Development and determination of method for gut damage evaluation in springtailTrials with trypan blue were performed using different staining plates and depuration plates to determine the optimum protocol for assessing gut damage in F. candida. The depuration process removes surplus trypan blue dye, which did not stain gut barriers. This study considered and examined the following points using Brilliant Blue FCF dye (Junsei Chemical Co., Ltd., Tokyo, Japan) prior to application of the trypan blue (MilliporeSigma,, St. Louis, MO, USA):
1) Necessity of agar for determining the state of media: comparing the presence/absence of 0.8% agar (MilliporeSigma,) on staining plates.
2) Necessity of sugar to accelerate trypan blue intake: comparing the presence/absence of sugar (5%) on staining plates.
3) Optimum concentration of staining dye: comparing 0.1% and 1% concentrations on staining plates.
4) Optimum dye-staining period: Time-dependent staining rates were compared for 5 to 120 min.
5) Optimum depuration plate: comparing agar plate with sugar versus plaster plate with yeast.
6) Optimum dye depuration period: Time-dependent depuration rates were compared on depuration plates for 5 to 300 min.
Detailed ingredients for the tested staining and depuration plates are shown in Table S1. Blue dye was used as dye control. In this optimization test, ten adult F. candida (age 28–37 days) were tested per plate, and a total of 3 to 12 plates were run for each condition. The staining of damaged gut barrier in F. candida was evaluated under a stereomicroscope and then expressed as a staining ratio in ten F. candida individuals. All springtails were cross-checked by two of the authors.
Applicability Experiment Using Model Chemicals
The applicability of the developed trypan blue agar plate with sugar method was evaluated using two heavy metals (copper [Cu] and chromium [Cr]). Cu and Cr were selected as model chemicals because they are common soil pollutants with high ecotoxicity (Boeri et al. 2017, Fisker et al. 2013, Kim andAn 2014, Kwak et al. 2014a).
F. candida were exposed to Cr and Cu by means of a food exposure experiment. First, 5 mg of baker’s yeast was placed at the center of a Petri dish (60 × 15 mm) containing an 8:1 ratio of plaster of Paris and activated charcoal powder (Ducksan, Seoul, South Korea). Next, CuSO4·5H2O (MilliporeSigma) and K2Cr2O7 (MilliporeSigma) were dissolved in distilled water, and 30 µL of each test solution was added to yeast to prepare an exposure concentration of 600 mg Cu or Cr/kg yeast. The blank was treated with 30 µL of distilled water, while the negative control was preparing by adding 100 mg Ca/kg yeast using CaCO3 (Daejung, Gyeonggi-do, South Korea). Finally, 30 adult F. candida (28 ± 1 days) were exposed per exposure plate (Petri dish, 60 × 15 mm) containing the plaster of Paris and activated charcoal mixture. In total, 12 exposure plates for the blank, 3 exposure plates for the negative control, 8 exposure plates for the Cu-exposed groups, and 7 exposure plates for Cr-exposed groups were kept in darkness at 20 ℃ for 10 days.
Subsequently, ten survivors from each exposure plate were moved to one trypan blue agar plate with sugar (containing 0.1% trypan blue, 0.8% agar, and 5% sugar) and stained for 15 min (at 20 ℃ under 271–611 lux light). After 15 min of staining, the ten F. candida individuals were moved to one depuration plate (containing 0.8% agar and 10% sugar) and then maintained for 3 h (20 ℃, 271–611 lux light). Following a 3 h depuration period, the number of F. candida per depuration plate that were successfully stained by trypan blue were counted under a stereoscope, and the percentage of gut damage was analyzed using the staining ratio in ten F. candida individuals. A total of 27 replicates for blank groups, 6 replicates for negative controls, 14 replicates for Cu-exposed groups, and 11 replicates for Cr-exposed groups were investigated to determine the applicability of the experimental protocol for assessing gut damage by heavy metal (Cr or Cu) exposure. Five replicates were run for blue dye controls. All the springtails were cross-checked by two of the authors.
Ultrastructural alterations were also analyzed using field emission-transmission electron microscopy (FE-TEM) (JEM-2100F; Jeol Ltd., Tokyo, Japan). F. candida were fixed in a mixture of 4% formaldehyde and 1% triton X-100 (MilliporeSigma) for 10 min and then in 2.5% glutaraldehyde for 2 h. FE-TEM samples were prepared by following (Kwak et al. 2014b). Briefly, the fixed F. candida was rinsed with PBS buffer (pH 7.4) and stained with 1% osmium in PBS buffer for 1 h. The stained F. candida was then rinsed with PBS buffer, dehydrated with ethanol (50 to 99.9%), and embedded with a series of propylene oxide and resin mixture (2:1 to 1:2). Finally, the resin-embedded blocks were sectioned and analyzed using the FE-TEM.
Statistical analysis
Statistical differences were analyzed based on the one-way ANOVA with Bonferroni test using the OriginPro (ver. 8; OriginLab Corporation, North Hampton, MA, USA).