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Wiley Online Library : Morphological abnormalities in gall-forming aphids in a radiation-contaminated area near Fukushima Daiichi: selective impact of fallout?
Shin-ichi Akimoto*
Article first published online: 13 JAN 2014
DOI: 10.1002/ece3.949
© 2014 The Author. Ecology and Evolution published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Abstract
To evaluate the impact of fallout from the Fukushima Daiichi Nuclear Power Plant accident on organisms, this study compared the morphology and viability of gall-forming aphids between the Fukushima population and control populations from noncontaminated areas. This study, in particular, focused on the morphology of first-instar gall formers derived from the first sexual reproduction after the accident. Of 164 first instars from Tetraneura sorini galls collected 32 km from Fukushima Daiichi in spring 2012, 13.2% exhibited morphological abnormalities, including four conspicuously malformed individuals (2.4%). In contrast, in seven control areas, first instars with abnormal morphology accounted for 0.0–5.1% (on average, 3.8%). The proportions of abnormalities and mortality were significantly higher in Fukushima than in the control areas. Similarly, of 134 first instars from T. nigriabdominalis galls, 5.9% exhibited morphological abnormalities, with one highly malformed individual. However, of 543 second-generation larvae produced in T. sorini galls, only 0.37% had abnormalities, suggesting that abnormalities found in the first generation were not inherited by the next generation. Although investigation is limited to one study site, this result suggests that radioactive contamination had deleterious effects on embryogenesis in eggs deposited on the bark surface, but a negligible influence on the second generation produced in closed galls. Furthermore, analysis of both species samples collected in spring 2013 indicated that the viability and healthiness of the aphids were significantly improved compared to those in the 2012 samples. Thus, the results of this study suggest the possibility that a reduced level of radiation and/or selection for radiation tolerance may have led to the improved viability and healthiness of the Fukushima population.
Introduction
There is increasing interest in the effects of radionuclides released from nuclear power plant accidents on the biodiversity and genetics of wildlife (Møller and Mousseau 2006; Yablokov et al. 2009; Mousseau and Møller 2012a; Møller et al. 2013). Even 20 years after the 1986 Chernobyl nuclear disaster, bird and arthropod species abundances are negatively correlated with the level of radioactivity at ground level (Møller and Mousseau 2007, 2009). It is reasonable to suppose that the largest impact on biodiversity occurs immediately after an accident through the large amount of fallout. Alternatively, it is also possible that mutations accumulate over time and express deleterious effects several years after the accident in contaminated areas (Møller and Mousseau 2006). At present, we have limited knowledge about the effects of radiation on the wildlife. In the case of the Chernobyl nuclear disaster, the initial impacts on the abundance, phenotypes, and genetics of animal and plant species were not fully assessed (Yablokov et al. 2009; Møller et al. 2013). The Fukushima Daiichi Nuclear Power Plant exploded in mid-March 2011 and released 11,347 PBq of radionuclides, including 160 PBq of iodine-131, 15 PBq of cesium-137, and 0.14 PBq of strontium-90 into the atmosphere (Nuclear Emergency Response Headquarters, Government of Japan. 2011). An area northwest of the power plant was covered with fallout and highly contaminated by radioactive substances (Nuclear Regulation Authority, 2011a). For the Fukushima Daiichi accident, a vast area of contamination, including several landscapes, can be surveyed without legal restrictions, except within a 20-km radius of the power plant, and thus, there are opportunities to detect initial impacts of radionuclides on individual species and the ecosystem. Therefore, ecological and genetic investigations in the Fukushima area are an urgent issue.
Several researchers and governmental agencies have started evaluating the levels of radionuclides accumulated in wild animals and plants in the Fukushima area (e.g., Nakanishi and Tanoi 2013). Ishida (2013) started a long-term survey on the abundance of birds and mammals and confirmed a high level of radioactive contamination in bird feathers in the first year. Møller et al. (2012, 2013) conducted field censuses of birds and several insects and reported a significant negative correlation between densities and background radiation level. Hiyama et al. (2012) reported that the lycaenid butterfly Zizeeria maha that emerged in 2011 in contaminated areas exhibited morphological abnormalities and that these abnormalities were inherited by the next generation, which exhibited a higher proportion of abnormalities than the first generation. Hiyama et al.'s report is the first example of phenotypic abnormalities in the Fukushima area, so it is difficult to understand whether the occurrence of phenotypic abnormalities is common to the populations of other animals and plants in the Fukushima area or not. Thus, phenotypic and genetic studies of other systems might be encouraged.
