Nico Posnien

Divergent functions of orthodenticle, empty spiracles and buttonhead in early head patterning of the beetle Tribolium castaneum (Coleoptera).

The head gap genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) are required for metamerization and segment specification in Drosophila. We asked whether the function of their orthologs is conserved in the red flour beetle Tribolium castaneum which in contrast to Drosophila develops its larval head in a way typical for insects. We find that depending on dsRNA injection time, two functions of Tc-orthodenticle1 (Tc-otd1) can be identified. The early regionalization function affects all segments formed during the blastoderm stage while the later head patterning function is similar to Drosophila. In contrast, both expression and function of Tc-empty spiracles (Tc-ems) are restricted to the posterior part of the ocular and the anterior part of the antennal segment and Tc-buttonhead (Tc-btd) is not required for head cuticle formation at all. We conclude that the gap gene like roles of ems and btd are not conserved while at least the head patterning function of otd appears to be similar in fly and beetle. Hence, the ancestral mode of insect head segmentation remains to be discovered. With this work, we establish Tribolium as a model system for arthropod head development that does not suffer from the Drosophila specific problems like head involution and strongly reduced head structures.

RNAi in the Red Flour Beetle (Tribolium)

INTRODUCTION Tribolium castaneum is exceptionally amenable to gene knockdown by RNA interference (RNAi) which, in this insect, is systemic (spreading throughout the organism and to the next generation), highly penetrant, and able to phenocopy genetic null phenotypes. Hence, any gene function can be knocked down at any stage in (apparently) all tissues upon injection of double-stranded RNA (dsRNA). The RNAi effect is elicited both in the injected animal and, if female pupae or adults have been injected, transferred to the offspring. Embryonic RNAi (eRNAi) usually generates the strongest phenotypes in the injected individual, but suffers from elevated lethality caused by injection injury. Pupal RNAi (pRNAi), in which female pupae are injected and phenotypes scored in the offspring, is the easiest to perform. However, in some cases, the knockdown of a gene leads to sterility of the injected female. This problem can be circumvented in many cases by injecting adult females (aRNAi) or using eRNAi. In order to interfere with processes during metamorphosis, injection into last-stage larvae is used (lRNAi). Up to two genes in a single experiment have been successfully knocked down via RNAi. The inclusion of more than two genes usually leads to a dilution effect, which lowers phenotypic strength. This protocol describes the production of dsRNA from a polymerase chain reaction (PCR) template, injection procedures for each Tribolium life stage, and important controls for effective analysis.

Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development.

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.

The insect upper lip (labrum) is a nonsegmental appendage‐like structure

SUMMARY The insect upper lip—the labrum—is a lobe‐like structure anterior to the mouth opening. Whether the labrum represents a fused pair of segmental appendages or evolved independently is heavily debated. Here, we identify additional similarities of the regulatory gene network active in labrum and trunk appendages. However, we do not find a labral parasegment boundary and we show that labral Tc‐Dll expression is independent of Tc‐wg and Tc‐hh signals. In contrast, Tc‐Dll expression in all trunk appendages does require these signals. Finally, we identify crucial differences between the location of the labrum and trunk appendages: the labrum develops in median rather than lateral tissues and is part of an anterior nonsegmental tissue marked by and dependent on Tc‐six3 activity. To reconcile these seeming contradictory results, we propose that the genetic network evolved in either labrum or trunk appendages and became redeployed at a novel location to form the other structure.

Asymmetrically expressed axin required for anterior development in Tribolium

Canonical Wnt signaling has been implicated in an AP axis polarizing mechanism in most animals, despite limited evidence from arthropods. In the long-germ insect, Drosophila, Wnt signaling is not required for global AP patterning, but in short-germ insects including Tribolium castaneum, loss of Wnt signaling affects development of segments in the growth zone but not those defined in the blastoderm. To determine the effects of ectopic Wnt signaling, we analyzed the expression and function of axin, which encodes a highly conserved negative regulator of the pathway. We found Tc-axin transcripts maternally localized to the anterior pole in freshly laid eggs. Expression spread toward the posterior pole during the early cleavage stages, becoming ubiquitous by the time the germ rudiment formed. Tc-axin RNAi produced progeny phenotypes that ranged from mildly affected embryos with cuticles displaying a graded loss of anterior structures, to defective embryos that condensed at the posterior pole in the absence of serosa. Altered expression domains of several blastodermal markers indicated anterior expansion of posterior fates. Analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the effects of Tc-axin depletion are mediated through this pathway and that Wnt signaling must be inhibited for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt signaling must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt activity in defining AP polarity and patterning in metazoan development.

