Wheat Flour

Adult females were collected in February 2013 (rainy season) in the community of Zungarococha (03º49′32.40″S, 73º21′00.08″W), 18 km southwest of Iquitos, Peru. Active transmission of P. falciparum and P. vivax historically occurs in this area [26 (link)], where An. darlingi has been incriminated as the main vector [12 (link)]. Adult females were captured hourly from 18:00 to 22:00 using protected human landing collections [25 ]. Anopheles darlingi females collected were placed in carton cups (250 ml) covered with nylon mesh, provided 10 % sugar solution and taken to the NAMRU-6 insectary in Iquitos. Here, cow, or chicken blood was offered through glass membrane feeders (3.8 cm outer diameter) to induce oviposition and obtain F₁ adults following previously described procedures [21 , 27 (link)]. Briefly, one wing of each blood-engorged female was cut and then females were placed individually in vials for egg laying. Eggs were inundated with water within 24 h and placed in plastic trays (26.5 × 16.5 cm). Larvae were transferred into plastic trays containing a mix of water from natural breeding sites and filtered water and fed a mixture of wheat flour and fish-meal with quantities increasing with larval development from 0.14 mg/larva (first instar) to 0.5 mg/larva (fourth instar). Pupae were transferred into plastic containers with water (200 pupae/container) and placed in screen cages for adult eclosion. F₁ adults were used in natural copulation induction assays and for optimization of oviposition and larvae rearing. Morphological species identification of wild-caught and F₁ adult Anopheles darlingi was conducted using keys for Neotropical Anopheles [28 , 29 ]. Molecular verification of F₁₀An. darlingi adults was confirmed using mitochondrial cytochrome c oxidase I (COI) gene sequences, following the standard protocol of the Mosquito Barcoding Initiative [30 (link)].

Fluorescence data were exported from iCycler software using the "Reports" function within the "PCR Standard Curve" menu. To convert the DNA quantity in pg determined in a PCR tube to the concentration of fungal DNA in the plant sample (μg DNA per kg plant material), the value obtained by real-time PCR had to be multiplied with a factor of 33.333 and 66.667 for wheat flour and maize debris, respectively. (Different factors for wheat and maize samples were needed because of the difference in the sample size used in the optimized extraction protocol, see section Upscaled DNA extraction from plant material above).

Cornstarch (CS), wheat starch (WS), and wheat flour (WF) were used as the carbohydrate sources for the three experimental diets, and the inclusion level was the same as 45%, respectively. Besides that, the three diets had the same composition and levels of energy (19.5 kJ g⁻¹ DM), protein (40.0%), and lipids (6.4%). The detailed feed formulations and their chemical analysis are shown in Table S1. Before pelleting, all the ingredients were well ground, passed through 40 mesh (mesh size: 0.25 mm) sieves, evenly mixed, and then made into the pellets as 2 mm diameter to fit the size (3.02 ± 0.82 g) of the experiment fish.

There were three strains of gibel carp in the present study, and they were all from the hatchery of the Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan, Hubei, China. Before the formal trial, a prefeeding period was conducted for 4 weeks to acclimate to the feed and rearing conditions. Then, three strains of the experimental fish were pooled after 24 h starvation. Fish of each strain with the similar weight (3.02 ± 0.82 g) were selected, weighted, and distributed into round fiberglass tanks at the density as 30 fish per tank. Each strain had 9 tanks, respectively, fed by cornstarch (CS) diet, wheat starch (WS) diet, and wheat flour (WF) diet in triplicate, and there were 27 tanks in total (3 strains^∗ 3 diets^∗ 3 duplication). The fish were fed to apparent satiation twice a day at 08 : 30 am and 14 : 30 pm and reared in the circulating water system: water volume of each tank was 300 L; diameter of tank was 100 cm; water flow rate was 1.8 L min⁻¹; water temperature was 25.5 ± 1.5°C; light intensity was approximately 3 μmol m⁻² s⁻¹ at the water surface; light period was from 08 : 00 am to 20 : 00 pm; water dissolved oxygen was more than 5 mg L⁻¹; water ammonia nitrogen was less than 0.5 mg L⁻¹; and water residual chloride was less than 0.01 mg L⁻¹ (weekly monitored).
The feeding trial lasted for 8 weeks. Then, all fish in each tank were weighed after 24 h of food deprivation, and 4 fish per tank were randomly sampled and frozen at -20°C for whole-body composition analysis. The rest of the fish continued refeeding for one more week; 3 fish/tank were randomly sampled at 6 h after the last meal. They were anesthetized by MS-222 (100 mg L⁻¹ tricaine methane sulfonate, Argent Chemical Laboratories Inc., Redmond, WA, USA). After that, blood was collected and then centrifuged at 3000 g, 4°C, 15 min, to obtain the plasma; fresh liver, middle intestine, and muscle (biopsies) of the fish were separated on the ice, immediately frozen in liquid nitrogen, and then kept at -80°C for further analysis.