In attempts to clarify the effects of radiation on mammals, birds, and arthropods, phenotypic abnormalities have been evaluated based on adults (Møller 1993, 2002; Møller and Mousseau 2006; Møller et al. 2007; Yablokov et al. 2009) except in a few studies (Williams et al. 2001). However, the effects of radiation emerge most strongly in hatchlings or newborns, resulting in mortality and phenotypic abnormalities, as shown in irradiation experiments (Elbadry 1965; Tilton et al. 1966; McGregor and Newcombe 1968; Vereecke and Pelerents 1969; Matranga et al. 2010) as well as field studies in Chernobyl (Krivolutzkii and Pokarzhevskii 1992; Krivolutsky 1995). When exposed to high radiation, hatchlings or newborns often fail to grow, with few individuals reaching adulthood. Therefore, it is possible to overlook the emergence of abnormalities if the research focus is limited to adult phenotypes only (Møller 1997). The present study focuses on gall-forming aphids and reports the extent to which morphological abnormalities emerge in first-instar aphids derived from the first sexual reproduction after the Fukushima Daiichi nuclear accident.
Aphids are tiny, soft-bodied insects that reproduce asexually from spring to autumn, with many consecutive generations (Dixon 1998). The use of aphids for a bioassay of radiation appears to have three merits. First, aphids always maintain high reproduction rates, with numerous developing embryos in their abdomens (Dixon 1998). Reports indicate that rapidly developing embryos are readily affected by radiation (Russell and Russell 1952; Vereecke and Pelerents 1969; Cerutti 1974). After the fallout that occurred in mid-March 2011, aphids continued to reproduce asexually under radioactive contamination until autumn, so that aphid embryos likely were damaged by radiation. Genetic damages, if any, would be inherited and accumulated through clonal lines. Through sexual reproduction in autumn 2011, damaged genes may have been genetically recombined to the next generation, leading to a release of a large phenotypic variation (Lynch and Gabriel 1983). Second, as hemimetabolous insects, aphid first instars possess several metrical characters, which would facilitate the detection of morphological abnormalities. So far, aphid first instars have been reported to exhibit phenological and morphological traits that well reflect genetic and environmental variation (Akimoto 1988, 1990, 1998, 2006; Komatsu and Akimoto 1995; Akimoto and Yamaguchi 1997; Komazaki 1998). Third, in gall-forming aphids, first-instar larvae induce leaf galls, in which they develop and produce offspring (Blackman and Eastop 1994). Thus, using aphid species that induce closed galls, the performance and mortality of gall formers can be evaluated without the disturbance of predators (Whitham 1978; Akimoto and Yamaguchi 1994; Wool 2004; Aoyama et al. 2012). If gall formers die in the gall, we can examine the morphology, and if they show some deformation, we can exclude the possibility of predation as a cause (Ballengée and Sessions 2009; Bowerman et al. 2010). The presence of malformed first instars in gall-forming aphids has been reported previously (Akimoto 1985a).
In the present study, I collected closed galls of Tetraneura aphid species from leaves of Ulmus davidiana var. japonica at a point 32 km from the Fukushima Daiichi Nuclear Power Plant and inspected the morphology of the gall formers at the first stadium. I report frequent occurrences of morphological abnormalities and mortalities in two gall-forming aphids in the Fukushima area by comparing them with other populations in noncontaminated areas. Although investigation is limited to one site in Fukushima, the results from two species corroborate the possibility of an initial, detrimental impact of fallout.
Materials and Methods
Life cycle of Tetraneura aphids
Tetraneura species are host-alternating and associated with two kinds of plants: elm trees Ulmus spp. as the primary host and gramineous plants as the secondary host (Blackman and Eastop 1994; Wool 2004). In spring, after hatching from overwintered eggs on the trunks of elm trees, first instars move to developing new leaves and start gall formation. The first instar (gall former) settles on the underside of a new leaf and, using its stylet, stimulates one point of the leaf. The stimulated plant tissues rapidly proliferate upward, encasing the gall former inside, to form a closed, hollow gall. During gall formation, the gall formers continuously stimulate the tissues without moving. The gall former develops inside the gall and parthenogenetically produces second-generation larvae (Fig. S1). The second generations develop into winged adults, which emerge from the cracked galls in mid-June and migrate to gramineous plants to produce third-generation larvae. These larvae move to the roots, where they develop into wingless adults. On the roots, several generations of wingless adults repeat reproduction from early summer until autumn, when winged adults emerge and return to elm trees. Winged adults produce wingless males and sexual females on the trunks of elm trees. After copulation, the sexual female oviposits a single egg in a crevice of the bark. Eggs overwinter until the next spring.
Gall collection in Fukushima
I collected a total of 284 galls of Tetraneura aphid species from 1 U. davidiana var. japonica tree at Kawamata Town, Fukushima Prefecture (37o 35′ N, 140 o 42′ E) on 3 June 2012. Leaves with galls were haphazardly collected from the tree, and all galls on the collected leaves were preserved in 80% ethanol. Galls of Tetraneura species are bean or horn shaped, a maximum of 25 mm in height, and formed in various positions on a leaf. On one leaf, 10 or more galls of two or more species are sometimes formed. The sample aphids were collected on roadsides that are not included in special reservation areas, so that no specific permissions were required for the collections. The aphid species collected are not designated as endangered or protected species.
After fixation, all galls were dissected under a binocular microscope. When the gall former gave birth to larvae, the adult and the cast-off skin of the first-instar gall former were collected from the gall and mounted on a glass slide. In the Eriosomatinae, the cast-off skin of the first-instar gall former is strongly sclerotized and characterized by species-specific features (Aoki 1975; Akimoto 1983, 1985b). The second-generation larvae produced in a gall were preserved in a vial of 80% ethanol, and all the larvae were later inspected for morphological abnormality. When the gall former was immature, only the cast-off skin of the first instar was mounted on a glass slide. When the gall former died in the gall for some reason, it was mounted for morphological examination.
Before mounting, first-instars' cast-off skins and adult gall formers were kept in 10% KOH for 1 or 2 days. The exoskeleton of the adult or first instar was rinsed in 80% ethanol with a small amount of acetic acid and dehydrated in carboxylol (phenol:xylol = 4:1) for 15 min and then in xylol for 10 min. The dehydrated samples were mounted on a glass slide with Canada balsam. All of the mounted specimens are preserved in the Laboratory of Systematic Entomology, Graduate School of Agriculture, Hokkaido University. No permits were required for the described study, which complied with all relevant regulations.
Gall collection in other areas
Prior to the Fukushima Daiichi nuclear accident, no specimens had been collected in or around the Fukushima area. However, numerous specimens of Tetraneura species were collected before the accident in several localities of Japan and are preserved in the Laboratory of Systematic Entomology, Graduate School of Agriculture, Hokkaido University (Table 1). These specimens were used for comparison with the Fukushima specimens. In 2012, I collected Tetraneura aphids in Kashiwa, Chiba Prefecture (35°53′N, 139°56′E), Sapporo, Hokkaido (43°4′N, 141°20′E), and Iwamizawa, Hokkaido (43°11′N, 141°46′E), as control specimens. The sample aphids were collected on public parks and a university campus that are not included in special reservation areas, so that no specific permissions were required for the collections.
Gall collection, the examination of the gall contents, and the preparation of slide-mounted specimens were conducted as in the Fukushima samples. Where first-instar larvae were sampled, I haphazardly collected developing host buds infested with the first instars and preserved them in vials of 80% ethanol. Later, first instars were removed from the buds and mounted on glass slides.
Morphological abnormalities and mortality
All mounted specimens of two common species, T. sorini and T. nigriabdominalis, were examined under a microscope (Axiophoto, Carl Zeiss, Oberkochen, Germany) for the morphological abnormalities and mortality. Because first-instar gall formers from all the samples were available for the microscopic inspection, the present study focused specifically on comparisons of first-instar morphology; 167 T. sorini first instars and 136 T. nigriabdominalis first instars from the Fukushima area were compared with 1559 T. sorini first instars from seven control areas and 1677 T. nigriabdominalis first instars from six control areas, respectively. For all the mounted specimens, including dead individuals, I inspected the morphology of legs, antennae, rostra, and tergites. The first instars of T. sorini and T. nigriabdominalis were approximately 0.90 ± 0.099 (SD) mm and 0.72 ± 0.028 (SD) mm in body length, respectively.
Several morphological abnormalities, ranging from slight variants to serious deformations, were found among first-instar gall formers. The abnormalities were classified into three categories, and the proportion of each category was calculated for samples in each area. The levels of abnormalities were determined irrespective of mortality; that is, dead first instars may be considered normal morphologically, while first instars in any of three abnormality categories may molt to the next larval stadium. Categorization of abnormal first instars was conducted for demonstrating the peculiarity of the Fukushima population, but in statistical tests, first instars with any abnormal morphology were pooled. Mortality was assessed for each gall former at the time galls were collected.
Comparison of growth performance
To examine whether growth performance in the Fukushima samples was reduced or not, the growth rate of gall formers was compared between the Fukushima and Iwamizawa samples collected in early June 2012. Based on the cast-off skin of the first instar and the adult collected from each gall, I measured the lengths of larval and adult hind femurs of the same individual using an eyepiece micrometer installed on the microscope. The regression line of adult femur length on larval femur length demonstrates the mode of growth in each population. A steeper slope of the regression line indicates higher growth performance. Thus, ANCOVA (Sokal and Rohlf 1995) was applied to detect the difference in the regression slope or the intercept between the two populations. Galls from Fukushima were collected from one host tree; galls from Iwamizawa were collected from three host trees and pooled in the analysis.
