Butterflies and larval rearing

Throughout this paper, the cabbage white butterfly P. rapae (Linnaeus, 1758) was used. The Japanese subspecies of this butterfly species are known as P. rapae crucivora Boisduval, 183680. Larvae of this butterfly eat cruciferous plants and prefer cultivated cabbage. All rearing procedures were performed in our laboratory, University of the Ryukyus, Okinawa, Japan. Throughout the rearing procedure, we controlled lighting under L18:D6 conditions at 25–27 °C.

We collected female adult individuals from Yomitan Village and Nanjo City, Okinawa-jima Island, the Ryukyu Archipelago, Japan. Eggs were collected in the laboratory in Okinawa from these field-collected females (n = 9). Egg-collecting was attempted 4 times (Table S6). We used an insect rearing cage (300 × 740 × 300 mm) (MegaView Science, Talchung, Taiwan), in which cabbage leaves (cultivated in Okinawa for control rearing) and female butterflies were placed together with some flowers for nectar such as Dahlberg daisy Thymophylla tenuiloba, sweet alyssum Lobularia maritima, and Spanish needle Bidens pilosa (Fig. 1d,e). Cabbage leaves inside the cage, on which eggs were deposited, were replaced with new ones daily until the female butterflies died (2–4 days). The egg period was approximately 3 days, after which larvae hatched. Larvae ate noncontaminated cabbage leaves on which they were born until the fifth day after egg collection. Then, the first-instar larvae were randomly allocated to one of the four groups to feed the contaminated or control cabbage leaves. Each group of larvae were fed the cabbage leaves of different contamination levels. Larvae from the same females were evenly allocated to all cabbage groups to avoid genetic bias among these groups. Therefore, any effects are attributable to cabbage quality and possibly the level of radioactive materials in the cabbage leaves, although nonradioactive materials could also contribute to the effects. Each group started with approximately 158 individuals for the rearing experiment and 28 individuals for hemolymph collection (Table S6). Throughout this study, only soft cabbage leaves inside the cabbage heads were used for egg collection and larval rearing procedures without washing.

The rearing experiment for the developmental factors and the forewing sizes were performed 3 times independently under the same protocol (trial numbers 1, 2, and 4). Developmental factors such as the total normality rate (see below) were calculated for each trial and were averaged using data from three trials. The forewing sizes were also averaged in this way, but the individual size data were also used for statistical analyses. The hemolymph collection was attempted twice (trial numbers 3 and 4) under the same protocol, and the percentages of hemocytes (see below) were averaged using these two trials.

For rearing, two types of plastic containers were used depending on larval size and density: a cylindrical container (55 mm height × 101 mm in diameter) and a square prism container (57 mm height × 168 mm width × 168 mm depth). Container cleaning and feeding processes were carried out every day or every two days, and simultaneously, dead bodies were identified, if any, and the number of individuals were counted. At the prepupal stage, each individual was independently transferred to a cylindrical plastic container (55 mm height × 101 mm in diameter) and was numbered individually. After eclosion inside the container, adults were readily frozen.

Soil and cabbage cultivation

We cultivated the miniature cabbage “F 1 Yokamaru” (Kokkaen, Osaka, Japan), a commercially available Japanese cultivar of the cabbage Brassica oleracea var. capitate, from seeds. Seeds for cultivation were used from a single commercial package, irrespective of the soil group, to minimize genetic variability as much as possible. We collected soils from Okinawa and Fukushima at the following localities: (1) Nanjo City, Okinawa Prefecture, as a control, (2) Ohara, Minami-soma City, Fukushima Prefecture, (3) Baba, Minami-soma City, Fukushima Prefecture, and (4) Iitate Village, Fukushima Prefecture. Four plastic planters (345 mm height × 520 mm width × 260 mm depth) per locality group were filled with the wild-collected soils mixed with a small amount of natural fertilizer.

During the early stages of cultivation, we used a kit including a nursery pot and soil (Jiffy 7) and a small greenhouse for seedlings (Moerdijk, Netherlands). We seeded 3 seeds per pot, and when 3 or 4 true leaves were formed, only one seedling was saved. When seedlings had 5 or 6 true leaves, the seedlings were planted in the planter containing the field-collected soils. Before the planting process, the field-collected soils were mixed with 100 g of fertilizer MagAmp K (HYPONeX, Osaka, Japan) per planter. Three and 6 weeks after the planting, 140 g of additional fertilizer My Garden (Sumitomo Chemical Garden Products, Tokyo, Japan) per planter was added. These seedling processes were performed in August and September with a one-month interval to accommodate 4 rearing attempts.

