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BACKGROUND Prevention is the first line of defense in mitigating losses of post-harvest crops. Long-lasting insecticide treated (LLIN) could be used in food facilities to expose insects to insecticide at different areas within a facility. Prior research has shown that single short exposures reduce movement and longer exposures increase mortality for stored-product insect pests, but we do not know how multiple short duration exposures and biotic and abiotic conditions affect insects exposed to LLIN. Here, we repeatedly exposed red flour beetles, Tribolium castaneum, to LLIN to assess the cumulative effects. We also examined the effects of beetle age and time of day during exposure, and temperature, humidity and food availability during recovery after a single exposure to LLIN. RESULTS We found that four repeated 10-min exposures had similar knockdown effects as a single 30-min exposure. We also found that beetles were more affected when aged 1-6 days versus 14-20 days or were exposed at mid- or late in the day versus earlier in the day. Higher recovery levels were observed with food and at higher relative humidity. In addition, older beetles were more active than younger beetles during exposure, which could reduce time in contact with netting and partially explain why older beetles tended to be less affected. CONCLUSION Some individuals can recover after exposure to LLIN, dependent on exposure duration and environmental factors, but our study shows that sublethal effects likely persist and future work should consider the physiology of T. castaneum before, during, and after exposure to LLIN. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Few transcription factors have been identified in C4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves. 35S::SbMyb60 was associated with the transcriptional activation of genes involved in aromatic amino acid, S-adenosyl methionine (SAM) and folate biosynthetic pathways. The high coexpression values between SbMyb60 and genes assigned to these pathways indicate that SbMyb60 may directly induce their expression. In addition, 35S::SbMyb60 altered the expression of genes involved in nitrogen (N) assimilation and carbon (C) metabolism, which may redirect C and N towards monolignol biosynthesis. Genes linked to UDP-sugar biosynthesis and cellulose synthesis were also induced, which is consistent with the observed increase in cellulose deposition in the internodes of 35S::SbMyb60 plants. However, SbMyb60 showed low coexpression values with these genes and is not likely to be a direct regulator of cell wall polysaccharide biosynthesis. These findings indicate that SbMyb60 can activate pathways beyond monolignol biosynthesis, including those that synthesize the substrates and cofactors required for lignin biosynthesis.

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses.