Excess melanin precursors rescue defective cuticular traits in stony mutant silkworms probably by upregulating four genes encoding RR1-type larval cuticular proteins.

Melanin and cuticular proteins are vital cuticle components in insects. Cuticular defects caused by mutations in cuticular protein-encoding genes can obstruct melanin deposition. The effects of changes in melanin on the expression of cuticular protein-encoding genes, the cuticular and morphological traits, and the origins of these effects are unknown. We found that the cuticular physical characteristics and the expression patterns of larval cuticular protein-encoding genes markedly differed between the melanic and non-melanic integument regions. By using four p multiple-allele color pattern mutants with increasing degrees of melanism (+p, pM, pS, and pB), we found that the degree of melanism and the expression of four RR1-type larval cuticular protein-encoding genes (BmCPR2, BmLcp18, BmLcp22, and BmLcp30) were positively correlated. By modulating the content of melanin precursors and the expression of cuticular protein-encoding genes in cells in tissues and in vivo, we showed that this positive correlation was due to the induction of melanin precursors. More importantly, the melanism trait introduced into the BmCPR2 deletion strain Dazao-stony induced up-regulation of three other similar chitin-binding characteristic larval cuticular protein-encoding genes, thus rescuing the cuticular, morphological and adaptability defects of the Dazao-stony strain. This rescue ability increased with increasing melanism levels. This is the first study reporting the induction of cuticular protein-encoding genes by melanin and the biological importance of this induction in affecting the physiological characteristics of the cuticle.

Transcriptomic analysis reveals the mechanism of thermosensitive genic male sterility (TGMS) of Brassica napus under the high temperature inducement.

BACKGROUND: The thermo-sensitive genic male sterility (TGMS) of Brassica napus facilitates reproductive researches and hybrid seed production. Considering the complexity and little information about the molecular mechanism involved in B. napus TGMS, comparative transcriptomic analyses were peroformed for the sterile (160S-MS) and fertile (160S-MF) flowers to identify potential crucial genes and pathways associated with TGMS. RESULTS: In total, RNA-seq analysis showed that 2202 genes (561 up-regulated and 1641 down-regulated) were significantly differentially expressed in the fertile flowers of 160S-MF at 25 °C when compared the sterile flower of 160S-MS at 15 °C. Detailed analysis revealed that expression changes in genes encoding heat shock proteins, antioxidant, skeleton protein, GTPase and calmodulin might be involved in TGMS of B. napus. Moreover, gene expression of some key members in plant hormone signaling pathways, such as auxin, gibberellins, jasmonic acid, abscisic acid, brassinosteroid signalings, were significantly surppressed in the flowers of 160S, suggesting that these genes might be involved in the regulation in B. napus TGMS. Here, we also found that transcription factor MADS, NFY, HSF, MYB/C and WRKY might play a crucial role in male fertility under the high temperature condition. CONCLUSION: High temperature can significant affect gene expression in the flowers. The findings in the current study improve our understanding of B. napus TGMS at the molecular level and also provide an effective foundation for male fertility researches in other important economic crops.

Global Transcriptome Sequencing Reveals Molecular Profiles of Summer Diapause Induction Stage of Onion Maggot, Delia antiqua (Diptera: Anthomyiidae).

The onion maggot, Delia antiqua, is a worldwide subterranean pest and can enter diapause during the summer and winter seasons. The molecular regulation of the ontogenesis transition remains largely unknown. Here we used high-throughput RNA sequencing to identify candidate genes and processes linked to summer diapause (SD) induction by comparing the transcriptome differences between the most sensitive larval developmental stage of SD and nondiapause (ND). Nine pairwise comparisons were performed, and significantly differentially regulated transcripts were identified. Several functional terms related to lipid, carbohydrate, and energy metabolism, environmental adaption, immune response, and aging were enriched during the most sensitive SD induction period. A subset of genes, including circadian clock genes, were expressed differentially under diapause induction conditions, and there was much more variation in the most sensitive period of ND- than SD-destined larvae. These expression variations probably resulted in a deep restructuring of metabolic pathways. Potential regulatory elements of SD induction including genes related to lipid, carbohydrate, energy metabolism, and environmental adaption. Collectively, our results suggest the circadian clock is one of the key drivers for integrating environmental signals into the SD induction. Our transcriptome analysis provides insight into the fundamental role of the circadian clock in SD induction in this important model insect species, and contributes to the in-depth elucidation of the molecular regulation mechanism of insect diapause induction.

