Rethinking temperature effects on leaf growth, gene expression and metabolism: Diel variation matters.

Plants have evolved to grow under prominently fluctuating environmental conditions. In experiments under controlled conditions, temperature is often set to artificial, binary regimes with constant values at day and at night. This study investigated how such a diel (24 hr) temperature regime affects leaf growth, carbohydrate metabolism and gene expression, compared to a temperature regime with a field-like gradual increase and decline throughout 24 hr. Soybean (Glycine max) was grown under two contrasting diel temperature treatments. Leaf growth was measured in high temporal resolution. Periodical measurements were performed of carbohydrate concentrations, carbon isotopes as well as the transcriptome by RNA sequencing. Leaf growth activity peaked at different times under the two treatments, which cannot be explained intuitively. Under field-like temperature conditions, leaf growth followed temperature and peaked in the afternoon, whereas in the binary temperature regime, growth increased at night and decreased during daytime. Differential gene expression data suggest that a synchronization of cell division activity seems to be evoked in the binary temperature regime. Overall, the results show that the coordination of a wide range of metabolic processes is markedly affected by the diel variation of temperature, which emphasizes the importance of realistic environmental settings in controlled condition experiments.

Novel Experimental Methods for the Investigation of Hermetia illucens (Diptera: Stratiomyidae) Larvae.

Large-scale insect rearing for food and feed production can be improved by understanding diet digestion and host-microbe interactions. To examine these processes in black soldier fly (Hermetia illucens L.; Diptera: Stratiomyidae) larvae, two protocols were developed. Protocol 1 describes a method to produce viable, sterile black soldier fly larvae and a gentle method for diet sterilization. Sterile black soldier fly larvae can be used to study the diverse role of microbes in larval development. Nutrient requirements of sterile black soldier fly larvae are met only through diet. Viable sterile black soldier fly larvae were consistently generated using a four-step treatment with alternating immersions of eggs for 2 min each in ethanol (70%) and sodium hypochlorite (0.6%), over two cycles. A nonthermal method of diet sterilization, namely high-energy electron beam (HEEB) treatment, was introduced. Subsequently, growth of sterile black soldier fly larvae was observed on the HEEB-treated diets (40, 60, and 40% of replicates with poultry feed, liver pie, and an artificial diet, respectively) but not on autoclaved diets. In Protocol 2, we propose a novel method to collect frass from individual larvae. We then measured the metabolites in frass, using high-pressure liquid chromatography. Results on metabolites confirmed the influence of digestion. For instance, succinate increased from 1 to 2 and 7 µmol/g sample from diet to gut homogenate and frass, respectively. The collection method is a promising tool to estimate the diet and nutrient requirements of black soldier fly larvae, thus increasing the performance and reliability of black soldier fly larvae rearing. We discuss in detail the possible applications and limitations of our methods in black soldier fly larvae research.