Comparison between the 2012 and 2013 samples from Fukushima
To evaluate temporal changes in the viability and healthiness of the Fukushima population, Tetraneura galls were again collected from the same elm tree from which the 2012 sample was collected, on 21 May 2013. For the 2013 samples, 50 and 347 gall formers of T. sorini and T. nigriabdominalis, respectively, were mounted on glass slides. The proportions of morphological abnormality and mortality were evaluated based on the gall formers at the first to the last stadium. By sampling galls from the same tree in both years, it is possible to remove the effects of plant factors on the morphology and viability of gall formers.
Logistic regression was used to test whether the proportions of morphological abnormality and mortality changed or not between the 2 years. In the model, the proportions of healthy, abnormal, and dead gall formers were treated as the dependent variable, while species (T. sorini or T. nigriabdominalis) and years (2012 or 2013) were specified as independent variables. The interaction between years and species was also included in the model.
Measurements of radioactivity
Radioactivity levels were measured at the time of gall collection using Geiger counters (SOEKS-01M [SOEKS, Russia] and PA-1000 Radi [Horiba, Kyoto]). Measurement was conducted at a height of 1 m and at the ground level near the tree from which galls were collected. For the other areas in 2012 (Kashiwa, Sapporo, and Iwamizawa), I used the published data from the document “Monitoring information of environmental radioactivity level” by the Nuclear Regulation Authority, Government of Japan (http://radioactivity.nsr.go.jp/map/ja/).
Results
Categorization of abnormalities
From the 284 galls collected in Fukushima, four species of gall-forming Tetraneura were found, including 162 T. sorini, 118 T. nigriabdominalis, 3 T. radicicola, and 1 undescribed species. Galls occasionally contained two or more gall formers inside (of the T. sorini and T. nigriabdominalis galls, 3.7% and 9.3%, respectively). From the T. sorini galls, 167 gall formers were found, and 76 (71.7% of gall formers that attained at least the second instar) gave birth to larvae. From the T. nigriabdominalis galls, 136 gall formers were found, and only 1 (2.2%) gave birth to larvae.
I detected several morphological abnormalities in the first-instar gall formers of the two species both from Fukushima and from the other areas. Slight abnormalities (level 1) included the atrophy or bending of 1 leg (Fig. S2A,B), small ganglia on the ventral surface (Fig. S2C), partial fusion of adjacent abdominal tergites (Fig. S2D), and tissue necrosis in 1 leg or antenna (Fig. S3A,B). Tissue necrosis that occurred inside an appendage resulted in the partial or complete loss of that appendage after molting. Level 2 abnormalities included the atrophy or bending of 2 legs and tissue necrosis in two or more appendages. I categorized the complete or partial loss of one appendage of first instars as level 2 (Fig. S3C,D). Such first instars appear to have already lost an appendage at their hatching. Intense level 3 abnormalities included the loss of two or more appendages, the loss of 1 leg and atrophy of another leg, the appearance of new features, and conspicuous asymmetry in bilateral characters. These abnormalities were common in the two species.
At the time of gall collection in Fukushima on 3 June 2012, the radiation dose was 4.0 μSv/h at 1 m and 6.0 μSv/h at ground level. In contrast, the radiation doses in Kashiwa, Sapporo, and Iwamizawa were 0.117, 0.038, and 0.040 μSv/h, respectively.
Abnormalities and mortality in T. sorini
Of the 167 T. sorini first instars collected in Fukushima, 13.2% exhibited any of the abnormalities (Fig. 1). Of the seven control areas, the samples from Ukiha, Fukuoka Prefecture, exhibited no abnormalities. Of the samples from Sapporo in 1990, 5.1% exhibited level-1 abnormalities, and this value was the maximum found in the control areas (Fig. 1). The incidence of morphological abnormalities in Fukushima was significantly higher than that in other areas (Table 2; on average, 3.8% for other areas). The Fukushima samples were peculiar because they contained four level-3 malformed individuals (Fig. 1). One of the individuals had a bifurcated abdomen with 2 caudae (Fig. 2). Despite this intense malformation, this individual molted successfully four times and attained adulthood with two caudae. Although larviposition was not confirmed, this individual contained mature embryos in its abdomen. The second example (Fig. 3A) was found dead in an incipient gall. This individual had an empty, distended abdomen and a projection on the joint of the mid-femur and tibia; this projection bore several setae, suggesting that the projection was a homologous leg structure. The third example (Fig. 3B) was also found dead in a gall and bore a large protuberance on the abdomen. In addition, it had another solid protuberance at the base of the mid-femur. The last example (Fig. 4A), which was also found dead in a gall, had 1 hind leg with apical segments missing due to necrosis and another hind leg that was atrophied.
