bioRxiv preprint doi: https://doi.org/10.1101/383059; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 [Title] 2 3 Brood-holding causes workers to pay attention to the queen in the carpenter 4 ant Camponotus japonicus 5 6 7 Author’s name: 8 Kenji Hara 9 10 Address: 11 Life Science, Tokyo Gakugei University, 4-1-1, Nukui-Kitamachi, 12 Koganei-shi, Tokyo 184-8501, Japan. 13 14 Abbreviated form of the title: 15 Attention of the ant workers to the queen 16 17 Name and address for correspondence: 18 Kenji Hara (Ph.D.) 19 Life Science, Tokyo Gakugei University, 4-1-1, Nukui-Kitamachi, 20 Koganei-shi, Tokyo 184-8501, Japan. 21 Tel/FAX; +81-42-329-7522 22 e-mail; [email protected] 23 1 bioRxiv preprint doi: https://doi.org/10.1101/383059; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 [Abstract] 2 3 Brood accumulation, a fundamental behavior of offspring care in the carpenter ant 4 Camponotus japonicus, is driven by alternation of 'holding run' and 'empty-handed 5 run' behaviors. In the holding run, a worker holds a brood with her mandibles and 6 carries it to the queen (holding run). After releasing it beside the queen, she hurries 7 back to another brood (empty-handed run). To address the motivation for the 8 brood-accumulation task, in this study, I observed these behaviors under 9 experimental conditions. When workers performed the task in a situation that 10 involved selection between their own and unfamiliar queens, they ran in 11 significantly more restrictive ways during the holding run than during the 12 empty-handed run. Hence, 'holding' represents a different motivational state than 13 'empty-handed'. In a second experiment, the workers were suddenly presented with 14 an unfamiliar floor during the task. Regardless of whether they were holding or 15 empty-handed, their running traces on the familiar floor were simple, whereas on 16 the unfamiliar floor they were more complex. These results show that holding 17 workers would pay attention to the queen, exploiting cues on the floor to restrict 18 their responses to the queen. 19 20 [Key words] 21 22 selective attention, olfaction, reiteration, social behavior, insect 23 2 bioRxiv preprint doi: https://doi.org/10.1101/383059; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 [Introduction] 2 3 The behavioral complexity of insects has been demonstrated in behavioral 4 and physiological studies of many species. Insects' adaptive actions are based on 5 reflexes and/or the internal patterns, modulated by their response to sensory 6 stimuli. Motivation is a process that leads to the formation of behavioral intentions, 7 i.e., it sets the aim of the behavior. In a behavior that is accomplished by combining 8 multiple actions together, there is thought to be a different motivation for every 9 action. For example, desert ants, Cataglyphis fortis, use path integration (PI) as 10 their main mode of navigation (Wehner, 2003). Their foragers continuously measure 11 directions and distances both when going out to a feeding site (outbound) and when 12 returning to the nest (inbound), and then form outbound and inbound vectors, 13 respectively, by integrating these two quantities. Utilization and switching of the 14 vectors associated with the motivation allows the ants to perform the adaptive 15 behavior (Collett et al., 1999; Merkle and Wehner, 2008). The appropriate change in 16 motivation is necessary to achieve the goal resulting from a chain of actions. 17 In social insects, brood care by sterile workers plays important roles in 18 maintenance of highly sophisticated communities. In the carpenter ant Camponotus 19 japonicus, nestmates divide labor according to their ages, and brood care is the 20 initial task after emergence. Nurse workers repeatedly carry scattered broods and 21 accumulate them beside the queen (Hara, 2013), who affects brood growth by 22 producing and secreting various substances (Holman, 2010; Motais de Narbonne et 23 al., 2016). Therefore, brood-accumulation behavior is fundamental to the altruistic 24 behavior responsible for rearing of kin broods by sterile workers. 