Larval RNA Interference in the Red Flour Beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.     

RAPD-based genetic linkage maps of Tribolium castaneum.

A genetic map of the red flour beetle (Tribolium castaneum) integrating molecular with morphological markers was constructed using a backcross population of 147 siblings. The map defines 10 linkage groups (LGs), presumably corresponding to the 10 chromosomes, and consists of 122 randomly amplified polymorphic DNA (RAPD) markers, six molecular markers representing identified genes, and five morphological markers. The total map length is 570 cM, giving an average marker resolution of 4.3 cM. The average physical distance per genetic distance was estimated at 350 kb/cM. A cluster of loci showing distorted segregation was detected on LG9. The process of converting RAPD markers to sequence-tagged site markers was initiated: 18 RAPD markers were cloned and sequenced, and single-strand conformational polymorphisms were identified for 4 of the 18. The map positions of all 4 coincided with those of the parent RAPD markers.     

A genetic maternal effect on egg surface in Tribolium castaneum.

Females homozygous for an autosomal recessive gene, wd, produce "weird" eggs that are dry when laid. The wd gene has some important population consequences and also provides a very useful example of a genetic maternal effect for laboratory courses.     

Neuropeptide precursors in Tribolium castaneum

Neuropeptides and neurohormones are among the more diverse and functionally important classes of cell-to-cell signaling molecules involved in animal development and behavior. Less is known about the hormones and neuropeptides of the red flour beetle, Tribolium castaneum, than many other insects. However, the genomic information becoming available from this organism presents an opportunity to identify multiple neuropeptide and hormone genes, and hence their associated protein precursors. Using similarity-based prediction, we report new neuropeptides and hormone precursors from T. castaneum, bringing the number of annotated precursors to 37. We identified one prohormone (SVDPIDGDLIG-containing) having little similarity to other insect prohormones. The conversion of the protein precursors into bioactive peptides requires a suite of processing enzymes and a number of enzymatic steps; using the web-based NeuroPred application and similarity-based bioinformatics approaches, we predict 132 likely peptides that may result from the enzymatic processing of these gene products.     

Large diversity of the piggyBac-like elements in the genome of Tribolium castaneum

The piggyBac transposable element (TE), originally discovered in the cabbage looper, Trichoplusia ni, has been widely used in insect transgenesis including the red flour beetle Tribolium castaneum. We surveyed piggyBac-like (PLE) sequences in the genome of T. castaneum by homology searches using as queries the diverse PLE sequences that have been described previously. The search yielded a total of 32 piggyBac-like elements (TcPLEs) which were classified into 14 distinct groups. Most of the TcPLEs contain defective functional motifs in that they are lacking inverted terminal repeats (ITRs) or have disrupted open reading frames. Only one single copy of TcPLE1 appears to be intact with imperfect 16bp ITRs flanking an open reading frame encoding a transposase of 571 amino acid residues. Many copies of TcPLEs were found to be inserted into or close to other transposon-like sequences. This large diversity of TcPLEs with generally low copy numbers suggests multiple invasions of the TcPLEs over a long evolutionary time without extensive multiplications or occurrence of rapid loss of TcPLEs copies.     

The karyosphere capsule in Tribolium castaneum oocytes

The structure and composition of the karyosphere (karyosome) capsule of the laboratory insect, Tribolium castaneum, were studied with the use of electron and immunoelectron microscopy. Eight stages that characterized the period of oocyte growth have been established basing on the study of nuclear structure dynamics. At the diplotene stage, T. castaneum oocyte chromosomes are being united early to form a compact karyosphere; however, prominent chromatin condensation does not occur at the same time. The process of karyosphere formation is accompanied by the development of a spacious extrachromosomal capsule surrounding chromatin. The capsule consists of a fibrous material of different morphological types. The most prominent molecular components of T. castaneum karyosphere capsule are represented by the proteins of nuclear matrix, including F-actin and lamin B. Apart from the structural proteins, immunocytochemical approach allowed revealing Sm proteins of small nuclear (sn) RNPs and “mature” snRNAs with 2,2,7-trimetyl guanosine (TMG) cap at the 5’-end of their molecules. These data may serve as a base for further broadening of the conception about the functions of the karyosphere capsule as a specialized oocyte nuclear domain. We believe that T. castaneum karyosphere capsule plays not only a structural role, but may be involved directly in the processes related to gene expression.     

AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum)

The red flour beetle (Tribolium castaneum) is a major pest of stored grain and grain products and a popular model species for a variety of ecological, evolutionary, and developmental biology studies. Development of a linkage map based on reproducible and highly polymorphic molecular markers would greatly facilitate research in these disciplines. We have developed a genetic linkage map using 269 amplified fragment length polymorphism (AFLP) markers. Ten previously known random amplified polymorphic DNA (RAPD) markers were used as anchor markers for linkage group assignment. The linkage map was constructed through genotyping two independent F(2) segregating populations with 48 AFLP primer combinations. Each primer combination generated an average of 4.6 AFLP markers eligible for linkage mapping. The length of the integrated map is 573 cM, giving an average marker resolution of 2.0 cM and an average physical distance per genetic distance of 350 kb/cM. A cluster of loci on linkage group 3 exhibited significant segregation distortion. We have also identified six X-linked and two Y-linked markers. Five mapped AFLP fragments were sequenced and converted to sequence-tagged site (STS) markers.     

