Cynthia L. Goodman

Insect cell culture and applications to research and pest management

Building on earlier research, insect cell culture began with the successful establishment of one cell line from pupal ovarian tissue. The field has grown to the extent that now over 500 insect cell lines have been established from many insect species representing numerous insect orders and from several different tissue sources. These cell lines are used as research tools in virology, in studies of signaling mechanisms to study insect immunity, hemocyte migration, and to test hypotheses about gene expression, and in screening programs designed to discover new insecticide chemistries. Virology research is revealing fundamentally new information on virus/host cell interactions. Studies in gene expression are uncovering signal transduction pathways that are new to insect science. Research is leading to the development of high-speed screening technologies that are essential in the search for new insect pest management tools. A few insect cell lines are, in routine industrial processes, designed to produce proteins of biomedical significance. Both primary cell cultures and established lines are used in basic biological studies to reveal how insect cells work. This review is designed to briefly cover the history of insect cell culture, recount some recent advances in the field, and offer a vision of the future of insect cell culture.

Prostaglandins A1 and E1 influence gene expression in an established insect cell line (BCIRL-HzAM1 cells)

Prostaglandins (PGs) and other eicosanoids exert important physiological actions in insects and other invertebrates, including influencing ion transport and mediating cellular immune defense functions. Although these actions are very well documented, we have no information on the mechanisms of PGs actions in insect cells. Here we report on the outcomes of experiments designed to test our hypothesis that PGs modulate gene expression in an insect cell line established from pupal ovarian tissue of the moth Helicoverpa zea (BCIRL-HzAM1 cells). We treated cells with either PGA(1) or PGE(1) for 12 or 24h then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by densitometry revealed substantial changes in protein expression in some of the protein spots we analyzed. These spots were processed for mass spectrometric analysis by MALDI TOF/TOF, which yielded in silico protein identities for all 34 spots. The apparent changes in three of the proteins were confirmed by semi-quantative PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. The 34 proteins were sorted into six categories, protein actions, lipid metabolism, signal transduction, protection, cell functions and metabolism. The findings support the hypothesis that one mechanism of PG action in insect cells is the modulation of gene expression.

Providence virus: a new member of the tetraviridae that infects cultured insect cells

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera)

SummaryThe in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell lines. Two baculoviruses with host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolyhedrovirus, AcMNPV) and the celery looper Anagrapha falcifera (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AMI and TN-CLI cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV.

Baculovirus infection influences host protein expression in two established insect cell lines

We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus-host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and semi-permissive to H. zea single nucleopolyhedrovirus (HzSNPV). Hz-AM1 cells are non-permissive to AcMNPV. We challenged each cell line with baculovirus infection and after 24h determined protein identities by MALDI TOF/TOF mass spectrometry. For Hv-AM1 cells, 21 proteins were identified, and for Hz-AM1 cells, 19 proteins were newly identified (with 8 others having been previously identified). In the permissive relationship, 18 of the proteins changed in expression by 70% or more in AcMNPV infected Hv-AM1 cells as compared with non-infected controls; 12 were significantly decreased and 6 cellular proteins were significantly increased. We also identified 3 virus-specific proteins. In the semi-permissive infections, eight proteins decreased by 2-fold or more. Non-permissive interactions did not lead to substantial changes in host cell protein expression. We hypothesize that some of these proteins act in determining host cell specificity for baculoviruses.

Wheat germ oil in larval diet influences gene expression in adult oriental fruit fly

Culture medium supplemented with wheat germ oil (WGO) causes physiological reactions, such as increased fecundity and mobility, in some insects. Although the impact of WGO on insect physiology is important, the mechanisms of these actions are poorly understood. In this paper, we test the hypothesis that the addition of WGO to medium developed for larval oriental fruit flies modulates gene expression in the corresponding adults. We separately reared larvae of Bactrocera dorsalis on diets lacking or supplemented with WGO, and analyzed for expressed proteins in the resulting adult males and females by 2D-electrophoresis. Analysis of the gels revealed significant changes in expression levels of >70 proteins, 64 of which were identified by mass spectrometric analysis on MALDI-TOF/TOF. Apparent changes in expression levels for 6 of these proteins were confirmed by quantitative real-time PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. These findings support the hypothesis that one mechanism of WGO actions in insect nutrition is the modulation of gene expression.

Establishment and characterization of an Ostrinia nubilalis cell line, and its response to ecdysone agonists.

