A universal diet for corn rootworms (Diabrotica spp.) capable of rearing both specialist and generalist insect species including the western corn rootworm, the northern corn rootworm (D. barberi Smith & Lawrence), and the southern corn rootworm (D. undecimpunctata howardi Barber) was utilized (19). This artificial diet also supports the development of the Mexican corn rootworm (D. virgifera zeae Krysan & Smith) (Huynh et al., unpublished). The universal diet, detailed in Huynh, Hibbard, Vella, Lapointe, Niedz, Shelby and Coudron (20), is comprised of 15 ingredients (e.g., wheat germ, whole egg powder, cellulose, vitamin mix, salt mix, preservatives, water) that are commonly used in many insect diets (5). A single batch of its dry mixture, prepared by Frontier Agricultural Sciences, was used for all experiments.

Adults of a non-diapausing strain of the western corn rootworm were maintained at the USDA-ARS PGRU as described previously (21). This strain, originally established from eggs purchased from Crop Characteristics (Farmington, MN), has been maintained on a non-transgenic maize line (Viking 60-01N, Albert Lea Seed, Albert Lea, MN) for multiple generations (22). The adult beetles were reared on an artificial diet (23) and water in 30 × 30 × 30 cm cages (BugDorm^®, BioQuip Products Inc., Rancho Dominguez, CA). Petri dishes (9-cm, Fisher Scientific, Pittsburgh, PA) containing 80-mesh sieved soil as an ovipositional medium were placed in the rearing cages to collect eggs (24). After the eggs were collected, the egg dishes were incubated at 25 ± 1°C in darkness for prompt use or were stored at 8 ± 1°C for later use in an incubator (Percival, Perry, IA) (10).

Before conducting the bioassays, the egg dishes were incubated at 25 ± 1°C in darkness in a Percival incubator. Once several eggs had hatched, the eggs were surface-sterilized according to previously described procedures (25, 26) with modifications. Briefly, the mixtures of eggs and soil were rinsed through a 250-µm, 60-mesh sieve (Hogentogler & Co. Inc., Columbia, MD) to remove soil. The eggs retained in the sieve were collected into a 50 mL beaker using a stream of tap water. The eggs were then rinsed with tap water to remove neonates and hatched egg cases that floated to the water’s surface. Next, the eggs were treated with undiluted Lysol (10 ml, Clean & Fresh Multi-Surface Cleaner, Reckitt Benckiser, Parsippany, NJ) for 3 min, followed by a triple rinse with purified water to remove the Lysol solution. The eggs were then rinsed with 10% formaldehyde solution (10 ml, HT501128, Sigma Aldrich, St. Louis, MO). After removing the formaldehyde solution, the eggs were triple rinsed with purified water and were dispensed to a coffee filter paper (Pure Brew, Rockline Industries, Sheboygan, WI). The filter paper was then placed inside a container with a lid (16-oz, LG8RB-0090 & DM16R-0090, Solo Cup Company, Lake Forest, IL) and incubated at 25 ± 1°C in darkness in the Percival incubator. Larvae that hatched within < 24 h were used for the insect bioassays.

The eggs can be surface-sterilized to obtain larvae for multiple days. After the initial egg treatment, in the following days, the eggs were washed with 10% formaldehyde solution for 3 min, followed by a triple rinse with purified water. The eggs were then dispended to a new filter paper in a 16-oz container and were incubated as described above to collect the newly hatched larvae (< 24 h old).

The artificial diet was prepared in 96-well plates by Frontier Agricultural Sciences using a procedure described previously (20) with modifications. Briefly, agar was added to purified water and the agar solution was boiled using a microwave until the agar was completely melted. The dry mixture was poured into the agar solution when it cooled to approximately 65°C. The plates (96-well plate, 3370, Corning Inc., Corning, NY) were then filled with diet solutions (200 µl per well) using a repeater pipette (HandyStep^® S, Brandtech, Essex, CT). The diet plates were sealed with an adhesive sealer (AB-0558, Thermo Scientific™, Waltham, MA). All fills occurred in a laminar flow hood (1800 Series, Thermo Scientific™) to prevent incidental contamination. All filled diet plates were prepared at the same time.

The completed diet plates were allowed to be exposed to varying temperature conditions experienced during transit for 24 hours from Frontier Agricultural Sciences to the USDA-ARS PGRU. The filled diet plates were packed in separate Styrofoam shipping boxes filled with either hot packs, cool packs, ice packs, or dry ice to mimic varying potential environmental conditions experienced during transit. Five different temperatures ranging from 0°C to 30°C were targeted. During the transit, the box temperatures were monitored at one minute intervals using a data logger (UX100-003, Onset, Bourne, MA) placed inside each box. Upon receipt, the diet plates were immediately stored at 4°C for up to 28 days. At 5 durations after storage (1, 7, 14, 21, and 28 days), the quality of the prepared diet was evaluated by life history parameters (survival, molting, and weight) of the western corn rootworm larvae reared on diets.

The completed diet was prepared in 96-well plates (3370, Corning Inc.) at the USDA-ARS PGRU as described previously (20) using the same dry diet batch as used for the transit experiment. After the completed diet plates were made, the diet plates were immediately stored at 5 different fixed temperatures (-20, 4, 22, 25, and 33°C) for 5 durations (1, 7, 14, 21, and 28 days). Diet assays were conducted to evaluate the effects of these fixed temperatures on diet quality immediately after each predetermined duration. The quality of the artificial diet was assessed via the evaluation of life history parameters (weight, molting, and survival) of the western corn rootworm larvae reared on the diets.

