Olfactometers can test dung beetle olfactory response and diel activity: a case study in South Korea.

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Suk Young Hong, Minwoo Oh, Eun Ju Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6232839/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Laboratory experiments are widely used to understand insect ecology and behavior. One of the taxa frequently studied this way is the dung beetle to test olfactory responses to resources using olfactometers. Diel activity is another frequently investigated characteristic of dung beetles, but this was usually done with field experiments. The disadvantages of this are that it is labor- and resource-intensive and that weather conditions can influence the results. To address this, we propose that diel activity can also be measured with olfactometers in addition to olfactory response. A four-trap olfactometer was designed to test for inter-trophic preference between carnivore, herbivore, omnivore dung, and control (no dung). Intra-trophic preference was also examined to check differences between mammals of a single feeding guild. The diel activity was examined by checking the traps every three hours from 07:30 to 19:30. Six experiments with different combinations of dung were conducted on six dung beetle species. The six species were chosen based on their resource preference assessed from field experiments, which were expected to be mimicked in the laboratory experiment. The results for the olfactory preference were unclear and did not resemble the results of the field experiment, possibly due to limitations in olfactometer design. However, more accurate results were produced for diel activity, suggesting that using olfactometers to measure diel activity is possible. Conducting two experiments simultaneously would save time and resources and ultimately contribute to enhancing our understanding of dung beetle morphology, evolutionary history, and life cycle. Activity pattern Behavior bioassay Insect ecology Laboratory experiment Resource partitioning Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction To understand insect ecology and behavior, controlled experiments in laboratory conditions have been widely used. There are many advantages of conducting experiments in laboratory settings to natural settings. First and foremost, it is possible to control for desired conditions while excluding unwanted ones. Plus, replicates of the same conditions can be made to increase the sample size. Second, experiments can be conducted regardless of weather conditions so that experiments can be done on schedule. Also, the results are less likely to be influenced by the weather. The downside is that there is a possibility of introducing unexpected biases, due to apparatus design, experiment design, or insect behavior (Roberts et al., 2023 ). Nevertheless, laboratory experiments have allowed us to gain valuable insights into the world of insects. One group of insects studied extensively in laboratory settings are dung beetles (Geotrupidae, Scarabaeinae, and Aphodiinae). They have been tested to check the olfactory response to different types of dung (Dormont et al., 2004 ; Dormont et al., 2007 ; Verdú et al., 2007 ; Giménez Gómez et al., 2021 ; Perera et al., 2022 ), olfactory responses to different single or multiple volatile organic compounds (VOCs) (Inouchi et al., 1988 ; Frank et al., 2018 ), ecosystem services (Bang et al., 2005 ; Nervo et al., 2014 ; deCastro-Arrazola et al., 2020 ), and reproduction and development (Finn & Giller, 2002 ; Bang et al., 2003 ; Bang et al., 2007 ). These experiments were often conducted in tandem with field experiments to ascertain findings made in the field. Olfactory responses of dung beetles are tested using olfactometers, an apparatus used to study how insects respond to volatile chemical compounds. In the case of dung beetles, their ability to differentiate between dung types (Dormont et al., 2004 ; Dormont et al., 2007 ) and their preference (Dormont et al., 2010 ; Salomão et al., 2018 ; Giménez Gómez et al., 2021 ; Perera et al., 2022 ) have been investigated. Other experiments have sought to “look outside the dung” and explored dung beetle preference for acorns (Verdú et al., 2007 ), and rotting meat, arthropod carcasses, fungi (Giménez Gómez et al., 2021 ). The results of these studies were similar to field experiments, in some cases with higher accuracy (Giménez Gómez et al., 2021 ). This shows that olfactometers are versatile and accurate tools for testing dung beetle preference. In addition to olfactory responses, it may be possible to test dung beetle diel activity using olfactometers. Many dung beetles burrow into the ground when they are not searching for dung (Houston & McIntyre, 1985 ; Caveney et al., 1995 ). Depending on the time of