赤拟谷盗微卫星多态性的研究

为了研究赤拟谷盗种群的遗传结构,采用微卫星分子标记技术,对4个不同地理种群共120个个体的遗传多样性水平及种群遗传结构进行了研究.通过提取基因组DNA,用5对微卫星引物进行PCR扩增、电泳分析、凝胶成像系统分析,结果表明CB337218、DN648427、DN647734、EB754173、EB750288这5个位点的等位基因数分别为4、4、3、6和3, 4个不同地理种群赤拟谷盗的遗传杂合度平均值为0.4379,4个不同地理种群赤拟谷盗间的多态信息含量各在0.3771～0.5089之间,都达到中度多态性或高度多态性水平.为进一步研究赤拟谷盗的成灾机理提供了分子遗传学的基础.     

Epistatic contributions to quantitative traits in Tribolium castaneum

Summary Triple-testcross experiments were used to analyze epistatic contributions to % hatchability of eggs, age of pupation, number of eggs laid in 24-hour period, and survival from hatching to day 35. Seven diverse inbred lines and the F₁ produced by crossing the two tester lines were examined for the presence of epistasis. There was evidence of epistasis for each of the 4 traits in at least one of the 8 lines tested. Epistasis was a major source of variation in survival in all of the lines tested.     

Cytogenetics of chromosome rearrangements in Tribolium castaneum.

The karyotype of the red flour beetle, Tribolium castaneum, was reexamined and improved by restriction enzyme banding with HpaII. After this treatment, each of the 10 chromosomes were identified in spermatogonial metaphase cells and 3 of the 8 autosomal bivalents and the XY pair were identified in spermatocyte metaphase I nuclei. Based on centromere position, relative length, and banding pattern, probable correlations between some of the mitotic chromosomes and some of the metaphase I bivalents were ascertained. Thus improved, the karyotypes of beetles harboring genetically defined translocations were investigated. Spermatocyte metaphase I nuclei were most informative, as normal chromosome pairing was visibly disrupted by rearrangements. Bivalents associated with each rearrangement were identified. Results demonstrated that each of the five best defined T. castaneum linkage groups corresponds to a different chromosome and established correspondence between bivalents and linkage groups 1-4. The relevance of these findings is discussed with regard to Tribolium genetics and evolution.     

Mutational variance for pupa weight in Tribolium castaneum

Summary Directional selection for heavier pupa weight in Tribolium castaneum was practiced for 18 generations in two replicates of an inbred line, each separately maintained in small population cages for more than 90 generations. Mutational variance was estimated in two ways, based on Hill's (1982a) prediction equation for response to directional selection where an equilibrium state between effective population size and variation created by new mutation is assumed. Estimates of mutational variance based on response to selection in a selected population and from a sire-offspring regression analysis in an unselected control population were in strong agreement within each replicate population. Significant differences between the two replicates were observed. Estimates of the ratio of mutational variance to environmental variance ranged from 0.0002 to 0.0012, depending upon the assumptions made about effective population sizes maintained in the two replicate lines. Estimates of realized heritability from the 18 generations of selection were 0.23±0.02 and 0.12±0.02 in the two replicates. The results support the hypothesis that mutation may have played a significant role in supplying useful genetic variation for long-continuing response to selection for this trait in experiments reported earlier.     

Epistatic contributions to quantitative traits in Tribolium castaneum

Summary Triple-testcross experiments were used to analyze epistatic contributions to larva weight, pupa weight, pupa width and adult weight in Tribolium castaneum. Seven diverse inbred lines and the F₁. produced by crossing the two tester lines were examined for indications of epistasis. Larva weight was the only trait for which no significant epistasis was detected. There was significant epistasis for pupa weight in three of the inbred lines; for pupa width in four of the inbred lines; for adult weight in five of the inbred lines. Only one inbred line and the F₁ line failed to exhibit significant epistasis for any trait. Each inbred line had a unique pattern of epistasis, suggesting that a number of different loci were contributing to the detected epistasis.This paper (No. 76-5-158) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Individual and group selection for productivity in Tribolium castaneum

Summary The usefulness of a non-random group selection method in the improvement of a character influenced by interactions among individuals has been experimentally tested in a population of Tribolium castaneum. The selected trait —number of adults produced in a fixed period of time —showed a clear increase after 11 generations of selection. This increase is related to a reduction in developmental time, and it was specific for the population structure in which selection was applied, vanishing when this structure was altered.