25 The unit of brood-accumulation behavior consists of four sequential 26 elements: (1) The worker recognizes a brood by touching it with the antennae, and 27 then picks it up with her mandibles ('pickup'). (2) The worker starts an inbound run 28 while holding a brood ('holding'). (3) The worker recognizes the queen by touching 29 with the antennae, and then places the brood beside her ('release'). (4) The worker 30 turns her back on the queen and starts the outbound run ('empty-handed'). 31 Repetition of the unit results in gathering of broods near the queen. 32 To address the physiological mechanisms underlying brood accumulation behavior, 33 the workers performed tasks under two experimental conditions: a situation that 34 involved selection between unfamiliar (UQ) and fostered queens (FQ) (Experiment 35 1), and a situation in which the familiar floor disappeared (Experiment 2). The data 36 were analyzed to compare 'holding' and 'empty-handed' behaviors. I discuss the 3 bioRxiv preprint doi: https://doi.org/10.1101/383059; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 results from the standpoint of attention-like phenomena of workers engaging in the 2 brood-accumulation task. 3 4 4 bioRxiv preprint doi: https://doi.org/10.1101/383059; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 [Materials and methods] 2 3 Animals 4 5 Experiments were performed on the laboratory-reared workers and queens of 6 the carpenter ant Camponotus japonicus. The laboratory colonies were found and 7 maintained as previously described (Hara, 2002). Founding queens were collected 8 from 2004 to 2016 in Tokyo, Japan. In order to obtain the healthy data, the workers 9 and queens were used from the colonies within two years from the foundation. 10 To prepare the experimental workers, the pupae isolated from their birth 11 colonies were removed from the cocoon and were incubated individually in the 12 96-well culture plate with U bottom (Ishii et al., 2005). Shortly after emergence, 13 they were marked individually with cloth threads of different colors tied between 14 their petiole and gaster and were transferred to the foster colonies (Hara, 2003). On 15 acceptance by the natural ants and performance of the social activities, availability 16 of those workers was decided for this experiment. 17 18 Experimental design and trial procedure 19 20 Experiment 1: The brood-accumulation behaviors of the workers were 21 recorded under the selective condition between the foster queen (FQ) and an 22 unfamiliar queen (UQ). As details of the experimental procedure used for recording 23 the behavior were given in a previous paper (Hara, 2003), only a brief outline is 24 described below. An acrylic box was used for the test consisting of three rooms and 25 the central space (Fig. 1A). Each room is connected to the central space by a 26 doorway that allowed the workers free access to all rooms. A distance between the 27 corners of the central space (red dots in Fig. 1A) was 2.7cm. Two queens and broods 28 were put in the rooms of the box, Q1, Q2 and B, respectively. FQ was put in the 29 room Q1 or Q2, and UQ was put in the other room. After acclimation for 15 min, the 30 trial lasted 60 min. To avoid possible bias resulting from the UQ accidentally having 31 the 'colony labels' similar to those of FQ, the preliminary check were performed 32 every pair, with a control worker from the foster colony. 33 Experiment 2: To show an experimental worker the unfamiliar ground 34 condition suddenly on the trial, the following apparatus was used (Fig.1B); Two 35 plastic dishes (35 mm in diameter) were put in an acrylic box (252×345 mm). Broods 36 were set in the one designated as "B" in Fig. 1B and queen was in the other "Q". A 5 bioRxiv preprint doi: https://doi.org/10.1101/383059; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 thread was attached to the dish "Q" in order to operate it from outside the box. FQ 2 was introduced in the apparatus and then a worker was allowed to explore the 3 inside for 15 min. After finishing such acclimation, five broods from the foster colony 4 were introduced into the dish "B" and then, the worker was put back into it. The 5 behavior had been recorded for 60 min by the video camera since the worker picked 6 a brood up. 7 The preliminary and the final tests were carried out sequentially with each 8 worker. The distance between the dishes "Q" and "B" (B-Q distance) was constant at 9 5 cm through the preliminary trial.