Real‐time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real‐Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL‐TcA‐CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV‐A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up‐regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real‐time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.     

The putative‐farnesoic acid O‐methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre‐imaginal life expression

Farnesoic acid O‐methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative‐FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real‐Time PCR in medfly pre‐imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre‐imaginal putative‐FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well‐known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control. © 2010 Wiley Periodicals, Inc.     

Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast

The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes. Received: 29 June 1999 / Accepted: 27 October 1999     

A new cell line from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially expressed genes

A new cell line, designated IOZCAS‐Spex XI, was established from the pupal ovaries of Spodoptera exigua (Lepidoptera: Noctuidae) in TNM‐FH medium containing 10% foetal bovine serum. The spherical cells were predominant among the various cell types. The population‐doubling time during the logarithmic phase of growth was 81.7 h. It was confirmed that the cell line originated from S. exigua by DAF‐PCR technique. Analysis of susceptibility to baculovirus showed that the new cell line was susceptible to S. exigua nucleopolyhedrovirus (SeNPV), Autographa californica multiple NPV (AcMNPV) and slightly susceptible to S. litura NPV (SpltNPV), while not permissive to Helicoverpa armigera NPV and Hyphantria cunea NPV (HcNPV). Real‐Time PCR analysis was carried out to compare some differentially expressed genes between the cell line and the primary culture. The result showed that marked significant differences were observed in the expression of the genes of SUMO‐1 activating enzyme, BCCIP‐like protein, 10 kDa HSP, CypA, receptor for activated PKC, PDI‐like protein ERp57, ALDH, DEAD box ATP‐dependent RNA helicase‐like protein (P < 0.01), while a significant difference was obtained in the expression of GST gene between the cell line and the primary culture (P < 0.05).     

Expression of homeobox genes in oral squamous cell carcinoma cell lines treated with all‐trans retinoic acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All‐trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA‐mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA‐sensitive SCC‐25 cells compared to atRA‐resistant SCC‐9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC‐25 cells but not in SCC‐9 cells. Gene expression levels were confirmed for seven of these genes by RT‐qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC‐25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA‐dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on day 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437–1444, 2010. © 2010 Wiley‐Liss, Inc.     

Genome size estimation with quantitative real‐time PCR in two Tephritidae species: Ceratitis capitata and Bactrocera oleae

The medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae belong to the Tephritidae family of Diptera, a family whose members cause severe damages in agriculture worldwide. For such insect pests, the utmost concern is their population control. The sterile insect technique (SIT) has been used in the Tephritidae family with varying degrees of success. Its efficient use usually depends on the development of genetic sexing strains and the release of only male flies. However, such advances are based on modern genetic, molecular and genomic tools. The medfly is clearly the prototype of the family, since such tools have advanced considerably, which has resulted in effective SIT efforts around the world. A whole‐genome sequencing project of this insect is already underway. In contrast, similar tools in the olive fly lag behind, even though the insect is considered a promising candidate for a next SIT target. An accurate estimate of genome size provides a preliminary view of genome complexity and indicates possible difficulties in genome assembly in whole‐genome projects. Taking advantage of a quantitative real‐time PCR approach, we determined the genome size of these two species C. capitata and B. oleae as 591 Mb (CI range: 577–605 Mb) and 322 Mb (CI range: 310–334 Mb) respectively.     

In vitro rearing of Edovum puttleri,an egg parasitoid of the Colorado potato beetle,on artificial diets: Effects of insect cell line‐conditioned medium