day when they surface and become active, they are categorized into diurnal, nocturnal, and crepuscular species. While many species were found to be active around noon (Krell-Westerwalbesloh et al., 2004 ), interspecific differences exist. The differences may stem from physiological factors such as body temperature requirements, competition in the form of temporal resource partitioning, or both (Krell-Westerwalbesloh et al., 2004 ; Feer & Pincebourde, 2005 ). Information on diel activity and its cause can give us clues about dung beetle morphology, evolutionary history, and life cycle (Tocco et al., 2021 ; Ribeiro et al., 2024 ), which can help us speculate the effects that climate change will have on dung beetles. But how do dung beetles know when to search for resources? Flight chambers were used in laboratory experiments to understand the mechanism behind dung beetle flight behavior (Houston & McIntyre, 1985 ; Caveney et al., 1995 ). In these experiments, dung beetles were placed in a shallow basin with loam, where they would bury themselves. Afterward, the basin was placed inside the flight chamber, and the dung beetles would emerge and eventually fly when conditions were right. However, most experiments on dung beetle diel activity have been conducted in the field. Several pitfall traps or dung pats are deployed in the field, and they are checked at regular intervals (e.g., four hours) or at different times of the day (e.g., dawn, morning, noon, afternoon, dusk, night, etc.) depending on the research question (Krell-Westerwalbesloh et al., 2004 ; Feer & Pincebourde, 2005 ; Lobo & Cuesta, 2021 ). However, this is often very resource-intensive. More dung is required to attract dung beetles in outside settings, where 250 g to 1 kg of dung was used per trap/pat (Krell-Westerwalbesloh et al., 2004 ; Kamiński et al., 2015 ; Lobo & Cuesta, 2021 ) compared to 15–50 g of dung in indoor settings (Dormont et al., 2004 ; Salomão et al., 2018 ). Plus, it is very labor-intensive as many traps/pats must be deployed to obtain a sufficient sample size. Then, they need to be revisited at each interval to collect the dung beetles (Krell-Westerwalbesloh et al., 2004 ; Lobo & Cuesta, 2021 ). In addition, bad weather conditions, such as rain, can make it difficult to conduct the experiment on schedule and possibly affect the results. Olfactometers could combine the functions of the traps used in outdoor settings and flight chambers. This way, both olfactory response and diel activity can be measured simultaneously. It has the advantages of being less resource- and labor-intensive while allowing the experiment to be conducted regardless of the weather. In this study, such an olfactometer was designed and built to assess the olfactory responses and the diel activity of dung beetles in South Korea. These species’ olfactory responses have only been tested in the field (unpublished data; Table A1), while there are no records of their diel activity. To ascertain that olfactometers can be used to serve dual purposes, the accuracy of the olfactometer in measuring olfactory response and diel activity will be assessed in this study. To do this, three hypotheses were tested. First, dung beetles will exhibit similar olfactory responses to the field results. Second, differences in dung preference will exist within the same mammal feeding guild. Third, dung beetles will be active at specific times of the day, with many species active at noon. 2. Methods 2.1. Arena design The olfactometer designs were adapted from Perera et al. ( 2022 ) in the use of the square shape and pitfall traps, and Verdú et al. ( 2007 ) in the use of a control trap with no dung and the use of a buffer in the center. This study used soil instead of leaf litter as a buffer, as used in Houston and McIntyre ( 1985 ), so that dung beetles can bury themselves outside their activity period. The olfactometer was made of a 5 mm acrylic sheet, assembled in a cube shape with dimensions of 600 mm × 600 mm × 400 mm (width, length, height) with the top and bottom open. A “field” for dung beetles to search for dung was made with a 5 mm acrylic sheet, placed 120 mm above the ground. A 10 mm foam board was placed above the acrylic sheet to prevent dung beetles from slipping. The floor had five holes where five \(\:\varnothing\:\) 100 mm beakers were placed. The beaker in the center was the “starting point” for the dung beetles and was filled with soil. The four beakers in each corner had funnels and roof structures that acted as pitfall traps. The roof structures held broth bags filled with dung baits. A mesh cover was placed on the top of the arena to prevent dung beetles from escaping. The sides of the arena were bordered with cardboard, and blinds in the laboratory were lowered so that direct sunlight could not enter the arena. Artificial light was used to supplement natural light in the relatively dark indoor setting during the day (Fig. 1 ). An “IKEA TRÅDFRI LED bulb E26 1055 lumen” was used for lighting. The color temperature was adjusted to 2700K from 07:30 ~ 10:30 and 16:30 ~ 19:30, and 4000 K from 10:30 ~ 16:30. Airflow within the arena was checked with a smoke test. Because the roof structure was made of foam boards that may absorb odors, one type of dung was assigned for each roof structure. Each corner of the arena was assigned a mammal feeding guild (carnivore, herbivore, omnivore) and control (no dung) to minimize the possibility of mixing up different odors. 