The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.     

Determinants of virulence for the parasite Nosema whitei in its host Tribolium castaneum

For many parasites, especially those that obligately kill the host for transmission, host age is crucially important to determine success. Here, we have experimentally investigated this relationship with the microsporidian parasite, Nosema whitei, in its host, the Red Flour Beetle, Tribolium castaneum. We find that infection is only possible in young larvae and that spore load at the time of transmission (i.e., host death) correlates with host body size. The data suggested that an infection by N. whitei prolongs the life span of the infected larva and prevents them from pupation. Together, virulence to the host and success for the parasite is mainly determined by the host age at infection. The patterns are consistent with theoretical predictions for obligate killer parasites.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum

As invertebrates lack the molecular machinery employed by the vertebrate adaptive immune system, it was thought that they consequently lack the ability to produce lasting and specific immunity. However, in recent years, it has been demonstrated that the immune defence of invertebrates is by far more complicated and specific than previously envisioned. Lasting immunity following an initial exposure that proves protection on a secondary exposure has been shown in several species of invertebrates. This phenomenon has become known as immune priming. In the cases where it is explicitly tested, this priming can also be highly specific. In this study, we used survival assays to test for specific priming of resistance in the red flour beetle, Tribolium castaneum, using bacteria of different degrees of relatedness. Our results suggest an unexpected degree of specificity that even allows for differentiation between different strains of the same bacterium. However, our findings also demonstrate that specific priming of resistance in insects may not be ubiquitous across all bacteria.     

Identification and characterization of novel ER-based hsp90 gene in the red flour beetle,Tribolium castaneum

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0487-y) contains supplementary material, which is available to authorized users.     

Effects of assortative mating for pupa weight on genetic parameters of unselected Tribolium castaneum

Summary Effects of random (R) or positive assortative (A) mating for pupal weight (PW) on genetic parameters of pupation time (PT), pupal and larval weights (LW) were studied in unselected populations of Tribolium castaneum. Two groups, each with 50 males mated to 100 females in each of 5 replicates, were either R-mated or A-mated for 3 generations. Genetic parameters were estimated from covariances between sibs (R group) or by an iterative method (A group). Estimates of heritability in R and A groups were 0.30±0.12 and 0.39±0.02 (PW); 0.26±0.13 and 0.49±0.04 (LW); and 0.39±0.10 and 0.25±0.03 (PT). Estimates of genetic correlations in the R group were –0.21±0.23 (PW and LW); 0.45±0.10 (PW and PT); and –0.77±0.14 (LW and PT). Those in the A group were 0.27±0.10 (PW and LW); 0.15±0.14 (PW and PT); the genetic correlation between LW and PT was not estimable in this group. Within-family variances (grams squared) of PW by generation (1, 2, and 3) were, respectively: 0.048 (R) and 0.047 (A); 0.054 (R) and 0.041 (A); and 0.050 (R) and 0.046 (A). In agreement with theory, estimates of heritability of PW and LW were larger in the A group. Estimates of genetic correlations in the A group were inconsistent with expectations from theory. Assortative mating tended to decrease within-family variance of PW.     

Pesticidal and pest repellency activities of rhizomes of Drynaria quercifolia (J. Smith) against Tribolium castaneum (Herbst)

Background

Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest.

Results

Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm² and significant pest repellency activity at doses 0.94 to 0.23 mg/cm². No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid.

Conclusion

Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifolia is useful in controlling T. castaneum of stored grain and flour-based products.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-51) contains supplementary material, which is available to authorized users.     

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.     

Genome-wide analysis of tandem repeats in Tribolium castaneum genome reveals abundant and highly dynamic tandem repeat families with satellite DNA features in euchromatic chromosomal arms

Although satellite DNAs are well-explored components of heterochromatin and centromeres, little is known about emergence, dispersal and possible impact of comparably structured tandem repeats (TRs) on the genome-wide scale. Our bioinformatics analysis of assembled Tribolium castaneum genome disclosed significant contribution of TRs in euchromatic chromosomal arms and clear predominance of satellite DNA-typical 170 bp monomers in arrays of ≥5 repeats. By applying different experimental approaches, we revealed that the nine most prominent TR families Cast1–Cast9 extracted from the assembly comprise ∼4.3% of the entire genome and reside almost exclusively in euchromatic regions. Among them, seven families that build ∼3.9% of the genome are based on ∼170 and ∼340 bp long monomers. Results of phylogenetic analyses of 2500 monomers originating from these families show high-sequence dynamics, evident by extensive exchanges between arrays on non-homologous chromosomes. In addition, our analysis shows that concerted evolution acts more efficiently on longer than on shorter arrays. Efficient genome-wide distribution of nine TR families implies the role of transposition only in expansion of the most dispersed family, and involvement of other mechanisms is anticipated. Despite similarities in sequence features, FISH experiments indicate high-level compartmentalization of centromeric and euchromatic tandem repeats.     