During the early stages of cultivation, planters were covered with fine-meshed nets to avoid insect damage. No insecticide was used throughout the cultivation period. Planters of the 3 soil groups from Fukushima Prefecture were placed in a plastic greenhouse in Minami-soma City, Fukushima Prefecture. Planters of the Okinawa soil group were placed in Nishihara Town, Okinawa Prefecture. The seeding cultivation was performed during the same period among 4 groups, and the Fukushima and Okinawa cabbage heads were harvested on the same day. The Fukushima cabbage was immediately sent to our laboratory in Okinawa under refrigeration. During the transportation period, the Okinawa cabbage was also stored in a refrigerator. During the feeding period, leaves were detached from the heads as necessary, and the remaining parts were stored in a refrigerator.

Our protocol eliminated the adsorption of radioactive materials on the surface of the cabbage leaves by two means. First, the planters were covered with fine-meshed nets during the early period of cultivation and were placed in a green house. Second, the outer leaves of the cabbage heads were not given to larvae. Only young leaves inside the cabbage heads were given to larvae.

Developmental factors and morphological abnormalities

We obtained the following 5 developmental factors: the pupal eclosion rate (the number of individuals who eclosed among the number of pupae), the adult achievement rate (the number of individuals who successfully eclosed among the number of starting larvae used), the total normality rate (the number of adults without any noticeable morphological abnormality among the number of starting larvae), the larval period [days] (from the day of egg deposition to the pupation day including the prepupal stage; dead individuals during this period were not included), and the pupal period [days] (from the pupation day to the eclosion day; dead individuals during this period were not included, but individuals of eclosion failure were included) (Table S2). Morphological abnormalities were checked with the naked eye or using a conventional stereomicroscope. All individuals that successfully eclosed (individuals for which all body parts were escaped from the pupal case) were subjected to abnormality checks except for the individuals subjected to hemocyte counting. The adult abnormality rate (the number of abnormal adult individuals among the individuals that eclosed), which was reflected in the total normality rate, were also used when appropriate (Fig. 2d, Table S3). For the abnormality check, attention was paid to the following body parts: wing shape, appendages (legs, antennae, palpi, and proboscises), compound eyes, trunk (thorax and abdomen), and sexual organ at the tip of the abdomen (valva). Each type of abnormalities was given scores based on the frequency of that abnormality type across all samples, resulting in adult abnormality score for each group (see Statistical analyses; Fig. 2e, Table S3). It appeared that the color patterns were highly variable among individuals in this species despite exhibiting a small number of pattern elements. Variations in black spots and yellow areas in size and shape were considered within the normal range for this species. Abnormal structures were photographed using a high-resolution Keyence VHX-1000 digital microscope (Osaka, Japan).

Forewing size measurements

The adult male and female forewing sizes were measured from the wing base to the apical point using a desktop digital microscope SKM-S30A-PC and its associated software SK measure (Saitoh Kougaku, Yokohama, Japan). The forewing was placed under a glass slide when necessary to make it flat. Both right and left forewing sizes were measured whenever possible, and the mean value was considered the final forewing size. In rare cases, only one forewing was intact. In that case, either right or left forewing size was considered the final forewing size.

Hemocytological examinations

Larvae were reared for hemocytological examinations in the same way as above. Immediately after prepupation when the larva stopped moving, a dorsal side of an abdominal segment was cut to a depth of 1–2 mm. Bleeding hemolymph samples (4 μL from each individual) were collected. When the sample volume was less than 4 μL, an additional incision was made at a dorsal side of a different abdominal segment. The 4-μL hemolymph sample was readily mixed with 16 μL of Turk’s stain solution (Nakalai Tesque, Kyoto, Japan) to stain nuclei of hemocytes in light purple for cell counting. The mixture was then injected into two poring sites of a disposable OneCell® counter (OneCell, Nagahama, Shiga, Japan). After waiting one or two minutes for cell settlement, the cell counter was set under a high-resolution Keyence VHX-1000 digital microscope (Osaka, Japan), and images for all 8 counting compartments per sample were obtained. The number of hemocytes were then counted in these images using the cell counter function of ImageJ81. Cells having a contact with borderlines of compartments were not counted, according to the manufacturer’s protocol.

Hemocytes were classified into 3 cell types (granulocyte, plasmatocyte, and prohemocyte) based on their cellular morphology and staining patterns, according to Wago and Kitano (1985)59. Plasmatocytes are relatively large (8–15 μm in diameter) and flat cells with variable shapes that have round cytoplasmic inclusions, lamellipodia, and filopodia. Granulocytes are medium-sized (5–10 μm in diameter) and round or oval cells that have round cytoplasmic inclusions (more than plasmatocytes) and filopodia but no lamellipodia. Prohemocytes are relatively small (4–6 μm in diameter) and round or oval cells that have no cytoplasmic inclusions and no filopodia. Their percentages among all hemocytes were obtained, and these 3 factors were examined for possible correlations with radioactivity concentrations of radiocesium (134Cs + 137Cs), radiopotassium (40K), and the summation of both radiocesium and radiopotassium (134Cs + 137Cs + 40K). Hemocyte percentages were also paired with 5 developmental factors and male and female forewing sizes for correlation analyses.