2.2. Experiment Six experiments were conducted with six dung beetle species (Table 1 ). Experiments one through four compared dung from mammals of three trophic guilds: omnivore, herbivore, and carnivore. It was based on the assumption that dung from mammals of the same trophic guilds has a similar attraction to dung beetles (Hanski & Cambefort, 1991 ; Martín-Piera & Lobo, 1996 ). However, experiments five and six were carried out to verify this assumption, which is why comparisons between two omnivores and two herbivores were made, respectively. A control trap without any bait was also used in all experiments to test if dung beetles are trapped by factors other than olfactory response. Before the main experiments, three pilot studies were conducted with Copris tripartitus using boar, cattle, and leopard cat dung. Table 1 (a) List of species used for this experiment and (b) combinations of mammal dung used for the lab experiment. Six experiments were carried out using dung from different mammals. All six species were used for all six experiments. Hyphens (-) indicate no dung for the respective dung type. (a) Species (b) Experiment # Species name # Herbivore Omnivore Carnivore Control 1 Phelotrupes auratus 1 cattle boar leopard cat - 2 Sisyphus schaefferi 2 water deer boar leopard cat - 3 Copris tripartitus 3 cattle raccoon dog leopard cat - 4 Onthophagus fodiens 4 water deer raccoon dog leopard cat - 5 Onthophagus rugulosus 5 - raccoon dog / boar - - 6 Aphodius rectus 6 cattle / water deer - - - 2.3. Test subjects and materials The six dung beetles chosen for the experiment were Phelotrupes auratus , Sisyphus schaefferi , C. tripartitus , Onthophagus fodiens , Onthophagus rugulosus and Aphodius rectus . The omnivore dung was from boar ( Sus scrofa ) and raccoon dog ( Nyctereutes procyonoides ), the herbivore dung was from cattle ( Bos taurus ) and water deer ( Hydropotes inermis ), and the carnivore dung was from Leopard cat ( Prionailurus bengalensis ). All the dung beetles and mammals were native to South Korea. Dung beetles with varying dung preferences were chosen, which were identified with the indicator species analysis (De Cáceres & Legendre, 2009 ) with data from field experiments (unpublished data; Table A1). A. rectus and O. rugulosus were attracted to boar dung, C. tripartitus to boar and cattle dung, S. schaefferi to boar and leopard cat dung, and O. fodiens and P. auratus to all types of dung. All species were collected at Yeoncheon-gun, Gyeonggi-do province, except for C. tripartitus , which could not be captured in the wild due to its status as an endangered species (NIE, 2023 ). As such, they were brought from Jeju Technopark Biodiversity Research Institute via a research loan for the period of the experiment. All dung beetles were kept in cages half-filled with soil when not used for the experiment. For each experiment, 20–30 individuals of dung beetles were used, which was determined with the power test (Table A2). The power test was done using the “pwr” package (Champely, 2020 ). Although the use of different individuals per test is suggested (Roberts et al., 2023 ), same individuals were used multiple times for the experiment because of the difficulty of collecting endangered species C. tripartitus, O. rugulosus , and S. schaefferi (Bae et al., 2013 ). Dung from mammals was collected and portioned into bags of 30 \(\:\:\pm\:\:\) 3 grams. Boar dung was collected at a farm in Taebaek-si, Gangwon-do province. Cattle dung was collected at Seoul National University Animal Farm in Pyeongchang-gun, Gangwon-do province. Leopard cat dung was collected at Seoul Zoo. Raccoon dog feces were collected from Seoul Wildlife Center when raccoon dogs were rescued and in rehabilitation. Water deer dung was collected near the field experiment sites in Yeoncheon-gun, Gyeonggi-do province. They were frozen until use. 2.4. Procedure Before the experiment, the beaker in the center was half-filled with soil, and the four beakers on the sides were filled to 5–10 mm, depending on the size of the species. Then, frozen dung was put into broth bags and tied underneath the roof structure. 