Tubulin superfamily genes in Tribolium castaneum and the use of a Tubulin promoter to drive transgene expression

The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum alpha-Tubulin1 (TcalphaTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcalphaTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages.     

Maxillopedia is the Tribolium ortholog of proboscipedia

SUMMARY Null mutations in the Drosophila melanogaster homeotic gene proboscipedia ( pb ) cause transformation of the adult labial palps to legs. The similar phenotype produced by mutations in the Tribolium castaneum homeotic complex (HOMC) gene maxillopedia ( mxp ) has led to suggestions that the two genes may be orthologous. We have cloned the Tribolium ortholog of pb , which predicts a protein with a homeodomain identical to that of Drosophila Pb. The two proteins also share several additional regions of identity, including an N-box, a motif unique to Pb orthologs. We have identified a frameshift mutation within Tribolium pb associated with an mxp null mutation, demonstrating that Tribolium pb corresponds to the mxp genetic locus. Thus, we will refer to the cloned gene as mxp . In addition, we have begun to construct a molecular map of the Tribolium HOMC. Two overlapping BAC clones which span the mxp locus also include the Tribolium labial ortholog ( Tclabial   ) and part of Tczerknüllt , indicating that the order of these genes in the HOMC is conserved between Drosophila and Tribolium.     

Comparative induction of somatic eye-color mutations and sex-linked recessive lethals in Drosophila melanogaster by tryptophan pyrolysates

The mutagenicities of the products of pyrolysis of tryptophan, Trp-P-1 and Trp-P-2, on Drosophila melanogaster were examined by measuring the effects of these compounds in inducing recessive lethals and somatic eye-color mutations. Since negative results have already been obtained by the standard procedure in males, Trp-P-1 and Trp-P-2 (0.75 to 6 mg/ml) in sucrose solution were given to females for assay of recessive lethal mutations in X-chromosomes. These compounds caused a marginal increase above the control level in the mutation frequency. For the assay of effects on somatic eye-color mutations, Trp-P-1 (200 and 400 ppm) and Trp-P-2 (400 and 800 ppm) were fed to male larvae of a tester strain carrying a genetically unstable marker set of z and w+ on the X-chromosome. These compounds caused dose-dependent increases above the control level in somatic eye-color mutations in adults. It is concluded that, under the conditions used, the somatic eye-color mutation system was more sensitive than the recessive lethal system to the mutagenic effects of tryptophan pyrolysates.     

Introduction of the Transposable Element Mariner into the Germline of Drosophila Melanogaster

A chimeric white gene (wpch) and other constructs containing the transposable element mariner from Drosophila mauritiana were introduced into the germline of Drosophila melanogaster using transformation mediated by the P element. In the absence of other mariner elements, the wpch allele is genetically stable in both germ cells and somatic cells, indicating that the peach element (i.e., the particular copy of mariner inserted in the wpch allele) is inactive. However, in the presence of the active element Mos1, the wpch allele reverts, owing to excision of the peach element, yielding eye-color mosaics and a high rate of germline reversion. In strains containing Mos1 virtually every fly is an eye-color mosaic, and the rate of wpch germline reversion ranges from 10 to 25%, depending on temperature. The overall rates of mariner excision and transposition are approximately sixfold greater than the rates in comparable strains of Drosophila simulans. The activity of the Mos1 element is markedly affected by position effects at the site of Mos1 insertion. In low level mosiac lines, dosage effects of Mos1 are apparent in the heavier level of eye-color mosaicism in Mos1 homozygotes than in heterozygotes. However, saturation occurs in high level mosaic lines, and then dosage effects are not observed. A pBluescribe M13+ plasmid containing Mos1 was injected into the pole plasm of D. melanogaster embryos, and the Mos1 element spontaneously integrated into the germline at high efficiency. These transformed strains of D. melanogaster presently contain numerous copies of mariner and may be useful in transposon tagging and other applications.     

A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species.     

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.     

Insertion and Excision of the Transposable Element Mariner in Drosophila

The transposable element mariner is active in both germline and somatic cells of Drosophila mauritiana. Activity of the element is greatly enhanced in the presence of Mos1, a genetic factor identified as an autonomous copy of mariner. A strain of D. mauritiana containing Mos1 and other copies of mariner was used to initiate a screen for visible mutations. More than 20 mutations were obtained, including alleles of white, yellow and vermilion. Six alleles were characterized at the molecular level, and all were found to contain a mariner element inserted into the affected gene. Four insertions into the white locus were sequenced to determine the exact site of insertion of mariner. There appears to be little sequence specificity requirement for mariner insertion, other than an absolute requirement for the dinucleotide TA, which is duplicated upon insertion. Sequences of phenotypically wild-type germline and somatic revertants obtained from various white alleles, including the previously isolated wpch allele, were obtained using the polymerase chain reaction. Mariner excision is imprecise in both germline and soma, and the most frequent excision events are the same in the two tissues. Mutant derivatives of wpch were also studied, and were found to exhibit a wide range of molecular structures and phenotypes.     

Larval RNAi in Drosophila?

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi.     