Effects of conditioned media prepared from cell lines derived from 11 insect species (six families, three orders) on the in vitro growth and development of the egg parasitoid Edovum puttleri were investigated. The parasitoid exhibited significantly different responses to the various insect cell line‐conditioned media that were incorporated into the artificial diets. When cell lines were derived from embryos, higher percentages of 3rd instars and prepupae were observed than when cell lines were derived from fat body or ovaries. Medium conditioned with cell line IPLB‐CPB2 derived from the embryos of the Colorado potato beetle produced the best result. Preconditioning time was important. In general, 5 days of preconditioning appeared to be optimal. The growth‐ and development‐promoting effect may have resulted from growth factors or growth‐supporting factors produced/ released by the insect cell lines into the culture medium. Upon storage at 0–4°C for 7–14 days, the ability of cell line‐conditioned medium to promote development beyond the second instar was greatly reduced (approximately 10–55%). Our studies demonstrated that to support the in vitro growth and development of E. puttleri, insect hemolymph could be successfully replaced with insect cell line‐conditioned medium. These findings should facilitate the development of a cost‐effective mass‐rearing system for E. puttleri and/or other parasitoids. Arch. Insect Biochem. Physiol. 40:173–182, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Actin Genes in the Mediterranean Fruit Fly, Ceratitis Capitata

We have undertaken the study of actin gene organization and expression in the genome of the Mediterranean fruit fly (medfly), Ceratitis capitata. Actin genes have been extensively characterized previously in a wide range of eukaryotic organisms, and they have valuable properties for comparative studies. These genes are typically highly conserved in coding regions, represented in multiple copies per genome and regulated in expression during development. We have isolated a gene in the medfly using the cloned Drosophila melanogaster 5C actin gene as a probe. This medfly gene detects abundant messages present during late larval and late pupal development as well as in thoracic and leg tissue preparations from newly emerged adults. This pattern of expression is consistent with what has been seen for actin genes in other organisms. Using either the D. melanogaster 5C actin gene or the medfly gene as a probe identifies five common cross reacting EcoRI fragments in genomic DNA, but only under less than fully stringent hybridization conditions.     

Conserved roles of Osiris genes in insect development,polymorphism and protection

Much of the variation among insects is derived from the different ways that chitin has been moulded to form rigid structures, both internal and external. In this study, we identify a highly conserved expression pattern in an insect‐only gene family, the Osiris genes, that is essential for development, but also plays a significant role in phenotypic plasticity and in immunity/toxicity responses. The majority of Osiris genes exist in a highly syntenic cluster, and the cluster itself appears to have arisen very early in the evolution of insects. We used developmental gene expression in the fruit fly, Drosophila melanogaster, the bumble bee, Bombus terrestris, the harvester ant, Pogonomyrmex barbatus, and the wood ant, Formica exsecta, to compare patterns of Osiris gene expression both during development and between alternate caste phenotypes in the polymorphic social insects. Developmental gene expression of Osiris genes is highly conserved across species and correlated with gene location and evolutionary history. The social insect castes are highly divergent in pupal Osiris gene expression. Sets of co‐expressed genes that include Osiris genes are enriched in gene ontology terms related to chitin/cuticle and peptidase activity. Osiris genes are essential for cuticle formation in both embryos and pupae, and genes co‐expressed with Osiris genes affect wing development. Additionally, Osiris genes and those co‐expressed seem to play a conserved role in insect toxicology defences and digestion. Given their role in development, plasticity, and protection, we propose that the Osiris genes play a central role in insect adaptive evolution.     

Involvement of the MyD88‐independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7

Burkholderia pseudomallei is a facultative intracellular Gram‐negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O‐antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88‐dependent (iκbζ, il‐6 and tnf‐α), but not of the MyD88‐independent (inos, ifn‐β and irg‐1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant‐infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88‐independent pathway in B. pseudomallei‐infected macrophages, the expression of TBK1, an essential protein in the MyD88‐independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant‐infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88‐independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN‐γ upregulated gene expression downstream of the MyD88‐independent pathway, and interfered with intracellular survival in both wild type and tbk1‐knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88‐independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN‐γ regulates gene expression through the TBK1‐independent pathway.     

Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression

Site‐specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)‐mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single‐copy integration clones were then co‐transfected with both Flp‐containing plasmid and an EGFP‐containing targeting cassette. Successful cassette exchange was suggested by emergence of G418‐resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re‐usable platform to produce multiple recombinant proteins for fundamental and applied research. Biotechnol. Bioeng. 2012; 109: 2836–2844. © 2012 Wiley Periodicals, Inc.     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.