Complete mitochondrial genomes of Anopheles stephensi and An. dirus and comparative evolutionary mitochondriomics of 50 mosquitoes.

To better understand the phylogeny and evolution of mosquitoes, the complete mitochondrial genome (mitogenome) of Anopheles stephensi and An. dirus were sequenced and annotated, and a total of 50 mosquito mitogenomes were comparatively analyzed. The complete mitogenome of An. stephensi and An. dirus is 1,5371 bp and 1,5406 bp long, respectively. The main features of the 50 mosquito mitogenomes are conservative: 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, positive AT-skew and negative GC-skew. The gene order trnA-trnR in ancestral insects is rearranged. All tRNA genes have the typical clover leaf secondary structure but tRNA Ser . The control regions are highly variable in size. PCGs show signals of purifying selection, but evidence for positive selection in ND2, ND4 and ND6 is found. Bayesian and Maximum Likelihood phylogenetic analyses based on all PCG nucleotides produce an identical tree topology and strongly support the monophyly of subgenera Cellia, Anopheles, Keterszia and Nyssorhynchus, the sister relationship of the subgenera Nyssorhynchus and Keterszia, and Cellia and Anopheles. The most recent ancestor of the genus Anopheles and Culicini + Aedini exited ~145 Mya ago. This is the first comprehensive study of mosquito mitogenomes, which are effective for mosquito phylogeny at various taxonomic levels.

Suppression of Laccase 2 severely impairs cuticle tanning and pathogen resistance during the pupal metamorphosis of Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Phenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear. METHODS: The association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls. RESULTS: During the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage. CONCLUSIONS: To our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies.

The genome, transcriptome, and proteome of the nematode Steinernema carpocapsae: evolutionary signatures of a pathogenic lifestyle.

The entomopathogenic nematode Steinernema carpocapsae has been widely used for the biological control of insect pests. It shares a symbiotic relationship with the bacterium Xenorhabdus nematophila, and is emerging as a genetic model to study symbiosis and pathogenesis. We obtained a high-quality draft of the nematode's genome comprising 84,613,633 bp in 347 scaffolds, with an N50 of 1.24 Mb. To improve annotation, we sequenced both short and long RNA and conducted shotgun proteomic analyses. S. carpocapsae shares orthologous genes with other parasitic nematodes that are absent in the free-living nematode C. elegans, it has ncRNA families that are enriched in parasites, and expresses proteins putatively associated with parasitism and pathogenesis, suggesting an active role for the nematode during the pathogenic process. Host and parasites might engage in a co-evolutionary arms-race dynamic with genes participating in their interaction showing signatures of positive selection. Our analyses indicate that the consequence of this arms race is better characterized by positive selection altering specific functions instead of just increasing the number of positively selected genes, adding a new perspective to these co-evolutionary theories. We identified a protein, ATAD-3, that suggests a relevant role for mitochondrial function in the evolution and mechanisms of nematode parasitism.

The mitochondrial genomes of Culex tritaeniorhynchus and Culex pipiens pallens (Diptera: Culicidae) and comparison analysis with two other Culex species.

BACKGROUND: Culex tritaeniorhynchus and Culex pipiens pallens are the major vectors of the Japanese encephalitis virus and Wuchereria bancrofti, the causative agent of filariasis. The knowledge of mitochondrial genomes has been widely useful for the studies on molecular evolution, phylogenetics and population genetics. METHODS: In this study, we sequenced and annotated the mitochondrial (mt) genomes of Cx. tritaeniorhynchus and Cx. p. pallens, and performed a comparative analysis including four known mt genomes of species of the subgenus Culex (Culex). The phylogenetic relationships of Cx. tritaeniorhynchus, Cx. p. pallens and four known Culex mt genome sequences were reconstructed by maximum likelihood based on concatenated protein-coding gene sequences. RESULTS: Culex tritaeniorhynchus and Cx. p. pallens mt genomes are 14,844 bp and 15,617 bp long, both consists of 13 PCGs, 22 tRNAs, 2 rRNAs and 1 CR (not sequenced for Cx. tritaeniorhynchus). The initiation and termination codons of PCGs are ATN and TAA, respectively, except for COI starting with TCG, and COI and COII terminated with T. tRNAs have the typical clover-leaf secondary structures except for trnS ((AGN)) that is lacking the DHU stem. 16S rRNA and 12S rRNA secondary structures were drawn for the first time for mosquito mt genomes. The control region of Cx. p. pallens mt genome is 747 bp long and with four tandem repeat structures. Phylogenetic analyses demonstrated that the mt genome of Cx. tritaeniorhynchus was significantly separated from the remaining five mt genomes of Culex spp. Culex p. pipiens, Cx. p. pallens and Cx. p. quinquefasciatus formed a monophyletic clade with Cx. p. quinquefasciatus linked in the middle of the clade, and Cx. p. pallens should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus. CONCLUSIONS: The mt genomes of Cx. tritaeniorhynchus and Cx. p. pallens share the same gene composition and order with those of two other Culex species. Culex p. pallens of the Pipiens complex should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus investigated. We enriched the Culex mt genome data and provided a reference basis for further Culex mt genome sequencing and analyses.