24–30 individuals of dung beetles were placed in the center beaker and covered up with soil to the top. Only one species of dung beetle was used per experiment, and in the case of dung beetles whose sex could be distinguished easily ( C. tripartitus, O. rugulosus, O. fodiens ), the sex ratio was matched to approximately 1:1. In between the experiments, the roof structure, plastic funnels, beakers, and the floor board were washed to get rid of the odors from the previous experiment. The roof structure and floor boards were washed with tap water, while the plastic funnels and beakers were washed thoroughly with detergent. Each experiment started at noon or after 19:30, depending on the activity pattern of the species. The beaker in the center was covered with an object above the soil for approximately 30 minutes so that dung beetles could get accustomed to the environment. The traps were checked every three hours from 07:30 to 19:30. The experiment was carried out for 48 hours, but when less than 20 individuals fell into the traps within this time, the experiment continued until 20 individuals made their choice. The experiments for C. tripartitus, O. rugulosus, O. fodiens, P. auratus , and A. rectus were conducted from 24 September to 5 November in 2023, while the experiments for S. schaefferi were conducted from 8 July to 26 July in 2024. Due to the seasonality of dung beetles, experiment six for O. fodiens and experiments five and six for A. rectus could not be conducted. 2.5. Data analysis 2.5.1. Dung preference The results of experiments one through four were used to find the trophic preference of dung beetles. The results of the four experiments were used to fit generalized linear models (GLM) for the number of individuals that fell into each trap. A Poisson distribution with link function log was used, and the mammal trophic guild was used as the fixed variable. For each model, residual fit was assessed with the DHARMa package (Hartig, 2024 ), and the Analysis of Variance (ANOVA) was performed to test for statistical differences. Then, pairwise comparisons between trophic guilds were conducted using the emmeans package (Lenth, 2024 ). The p-values of the pairwise comparisons were adjusted using the Tukey method. The results of experiments five and six were used to find the preference of dung beetles within the same mammal trophic guild, i.e., herbivore and omnivore. A GLM was fitted for each experiment, using the same methods employed for experiments one through four, except that mammal species were used as the fixed variable. All statistical analyses were conducted using R (R Core Team, 2023 ). 2.5.2. Diel activity The results of all six experiments were pooled to analyze the diel activity of each dung beetle species. First, whether dung beetles were nocturnal or diurnal (day/night) was assessed. This was done by comparing the number of dung beetles caught in the traps at 07:30 (night) to the sum of individuals caught at other times, i.e., 10:30, 13:30, 16:30, and 19:30 (day). Second, dung beetle activity during different hours of the day (daytime activity) was compared to find out when dung beetles were most active. The comparison was made between the number of individuals caught at 10:30, 13:30, 16:30, and 19:30. For both cases, GLMs were fitted using Poisson distribution with link function log, using the activity pattern or activity time as fixed variables. However, the negative binomial distribution was employed when residuals were poorly fit, using the MASS package (Venables & Ripley, 2002 ). The consecutive methods were identical to that used for dung trophic preference. 3. Results 3.1. Pilot experiment The three pilot studies conducted with C. tripartitus using boar, cattle, and leopard cat dung exhibited precise results. Preference for cattle dung was pronounced, while close to zero individuals were found in control traps (Table A3). As such, the main experiment was initiated. 3.2. Dung preference The first hypothesis that dung beetles exhibit similar olfactory response to the field experiment was not true for most dung beetles in this experiment (Fig. 2 ). The second hypothesis that dung beetles will have preference for different mammals of the same trophic guild was valid only for P. auratus , while it was only marginally true for C. tripartitus (Fig. 3 ). P. auratus was a generalist in the field experiment. However, in the olfactometer bioassay, it displayed significant differences in preference for dung from different feeding guilds (Table A4a). Pairwise comparisons showed significant differences between omnivore dung and control, omnivore dung and carnivore dung, and herbivore dung and control (Fig. 2 a, Table A5a). The difference within a single trophic guild was not statistically significant between omnivores. However, it was significant for herbivores, with water deer dung preferred over cattle dung (Fig. 