Enhanced genome assembly and a new official gene set for Tribolium castaneum.

BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.

Functional characterization of the Drosophila suzukii pro-apoptotic genes reaper, head involution defective and grim.

Apoptosis is a fundamental process for the elimination of damaged or unwanted cells, and is a key aspect of development. It is triggered by pro-apoptotic genes responding to the intrinsic pathway that senses cell stress or the extrinsic pathway that responds to signals from other cells. The disruption of these genes can therefore lead to developmental defects and disease. Pro-apoptotic genes have been studied in detail in the fruit fly Drosophila melanogaster, a widely-used developmental model. However, little is known about the corresponding genes in its relative D. suzukii, a pest of soft fruit crops that originates from Asia but is now an invasive species in many other regions. The characterization of D. suzukii pro-apoptotic genes could lead to the development of transgenic sexing strains for pest management. Here, we describe the isolation and characterization of the pro-apoptotic genes reaper (Dsrpr), head involution defective (Dshid) and grim (Dsgrim) from a laboratory strain of D. suzukii. We determined their expression profiles during development, revealing that all three genes are expressed throughout development but Dsrpr is expressed most strongly, especially at the pupal stage. Functional analysis was carried out by expressing single genes or pairs (linked by a 2A peptide) in S2 cell death assays, indicating that Dsgrim and Dshid are more potent pro-apoptotic genes than Dsrpr, and the lethality can be significantly enhanced by co-expression of two genes. Therefore, the binary or multiple expression of different pro-apoptotic genes can be considered to build an efficient transgenic sexing system in D. suzukii.

Identification and characterization of four Drosophila suzukii cellularization genes and their promoters.

BACKGROUND: The spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product. RESULTS: Here, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5' flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells. CONCLUSIONS: The similarities in the gene coding and 5' flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

A morphological novelty evolved by co-option of a reduced gene regulatory network and gene recruitment in a beetle.

The mechanisms underlying the evolution of morphological novelties have remained enigmatic but co-option of existing gene regulatory networks (GRNs), recruitment of genes and the evolution of orphan genes have all been suggested to contribute. Here, we study a morphological novelty of beetle pupae called gin-trap. By combining the classical candidate gene approach with unbiased screening in the beetle Tribolium castaneum, we find that 70% of the tested components of the wing network were required for gin-trap development. However, many downstream and even upstream components were not included in the co-opted network. Only one gene was recruited from another biological context, but it was essential for the anteroposterior symmetry of the gin-traps, which represents a gin-trap-unique morphological innovation. Our data highlight the importance of co-option and modification of GRNs. The recruitment of single genes may not be frequent in the evolution of morphological novelties, but may be essential for subsequent diversification of the novelties. Finally, after having screened about 28% of annotated genes in the Tribolium genome to identify the genes required for gin-trap development, we found none of them are orphan genes, suggesting that orphan genes may have played only a minor, if any, role in the evolution of gin-traps.

Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

BACKGROUND: Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening. RESULTS: We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites. CONCLUSIONS: Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

The evolution of the knirps family of transcription factors in arthropods.

The orphan nuclear receptor gene knirps and its relatives encode a small family of highly conserved proteins. We take advantage of the conservation of the family, using the recent prevalence of genomic data, to reconstruct its evolutionary history, identifying duplication events and tracing the intron-exon structure of the genes over evolution. Many arthropod species have two or three members of this family, but the orthology between members is unclear. We have analyzed the protein coding sequences of members of this family from 15 arthropod species covering all four main arthropod classes, including a total of 28 genes. All members of the family encode a highly conserved 94 amino acid core sequence, part of which is encoded by a single invariant exon. We find that many of the automated predictions of these genes contain errors, while some copies of the gene were not uncovered by automated pipelines, requiring manual corrections and curation. We use the coding sequences to present a phylogenetic analysis of the knirps family. Our analysis indicates that there was a duplication of a single ancestral gene in the lineage leading to insects, which gave rise to two paralogs, eagle and knirps-related. Descendants of this duplication can be identified by the presence or absence of a short protein-coding motif. Independent, lineage-specific duplications occurred in the two crustaceans we sampled. Within the insects, the knirps-related gene underwent further lineage-specific duplications, giving rise to--among others--the Drosophila gap gene knirps.

RNAi phenotypes are influenced by the genetic background of the injected strain.

BACKGROUND: RNA interference (RNAi) is a powerful tool to study gene function in organisms that are not amenable to classical forward genetics. Hence, together with the ease of comprehensively identifying genes by new generation sequencing, RNAi is expanding the scope of animal species and questions that can be addressed in terms of gene function. In the case of genetic mutants, the genetic background of the strains used is known to influence the phenotype while this has not been described for RNAi experiments. RESULTS: Here we show in the red flour beetle Tribolium castaneum that RNAi against Tc-importin α1 leads to different phenotypes depending on the injected strain. We rule out off target effects and show that sequence divergence does not account for this difference. By quantitatively comparing phenotypes elicited by RNAi knockdown of four different genes we show that there is no general difference in RNAi sensitivity between these strains. Finally, we show that in case of Tc-importin α1 the difference depends on the maternal genotype. CONCLUSIONS: These results show that in RNAi experiments strain specific differences have to be considered and that a proper documentation of the injected strain is required. This is especially important for the increasing number of emerging model organisms that are being functionally investigated using RNAi. In addition, our work shows that RNAi is suitable to systematically identify the differences in the gene regulatory networks present in populations of the same species, which will allow novel insights into the evolution of animal diversity.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

A transgenic female killing system for the genetic control of Drosophila suzukii.