Insight into the possible mechanism of the summer diapause of Delia antiqua (Diptera: Anthomyiidae) through digital gene expression analysis.

The onion fly, Delia antiqua, is a major underground agricultural pest that can enter pupal diapause in the summer and winter seasons. However, little is known about its molecular regulation due to the lack of genomic resources. To gain insight into the possible mechanism of summer diapause (SD), high-throughput RNA-Seq data were generated from non-diapause (ND) and SD (initial, maintenance and quiescence phase) pupae. Three pair-wise comparisons were performed and identified, 1380, 1471 and 435, and were significantly regulated transcripts. Further analysis revealed that the enrichment of several functional terms related to juvenile hormone regulation, cell cycle, carbon hydrate and lipid metabolism, innate immune and stress responses, various signalling transductions, ubiquitin-dependent proteosome, and variation in cuticular and cytoskeleton components were found between ND and SD and between different phases of SD. Global characterization of transcriptome profiling between SD and ND contributes to the in-depth elucidation of the molecular mechanism of SD. Our results also offer insights into the evolution of insect diapause and support the importance of using the onion fly as a model to compare the molecular regulation events of summer and winter diapauses.

Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua (Diptera: Anthomyiidae).

Trehalose represents the main hemolymph sugar in many insects, and it functions in energy metabolism and protection in extreme environmental conditions. To gain an insight into trehalose functions in Delia antiqua diapausing pupae, genes encoding trehalose-6-phosphate synthase (TPS), trehalose-6-phosphatase (TPP) and trehalase (TRE) were identified and characterized. Analysis of the deduced amino acid sequences indicated that these genes were highly similar to each homolog from Diptera insects. Gene expressions and their enzyme activities were also investigated. The differential expressions of TPS and TPP shared very similar trends for summer and winter diapausing pupae. Their enzyme activities were consistent with the gene expressions. Trehalose concentrations in summer- and winter-diapausing pupae were lower at the initial phase (4.37-5.09µg/mg) but increased gradually and peaked in the maintenance phase (10.59-14.36µg/mg); the concentrations then declined in the quiescence phase. We speculated that a higher trehalose content during the maintenance stage may contribute to protein and/or biological membrane stabilization in winter or to desiccation resistance in the summertime. Diapause termination requires a decrease in the trehalose concentration to promote pupal-adult development. The glucose content also varied during the diapausing processes. Our results provide an overview of the differential expression levels of trehalose metabolic enzymes, confirming the important roles of trehalose in diapausing pupae of the onion maggot. Further work remains to explore its actual functions.

Macroevolutionary trends of atomic composition and related functional group proportion in eukaryotic and prokaryotic proteins.

To fully explore the trends of atomic composition during the macroevolution from prokaryote to eukaryote, five atoms (oxygen, sulfur, nitrogen, carbon, hydrogen) and related functional groups in prokaryotic and eukaryotic proteins were surveyed and compared. Genome-wide analysis showed that eukaryotic proteins have more oxygen, sulfur and nitrogen atoms than prokaryotes do. Clusters of Orthologous Groups (COG) analysis revealed that oxygen, sulfur, carbon and hydrogen frequencies are higher in eukaryotic proteins than in their prokaryotic orthologs. Furthermore, functional group analysis demonstrated that eukaryotic proteins tend to have higher proportions of sulfhydryl, hydroxyl and acylamino, but lower of sulfide and carboxyl. Taken together, an apparent trend of increase was observed for oxygen and sulfur atoms in the macroevolution; the variation of oxygen and sulfur compositions and their related functional groups in macroevolution made eukaryotic proteins carry more useful functional groups. These results will be helpful for better understanding the functional significances of atomic composition evolution.