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onlinelibrary.wiley.com/doi/10.1002/ece3.949/full
Shin-ichi Akimoto*
Article first published online: 13 JAN 2014
DOI: 10.1002/ece3.949
© 2014 The Author. Ecology and Evolution published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Abstract
To evaluate the impact of fallout from the Fukushima Daiichi Nuclear Power Plant accident on organisms, this study compared the morphology and viability of gall-forming aphids between the Fukushima population and control populations from noncontaminated areas. This study, in particular, focused on the morphology of first-instar gall formers derived from the first sexual reproduction after the accident. Of 164 first instars from Tetraneura sorini galls collected 32 km from Fukushima Daiichi in spring 2012, 13.2% exhibited morphological abnormalities, including four conspicuously malformed individuals (2.4%). In contrast, in seven control areas, first instars with abnormal morphology accounted for 0.0–5.1% (on average, 3.8%). The proportions of abnormalities and mortality were significantly higher in Fukushima than in the control areas. Similarly, of 134 first instars from T. nigriabdominalis galls, 5.9% exhibited morphological abnormalities, with one highly malformed individual. However, of 543 second-generation larvae produced in T. sorini galls, only 0.37% had abnormalities, suggesting that abnormalities found in the first generation were not inherited by the next generation. Although investigation is limited to one study site, this result suggests that radioactive contamination had deleterious effects on embryogenesis in eggs deposited on the bark surface, but a negligible influence on the second generation produced in closed galls. Furthermore, analysis of both species samples collected in spring 2013 indicated that the viability and healthiness of the aphids were significantly improved compared to those in the 2012 samples. Thus, the results of this study suggest the possibility that a reduced level of radiation and/or selection for radiation tolerance may have led to the improved viability and healthiness of the Fukushima population.
Introduction
There is increasing interest in the effects of radionuclides released from nuclear power plant accidents on the biodiversity and genetics of wildlife (Møller and Mousseau 2006; Yablokov et al. 2009; Mousseau and Møller 2012a; Møller et al. 2013). Even 20 years after the 1986 Chernobyl nuclear disaster, bird and arthropod species abundances are negatively correlated with the level of radioactivity at ground level (Møller and Mousseau 2007, 2009). It is reasonable to suppose that the largest impact on biodiversity occurs immediately after an accident through the large amount of fallout. Alternatively, it is also possible that mutations accumulate over time and express deleterious effects several years after the accident in contaminated areas (Møller and Mousseau 2006). At present, we have limited knowledge about the effects of radiation on the wildlife. In the case of the Chernobyl nuclear disaster, the initial impacts on the abundance, phenotypes, and genetics of animal and plant species were not fully assessed (Yablokov et al. 2009; Møller et al. 2013). The Fukushima Daiichi Nuclear Power Plant exploded in mid-March 2011 and released 11,347 PBq of radionuclides, including 160 PBq of iodine-131, 15 PBq of cesium-137, and 0.14 PBq of strontium-90 into the atmosphere (Nuclear Emergency Response Headquarters, Government of Japan. 2011). An area northwest of the power plant was covered with fallout and highly contaminated by radioactive substances (Nuclear Regulation Authority, 2011a). For the Fukushima Daiichi accident, a vast area of contamination, including several landscapes, can be surveyed without legal restrictions, except within a 20-km radius of the power plant, and thus, there are opportunities to detect initial impacts of radionuclides on individual species and the ecosystem. Therefore, ecological and genetic investigations in the Fukushima area are an urgent issue.
Several researchers and governmental agencies have started evaluating the levels of radionuclides accumulated in wild animals and plants in the Fukushima area (e.g., Nakanishi and Tanoi 2013). Ishida (2013) started a long-term survey on the abundance of birds and mammals and confirmed a high level of radioactive contamination in bird feathers in the first year. Møller et al. (2012, 2013) conducted field censuses of birds and several insects and reported a significant negative correlation between densities and background radiation level. Hiyama et al. (2012) reported that the lycaenid butterfly Zizeeria maha that emerged in 2011 in contaminated areas exhibited morphological abnormalities and that these abnormalities were inherited by the next generation, which exhibited a higher proportion of abnormalities than the first generation. Hiyama et al.'s report is the first example of phenotypic abnormalities in the Fukushima area, so it is difficult to understand whether the occurrence of phenotypic abnormalities is common to the populations of other animals and plants in the Fukushima area or not. Thus, phenotypic and genetic studies of other systems might be encouraged.
