secreted proteins that are the most abundant such enzymes in molting fluid and/or integument, and represent the prototype enzyme of the family, with a single copy each of the catalytic domain and chitin-binding domain (ChBD) connected by an S/T-rich linker polypeptide. Group II chitinases are unusually larger-sized secreted proteins that contain multiple catalytic domains and ChBDs. Group III chitinases contain two catalytic domains and are predicted to be membrane-anchored proteins. Group IV chitinases are the most divergent. They usually lack a ChBD and/or an S/T-rich linker domain, and are known or predicted to be secreted proteins found in gut or fat body. Group V proteins include the putative chitinase-like imaginal disc growth factors (IDGFs). In each of the three insect genomes, multiple genes encode group IV and group V chitinase-like proteins. In contrast, groups I-III are each represented by only a singe gene in each species.