Deciphering Proteomic Signatures of Early Diapause in Nasonia

Insect diapause is an alternative life-history strategy used to increase longevity and survival in harsh environmental conditions. Even though some aspects of diapause are well investigated, broader scale studies that elucidate the global metabolic adjustments required for this remarkable trait, are rare. In order to better understand the metabolic changes during early insect diapause, we used a shotgun proteomics approach on early diapausing and non-diapausing larvae of the recently sequenced hymenopteran model organism Nasonia vitripennis. Our results deliver insights into the molecular underpinnings of diapause in Nasonia and corroborate previously reported diapause-associated features for invertebrates, such as a diapause-dependent abundance change for heat shock and storage proteins. Furthermore, we observed a diapause-dependent switch in enzymes involved in glycerol synthesis and a vastly changed capacity for protein synthesis and degradation. The abundance of structural proteins and proteins involved in protein synthesis decreased with increasing diapause duration, while the abundance of proteins likely involved in diapause maintenance (e.g. ferritins) increased. Only few potentially diapause-specific proteins were identified suggesting that diapause in Nasonia relies to a large extent on a modulation of pre-existing pathways. Studying a diapause syndrome on a proteomic level rather than isolated pathways or physiological networks, has proven to be an efficient and successful avenue to understand molecular mechanisms involved in diapause.     

Methionine-rich storage protein gene in the rice stem borer, Chilo suppressalis, is expressed during diapause in response to cold acclimation

Gene expressions of acclimatized and non-acclimatized diapausing larvae were examined in Chilo suppressalis using a subtraction technique. A gene encoding a methionine-rich storage protein, CsSP1, was cloned and its complete cDNA sequence was determined. Potentially, CsSP1 encoded a 758-amino acid protein, with a calculated molecular weight of 88.8 kDa. The expression level of CsSP1 was higher in nondiapausing larvae than in diapausing ones. The CsSP1 expression was up-regulated in diapausing larvae when the temperature of cold acclimation was shifted to 5 degrees C. The up-regulated level was maintained at 40 days after incubation at 5 degrees C. In nondiapausing larvae, CsSP1 expression was down-regulated when the temperature was below developmental zero. Involvement of CsSP1 in diapause, cold tolerance acquisition and postdiapause development in C. suppressalis is discussed.     

In vitro modulation of cisplatin accumulation in human ovarian carcinoma cells by pharmacologic alteration of microtubules.

We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function of cAMP is to activate protein kinase A, it was conjectured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a prominent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human beta-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol increased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13*5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous formation of microtubule bundles and decreased expression of beta-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13*5.25 cells had altered sensitivity to tubulin-binding drugs, being hypersensitive to taxol and cross-resistant to colchicine. We conclude that pharmacologic alterations of tubulin enhance accumulation of DDP, and that the DDP-resistant phenotype in 2008/C13*5.25 cells is associated with tubulin abnormalities.     

Expression of the Hsp83 gene in response to diapause and thermal stress in the moth Sesamia nonagrioides

A full-length Hsp83, named SnoHsp83, cDNA from the corn stalk borer, Sesamia nonagrioides, was cloned and sequenced. Genomic analysis showed that the SnoHsp83 gene is unique. The size of the SnoHsp83 cDNA was found to be approximately 2.6 kb. The deduced polypeptide comprised 717 amino acid residues, with a molecular mass of 82.6 kDa. It contained all the highly conserved amino acid motifs that characterize the cytosolic members of the hsp90 family. We investigated the expression of SnoHsp83 gene in response to diapause and heat/cold stress. SnoHsp83 is constitutively expressed in non-diapausing larvae and is induced 15-fold by heat. SnoHsp83 displays a similar pattern to SnoHsc70 under diapause conditions, when extra larval moults occur. Our results indicate that the SnoHsp83 gene could be involved in the developmental process that occurs between two moults.     

Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site-directed recombination

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.     

Transgene‐mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication

RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose‐dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV‐B2) from the poly‐ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site‐directed recombination system to investigate the impact of transgene‐mediated RNAi pathway suppression on infections with SINV‐TR339eGFP and DENV2‐QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV‐B2 was constitutively expressed in midguts of sugar‐ and blood‐fed mosquitoes of transgenic line PUbB2 P61. B2 over‐expression suppressed RNA silencing of carboxypeptidase A‐1 (AeCPA‐1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV‐TR339eGFP or DENV2‐QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post‐bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV‐TR339eGFP. Following infection with DENV2‐QR94, midgut infection rates were significantly increased in the B2‐expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2‐QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi.     

Photoperiodic effects on diapause‐associated gene expression trajectories in European Leptinotarsa decemlineata populations

Behavioural and physiological changes during diapause, an important strategy of insects for surviving harsh seasonal conditions, have been intensively studied. The genetic and molecular mechanisms underpinning diapause development are less well known. We took a candidate gene approach to study prediapause gene expression patterns in the Colorado potato beetle (Leptinotarsa decemlineata), an invasive insect that has rapidly spread northwards to high seasonality environments. Newly eclosed beetles originating from southern (Italy) and northern (Russia) Europe were reared under short‐ [12 h light (L):12 h dark (D)] and long‐day (18L:6D) photoperiods for 10 days. This time period includes the sensitive period for the photoperiodic induction and initiation of diapause. Gene expression trajectories of 12 diapause‐related genes (regulatory, metabolic and stress‐resistance) were analysed from 0‐, 5‐ and 10‐day‐old beetles. Gene expression differences increased with age, deviating significantly between populations and photoperiods in 10‐day‐old beetles. The gene expression profiles, particularly those related to energy metabolism and stress‐resistance, indicate that beetles originating from Russia also prepare for diapause under the long‐day photoperiod and show qualitative differences in the diapausing phenotype. Our study shows that population‐dependent differences seen in behavioural and physiological traits connected with diapause in L. decemlineata are also evident in the expression trajectories of diapause‐related genes.     

Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes.

The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the beta subunit of tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six beta-tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa beta-tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific beta-tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.     

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.     

Identification of ecdysteroidogenic enzyme genes and their expression during pupal diapause in the cabbage armyworm,Mamestra brassicae

In this study, we identified ecdysteroidogenic enzymes in the cabbage armyworm, Mamestra brassicae, and demonstrated reduced expression of these genes during diapause. Some insects employ a temporary developmental arrest, diapause, to survive in severe environments. The titres of the moulting hormone ecdysteroid were reduced in diapause pupae of M. brassicae; therefore, ecdysteroidogenesis might be suppressed by a diapause‐specific mechanism. To clarify expression changes of ecdysteroidogenic enzyme genes during diapause in M. brassicae, we first identified the genes for seven ecdysteroidogenic enzymes: Neverland, Non‐molting glossy (Nm‐g), CYP307A1 (Spook), CYP306A1 (Phantom), CYP302A1 (Disembodied), CYP315A1 (Shadow) and CYP314A1 (Shade). Enzymatic assays using heterologous expression in Drosophila Schneider 2 (S2) cells and analysis of mRNA distribution indicated that the identified genes were ecdysteroidogenic enzymes of M. brassicae. Expression levels of these ecdysteroidogenic enzyme genes were compared between prothoracic glands in different pupal stages throughout diapause. Immediately after pupation, diapause‐destined pupae showed similar expression levels of ecdysteroidogenic enzyme genes to those of nondiapause pupae. All of these genes showed reduced gene expression after diapause initiation. Expression was immediately increased in diapause‐destined pupae at the postdiapause quiescence phase. These results indicate that reduced expression of ecdysteroidogenic enzyme genes suppresses ecdysteroidogenesis and maintains developmental arrest during diapause.