DOI: 10.13102/sociobiology.v62i1.116-119Sociobiology 62(1): 116-119 (March, 2015) Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Clonal composition of colonies of a eusocial aphid, Ceratovacuna japonica Introduction Altruistic behavior, in which sterile individuals benefit their kin individuals, has evolved in eusocial insects of some taxonomic groups (Trivers & Hare, 1976; Hölldobler & Wilson, 1990; Pike & Foster, 2008). Eusocial aphids produce sterile individuals (so-called soldiers) that protect their colony mates from predators (Stern & Foster, 1996; Pike & Foster, 2008). Soldiers have morphological, behavioral and physiological traits that have specialized functions against predators (Kutsukake et al., 2004; Hattori & Itino, 2008; Hattori et al., 2013a, b). For example, soldiers of the eusocial aphid Ceratovacuna japonica (Homoptera, Hormaphidinae) have longer horns and forelegs than non-soldiers (Hattori & Itino, 2008). When they encounter a predator, they grasp it with their forelegs and then stick their frontal horns into the body of the predator to kill it or to delay its predation (Hattori et al., 2013a). Kin selection theory predicts that such a reproductive division of labor is maintained by high genetic relatedness within a colony (Hamilton, 1964). In accordance with this Abstract High degrees of relatedness among colony mates and kin recognition ability are important factors maintaining eusociality because kin-selection favors eusociality when donor and recipient of altruistic acts are related to each other. However, in eusocial aphids, clone mixing between different colonies occurs frequently, suggesting a lack of kin recognition. Studies investigating the clonal composition of eusocial aphid colonies have focused on the aphid generation on the primary host plant (gall generation) and showed that clone mixing occurs frequently among galls (= nest). To test whether clone mixing also occurs in open colonies of eusocial aphids on their secondary host plants (open- colony generation), we carried out an amplified fragment length polymorphism analysis to investigate clonal composition within colonies of the eusocial aphid Ceratovacuna japonica. The results showed that clone mixing occurred frequently in open-colony generation. They suggest that not only relatedness but also other factors (e.g., ecological background) are important for maintaining eusociality in eusocial aphids. Sociobiology An international journal on social insects Shinshu University, Matsumoto, Nagano, Japan Article History Edited by Kleber Del-Claro, UNB, Brazil, Brazil Received 26 May 2014 Initial acceptance 30 June 2014 Final acceptance 05 August 2014 Keywords amplified fragment length polymorphism, clone mixing, kin recognition, sociality. Corresponding author Mitsuru Hattori Department of Biology, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan E-Mail: mtr.hattori@gmail.com hypothesis, some hymenopteran insects can recognize non- kin individuals and prevent them from invading the colony (Hölldobler & Wilson, 1990). Such kin recognition may generally help maintain eusociality (Hamilton, 1964 but see Abbot, 2009), because an ability of kin recognition can keep high relatedness between donor and recipient in altruistic interactions (Gadagkar 1985). However, some eusocial aphids (e.g., C. japonica, Ceratoglyphina bambusae, and Pseudoregma bambucicola) cannot recognize kin individuals (Aoki et al., 1991 Carlin et al., 1994; Shibao, 1999; for a review see Pike & Foster, 2008). Several eusocial aphids show a cyclic parthenogenesis which is composed of a single sexual generation and multiple parthenogenetic generations, and host alternation (i.e., seasonally migrating from the primary host to the secondary host vice versa) (Stern & Foster, 1996). In the primary host generation, aphid often construct gall (= nest) (Aoki & Kurosu, 2010). Several studies have shown that even within a eusocial aphid gall on the primary host (= nest), the degree of relatedness among colony mates is low (Abbot et al., 2001; Johnson et al., 2002; Wang et al., 2008). This low relatedness indicates that RESEARCH ARTICLE - SOCIAL