Genetics, development and composition of the insect head – A beetle’s view

Many questions regarding evolution and ontogeny of the insect head remain open. Likewise, the genetic basis of insect head development is poorly understood. Recently, the investigation of gene expression data and the analysis of patterning gene function have revived interest in insect head development. Here, we argue that the red flour beetle Tribolium castaneum is a well suited model organism to spearhead research with respect to the genetic control of insect head development. We review recent molecular data and discuss its bearing on early development and morphogenesis of the head. We present a novel hypothesis on the ontogenetic origin of insect head sutures and review recent insights into the question on the origin of the labrum. Further, we argue that the study of developmental genes may identify the elusive anterior non-segmental region and present some evidence in favor of its existence. With respect to the question of evolution of patterning we show that the head Anlagen of the fruit fly Drosophila melanogaster and Tribolium differ considerably and we review profound differences of their genetic regulation. Finally, we discuss which insect model species might help us to answer the open questions concerning the genetic regulation of head development and its evolution.

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BackgroundThe duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum.ResultsWe found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication.ConclusionsOur results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.

Single and Double Whole-Mount In Situ Hybridization in Red Flour Beetle (Tribolium) Embryos

INTRODUCTION The red flour beetle, Tribolium castaneum, has emerged as an important model system for studying the evolution of development. Studies with Tribolium complement the vast amount of research done with Drosophila. Developmental features that are conserved between Drosophila and Tribolium, such as body segmentation, are achieved by quite different means, and thus comparison of developmental mechanisms between these two insects can address many interesting questions concerning the evolution of morphology and other characters. Most in situ protocols used for Tribolium have been adapted from Drosophila studies. Whole-mount in situ hybridization is a standard technique to visualize the activity of genes in embryos. The single and double staining protocol presented here uses two nonfluorescent stains to reveal gene activity. The development of both stains can be monitored visually, allowing the strength of the signal to be adjusted as needed. Cells that express both of the genes under investigation are readily detected using a microscope. The use of EGTA during fixation increases the proportion of embryos that devitellinize upon methanol treatment.

A Comprehensive Reference Transcriptome Resource for the Common House Spider Parasteatoda tepidariorum

Parasteatoda tepidariorum is an increasingly popular model for the study of spider development and the evolution of development more broadly. However, fully understanding the regulation and evolution of P. tepidariorum development in comparison to other animals requires a genomic perspective. Although research on P. tepidariorum has provided major new insights, gene analysis to date has been limited to candidate gene approaches. Furthermore, the few available EST collections are based on embryonic transcripts, which have not been systematically annotated and are unlikely to contain transcripts specific to post-embryonic stages of development. We therefore generated cDNA from pooled embryos representing all described embryonic stages, as well as post-embryonic stages including nymphs, larvae and adults, and using Illumina HiSeq technology obtained a total of 625,076,514 100-bp paired end reads. We combined these data with 24,360 ESTs available in GenBank, and 1,040,006 reads newly generated from 454 pyrosequencing of a mixed-stage embryo cDNA library. The combined sequence data were assembled using a custom de novo assembly strategy designed to optimize assembly product length, number of predicted transcripts, and proportion of raw reads incorporated into the assembly. The de novo assembly generated 446,427 contigs with an N50 of 1,875 bp. These sequences obtained 62,799 unique BLAST hits against the NCBI non-redundant protein data base, including putative orthologs to 8,917 Drosophila melanogaster genes based on best reciprocal BLAST hit identity compared with the D. melanogaster proteome. Finally, we explored the utility of the transcriptome for RNA-Seq studies, and showed that this resource can be used as a mapping scaffold to detect differential gene expression in different cDNA libraries. This resource will therefore provide a platform for future genomic, gene expression and functional approaches using P. tepidariorum.