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Simple Summary: In the medical and pharmaceutical industries, insect cell lines are used to produce useful recombinant proteins, with their production necessitating the genetic engineering of recombinant insect viruses. Recombinant viruses can be used in experiments according to a virus quantification process. However, current virus quantification methods are complicated by their skill and experience requirements and long durations. In this study, we attempted to generate a novel transgenic insect cell line for the prompt and convenient quantification of insect viruses. By introducing a fluorescent gene, Sf9-QE, a transgenic insect cell line that rapidly emits high fluorescence in response to viral infection was generated. Using the Sf9-QE cell line, the quantification completion time was shortened by 4 to 6 days compared to existing insect cell lines, and convenient quantification was also facilitated by fluorescence photometry. The novel transgenic insect cell line generated, Sf9-QE, will be very useful in the production of and research on various medically and pharmaceutically applicable recombinant proteins, greatly contributing to human health and medical care. The following study was conducted to generate a transgenic Sf9 cell line for rapid and easy virus quantification in the baculovirus expression system (BES). The hr3 (homologous region 3) and 39K and p10 promoters were used as the expression structures to induce rapid and intense expression of the enhanced green fluorescent protein gene in cells in response to viral infection. Of 20 transgenic Sf9 cell lines generated using the piggyBac system, the cell line that showed the highest fluorescence expression in the shortest time in response to viral infection was selected and named Sf9-QE. The average diameter of the Sf9-QE cells was around 16 μm, which is 2 μm smaller than the average diameter of Sf9 cells, whereas the rate of cell proliferation was around 1.6 times higher in the Sf9-QE cells. Virus quantification using the Sf9-QE cell line did not produce significantly different results compared to the other cell lines; however, the time required for complete virus quantification was approximately 5.3 to 6.0 days for the Sf9-QE cells, which is around 4 to 6 days shorter than the time required for the other cell lines, enabling convenient and accurate virus quantification via fluorescence photometry within around 6.0 to 6.3 days. The properties of the Sf9-QE cells were stable for up to at least 100 passages. [ABSTRACT FROM AUTHOR]

Simple Summary: The pink bollworm, Pectinophora gossypiella, is one of the most damaging pests of cotton worldwide. Cotton has been genetically engineered to produce insect-killing proteins from the bacterium Bacillus thuringiensis (Bt) to control major lepidopteran pests, including the pink bollworm. The Bt proteins in genetically engineered crops are not toxic to people, other vertebrates, or most beneficial insects. Advantages of Bt crops can include pest suppression, improved yields, increased farmer profits, and decreased use of conventional insecticides. In the United States, Bt cotton, sterile moth releases, and other tactics were used to eradicate the pink bollworm. For more than 20 years, Bt cotton has been effective against pink bollworm in China. However, the benefits of Bt crops are reduced when pests evolve resistance, as exemplified by pink bollworm resistance to Bt cotton in India. For each of the two Bt proteins used widely in Bt cotton, the genetic basis of resistance is similar between resistance selected in the lab versus the field, regardless of the country of origin. The results suggest that lab selection can be useful for identifying genes likely to be important in field-evolved resistance to Bt crops and that differences in management practices among countries caused different outcomes. Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) control some important insect pests. However, evolution of resistance by pests reduces the efficacy of Bt crops. Here we review resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella, one of the world's most damaging pests of cotton. Field outcomes with Bt cotton and pink bollworm during the past quarter century differ markedly among the world's top three cotton-producing countries: practical resistance in India, sustained susceptibility in China, and eradication of this invasive lepidopteran pest from the United States achieved with Bt cotton and other tactics. We compared the molecular genetic basis of pink bollworm resistance between lab-selected strains from the U.S. and China and field-selected populations from India for two Bt proteins (Cry1Ac and Cry2Ab) produced in widely adopted Bt cotton. Both lab- and field-selected resistance are associated with mutations affecting the cadherin protein PgCad1 for Cry1Ac and the ATP-binding cassette transporter protein PgABCA2 for Cry2Ab. The results imply lab selection is useful for identifying genes important in field-evolved resistance to Bt crops, but not necessarily the specific mutations in those genes. The results also suggest that differences in management practices, rather than genetic constraints, caused the strikingly different outcomes among countries. [ABSTRACT FROM AUTHOR]