In attempts to clarify the effects of radiation on mammals, birds, and arthropods, phenotypic abnormalities have been evaluated based on adults (Møller 1993, 2002; Møller and Mousseau 2006; Møller et al. 2007; Yablokov et al. 2009) except in a few studies (Williams et al. 2001). However, the effects of radiation emerge most strongly in hatchlings or newborns, resulting in mortality and phenotypic abnormalities, as shown in irradiation experiments (Elbadry 1965; Tilton et al. 1966; McGregor and Newcombe 1968; Vereecke and Pelerents 1969; Matranga et al. 2010) as well as field studies in Chernobyl (Krivolutzkii and Pokarzhevskii 1992; Krivolutsky 1995). When exposed to high radiation, hatchlings or newborns often fail to grow, with few individuals reaching adulthood. Therefore, it is possible to overlook the emergence of abnormalities if the research focus is limited to adult phenotypes only (Møller 1997). The present study focuses on gall-forming aphids and reports the extent to which morphological abnormalities emerge in first-instar aphids derived from the first sexual reproduction after the Fukushima Daiichi nuclear accident.
Aphids are tiny, soft-bodied insects that reproduce asexually from spring to autumn, with many consecutive generations (Dixon 1998). The use of aphids for a bioassay of radiation appears to have three merits. First, aphids always maintain high reproduction rates, with numerous developing embryos in their abdomens (Dixon 1998). Reports indicate that rapidly developing embryos are readily affected by radiation (Russell and Russell 1952; Vereecke and Pelerents 1969; Cerutti 1974). After the fallout that occurred in mid-March 2011, aphids continued to reproduce asexually under radioactive contamination until autumn, so that aphid embryos likely were damaged by radiation. Genetic damages, if any, would be inherited and accumulated through clonal lines. Through sexual reproduction in autumn 2011, damaged genes may have been genetically recombined to the next generation, leading to a release of a large phenotypic variation (Lynch and Gabriel 1983). Second, as hemimetabolous insects, aphid first instars possess several metrical characters, which would facilitate the detection of morphological abnormalities. So far, aphid first instars have been reported to exhibit phenological and morphological traits that well reflect genetic and environmental variation (Akimoto 1988, 1990, 1998, 2006; Komatsu and Akimoto 1995; Akimoto and Yamaguchi 1997; Komazaki 1998). Third, in gall-forming aphids, first-instar larvae induce leaf galls, in which they develop and produce offspring (Blackman and Eastop 1994). Thus, using aphid species that induce closed galls, the performance and mortality of gall formers can be evaluated without the disturbance of predators (Whitham 1978; Akimoto and Yamaguchi 1994; Wool 2004; Aoyama et al. 2012). If gall formers die in the gall, we can examine the morphology, and if they show some deformation, we can exclude the possibility of predation as a cause (Ballengée and Sessions 2009; Bowerman et al. 2010). The presence of malformed first instars in gall-forming aphids has been reported previously (Akimoto 1985a).
In the present study, I collected closed galls of Tetraneura aphid species from leaves of Ulmus davidiana var. japonica at a point 32 km from the Fukushima Daiichi Nuclear Power Plant and inspected the morphology of the gall formers at the first stadium. I report frequent occurrences of morphological abnormalities and mortalities in two gall-forming aphids in the Fukushima area by comparing them with other populations in noncontaminated areas. Although investigation is limited to one site in Fukushima, the results from two species corroborate the possibility of an initial, detrimental impact of fallout.
Materials and Methods
Life cycle of Tetraneura aphids
Tetraneura species are host-alternating and associated with two kinds of plants: elm trees Ulmus spp. as the primary host and gramineous plants as the secondary host (Blackman and Eastop 1994; Wool 2004). In spring, after hatching from overwintered eggs on the trunks of elm trees, first instars move to developing new leaves and start gall formation. The first instar (gall former) settles on the underside of a new leaf and, using its stylet, stimulates one point of the leaf. The stimulated plant tissues rapidly proliferate upward, encasing the gall former inside, to form a closed, hollow gall. During gall formation, the gall formers continuously stimulate the tissues without moving. The gall former develops inside the gall and parthenogenetically produces second-generation larvae (Fig. S1). The second generations develop into winged adults, which emerge from the cracked galls in mid-June and migrate to gramineous plants to produce third-generation larvae. These larvae move to the roots, where they develop into wingless adults. On the roots, several generations of wingless adults repeat reproduction from early summer until autumn, when winged adults emerge and return to elm trees. Winged adults produce wingless males and sexual females on the trunks of elm trees. After copulation, the sexual female oviposits a single egg in a crevice of the bark. Eggs overwinter until the next spring.
Gall collection in Fukushima
I collected a total of 284 galls of Tetraneura aphid species from 1 U. davidiana var. japonica tree at Kawamata Town, Fukushima Prefecture (37o 35′ N, 140 o 42′ E) on 3 June 2012. Leaves with galls were haphazardly collected from the tree, and all galls on the collected leaves were preserved in 80% ethanol. Galls of Tetraneura species are bean or horn shaped, a maximum of 25 mm in height, and formed in various positions on a leaf. On one leaf, 10 or more galls of two or more species are sometimes formed. The sample aphids were collected on roadsides that are not included in special reservation areas, so that no specific permissions were required for the collections. The aphid species collected are not designated as endangered or protected species.