Translocation of bacteria from the gut to the eggs triggers maternal transgenerational immune priming in Tribolium castaneum

Invertebrates can be primed to enhance their protection against pathogens they have encountered before. This enhanced immunity can be passed maternally or paternally to the offspring and is known as transgenerational immune priming. We challenged larvae of the red flour beetle Tribolium castaneum by feeding them on diets supplemented with Escherichia coli, Micrococcus luteus or Pseudomonas entomophila, thus mimicking natural exposure to pathogens. The oral uptake of bacteria induced immunity-related genes in the offspring, but did not affect the methylation status of the egg DNA. However, we observed the translocation of bacteria or bacterial fragments from the gut to the developing eggs via the female reproductive system. Such translocating microbial elicitors are postulated to trigger bacterial strain-specific immune responses in the offspring and provide an alternative mechanistic explanation for maternal transgenerational immune priming in coleopteran insects.     

mummy/cystic encodes an enzyme required for chitin and glycan synthesis, involved in trachea, embryonic cuticle and CNS development--analysis of its role in Drosophila tracheal morphogenesis

Tracheal and nervous system development are two model systems for the study of organogenesis in Drosophila. In two independent screens, we identified three alleles of a gene involved in tracheal, cuticle and CNS development. Here, we show that these alleles, and the previously identified cystic and mummy, all belong to the same complementation group. These are mutants of a gene encoding the UDP-N-acetylglucosamine diphosphorylase, an enzyme responsible for the production of UDP-N-acetylglucosamine, an important intermediate in chitin and glycan biosynthesis. cyst was originally singled out as a gene required for the regulation of tracheal tube diameter. We characterized the cyst/mmy tracheal phenotype and upon histological examination concluded that mmy mutant embryos lack chitin-containing structures, such as the procuticle at the epidermis and the taenidial folds in the tracheal lumen. While most of their tracheal morphogenesis defects can be attributed to the lack of chitin, when compared to krotzkopf verkehrt (kkv) chitin-synthase mutants, mmy mutants showed a stronger phenotype, suggesting that some of the mmy phenotypes, like the axon guidance defects, are chitin-independent. We discuss the implications of these new data in the mechanism of size control in the Drosophila trachea.     

The Tribolium ortholog of knirps and knirps-related is crucial for head segmentation but plays a minor role during abdominal patterning

Segment formation in the long germ insect Drosophila is dominated by overlapping gap gene domains in the syncytial blastoderm. In the short germ beetle Tribolium castaneum abdominal segments arise from a cellular growth zone, implying different patterning mechanisms. We describe here the single Tribolium ortholog of the Drosophila genes knirps and knirps-related (called Tc-knirps). Tc-knirps expression is conserved during head patterning and at later stages. However, posterior Tc-knirps expression in the ectoderm is limited to a stripe in A1, instead of a broad abdominal domain covering segment primordia A2-A5 as in Drosophila. Tc-knirps RNAi yields only mild defects in the abdomen, at a position posterior to the abdominal Tc-knirps domain. In addition, Tc-knirps RNAi larvae lack the antennal and mandibular segments. These defects are much more severe than the head defects caused by combined inactivation of Dm-knirps and Dm-knirps-related. Our findings support the notion that the role of gap gene homologs in abdominal segmentation differs fundamentally in long and short germ insects. Moreover, the pivotal role of Tc-knirps in the head suggests an ancestral role for knirps as head patterning gene. Based on this RNAi analysis, Tc-knirps functions neither in the head nor the abdomen as a canonical gap gene.     

Structure, function and dynamics in the mur family of bacterial cell wall ligases

For bacteria, the structural integrity of its cell wall is of utmost importance for survival, and to this end, a rigid scaffold called peptidoglycan, comprised of sugar molecules and peptides, is synthesized and located outside the cytoplasmic membrane of the cell. Disruption of this peptidoglycan layer has for many years been a prime target for effective antibiotics, namely the penicillins and cephalosporins. Because this rigid layer is synthesized by a multi-step pathway numerous additional targets also exist that have no counterpart in the animal cell. Central to this pathway are four similar ligase enzymes, which add peptide groups to the sugar molecules, and interrupting these steps would ultimately prove fatal to the bacterial cell. The mechanisms of these ligases are well understood and the structures of all four of these ligases are now known. A detailed comparison of these four enzymes shows that considerable conformational changes are possible and that these changes, along with the recruitment of two different N-terminal binding domains, allows these enzymes to bind a substrate which at one end is identical and at the other has the growing polypeptide tail. Some insights into the structure-function relationships in these enzymes is presented.     

Effects of low-intensity microwave radiation on Tribolium castaneum physiological and biochemical characteristics and survival

The red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) is a widespread pest that lives in, and feeds on, wheat flour. Here, we studied the effects of low-intensity microwave radiation (LIMR; ≤2.0 kW/kg) on physiological and biochemical characteristics of T. castaneum, and compared them to the effects of heat conduction treatment, to provide a theoretical basis for using LIMR for pest control. Lethal model equations with respect to temperature were shown to provide acceptable fitting accuracy for the effects of LIMR treatment. Semi-lethal and lethal temperatures induced through LIMR (48 °C and 50 °C, respectively) for T. castaneum were lower than those induced through heat conduction (50 °C and 52 °C). When T. castaneum were subjected to LIMR, the insects’ moisture content, pH values, alkaline phosphatase and acetyl cholinesterase activity were all lower than when the insects were subjected to heat conduction. Peroxide values and total free amino acid content were higher, and protein subunits molecular weights were lower when T. castaneum were exposed to LIMR than to heat; moreover, after LIMR exposure, the amino acid composition of T. castaneum was changed and the insect's DNA was damaged.     

Both upstream and downstream intergenic regions are critical for the mob as tumor suppressor gene activity in Drosophila

The Drosophila mats gene plays a critical role in growth control. Using molecular genetic approaches we investigated how mats is regulated in development. A 2236-bp genomic sequence that contains entire mats including upstream and downstream intergenic regions can rescue mats mutant phenotypes, indicating that regulatory elements necessary for proper mats expression are mostly retained. However, constructs without the upstream or downstream intergenic region failed to rescue mats mutants, demonstrating the functional importance of these sequences. Moreover, mats expression is reduced in mats^e17, a mats allele with over one-third of the downstream intergenic region deleted. Consistent with a model that the downstream intergenic region is critical for mats activity, this sequence contains evolutionarily conserved elements and has enhancer activities.