Simple Summary: The efficiency of Lepidoptera RNA interference (RNAi) is highly varied among different species, different periods, and different genes. The stability of dsRNA is one of the important factors. DsRNA-degrading enzymes (dsRNases) are the key factors affecting the stability of dsRNA in insects. The efficiency of RNAi in diamondback moths was low and unstable. Furthermore, in vitro experiments, we found that dsRNA was completely degraded when incubated with the hemolymph or gut fluid of diamondback moths. Therefore, we hypothesized that the efficiency of RNAi in diamondback moths was decreased predominantly due to degradation of dsRNA by dsRNase. In this study, we identified four dsRNases in diamondback moths: PxdsRNase1 was mainly expressed in the hemolymph; and PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. PxdsRNase1, PxdsRNase2, and PxdsRNase3 were verified to be involved in the RNAi process in diamondback moths. In vitro, the recombinant protein of PxdsRNase1 degraded dsRNA completely and PxdsRNase3 cleaved dsRNA without complete degradation. Overall, our findings provided a fundamental basis for understanding the mechanism of dsRNase involvement in the RNAi process and using RNAi to control diamondback moths in the future. DsRNA-degrading enzymes (dsRNases) have been recognized as important factors in reducing RNA interference (RNAi) efficiency in different insect species. However, dsRNases in Plutella xylostella are still unknown. We identified the full-length cDNAs of PxdsRNase1, PxdsRNase2, PxdsRNase3, and PxdsRNase4. Gene expression profile showed that PxdsRNase1 was mainly expressed in the hemolymph; and that PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. The expression of PxCht (Chitinase of P. xylostella) in P. xylostella larvae injected with the mixture of dsPxCht (dsRNA of PxCht) and dsPxdsRNase1 (dsRNA of PxdsRNase1), dsPxdsRNase2 (dsRNA of PxdsRNase2), or dsPxdsRNase3 (dsRNA of PxdsRNase3) was significantly higher than that in the larvae injected with the mixture of dsGFP (dsRNA of green fluorescent protein gene, GFP) and dsPxCht; the transcription level of PxCht in the larvae feeding on the mixture of dsPxCht and dsPxdsRNase1, dsPxdsRNase2, or dsPxdsRNase3 was significantly higher than that in the larvae feeding on the mixture of dsPxCht and dsGFP. The recombinant protein of PxdsRNase1 degraded dsRNA rapidly, PxdsRNase3 cleaved dsRNA without complete degradation, and PxdsRNase2 could not degrade dsRNA in vitro. These results suggested that PxdsRNases1, PxdsRNases2, and PxdsRNases3 were involved in the dsRNA degradation to reduce RNAi efficiency with different mechanisms. [ABSTRACT FROM AUTHOR]

The spotted alfalfa aphid (Therioaphis trifolii (Monell)) is a known destructive pest that can significantly reduce alfalfa yields. Two differentially up-regulated alfalfa trypsin inhibitors 'Msti-94' and 'Msti-16' in transcriptome were verified in terms of their mRNA levels using RT-qPCR. The prokaryotic expression vector was constructed and its biological functions, including phenotypic and physiological responses, were verified through feeding spotted alfalfa aphids with active recombinant protein mixed with an artificial diet. Gene clone and gene prokaryotic expression confirmed that Msti-94 had a size of 651 bp, encoded 216 amino acids with a predicted protein weight of 23.5 kDa, and a pI value of 6.91. Similarly, the size of Msti-16 was 612 bp, encoded 203 amino acids, and had a predicted protein weight of 22.2 kDa with a pI value of 9.06. We concluded that both Msti-94 and Msti-16 acted as a stomach poison with survival rates reduced to 21.7% and 18.3%, respectively, as compared to the control, where the survival rate was significantly (p < 0.05) higher (60.0%). Aphid reproduction rates were significantly reduced, after 72 h of feeding, in both the Msti-94 and Msti-16 treatments compared to the controls. A concentration of 800 μg/mL (0.8 mg/mL) of recombinant protein and 5000 μg/mL (5 mg/mL) of recombinant expressing bacteria that inhibits the total protease, which ultimately disrupted the activity of trypsin, chymotrypsin, and aminopeptidase. [ABSTRACT FROM AUTHOR]