The bioassays were conducted as described previously (27). All materials used in the diet assays were surface-treated via exposure to UV light for 10 min in a biological cabinet (SG403, SterilGARD^® III Advance cabinet, Sanford, ME). Each treatment was a completed diet exposed to different temperatures and time intervals of storage. Each replicate included the artificial diet pipetted to a 12-well row of the 96-well plate. Each treatment was replicated 6 times in different diet plates. Each well was infested with one western corn rootworm neonate (< 24 h old) using a fine paintbrush. A sealing film (TSS-RTQ-100, Excel Scientific, Inc., Victorville, CA) was used to cover the plate. For ventilation, a hole was made in the sealing film over each well using a number zero insect pin. The plates were kept in a Percival incubator at 25°C in darkness for 7 days. Larval molting, survival, and evidence of contamination (fungi and bacteria) were recorded daily, whereas larval weight was measured at the end of the experiments. During the experiment, larvae were typically found on the surface of the diet. Larval molting was recorded by observing the presence of the old cuticles that the larvae shed during the molting process on the diet’s surface, while larvae were considered dead if they did not exhibit coordinated movement after being touched with the number zero insect pin. For larval dry weight, all live larvae in each treatment were pooled per replicate (12 possible) into 95% ethanol, dried in an oven (Binder GmbH, Tuttlingen, Germany) at 55°C for 48 h, and weighed using a micro balance (MSU6.6S-000-DM, Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany).

To determine survival and molting rates, the number of live larvae and successful larval molts from 1st to 2nd instar per replicate were divided by the initial number of larvae and multiplied by 100 to obtain percentages. The weight per larva (in mg) was calculated by dividing the dry weight by the number of live larvae per replicate. If all larvae for a replicate were dead, the larval weight for this replicate was recorded as 0.

The experiments were designed as a 2-factor factorial design (temperature × time). The life history data, including percent survival, weight, and percent molt to second instar, were analyzed separately using analysis of variance (ANOVA) in generalized linear mixed models (PROC GLIMMIX) in SAS 9.4 (SAS Institute, Cary, NC). Temperatures and time exposure were the fixed effects and replication was the random variable. Differences between treatments were determined using Fisher’s least significant difference (LSD) at p < 0.05. The percent variables (survival and molt) were arcsine square-root transformed and the numeric variable (weight) was square-root transformed prior to the analysis to meet the assumption of normality and homoscedasticity. The untransformed data were presented as MEAN ± SEM.

The diet plates arrived at the USDA-ARS PGRU (Columbia, MO) after departing approximately 24 hours from Frontier Agricultural Sciences (Newark, DE). During the transit, the complete diet was exposed to 5 different averaged temperatures of 6.6 ± 6.7°C, 9.7 ± 2.7°C, 18.8 ± 1.5°C, 22.5 ± 1.0°C, and 25.2°C ± 1.6°C with major temperature exposure from -2°C to 27°C ( Table 1 ; Figure 1 ). Upon receipt, all the diet plates were visually examined, and they were presented in good physical condition (i.e., gel matrix intact, no constituent separation, no excess moisture, no desiccation) and were stored at 4°C for up to 28 days.

Exposure to varying temperatures from -2°C to 27°C for approximately 24 hours during the transit had no significant impacts on the quality of the completed diet assessed by life history parameters measured (survival, molt, and weight) ( Table 2 ). However, the duration of storage at 4°C after shipping had significantly detrimental effects on the quality of the artificial diet for feeding the western corn rootworm larvae ( Table 2 ). With respect to time of storage with all varying temperatures combined ( Figure 2 ), significant reductions in larval molting and weight were observed for the completed diets stored within 7 days. There was no significant difference in larval weight and molting between 7 and 14 days after storage, whereas the lowest larval molting and weight were found at 21 and 28 days of storage, respectively. Larval survival remained above 95% at all time points evaluated, indicating that diet storage for up to 28 days did not adversely affect survival ( Figure 2 ). The interactions between temperature × time were not significantly different for any of the life history parameters ( Table 2 ).

The fixed temperatures, time of storage, and their interactions significantly impacted the quality of the completed diet for rearing the western corn rootworm larvae ( Table 3 ). With respect to the temperatures with all the time of storage combined ( Figure 3A ), averaged larval survival was over 95% for temperatures from -20°C to 25°C. In contrast, survival declined significantly to 60% for the highest temperature of 33°C. Temperatures of -20°C and 4°C had the highest larval weight and molting rates, followed by temperatures of 22°C and 25°C. The temperature of 33°C had the lowest rates for all examined parameters (weight, molting, and survival). Regarding storage duration with all temperatures combined ( Figure 3B ), the completed diet stored for 7 days had a significantly negative impact on larval molting and weight. A significant reduction in larval survival was found starting after 21 days of storage.

Figure 4 reveals the interactions between temperature and storage duration for each examined parameter. Over a 14-day storage period, there was no significant difference in larval survival on the completed diet stored at any temperature ( Figure 4A ). However, significant reductions in survival were observed primarily at the highest temperature (33°C), with no survival found after 21 days of storage ( Figure 4A ). Significant declines in molting began at all temperatures after 7 days of storage, except for the highest temperature (33°C) where reductions occurred after just 1 day of storage ( Figure 4B ). After 7 days of storage, diets stored at -20°C and 4°C supported greater larval molting compared to those stored at higher temperatures. Larval weight at the lowest two temperatures remained unaffected within 21 days of storage ( Figure 4 ).

No evidence of contamination (bacteria or fungi) was observed during all experiments, except for the temperature storage at 33°C. After 21 days of storage at 33°C, the completed diet was mostly contaminated with bacteria. This contamination contributed to 100% mortality of the insects fed on these diets ( Figure 4A ).