A family of chemoreceptors in Tribolium castaneum (Tenebrionidae: Coleoptera)

Chemoperception in invertebrates is mediated by a family of G-protein-coupled receptors (GPCR). To date nothing is known about the molecular mechanisms of chemoperception in coleopteran species. Recently the genome of Tribolium castaneum was sequenced for use as a model species for the Coleoptera. Using blast searches analyses of the T. castaneum genome with previously predicted amino acid sequences of insect chemoreceptor genes, a putative chemoreceptor family consisting of 62 gustatory receptors (Grs) and 26 olfactory receptors (Ors) was identified. The receptors have seven transmembrane domains (7TMs) and all belong to the GPCR receptor family. The expression of the T. castaneum chemoreceptor genes was investigated using quantification real- time RT-PCR and in situ whole mount RT-PCR analysis in the antennae, mouth parts, and prolegs of the adults and larvae. All of the predicted TcasGrs were expressed in the labium, maxillae, and prolegs of the adults but TcasGr13, 19, 28, 47, 62, 98, and 61 were not expressed in the prolegs. The TcasOrs were localized only in the antennae and not in any of the beetles gustatory organs with one exception; the TcasOr16 (like DmelOr83b), which was localized in the antennae, labium, and prolegs of the beetles. A group of six TcasGrs that presents a lineage with the sugar receptors subfamily in Drosophila melanogaster were localized in the lacinia of the Tribolium larvae. TcasGr1, 3, and 39, presented an ortholog to CO(2) receptors in D. melanogaster and Anopheles gambiae was recorded. Low expression of almost all of the predicted chemoreceptor genes was observed in the head tissues that contain the brains and suboesophageal ganglion (SOG). These findings demonstrate the identification of a chemoreceptor family in Tribolium, which is evolutionarily related to other insect species.     

Genetic and Cytogenetic Analysis of the 43a-E Region Containing the Segment Polarity Gene Costa and the Cellular Polarity Genes Prickle and Spiny-Legs in Drosophila Melanogaster

A cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster is presented. Within this interval 27 complementation groups have been identified by extensive F(2) screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described.     

A mariner-like element with a 5' lesion in Drosophila simulans

The unstable white-S2 (wS2) allele of the white gene occurred spontaneously in the S2 strain of Drosophila simulans. This mutation was caused by insertion of the submariner element, a mariner-like element with an abnormal tandem duplication of the 5' inverted terminal repeat (ITR). Although it has an incomplete ITR, submariner excises efficiently. The rate of somatic reversion, estimated by the number of eye-color mosaic flies, was 79.9%, and the reversion frequency in the germline was 0.6%. The change to the 5' ITR contributes to make this transposon precise excision.     

不同杀虫剂处理对赤拟谷盗P450基因诱导表达特性影响

害虫细胞色素P450基因可被杀虫剂迅速诱导,然而当前对不同杀虫剂处理下赤拟谷盗P450基因诱导表达特性的研究较少。本研究首先通过序列比对选取了来自不同家族的8个赤拟谷盗P450基因CYP4G7、CYP4Q4、CYP4BR3、CYP12H1、CYP6BK11、CYP9D4、CYP9Z5和CYP345A1,然后采用四种不同杀虫剂氯氰菊酯、氟氯氰菊酯、氯菊酯和吡虫啉对赤拟谷盗20 d幼虫进行生物测定,再根据生测结果以四个药剂亚致死剂量分别处理幼虫,并采用荧光定量PCR分析8个P450基因的表达特性。结果表明,CYP4G7和CYP345A1可以分别被氯氰菊酯(分别上调1.97倍和2.06倍)、氟氯氰菊酯(2.00倍和2.03倍)和氯菊酯(1.73倍和1.81倍)显著诱导,而CYP4BR3和CYP345A1可以被吡虫啉(分别上调1.99倍和1.93倍)显著诱导。本研究结果表明赤拟谷盗P450基因的显著诱导与基因家族类型以及农药品种有关。     

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.     

<Emphasis Type="Italic">Proboscipedia</Emphasis> represses distal signaling in the embryonic gnathal limb fields of<Emphasis Type="Italic"> Tribolium castaneum</Emphasis>

Orthologs of the Hox genes Sex combs reduced ( Scr) and proboscipedia ( pd) are active in the developing labial appendages of all insect species tested. The remarkable variation among insect gnathal structures, particularly in the distal podomeres, suggests two Hox genes may enhance the adaptive potential of gnathal appendage morphology. Functional studies in the fruitfly Drosophila melanogaster, the flour beetle Tribolium castaneum and the milkweed bug Oncopeltus fasciatus show that cooperation between Scr and pb has been generally conserved, but specific mechanisms have been altered during evolution. Cross-regulation of pb by Scr is evident in Drosophila and Tribolium, the more closely related of the three species, but not in Oncopeltus. In all three species, pb function is restricted to the distal podomeres, but details are only known for Drosophila and Oncopeltus, two species exhibiting specialized stylate-haustellate mouthparts. Drosophila pb is required for distal Scr expression, and to repress the appendage patterning genes dachshund and Distal-less ( Dll). Oncopeltus pb has the novel capacity to specify leg fates. Little is known about distal functions of Tribolium pb. Hypomorphic mutations of the Tribolium pb ortholog maxillopedia can be arranged in a graded phenotypic series of palp to leg transformations along both the proximodistal and dorsoventral axes. Mid-embryonic expression profiles of Tribolium pb, Scr, wingless ( wg) and Dll genes were examined in maxillopedia hypomorphic and null mutant backgrounds. Levels of pb and Scr are significantly reduced in the distal appendage field. Tribolium pb therefore positively regulates distal Scr expression, a role that it has in common with Drosophila pb. Tribolium wg is normally down-regulated in the distal domain of the embryonic gnathal appendage buds. It becomes activated distally in maxillopedia hypomorphs. Repression of wg by pb has not been reported in the labial imaginal discs of Drosophila. Alterations of Tribolium Scr and wg expression occur in Dll-expressing cells, however, unlike in Drosophila labial imaginal discs, Dll expression appears unaffected in pb hypomorphic backgrounds. We conclude that the Hox genes Sex combs reduced and proboscipedia control an appendage organizer and cell autonomous fate determination during embryonic labial palp development in Tribolium.