Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v68i2.5922Sociobiology 68(2): e5922 (June, 2021) Leaf-cutting ants of the genus Atta Fabricius, 1805 (Hymenoptera: Formicidae) are eusocial insects found exclu- sively in the Neotropical region (Hölldobler & Wilson, 1990). These ants build stable and long-lasting nests that protect themselves against abiotic and biotic factors, besides growing the fungus Leucocoprinus gongylophorus Heim, 1957, on which they feed (Sudd, 1982; Hölldobler & Wilson, 1990; Rocha et al., 2020). However, due to favorable conditions, like temperature and humidity, that these nests offer (Camargo et al., 2016), they also become favorable habitats for a multitude of inquilines, from invertebrates such as beetles, cockroaches, mites, crickets, dipterans and springtails (Waller & Moser, 1990; Dekoninck et al., 2007; Forti et al., 2007), to vertebrates such as frogs, lizards and snakes (Hölldobler & Wilson, 1990; Baer et al., 2009; Lima Barros et al., 2016). According to Hölldobler and Wilson (1990), Abstract Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) was first observed in early colonies of Atta sexdens leaf-cutting ants (Hymenoptera: Formicidae). The colonies were collected on February 6, 2019, from a transition area between the Atlantic Forest and the Cerrado, located in the municipality of Botucatu, São Paulo, Brazil. A total of four colonies collected had an average population density of 227 ± 212 C. innominatus individuals, and most of the latter were found in peripheral areas inside the fungus- garden-growing chamber of the colony. In addition, we observed a possible defensive behavior on the part of workers when C. innominatus individuals were present in the fungus garden chamber. Thus, this is the first record of C. innominatus living in association with early colonies of A. sexdens. Sociobiology An international journal on social insects Tarcísio Marcos M Mota Filho1, Kátia Kaelly Andrade Sousa1, Roberto S Camargo1, João Victor Lemos Cavalcante Oliveira2, Nadia Caldato1, Douglas Zeppelini2, Luiz Carlos Forti1 Article History Edited by Paulo Cristaldo, UFRPE, Brazil Received 20 October 2020 Initial acceptance 28 December 2020 Final acceptance 23 February 2021 Publication date 20 May 2021 Keywords Attini, Cyphoderus, Myrmecophily, Cyphoderinae. Corresponding author Tarcísio Marcos Macedo Mota Filho Laboratory of Social Insects-Pests Plant Protection Department School of Agronomic Sciences São Paulo State University Botucatu, São Paulo, Brasil. E-Mail: tarcisio972010@hotmail.com these ant guests are commonly known as myrmecophiles and may be present in ant nests temporarily engaging in predation or commensalism, or may also depend on such nests for a while or throughout their life cycle. Information about their ecology, behavior, life cycles and dispersion is scarce for most of these myrmecophilous species because they are difficult to observe and can only be found when specific studies are conducted (Dekoninck et al., 2007). Nonetheless, springtails are inquilines commonly reported and studied in nests of fungus-growing ants (Castaño- Meneses et al., 2017). Even a specific behavior known as jigging, performed by Cyphomyrmex costatus workers, Mann, 1922, and Myrmicocrypta buenzlii workers, Borgmeier, 1934, (Formicidae: Myrmicinae), has been reported as a defense strategy to keep springtails away from the fungus-garden- growing chamber (Weber, 1957, 1972; Kweskin, 2004). 1 - Laboratory of Social Insects-Pests, Plant Protection Department, School of Agronomic Sciences, São Paulo State University, Botucatu-SP, Brazil 2 - Laboratory of Collembola Systematics and Conservation, Biology Department, Center for Applied Biological and Social Sciences, Paraiba State University, Campus V, João Pessoa-PB, Brazil First Record of Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) in Early Colonies of the Leaf-Cutting Ant Atta sexdens SHORT NOTE ID mailto:tarcisio972010@hotmail.com https://orcid.org/0000-0002-2157-261X Tarcísio MM Mota Filho et al. – Cyphoderus innominatus in Early Colonies of Atta sexdens2 In this study, we identify, quantify and report the first species of springtails of the genus Cyphoderus living in early colonies of A. sexdens. In addition, we provide information on a possible defensive behavior performed by workers when springtails are present in their fungus garden chamber. The study design relied on sampling, six hundred colonies of A. sexdens (Hymenoptera: Formicidae), aged approximately four months old, were collected on February 6, 2019, in a transition area between the Atlantic Forest and the Cerrado, located in the municipality of Botucatu (22° 48’ 23.5” S 48° 25’ 52.8” W), São Paulo, Brazil. In the laboratory, a high population density of springtails was observed in four of the 600 collected colonies. These colonies were separated, and then, with the aid of an aspirator, all springtails present were removed by suction and stored in 98% alcohol. Afterwards, population density was quantified, and samples were taken for species identification. The identification was carried out by the Laboratory of Collembola Systematics and Conservation of the State University of Paraíba (UEPB). The methodology adopted for identification consisted of clearing and mounting of the specimens, following the procedures described by Jordana et al. (1997), with clearing being done using Nesbitt’s solution and a phase-contrast microscope (Zeiss Axioskop). Then, to identify the species, the classification key provided by Oliveira et al. (2017) was used. The specimens are deposited in the UEPB’s Soil Fauna Reference Collection [Coleção de Referência da Fauna de Solo] (CRFS). Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) was the species of springtails found living in association with early colonies of A. sexdens (Fig 1). Cyphoderus innominatus were found in four (0.67%) of the 600 collected colonies, with an average population density of 227 ± 212 individuals (Table 1). Most of the individuals were present in peripheral zones inside the fungus garden chamber of the colony, where some debris accumulated, but the springtails did not make direct contact with the ants (Fig 1B and video: https://youtu.be/EObSZYkZs30). The video showed that the springtails (https://youtu. be/EObSZYkZs30) were initially present in the fungus garden chamber of the ants, apparently without causing any disturbance. Until a medium worker (ant with head width of approximately 2.2 mm), between the 38th and 46th seconds, presented a defensive behavior against the springtails. With its jaws open, it jumped three consecutive times towards some springtails. However, the attacks were unsuccessful, and they managed to escape. The present study recorded, for the first time, the occurrence of C. innominatus living in association with early colonies of A. sexdens leaf-cutting ants collected in a transition area between the Atlantic Forest and the Cerrado. Previously, in Paraguay, springtails of the genus Cyphoderus had been reported living in colonies of A. sexdens subjected to laboratory conditions (Fowler, 1981). However, despite the occurrence of this genus being recorded, no species was identified. In addition, three other species of springtails had already been found in association with fungus growing carried out by leaf-cutting ants of the genera Atta and Acromyrmex, namely: Cyphoderus inaequalis with Acromyrmex octospinosus; Pseudosinella violenta (Lepidocyrtidae) with Atta texana; and Seira edmanni (Seiridae) with A. sexdens (Eidmann, 1937; Weber, 1958; Kistner, 1982; Waller & Moser, 1990; Castaño-Meneses et al., 2017). The presence of springtails of the genus Cyphoderus in early colonies of fungus-growing ants can be explained by the large amount of food found in these colonies; being myrmecophilous species, they feed on the mycelium of the fungus grown by the ants (Kistner, 1982). Moreover, colonies of fungus-growing ants also indirectly provide favorable conditions, such as temperature, moisture and protection from predators, to enable the growth, development and multiplication of the tenant population (Hughes et al., 2008). The average density of C. innominatus individuals found in the early colonies of A. sexdens was 227 ± 212 individuals. It is a relatively high density compared to the population density of workers (small, medium and large) from an early nest (up to 4 months) of A. sexdens, which, according to Camargo and Forti (2013), comprises approximately 121 individuals. According to Kweskin (2004), a high population density of springtails ends up consuming a large amount of the mycelium grown in the fungus garden, consequently disturbing the colony (Kweskin, 2004). Some behavioral acts performed by ants against these disturbances have been reported. Among said acts, jigging is a peculiar rhythmic swinging behavior, and there are two hypotheses as to why it is performed: alerting mates about disturbances, or expelling competitors (Weber, 1957;1972). However, this behavior was not observed in our study. On the other hand, the behavioral act also performed by ants in the presence of springtails in their fungus gardens, and which was observed in our study, is jumping, when ants jump and try to capture springtails with their jaws and legs. According to Kweskin (2004), this type of behavior seems to be exclusively meant for defense against invaders, and the same behavior was also performed by C. costatus workers when springtails approached their fungus garden. The actual reason and the efficiency of this behavioral act against these disturbances are still not well understood. In light of our findings, future studies should be carried out to elucidate the effect of the presence of C. innominatus in fungus gardens of A. sexdens, and whether their presence causes disturbances and induces defensive behaviors. Thus, our study provides important information about a species of myrmecophilous springtails that live in association with early colonies of fungus-growing ants, since we reported here, for the first time, the occurrence of C. innominatus in early colonies of A. sexdens. Furthermore, we provided information about a possible defensive behavior performed by A. sexdens workers when springtails are present in their fungus garden chamber. https://youtu https://youtu Sociobiology 68(2): e5922 (June, 2021) 3 Colony Number of individuals of Cyphoderus innominatus Mills, 1938 1 539 2 133 3 165 4 70 Mean ± standard deviation 227 ± 212 Table 1. Number of individuals of Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) found in early colonies of Atta sexdens (Hymenoptera: Formicidae). Fig 1. (a) Early nest of Atta sexdens (Hymenoptera: Formicidae); (b) Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) present in peripheral zone inside the fungus-garden-growing chamber of A. sexdens fungus in laboratory condition [temperature of 22 ± 2 ºC, relative humidity of 70 ± 10% and photoperiod of 14:10 h (L: D)]; (c) Side view of C. innominatus in 98% alcohol; (d) Slide view with Zeiss Axioskop microscope. Acknowledgments TMM Mota Filho and KKA Sousa thank the support of the Coordination for the Improvement of Higher Education Personnel [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior] – Brazil (CAPES) – Finance code 001. 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