3 a). Because no dung beetles fell into control traps, the comparison between control and other dung types were mostly significant (Fig. 3 a). S. schaefferi was attracted to omnivore and carnivore dung in field settings. But in laboratory settings, herbivore and omnivore dung were more attractive than carnivore dung, and less individuals were found in traps with carnivore dung than control (Fig. 2 b, Table A5a). This species did not exhibit preference to different mammals within a single trophic guild (Fig. 3 b). In the case of C. tripartitus , the results of the field experiment and the lab experiment matched. It was attracted to herbivore and omnivore dung, with statistical significance against control and carnivore dung (Fig. 2 c, Table A4a, A5a). Single trophic guild experiments showed that raccoon dog dung and water deer dung was preferred over control (Fig. 3 c). O. fodiens ’ lab experiment results also matched the field experiment, because it displayed generalist characteristics (Fig. 2 d, Table A4a, A5a). However, the comparison between control and other traps were also insignificant. This species did not display preference for different mammals within a single trophic guild (Fig. 3 d). O. rugulosus preferred omnivore dung in the field experiment. However, while the difference between trophic guilds was significant (Table A4a), pairwise comparisons only showed a significant difference between control and omnivore (Fig. 2 e, Table A5a). The difference within a single trophic guild did not yield significant results, but boar dung and water deer dung had more individuals than raccoon dog dung and cattle dung, respectively (Fig. 3 e). A. rectus was attracted to omnivore dung in the field experiment. However, this was different in laboratory conditions. Preference towards dung from different trophic guilds was exhibited (Table A4a), but herbivore dung and carnivore dung were preferred (Fig. 2 f, Table A4a, A5a). 3.3. Diel activity The third hypothesis that dung beetles would be active at specific times of the day was true for all the species tested. Also, as expected, many diurnal species were active at noon. All dung beetles except for C. tripartitus were diurnal (Fig. 4 , Table A8). For daytime activity, species had distinct patterns. P. auratus’ activity was spread relatively evenly throughout the day (Fig. 4 a, Table A4b, A5b). For S. schaefferi , activity peaked during noon, and declined sharply after 16:30 (Fig. 4 b, Table A4b, A5b). In comparison, O. rugulosus was most active later in the day, with peak activity at 16:30 and reduced activity at 10:30 (Fig. 4 e, Table A4b, A5b). The daytime activities of O. fodiens and A. rectus were similar, with activity peaks from noon to 16:30 (Fig. 4 d, 4 f, Table A4b, A5b). Because C. tripartitus was a nocturnal species, no significant relationship was observed for daytime activity (Fig. 4 c). 4. Discussion Contrary to the first and second hypotheses, the results obtained differed from the field experiments, and dung beetle olfactory response was not demonstrated for mammals of different trophic guilds nor within the same trophic guild. As assumed in the power test, it was expected that no dung beetles would fall into control traps. However, more dung beetles than expected were trapped in the control traps, obscuring the results. The main reason that dung beetles were found in control traps is because of flight activity, which caused them to fall into random traps. During the experiment, many dung beetles flew into the olfactometer wall or mesh cover and “accidentally” dropped into one of the traps (personal observation). This suggests that a change in olfactometer design is required to prevent these accidents. A good example is the design used by Verdú et al. ( 2007 ), which had dung beetles walk through a narrow tunnel at the sides of the central chamber to get to the source of the VOCs. This would require more effort from dung beetles, and result in reduced numbers at control traps. In fact, Verdú et al. ( 2007 ) demonstrated the feasibility of this design by showing that significantly more dung beetles were attracted to resources than control traps. The second reason is that the sun's direction may have presented problems with orientation. Dung beetles orient themselves with the direction of the sun, which was reported many times with “roller” dung beetles, or dung beetles that form dung balls and roll them away to nest elsewhere (Byrne et al., 2003 ; el Jundi et al., 2014 ). Although only S. schaefferi belonged to the roller group, all dung beetles used in this experiment moved towards the sun in the cages where they were kept before and in between experiments (personal observation). Despite the blinds and cardboards set up to prevent direct sunlight from entering the olfactometer, polarized light or light intensity