After fixation, all galls were dissected under a binocular microscope. When the gall former gave birth to larvae, the adult and the cast-off skin of the first-instar gall former were collected from the gall and mounted on a glass slide. In the Eriosomatinae, the cast-off skin of the first-instar gall former is strongly sclerotized and characterized by species-specific features (Aoki 1975; Akimoto 1983, 1985b). The second-generation larvae produced in a gall were preserved in a vial of 80% ethanol, and all the larvae were later inspected for morphological abnormality. When the gall former was immature, only the cast-off skin of the first instar was mounted on a glass slide. When the gall former died in the gall for some reason, it was mounted for morphological examination.
Before mounting, first-instars' cast-off skins and adult gall formers were kept in 10% KOH for 1 or 2 days. The exoskeleton of the adult or first instar was rinsed in 80% ethanol with a small amount of acetic acid and dehydrated in carboxylol (phenol:xylol = 4:1) for 15 min and then in xylol for 10 min. The dehydrated samples were mounted on a glass slide with Canada balsam. All of the mounted specimens are preserved in the Laboratory of Systematic Entomology, Graduate School of Agriculture, Hokkaido University. No permits were required for the described study, which complied with all relevant regulations.
Gall collection in other areas
Prior to the Fukushima Daiichi nuclear accident, no specimens had been collected in or around the Fukushima area. However, numerous specimens of Tetraneura species were collected before the accident in several localities of Japan and are preserved in the Laboratory of Systematic Entomology, Graduate School of Agriculture, Hokkaido University (Table 1). These specimens were used for comparison with the Fukushima specimens. In 2012, I collected Tetraneura aphids in Kashiwa, Chiba Prefecture (35°53′N, 139°56′E), Sapporo, Hokkaido (43°4′N, 141°20′E), and Iwamizawa, Hokkaido (43°11′N, 141°46′E), as control specimens. The sample aphids were collected on public parks and a university campus that are not included in special reservation areas, so that no specific permissions were required for the collections.
Gall collection, the examination of the gall contents, and the preparation of slide-mounted specimens were conducted as in the Fukushima samples. Where first-instar larvae were sampled, I haphazardly collected developing host buds infested with the first instars and preserved them in vials of 80% ethanol. Later, first instars were removed from the buds and mounted on glass slides.
Morphological abnormalities and mortality
All mounted specimens of two common species, T. sorini and T. nigriabdominalis, were examined under a microscope (Axiophoto, Carl Zeiss, Oberkochen, Germany) for the morphological abnormalities and mortality. Because first-instar gall formers from all the samples were available for the microscopic inspection, the present study focused specifically on comparisons of first-instar morphology; 167 T. sorini first instars and 136 T. nigriabdominalis first instars from the Fukushima area were compared with 1559 T. sorini first instars from seven control areas and 1677 T. nigriabdominalis first instars from six control areas, respectively. For all the mounted specimens, including dead individuals, I inspected the morphology of legs, antennae, rostra, and tergites. The first instars of T. sorini and T. nigriabdominalis were approximately 0.90 ± 0.099 (SD) mm and 0.72 ± 0.028 (SD) mm in body length, respectively.
Several morphological abnormalities, ranging from slight variants to serious deformations, were found among first-instar gall formers. The abnormalities were classified into three categories, and the proportion of each category was calculated for samples in each area. The levels of abnormalities were determined irrespective of mortality; that is, dead first instars may be considered normal morphologically, while first instars in any of three abnormality categories may molt to the next larval stadium. Categorization of abnormal first instars was conducted for demonstrating the peculiarity of the Fukushima population, but in statistical tests, first instars with any abnormal morphology were pooled. Mortality was assessed for each gall former at the time galls were collected.
Comparison of growth performance
To examine whether growth performance in the Fukushima samples was reduced or not, the growth rate of gall formers was compared between the Fukushima and Iwamizawa samples collected in early June 2012. Based on the cast-off skin of the first instar and the adult collected from each gall, I measured the lengths of larval and adult hind femurs of the same individual using an eyepiece micrometer installed on the microscope. The regression line of adult femur length on larval femur length demonstrates the mode of growth in each population. A steeper slope of the regression line indicates higher growth performance. Thus, ANCOVA (Sokal and Rohlf 1995) was applied to detect the difference in the regression slope or the intercept between the two populations. Galls from Fukushima were collected from one host tree; galls from Iwamizawa were collected from three host trees and pooled in the analysis.