The individuals used for hemolymph collection were not used for the rearing experiment to obtain developmental factors and forewing sizes (Table S6). The hemocytological data from the individual who ate cabbage leaves of a given dose were associated with the developmental and forewing size data from the same locality group. For this reason, for the correlation analyses of the hemocytological data including those for the forewing size correlations, the group-averaged values (and not the individual-associated values) were used after normalisation. The normalisation was performed by dividing the original data of a given trial by the Okinawa data of that trial. This normalisation supposedly eliminates strain-specific variability among samples. Since there were 3 rearing trials, 3 points from the Okinawa groups were all located at 1.00 in the scatter plots.

Radioactivity measurements

A Canberra GCW-4023 germanium semiconductor device (Meriden, CT, USA) was used to measure radioactivity of the cultivated cabbage leaves. The cabbage leaves (either remnants that were fed larvae or leaves that were not fed larvae but from the same cabbage head as the fed leaves, excluding the hard leaf veins that would not be eaten by larvae) were completely air-dried via long-time confinement within a sealed container together with a desiccating agent.

Additional cabbage leaf samples were prepared to obtain the dry rate. To do this, samples were weighted before and after the drying procedures. The dry rate was 7.63%, which was used to calculate radioactivity concentrations in wet weight. Dried samples were grinded into small pieces and put into a columnar plastic vial before radioactivity measurements. Depending on the height of samples in a vial, the following counting efficiencies were employed to calculate radioactivity concentrations: For 137Cs, 19.9% (7–8 mm), 19.5% (10 mm), and 14.6% (35 mm); for 40K, 8.35% (7–8 mm), 7.93% (10 mm), and 5.73% (35 mm). From measured values on a particular measuring day, the radioactivity values on the very first day of feeding radioactive leaves were calculated, which were considered the radioactivity concentration of the fed cabbage. Branching ratios for 137Cs (662 keV) and 40K (1461 keV) used were 85.1% and 10.7%, respectively68. Radioactivity concentrations of 134Cs were calculated based on the 137Cs measurements, assuming that the ratio of 137Cs and 134Cs was 1:1 on March 15, 2011. In the case of the Okinawa samples, the 134Cs peak at 605 keV was not detected at all, and the 137Cs peak detected at very small levels were likely from nuclear fallout from nuclear bomb experiments in the 1950s. Thus, 134Cs and 137Cs were considered nonexistent in the Okinawa samples.

Radioactivity concentrations of 137Cs in soils from Okinawa used for cabbage cultivation were measured using the Canbarra GCW-4023 as above, and those from Fukushima were measured using a NaI (TI) scintillation detector FNF-401 (OHYO KOKEN KOGYO, Fussa, Tokyo, Japan). From measured values on a particular day, radioactivity concentrations of the day of the first planting of seedlings were calculated, and these were considered the radioactivity concentrations of soils.

Statistical analyses

The statistical software R, version 3.2.1 (The R foundation for Statistical Computing, Vienna, Austria), was used to perform Pearson correlation analyses, Steel tests, and Spearman correlation analyses. For the Steel test shown in Fig. 2a, a value of 1 (successfully achieving the adult stage) or 0 (died before achieving the adult stage) was assigned to each individual. Similarly, for the Steel test shown in Fig. 2b, a value of 1 (successfully becoming a normal adult) or 0 (becoming an adult with morphological abnormality or died before achieving the adult stage) was assigned to each individual. For the Steel test shown in Fig. 2e, unique scores were assigned to each individual (Table S3), as calculated based on their frequencies after categorizing into 3 groups: the wing group, the head group (proboscis and palpus), and the body group (trunk and valva). In addition, the normal individuals were also assigned a unique score based on the same calculation. For example, the score for the wing abnormalities (12.0) was obtained as the total number of individuals examined (383) divided by the number of abnormalities (32). The scores for the Okinawa, Ohara, Baba, and Iitate groups were obtained by the summation of scores of all the individuals that belonged to those groups. When two different abnormalities were found in an individual, the summation of both scores was assigned to that individual.

For Spearman correlation analyses, individual-associated values were used for the forewing sizes when they were paired with radioactivity concentrations. However, the group-associated values were used to pair with the hemocytological factors because they were obtained from different individuals. For developmental and hemocytological factors, the original data of a given trial were divided by the Okinawa data of that trial for normalisation (indicated as “ratio” in Fig. 3). To avoid type I errors in performing the correlation analyses, Holm-adjusted P-values were obtained, those with P < 0.05 were considered significant, and these significant cases were reported with scatter plots in this study. However, the original non-corrected P-values were reported to understand inherent characteristics for their correlations.