SummaryA cell line derived from embryonic tissues of the European corn borer, Ostrinia nubilalis (UMC-OnE), was established in EX-CELL 401 medium containing 10% fetal bovine serum. The cells grew in suspension, and were mainly spherical in shape. The cell doubling times at the 17th and 79th passages were 56 and 36 h, respectively. DNA amplification fingerprinting showed that the DNA profile of the OnE cell line was different from that of the southwestern corn borer, Diatraea grandiosella (UMC-DgE), and that of the cotton bollworm, Helicoverpa zea (BCIRL-HZ-AM1). The OnE cell line was responsive to treatments of 20-hydroxyecdysone and the ecdysone agonists, methoxyfenozide (RH-2485) and tebufenozide (RH-5992). These compounds caused similar effects on the cells, which included cell clumping and decreased cell proliferation. The clumps were observed on the third day of incubation, and became larger after 7 d of incubation. After 168 h of incubation, methoxyfenozide and tebufenozide were 35 and 11 times more effective, respectively, in inhibiting proliferation of the OnE cells than was 20-hydroxyecdysone.

Prostaglandin A2 influences gene expression in an established insect cell line (BCIRL-HzAM1) cells

Prostaglandins (PGs) and other eicosanoids are oxygenated metabolites of arachidonic acid and two other C(20) polyunsaturated fatty acids. While most well studied in mammals, PGs exert important actions in insects and virtually all other invertebrates. We have been researching the mechanisms of PG actions in established insect cell lines and reported earlier that two PGs, PGA(1) and PGE(1), influence gene and protein expression in HzAM1 cells. Here we report on further experiments with three 2-series PGs, PGA(2), PGE(2) and PGF(2α). In separate experiments we treated cells with each of the three PGs for 12 and 24h and then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by Delta2D software showed that PGA(2) influenced expression of 60 proteins while PGE(2) and PGF(2α) treatments led to expression changes for only a few proteins. All spots representing changes in protein expression were processed for analysis by MALDI TOF/TOF mass spectrometry. Bioinformatic analysis of the resulting sequences yielded in silico identifications of all proteins. The apparent changes in some proteins were confirmed by quantitative PCR, which demonstrated that changes in protein expression were parallel to changes in mRNA expression. We assorted the proteins into functional categories, including 1/cell structure and function; 2/cell protection and immunity; 3/energetics and metabolism; 4/nucleotide processing; 5/protein action and processing and 6/signal transduction. These findings substantially extend our idea that one mechanism of PG actions in insect cells is the modulation of gene and protein expression.

Pupal X-ray irradiation influences protein expression in adults of the oriental fruit fly, Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis, is a pest of fruit in the Asia-Pacific region and also, due to quarantine restrictions, a threat to California fruit production. Area-wide suppression of B. dorsalis integrated several approaches including the sterile insect technique (SIT). SIT involves exposing juveniles to gamma radiation and releasing sterile males in substantial numbers, where they successfully compete for wild females. The resulting infertile eggs lead to reduction of the pest populations. Although these protocols are well documented, arising issues about the international transport and distribution of radioactive products is creating difficulties in use of radioactive sources for sterilizing radiation. This led to a shift toward use of X-ray irradiation, which also sterilizes male and female insects. However, use of X-ray technologies is in its infancy and there is virtually no information on the effects of irradiation, other than sterilization, at the physiological and molecular levels of fruit fly biology. We posed the hypothesis that sterilizing male oriental fruit flies via radiation treatment also influences protein expression in the flies. We found that exposing pupae to X-ray irradiation impacted expression of 26 proteins in adult females and 31 proteins in adult males. Seven proteins (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, larval cuticle protein 2, sarcoplasmic calcium-binding protein alpha-B and A chains, general odorant-binding protein 99b, polyubiquitin, and protein disulfide-isomerase) were impacted in both sexes. Some of the proteins act in central energy-generating and in pheromone-signal processing pathways; we infer that males sterilized by X-ray irradiation may be enfeebled in their ability to compete with wild males for females in nature.

Dietary wheat germ oil influences gene expression in larvae and eggs of the oriental fruit fly

Changes in animal nutrition, particularly essential dietary components, alter global gene expression patterns. Our goal is to identify molecular markers that serve as early indicators of the quality of insect culture media. Markers of deficient culture media will increase the efficiency of developing optimal systems for mass rearing beneficial insects and some pest species because decisions on culture media quality can be made without waiting through one or several life cycles. The objective of our current study is to discover molecular markers of essential dietary lipid deficiency in the oriental fruit fly, Bactrocera dorsalis. We reared groups of fruit flies separately on media either devoid of or supplemented with wheat germ oil (WGO) and analyzed gene expression in third instar larvae and F(1) eggs using 2D electrophoresis. Gel densitometry revealed significant changes in expression levels of genes encoding eight proteins in larvae and 22 proteins in eggs. We identified these proteins by using mass spectrometry (MALDI TOF/TOF) and bioinformatic analyses of the protein sequences. Among these, we identified one gene encoding the receptor of activated C Kinase 1 (RACK1) that increased in expression by 6.8-fold in eggs from adults that were reared as larvae on media supplemented with WGO. RACK1 is an essential component of at least three intracellular signal transduction pathways, making it a good molecular marker candidate of lipid deficiency in fruit flies and possibly many other insect species.