ARTRHOPODS M Hattori, T Yamamoto, T Itino Sociobiology 62(1): 116-119 (March, 2015) 117 clone mixing occurs due to the absence of kin discrimination and exclusion of non-kin individuals at the nest entrance. Therefore, we can hypothesize that clone mixing frequently occurs not only on primary hosts but also (even more frequently) on secondary hosts where free-living generation of aphids inhabits without a gall. In this study, to determine whether clone mixing occurs in colonies on secondary host plants, we surveyed the clonal composition of colonies of the eusocial aphid, C. japonica, by using amplified fragment length polymorphism (AFLP) analyses. Material and Methods The eusocial aphid Ceratovacuna japonica Takahashi is a eusocial aphid that parthenogenetically produces sterile “pseudoscorpion- like” first-instar soldiers, which defend their colony on secondary host plants. This species has one primary host plant, Styrax japonica Sieb. et Zucc. (Ebenales, Styracaceae), and several secondary host plants (Poaceae species; Sasa senanensis Rehd. in central Japan) (Aoki & Kurosu, 1991, 2010). Adult individuals on secondary hosts produce alate individuals in the fall, which disperse to the primary host plants on which they overwinter. However, in central Japan where this study was conducted, C. japonica aphids are rarely observed on primary hosts. This suggests an overwinter on the secondary host S. senanensis. In fact, we observed apparently overwintering individuals (alone or with a few mates on a leaf) in the fall and winter season on S. senanensis. In this paper, we define an aphid colony as an aggregation of aphid individuals on a single leaf of a secondary host plant. Field sampling Field sampling was conducted in an aphid population parasitizing a S. senanensis patch (about 10 m × 5 m in size) at the edge of a deciduous coniferous forest (dominated by Larix kaempferi [Pinales, Pinaceae]) at the foot of Mt. Norikura, Nagano, central Japan (1600 m above sea level; 36°8’16.64N, 137°36’24.5E). In August 2012, we randomly selected five aphid colonies, each consisting of 200–1000 aphid individuals, and randomly collected 10 soldiers and 10 adults from each colony. We preserved the collected specimens in 100% ethanol before AFLP analyses. Genetic analyses We extracted DNA from the ethanol-preserved aphids by using a “salting-out” protocol (Sunnucks & Hales, 1996). We performed AFLP analysis according to the method of Vos et al. (1995) with some modifications. Genomic DNA was digested with the restriction enzymes EcoRΙ and MseI at 37°C for 1.5 h. Double-stranded adaptors were then ligated to the ends of the digested DNA fragments at 20°C, overnight. We performed pre-selective amplification performed for 20 cycles, using a primer pair with one additional nucleotide on each restriction enzyme (MseI-C/EcoRI-A) with the following cycle profile: a 30-s DNA denaturing step at 94°C, a 1-min annealing step at 56°C, and a 1-min extension step at 72°C. We performed selective amplification performed for 30 cycles with the following cycle profile: a 30-s denaturing step at 94°C, a 20-s annealing step, and a 2-min extension step at 72°C. The annealing temperature of the first cycle was 66°C, then for each of the next 10 cycles, it was reduced by 1°C each cycle, and finally it was maintained at 56°C for the remaining 19 cycles. Selective polymerase chain reaction (PCR) amplifications were then conducted with a fluorescence-labeled primer combination: EcoRI-ACA (FAM)/MseI-CCG. A Dice TP600 PCR Thermal Cycler (Takara Bio, Shiga, Japan) was used with the AFLP Amplification Core Mix (Applied Biosystems, Foster City, CA, USA) for both the pre-selective and selective amplifications. AFLP fragments were detected with an ABI Prism 3130 automated sequencer (Applied Biosystems) and Gene Mapper software v.4.0 (Applied Biosystems). In this analysis, we selected 8 AFLP bands and obtained fragment data from 57 of the 100 collected aphid individuals. We did not include the fragment data from the remaining 43 individuals in the following analyses because the quality of the data from these specimens was low. To observe the clonal