Sucrose

Glucose, xylose, arabinose, sucrose, lactose, and glycerol were supplied from Bio Basic Canada Inc. Furfural, 5-hydroxymethyl Furfural (HMF), and 3,5-dinitrosalycilic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agroindustrial by-products (wheat bran and sugarcane bagasse) were kindly provided by a local food processing industry (White-rose group, Sfax, Tunisia). Almond shell, date palm leaves, and Posidonia oceanica balls were collected locally. All lignocellulosic residues are conserved at 4°C until use.
The new oleaginous yeast strain Y-MG1, isolated from rotten fruit, was identified in our previous work based on its internal transcribed spacer (ITS) sequence [21 (link)]. Y-MG1 strain was identified as being Rhodotorula mucilaginosa and was submitted in the National Strains Collection of Centre of Biotechnology of Sfax, CBS, Tunisia, under the accession number CTM-30138. The ITS sequence of Y-MG1 was also submitted to GenBank under the accession number ID: KX347596.1. This strain was stored at −80°C in sterilized glycerol-enriched solution containing 2% (w/v) glucose, 1% (w/v) yeast extract, 1% (w/v) bacto-peptone, and 20% (w/w) glycerol.

Based on chemically-induced transformation, protoplasts were treated with 60% polyethylene glycol (PEG) 4000 [19 (link)]. The 100 μl of protoplast suspension was added into 2 tubes (DNA+ and DNA- control). The linear fragment of gene deletion construct (about 500 ng) was added into DNA+ tube. The 25 μl of 60% PEG 4000 was added and then mixed by pipetting. The transformation tubes were incubated on ice at least 30 min. 1 ml of 60% PEG was added and rolled slowly to mix. Next, these tubes were incubated at room temperature for 30 min. Subsequently, 5 ml of STC were added into each tube and all tubes were spun at 4,500 rpm for 20 min. The pellet was suspended in 300 μl of STC. After the transformation, the protoplasts were plated on protoplast medium with 1.2 M sucrose (Bio basic Canada INC., Canada). The plates were incubated at 25°C for 12 d.

Drosophila larvae were exposed to nutritional stress for 3 days prior to presentation of stained Drosophila eggs. In order to induce nutritional stress, 30 larvae (hatched from synchronized Canton-S eggs) were reared on nutrient scarce medium composed of 4% (w/v) sucrose (BIO BASIC INC), 1.5% (w/v) bacteriological agar (Sigma-Aldrich), 0.5% (w/v) propionic acid (Apex^TM) and Nipagin (1 g/lit). A control population of 30 larvae was obtained from the same batch of synchronized eggs and maintained on standard cornmeal medium. Once larvae in both vials started foraging, 10 stained eggs were added to each vial. Larval consumption of eggs was observed under a stereomicroscope at regular intervals for up to 3 hours. Larvae were photographed after they started to aggregate around the eggs.