The spotted wing Drosophila (Drosophila suzukii) is an invasive pest of soft-skinned fruit crops. It is rapidly transmitted in Europe and North America, causing widespread agricultural losses. Genetic control strategies such as the sterile insect technique (SIT) have been proposed as environment-friendly and species-restricted approaches for this pest. However, females are inefficient agents in SIT programs. Here we report a conditional female-killing (FK) strategy based on the tetracycline-off system. We assembled sixteen genetic constructs for testing in vitro and in vivo. Twenty-four independent transgenic strains of D. suzukii were generated and tested for female-specific lethality. The strongest FK effect in the absence of tetracycline was achieved by the construct containing D. suzukii nullo promoter for early gene expression, D. suzukii pro-apoptotic gene hidAla4 for lethality, and the transformer gene intron from the Mediterranean fruit fly Ceratitis capitata for female-specific splicing. One strain carrying this construct eliminated 100% of the female offspring during embryogenesis and produced only males. However, homozygous females from these FK strains were not viable on a tetracycline-supplemented diet, possibly due to the basal expression of hidAla4. Potential improvements to the gene constructs and the use of such FK strains in an SIT program are discussed.

Characterization of the Drosophila suzukii ß2-tubulin gene and the utilization of its promoter to monitor sex separation and insemination.

The Drosophila melanogaster ß2-tubulin gene (Dm-ß2t) controls the function of microtubules in the testis and sperm, and has been evaluated for use in biocontrol strategies based on the sterile insect technique, including sexing and the induction of male sterility. The spotted-wing Drosophila (Drosophila suzukii) is native to eastern Asia but has spread globally as an invasive pest of fruit crops, so biocontrol strategies are urgently required for this species. We therefore isolated the ß2tubulin ortholog Ds-ß2t from the USA laboratory strain of D. suzukii and confirmed the presence of functional motifs by aligning orthologs from multiple insects. The developmental expression profile of Ds-ß2t was determined by RT-PCR using gene-specific primers and was similar to that of Dm-ß2t. We then isolated the Ds-ß2t promoter and used it to generate transgenic strains expressing a testis-specific fluorescent protein starting from the thirdinstar larvae. Efficient sexing was achieved based on fluorescence detection, and the transgenic males showed a similar survival rate to wild-type males. Fluorescence imaging and PCR were also used to confirm the insemination of wild-type females by transgenic males. We therefore confirm that D. suzukii strains expressing fluorescent markers under the control of the Ds-ß2t promoter can be used for sexing and the confirmation of mating, and we discuss the wider potential of the Ds-ß2t promoter in the context of genetic control strategies for D. suzukii.

Bicistronic expression and differential localization of proteins in insect cells and Drosophila suzukii using picornaviral 2A peptides.

Polycistronic expression systems in insects can be used for applications such as recombinant protein production in cells, enhanced transgenesis methods, and the development of novel pest-control strategies based on the sterile insect technique (SIT). Here we tested the performance of four picornaviral 2A self-cleaving peptides (TaV-2A, DrosCV-2A, FMDV 2A1/31 and FMDV 2A1/32) for the co-expression and differential subcellular targeting of two fluorescent marker proteins in cell lines (Anastrepha suspensa AsE01 and Drosophila melanogaster S2 cells). We found that all four 2A peptides showed comparable activity in cell lines, leading to the production of independent upstream and downstream proteins that were directed to the nucleus or membrane by a C-terminal nuclear localization signal (NLS) on the upstream protein and a poly-lysine/CAAX membrane anchor on the downstream protein. TaV-2A and DrosCV-2A were inserted into piggyBac constructs to create transgenic D. suzukii strains, confirming efficient ribosomal skipping in vivo. Interestingly, we found that the EGFP-CAAX protein was distributed homogeneously in the membrane whereas the DsRed-CAAX protein formed clumps and aggregates that induced extensive membrane blebbing. Accordingly, only flies expressing the DsRed-NLS and EGFP-CAAX proteins could be bred to homozygosity whereas expression of EGFP-NLS and DsRed-CAAX was lethal in the homozygous state. Our results therefore demonstrate that the 2A constructs and two novel targeting motifs are functional in D. suzukii, and that the combination of EGFP-NLS and DsRed-CAAX shows dosage-dependent lethality. These molecular elements could be further used to improve expression systems in insects and generate novel pest control strains.

RNAi Screen in Tribolium Reveals Involvement of F-BAR Proteins in Myoblast Fusion and Visceral Muscle Morphogenesis in Insects.

In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Our genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. We propose that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis.

A Large Scale Systemic RNAi Screen in the Red Flour Beetle Tribolium castaneum Identifies Novel Genes Involved in Insect Muscle Development.

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered ∼8,500 and ∼5,000 genes, respectively, of a total of ∼16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.

The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.