composition of the aphid colonies, we analyzed the fragment data of each aphid individual by conducting a principle coordinate analysis (PCoA) (Gower, 1966) with R Version 2.13.0 software (R Development Core Team, 2011). In this analysis, we considered specimens with identical PCoA scores to be clones. Results We found 15 clones (designated by the letters a–o) among 57 individuals from five colonies of C. japonica (Table 1, Fig 1). The number of clones per colony ranged from four to seven, and many clones occurred in several different colonies. For example, the ‘a’ clone was found in only colony 1, and the ‘o’ clone was found in all five colonies (Table 1). Each clone may consist of (1) only soldiers (first-instar larvae) (‘i’ and ‘m’ Table 1. Clonal composition of the five C. japonica colonies. Lowercase letters indicate identical clones. S, soldier; N, non-soldier adult. Colony No. Clone (number of individuals by caste*) 1 a (1S, 2N), c (2S), f (1N), g (1N), j (1N), m (1S), n (2N), o (1S) 2 b (1N), c (2N), d (1N), I (1S), j (1S), n (2S, 1N), o (3S, 4N)) 3 b (1S), c (1S), l (1N), n (N1), o (3N) 4 c (1S, 1N), e (1N), f (1N), g (1N), k (1N), n (1S), o (2S, 2N) 5 d (2S), h (1N), j (5N), o (2N) *S: Soldiers, N: non-soldier adults M Hattori, T Yamamoto, T Itino - Clonal composition of Ceratovacuna japonica colonies118 clones), (2) only non-soldier adults (‘f’, ‘g’ and ‘h’ clones), or (3) both soldiers and non-soldier adults (‘a’, ‘b’, ‘d’, ‘e’, ‘j’, ‘l’, ‘n’ and ‘o’ clones) (Table 1). Furthermore, PCoA showed that frequency of clone mixing did not relate to genetic similarity among clones (Table 1, Fig 1). For example, in colony 1, distant clones (e.g., ‘a’ and ‘j’) coexisted. Fig 1. Two-dimensional principal coordinate analysis based on the result of AFLP. PCoA Axis 1 and PCoA Axis 2 explained 26% and 21% of the total variance, respectively. Lower cases indicate clones (c.f., Table 1). Distance between lower cases shows a difference of fragment pattern in the result of AFLP. to fill a hole (Pike & Foster, 2004). Such behavior may be selected against predators and conspecific cheating intruders. On the other hand, surprisingly, C. japonica in the secondary host, cannot discriminate kin. Without kin recognition in the free-living, secondary host generation, the aphids may suffer intensive invasion by non-kin cheaters. Kin selection theory predicts that a high degree of relatedness among individuals within a colony is a mechanism for the maintenance of sociality (Hamilton, 1964). In fact, in the gall-forming social aphid Pemphigus obesinymphae (Homoptera, Pemphigidae), which does not have kin-recognition ability, intruders to non-kin colonies behave and develop selfishly; they rarely attack predators and are more than three times as likely to be reproductive as individuals that remain in their natal galls (Abbot et al., 2001). Theoretically, acceptance of intrusions by unrelated selfish individuals should gradually increase dominance of non-kin offspring, which may eventually break the sociality of this aphid species. However the eusociality of C. japonica is maintained despite the occurrence of clonal mixing as shown in this study. This fact implies the maintenance of eusociality in this species is not explained by relatedness only. For example, ecological background such as predation pressure may be important for the maintenance of eusociality in aphids because predation may select for colonies composed of only altruistic clones even if relatedness between clones is low. To understand how some individuals come to migrate to and to invade into other colonies and the precise mechanism that allows the maintenance of eusociality in C. japonica despite clonal mixing, future studies should observe the migration and competition of clone-identified individuals. Acknowledgments We thank Y. Nagano for advice on the AFLP analysis. This work was supported in part by the Nagano Society for the Promotion of Science (No. 24-1-9) to M. H., and by a Grant- in-Aid for Scientific Research (C-22570015) from the Japan Society for the Promotion of Science and Research to T. 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