Indomethacin and 20-hydroxyecdysone influence protein expression in a Spodoptera frugiperda nervous system cell line.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 µM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

Trade-Offs among Immune Mechanisms: Bacterial-Challenged Spodoptera frugiperda Larvae Reduce Nodulation Reactions during Behavioral Fever.

Insect innate immunity is composed of cellular and humoral reactions, the former acting via circulating hemocytes and the latter via immune signaling that lead to the production of antimicrobial peptides and phenol oxidase-driven melanization. Cellular immunity involves direct interactions between circulating hemocytes and invaders; it includes internalization and killing microbes (phagocytosis) and formation of bacterial-laden microaggregates which coalesce into nodules that are melanized and attached to body walls or organs. Nodulation can entail investing millions of hemocytes which must be replaced. We hypothesized that biologically costly hemocyte-based immunity is traded off for behavioral fevers in infected larvae of fall armyworms, Spodoptera frugiperda, that were allowed to fever. We tested our hypothesis by infecting larvae with the Gram-negative bacterium, Serratia marcescens, placing them in thermal gradients (TGs) and recording their selected body temperatures. While control larvae selected about 30 °C, the experimental larvae selected up 41 °C. We found that 4 h fevers, but not 2, 6 or 24 h fevers, led to increased larval survival. Co-injections of S. marcescens with the prostaglandin (PG) biosynthesis inhibitor indomethacin (INDO) blocked the fevers, which was reversed after co-injections of SM+INDO+Arachidonic acid, a precursor to PG biosynthesis, confirming that PGs mediate fever reactions. These and other experimental outcomes support our hypothesis that costly hemocyte-based immunity is traded off for behavioral fevers in infected larvae under appropriate conditions.

Next-generation cell lines established from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm, Spodoptera frugiperda (Sf), is a polyphagous lepidopteran herbivore that consumes more than 80 plant species, including many economically important crops, such as corn, soybeans, and sorghum. While already a serious pest in the Americas, it was recently introduced into Africa, India, and China. Because of its high economic costs in the New World and the continent-wide damage potentials in Africa, research to develop advanced pest management technologies is necessary. We are supporting this need by developing novel, next-generation insect cell lines from targeted tissues. Cell lines, such as these, will boost insecticide discovery programs and lead to innovative pest management solutions. Here, we report on the establishment of 16 new cell lines from larval S. frugiperda tissues: nine from the central nervous system, three from the aorta, and four from the testes. We confirmed the identities of the cell lines by DNA amplification fingerprinting polymerase chain reaction, determined their doubling times from growth curves, and described cell types via microscopy. We also developed 16 sublines from three neuronal cell lines.

Characterization of cell lines derived from the southern armyworm, Spodoptera eridania.

Spodoptera eridania (southern armyworm) is a polyphagous pest of many plants, including field crops, vegetables, fruits, and ornamentals. Larvae are leaf feeders, defoliating many crops in the tropics and subtropics of the western hemisphere. In this study, cell lines from S. eridania were established to support research focused on the development of advanced pest management technologies. We generated seven cell lines from larval tissues: three from nervous tissues, two from testes, and two from fat bodies. These cell lines have been passaged 18-57 times, indicating they are established lines. They are maintained in EX-CELL 420 or a combination of L15 + EX-CELL 420 media. The identities of the cell lines were confirmed by DAF-PCR and their doubling times ranged from 42 to 110 h. Microscopy indicated the presence of one or more morphologically distinct cell types in each cell line. We identified a catalase gene in all seven cell lines. H2O2 treatment suppressed the expression of catalase and led to a reduction in catalase activity. This is the first report of cell lines established from S. eridania, and these cell lines are now available to researchers worldwide on request.

Prostaglandin-mediated recovery from bacteremia delays larval development in fall armyworm, Spodoptera frugiperda.

Insect immunity includes a surveillance system that detects and signals infections, coupled with hemocytic and humoral immune functions. These functions are signaled and coordinated by several biochemicals, including biogenic amines, insect cytokines, peptides, and prostaglandins (PGs). The actions of these mediators are coordinated within cells by various forms of cross-talk among the signaling systems and they result in effective reactions to infection. While this is well understood, we lack information on how immune-mediated recovery influences subsequent juvenile development in surviving insects. We investigated this point by posing the hypothesis that PG signaling is necessary for larval recovery, although the recovery imposes biological costs, registered in developmental delays and failures in surviving individuals. Here, we report that nodulation responses to infections by the bacterium, Serratia marcescens, increased over time up to 5 h postinfection, with no further nodulation; it increased in a linear manner with increasing bacterial dosages. Larval survivorship decreased with increasing bacterial doses. Treating larvae with the PG-biosynthesis inhibitor, indomethacin, led to sharply decreased nodulation reactions to infection, which were rescued in larvae cotreated with indomethacin and the PG-precursor, arachidonic acid. Although nodulation was fully rescued, all bacterial challenged larvae suffered reduced survivorship compared to controls. Bacterial infection led to reduced developmental rates in larvae, but not pupae. Adult emergence from pupae that developed from experimental larvae was also decreased. Taken together, our data potently bolster our hypothesis.