Differential gene expression between summer and winter diapause pupae of the onion maggot Delia antiqua, detected by suppressive subtractive hybridization.

To gain a better understanding of the molecular mechanisms regulating pupal diapause of the onion maggot Delia antiqua, PCR-based suppressive subtractive hybridization was performed to identify genes involved in summer and/or winter diapause. A total of 209 unique sequences were obtained including 89 in forward library for winter diapausing pupae and 120 in the reverse library for summer diapausing pupae. 76.4% (68/89) and 68.3% (82/120) unique sequences had significant hits to non-redundant proteins database. Gene functional annotation showed these non-redundant sequences are involved in stress response and innate immunity, metabolism and energy, information processing and regulation, binding, food storage, morphogenesis and development, cell skeleton and cycle, protein synthesis and folding. Approximately 28.2% (59/209) transcripts showed no significant similarity to any other sequence in the public databases, probably representing unique genes of the onion maggot. Semi-quantitative RT-PCR revealed that the relative expression levels of 18 genes were comparable between summer and winter diapause. This study elucidates the temporal expression of diapause-related genes in onion maggot, also provides new insights into the differences in the physiological changes in summer and winter pupae. Functional characterization of some candidate genes will further enhance the understanding of the generating, maintaining, and breaking mechanism of diapause.

Genetic diversity and comparative analysis of gene expression between Heterorhabditis bacteriophora Az29 and Az36 isolates: uncovering candidate genes involved in insect pathogenicity.

Entomopathogenic nematode Heterorhabditis bacteriophora Az29 and Az36 isolates with different virulence against Popillia unipuncta and soil survival time were isolated from the Azorean archipelago (Portugal) and used for the study. RAPD analysis revealed a very low-level of genetic diversity (GD(axenic Az36 isolate)(axenic Az29 isolate)=0.2338±0.0541) between axenic Az29 and Az36 isolates, and a relative low-level of diversity (GD(Az36 isolate)(Az29 isolate)=0.3366±0.0471) between Az29 and Az36 isolates. To unravel the molecular differences, a suppressive subtractive hybridization library was constructed from the parasitic stage. Assembling 150 high quality ESTs produced 70 singletons and 17 contigs. BLAST analysis revealed that 48 ESTs showed significant similarity to known protein and 39 ESTs had no significant hits in the database, perhaps representing novel genes. Functional annotation revealed some of these genes to be involved in metabolism, cellular process and signaling, information storage and processing, stress response and host-parasite interactions. Genes with a role in the parasitism process were identified including lectin, metalloprotease, enolase, chitinase, surface-associated antigen, and as well as genes (aquaporin, Hsp70A, Hsp10 and Hsp20) essential for stresses tolerance. The work described here provides the molecular data necessary for investigating the fundamental molecular aspects of host-parasite interactions. Future investigations should be focused on determining the molecular mechanism of those genes in entomopathogenic nematode life cycle.

Transcripts analysis of the entomopathogenic nematode Steinernema carpocapsae induced in vitro with insect haemolymph.

Steinernema carpocapsae is an insect parasitic nematode widely used in pest control programs. The efficacy of this nematode in controlling insects has been found to be related to the pathogenicity of the infective stage. In order to study the parasitic mechanisms exhibited by this parasite, a cDNA library of the induced S. carpocapsae parasitic phase was generated. A total of 2500 clones were sequenced and 2180 high-quality ESTs were obtained from this library. Cluster analysis generated a total of 1592 unique sequences including 1393 singletons. About 63% of the unique sequences had significant hits (e

Identification and expression analysis of the Steinernema carpocapsae elastase-like serine protease gene during the parasitic stage.

A cDNA encoding elastase was isolated from Steinernema carpocapsae by suppression subtractive hybridization and rapid amplification of 5' cDNA ends. The predicted protein contained a 19-aa signal peptide, a 44-aa N-terminal propeptide, and a 264-aa mature protein with a predicted molecular mass of 28,949 Da and a theoretical pI of 8.88. BLAST analysis showed 27-35% amino acid sequence identity to serine proteases from insects, mammals, fish and other organisms. The Sc-ela gene contains three exons and two introns with at least two copies in the S. carpocapsae genome. Expression analysis indicated that the Sc-ela gene was upregulated during the initial parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental and fibrinolytic activities. Homology modeling showed that Sc-ELA adopts a two beta-barrel fold typical of trypsin-like serine proteases, and phylogenetic analysis indicates that Sc-ELA branched off early during elastase evolution.

Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae.

A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernema carpocapsae. The purified protease had an apparent molecular mass of 30kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K(m), V(max) and k(cat) values of 409microM/min, 0.389microM/min and 24.9s(-1), respectively. The protease was most active at pH 8.0 and 35 degrees C, and its proteolytic activity was almost completely reduced after incubation at 75 degrees C for 30min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.

Mitochondrial DNA mutations in cancer: a review.

As mitochondria participate in fundamental process of the cellular metabolism, recent research has addressed the role of mitochondria, and of mitochondrial DNA (mtDNA), in apoptosis, aging, and complex diseases. The association between mtDNA and cancer has been discussed since the beginning of the last century, and more recently, it has gained attention due to the observation of many somatic mutations in several types of cancers. In this review we describe those germinal mutations that have been associated to cancer, and present a compilation of somatic mutations that have been observed in different cancer tissues, describing relevant characteristics among them in a phylogenetic context. We also summarize the drawbacks and criticisms made towards the studies that report an association between mtDNA mutations and cancer, and discuss the experimental models used to analyse this relationship. Although many reported somatic mutations may actually be the outcome of laboratory artefacts, a considerable number could be authentic and may have a relationship with cancer development. In our compilation, we have observed 271 cancer mutations occurring in conserved positions of mtDNA, 70 of them appearing in more than one tumour. These mutations may be candidates to be used as cancer biomarkers, and deserve further investigation, perhaps through the use of experimental models and by an analysis of tumours of distinct grade to determine if the mutations arose early during tumourigenesis. Experiments with cybrids have been successfully used; however, models are needed in which specific mtDNA variants may be introduced into the same mitochondrial and cellular background.

Identification, characterization of functional candidate genes for host-parasite interactions in entomopathogenetic nematode Steinernema carpocapsae by suppressive subtractive hybridization.

Identifying parasitism genes encoding proteins secreted from nematodes is the key to understanding the molecular basis of nematode parasitism to insects. In this paper, a cDNA with two introns and three exons encoding a cysteine protease inhibitor was identified by screening a cDNA subtractive library constructed from the nematode, Steinernema carpocapsae, induced by Galleria mellonella hemolymph. The full-length cDNA contains an open reading frame encoding a 139-amino acid protein, designated Sc-cys, with a 19-residue signal peptide. The mature protein was predicted to have a molecular weight of 12,531.59 Da, a pI of 9.44, one disulfide bond, and three conserved domains believed to be important for the inhibition of cysteine proteases. In Basic Local Alignment and Search Tool analyses, the putative protein precursor displayed 26-42% identities to a multitude of cystatins or cystatin-like proteins. Phylogenetic analysis suggested the novel cystatin is likely a new member of the family 2 cystatins. Reverse northern blot, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses showed that the expression level of Sc-cys was upregulated substantially after induction by insect hemolymph. The specific analysis of genes encoding secretory proteins is providing a profile of putative parasitism genes expressed in S. carpocapsae throughout the parasitic cycle.

[Molecular cloning and expression of Attacin from housefly (Musca domestica)].

The antimicrobial peptides of insect are the main components of their non-specific immune system, and play a major role in the defense against the foreign disease-related microbes. In this report, a full length cDNA of Attacin, an insect antimicrobial peptide was cloned from housefly (Musca domestica) by homology cloning approach in combine with 3' and 5' RACE. Sequence analysis and phylogenetical study showed that this cDNA contained 778 nucleotides, with a 627 bp open reading frame (ORF) flanked with a 44 bp 5'UTR and a 107 bp 3'UTR. The encoded 208 amino acids housefly Attacin shared a high similarity of 50%-70% with that of the other dipterous insects. In addition, the phylogenetical analysis also indicated that the Attacin from housefly was in the same branch with those of other species, suggesting that they come from the same ancestor. The expression of Attacin transcript was measured by semi-quantitive RT-PCR. The results demonstrated that the expression of housefly Attacin is inducible, and that the level varies with the time of induction and the kinds of pathogens.