Comparison between the 2012 and 2013 samples from Fukushima
To evaluate temporal changes in the viability and healthiness of the Fukushima population, Tetraneura galls were again collected from the same elm tree from which the 2012 sample was collected, on 21 May 2013. For the 2013 samples, 50 and 347 gall formers of T. sorini and T. nigriabdominalis, respectively, were mounted on glass slides. The proportions of morphological abnormality and mortality were evaluated based on the gall formers at the first to the last stadium. By sampling galls from the same tree in both years, it is possible to remove the effects of plant factors on the morphology and viability of gall formers.
Logistic regression was used to test whether the proportions of morphological abnormality and mortality changed or not between the 2 years. In the model, the proportions of healthy, abnormal, and dead gall formers were treated as the dependent variable, while species (T. sorini or T. nigriabdominalis) and years (2012 or 2013) were specified as independent variables. The interaction between years and species was also included in the model.
Measurements of radioactivity
Radioactivity levels were measured at the time of gall collection using Geiger counters (SOEKS-01M [SOEKS, Russia] and PA-1000 Radi [Horiba, Kyoto]). Measurement was conducted at a height of 1 m and at the ground level near the tree from which galls were collected. For the other areas in 2012 (Kashiwa, Sapporo, and Iwamizawa), I used the published data from the document “Monitoring information of environmental radioactivity level” by the Nuclear Regulation Authority, Government of Japan (http://radioactivity.nsr.go.jp/map/ja/).
Results
Categorization of abnormalities
From the 284 galls collected in Fukushima, four species of gall-forming Tetraneura were found, including 162 T. sorini, 118 T. nigriabdominalis, 3 T. radicicola, and 1 undescribed species. Galls occasionally contained two or more gall formers inside (of the T. sorini and T. nigriabdominalis galls, 3.7% and 9.3%, respectively). From the T. sorini galls, 167 gall formers were found, and 76 (71.7% of gall formers that attained at least the second instar) gave birth to larvae. From the T. nigriabdominalis galls, 136 gall formers were found, and only 1 (2.2%) gave birth to larvae.
I detected several morphological abnormalities in the first-instar gall formers of the two species both from Fukushima and from the other areas. Slight abnormalities (level 1) included the atrophy or bending of 1 leg (Fig. S2A,B), small ganglia on the ventral surface (Fig. S2C), partial fusion of adjacent abdominal tergites (Fig. S2D), and tissue necrosis in 1 leg or antenna (Fig. S3A,B). Tissue necrosis that occurred inside an appendage resulted in the partial or complete loss of that appendage after molting. Level 2 abnormalities included the atrophy or bending of 2 legs and tissue necrosis in two or more appendages. I categorized the complete or partial loss of one appendage of first instars as level 2 (Fig. S3C,D). Such first instars appear to have already lost an appendage at their hatching. Intense level 3 abnormalities included the loss of two or more appendages, the loss of 1 leg and atrophy of another leg, the appearance of new features, and conspicuous asymmetry in bilateral characters. These abnormalities were common in the two species.
At the time of gall collection in Fukushima on 3 June 2012, the radiation dose was 4.0 μSv/h at 1 m and 6.0 μSv/h at ground level. In contrast, the radiation doses in Kashiwa, Sapporo, and Iwamizawa were 0.117, 0.038, and 0.040 μSv/h, respectively.
Abnormalities and mortality in T. sorini
Of the 167 T. sorini first instars collected in Fukushima, 13.2% exhibited any of the abnormalities (Fig. 1). Of the seven control areas, the samples from Ukiha, Fukuoka Prefecture, exhibited no abnormalities. Of the samples from Sapporo in 1990, 5.1% exhibited level-1 abnormalities, and this value was the maximum found in the control areas (Fig. 1). The incidence of morphological abnormalities in Fukushima was significantly higher than that in other areas (Table 2; on average, 3.8% for other areas). The Fukushima samples were peculiar because they contained four level-3 malformed individuals (Fig. 1). One of the individuals had a bifurcated abdomen with 2 caudae (Fig. 2). Despite this intense malformation, this individual molted successfully four times and attained adulthood with two caudae. Although larviposition was not confirmed, this individual contained mature embryos in its abdomen. The second example (Fig. 3A) was found dead in an incipient gall. This individual had an empty, distended abdomen and a projection on the joint of the mid-femur and tibia; this projection bore several setae, suggesting that the projection was a homologous leg structure. The third example (Fig. 3B) was also found dead in a gall and bore a large protuberance on the abdomen. In addition, it had another solid protuberance at the base of the mid-femur. The last example (Fig. 4A), which was also found dead in a gall, had 1 hind leg with apical segments missing due to necrosis and another hind leg that was atrophied.
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onlinelibrary.wiley.com/doi/10.1002/ece3.949/full
so whats happening in the Solomons, is haarp missing its mark?