gradients may have influenced dung beetle orientation (el Jundi et al., 2014 ). Dung beetles attempting to escape with this directional bias may randomly fall into one of the traps on the way. As such, it may be possible to address this with the design change suggested in the previous paragraph. Third, the olfactometer was made of materials that could retain odors from VOCs, unlike the recommended glass or polytetrafluoroethylene (PTFE) (Roberts et al., 2023 ). Measures were taken to prevent this from influencing the results, such as assigning a roof structure for each dung type and cleaning components of the olfactometer between experiments. However, because dung beetles possess acute olfactory senses (Dormont et al., 2010 ; Urrutia et al., 2022 ), there is a possibility that residual VOCs may have influenced dung beetle activity. Yet, because larger baits attract more diversity and abundance of dung beetles (Errouissi et al., 2004 ), it is probable that the dung baits had a far more significant influence on dung beetle response than residual VOCs. Also, experiment planning could have been improved. At the initial planning stage, it was assumed that no dung beetles would fall into the control traps. Therefore, the number of experiments and individuals used per experiment was insufficient for some species, especially ones prone to fall into control traps. In addition, dung from different mammals belonging to the same trophic guild may be different in attractiveness to dung beetles, as seen by the preference of P. auratus for water deer dung over cattle dung in this experiment. Similar results were reported in the past (Frank et al., 2018 ; Perera et al., 2022 ), suggesting that pooling data according to trophic guilds may not be advisable. Therefore, conducting multiple experiments with the same combination of dung is suggested. Overall, the results regarding the olfactory response of dung beetles were unclear and did not align with the findings from the field experiments. However, since several potential factors were identified, new experiments with enhanced olfactometer design and improved experimental planning should yield more meaningful results. In contrast to the olfactory response, diel activity yielded clearer results. The third hypothesis that dung beetles are active at specific times of the day and that many species will be active at noon was supported. The contrast between diurnal and nocturnal activity was particularly pronounced. Like many species belonging to the tribe Coprini, C. tripartitus is a nocturnal species (Hanski & Cambefort, 1991 ). This may be due to its large body size, which allows it to conserve heat in colder night temperatures (Lobo & Cuesta, 2021 ). Other species were clearly diurnal. However, many individuals of P. auratus and A. rectus were active at night, although it was statistically insignificant. While the reason for this is unclear, covering the center beaker for 30 minutes may not have been sufficient for these species to adapt to the new environment. Patterns in daytime activity were observed for most diurnal dung beetles. In line with the hypothesis, many species were active at noon, but each species had specific periods of activity. How the dung beetles could tell when to be active is not known, although it has been suggested that light intensity and temperature may be the reason (Wensler, 1974 ; Houston & McIntyre, 1985 ; Caveney et al., 1995 ). This experiment was conducted in an indoor setting where daytime temperatures were between 17.0°C and 25.5°C, which was higher than the threshold temperature suggested by Houston and McIntyre ( 1985 ) for Onitis alexis . In fact, many dung beetles were active between the lower temperature ranges between 17.0°C and 19.0°C during the experiment, likely because of the colder climates of South Korea compared to southern Africa where O. alexis is from. Therefore, the largest variation in the environment was in the light conditions. Although direct sunlight could not reach the interiors of the olfactometer, indirect sunlight was available. Dung beetles orient themselves with the position of the sun, but when it is obscured, they can use other cues, such as polarized light and changes in light intensity, to find their way (el Jundi et al., 2014 ). Similarly, dung beetles may be able to use these signs to discern the time of day, as polarized light changes with the sun’s position (Cronin & Marshall, 2011), and as light intensity changes throughout the day. In addition, although artificial light was used during the day, it does not seem to have influenced dung beetle diel activity. First, the light source was not nearly as bright as the sun. Although studies on the effect of artificial light during the day are limited, the fact that dung beetles are affected by light intensity (Wensler, 1974 ; Houston & McIntyre, 1985 ; Caveney et al., 1995 ; el Jundi et al., 2014 ) suggest similar brightness to the sun is required for artificial light to influence activity. Second, the results of this experiment also suggest little influence of artificial light. The color temperature of the artificial light was maintained at 4000K from 10:30 to 16:30, but S. schaefferi and O. rugulosus had significantly different numbers of individuals that were active between 10:30 ~ 13:30 and 13:30 ~ 16:30. However, it cannot be dismissed that the difference in active periods may be more pronounced without artificial light, nor could it be dismissed that species such as O. fodiens and A. rectus were not influenced. Therefore, additional experiments are required to confirm the effects of artificial light. Another factor that may influence dung beetle diel activity is the circadian rhythm. Houston and McIntyre ( 1985 ) and Wensler ( 1974 ) observed that dusk-crepuscular dung beetles emerged just below the soil surface before dusk. However, when the light conditions were manipulated, they would burrow back instead of taking flight. The authors suggested that circadian rhythm directs dung beetles to surface, while light conditions dictate flight. All in all, although the actual factor for dung beetle diel activity remains uncertain, the sun's contribution appears to be considerable. Comparing the results of olfactory response with diel activity, it was observed that diel activity produced much clearer results. This indicates that dung beetle diel activity can be measured indoors using olfactometers, allowing the disadvantages of field experiments to be mitigated. Moreover, in the South Korean context of this study, S. schaefferi and O. rugulosus are rare species not found in many places. One of the few places they could be found was in forests near the Demilitarized Zone (DMZ), where frequent activity may raise suspicion from military personnel and residents, making it difficult to conduct these experiments in the field. In such special cases, lab experiments are the only feasible option. With the appropriate olfactometer design and experiment planning, both dung beetle olfactory responses and diel activity can be measured effectively. Three key factors for this are given. First, sunlight should be available where the experiment is conducted, as dung beetles rely on it to perceive the time of day. Second, soil with sufficient depth should be available at the “starting point” so dung beetles can burrow until their activity period. Third, olfactometers should be designed to prevent dung beetles from falling into random traps, whether during an attempt to escape through flight or due to directional bias presented by the sun’s position. These principles will facilitate experiments that deepen our understanding of dung beetle morphology, evolutionary history, and life cycle (Tocco et al., 2021 ; Ribeiro et al., 2024 ). In the face of anthropogenic changes such as habitat loss, overexploitation, and climate change, this understanding will better prepare us for potential impacts on insects in the future. Declarations Acknowledgments This paper could not have been written without the help of many people and organizations, to whom we owe our greatest gratitude. Jeju Technopark Research Institute provided the endangered Copris tripartitus used in this experiment, where Minhee Ko kindly helped us. The staff at Seoul National University Animal farm kindly allowed us to collection of cattle dung at their farm. Leopard dung was collected with the help of Seoul Zoo. Doctor of Veterinary Medicine (D.V.M.) PhD So-Young Jung, and D.V.M. Ain Choi facilitated the administrations for dung collection, while zookeepers Seoungmin Choi and Sangha Lee collected leopard cat dung. The staff at Seoul Wildlife Center found time to help us collect raccoon dog dung, despite their busy schedule rescuing and rehabilitating wild animals. Lastly, Minseok Cho was a great help in collecting dung beetles and conducting the experiments. Funding: This research did not receive any grants or funding from external sources. Fieldwork was carried out with funds from the laboratory of plant ecology (EJ Lee’s lab at Seoul National University). Conflicts of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical approval: All applicable institutional and/or national guidelines for the care and use of animals were followed. Consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials The data was deposited in dryad under the reference number DOI: 10.5061/dryad.jm63xsjng. References Bae YJ, Cho YB, Park JY, Oh K-S, Econature (2013) vol 2). National Institute of Biological Resources Bang H-S, Lee J-H, Kwon OS, Na YE, Jang YS, Kim WH (2005) Effects of paracoprid dung beetles (Coleoptera: Scarabaeidae) on the growth of pasture herbage and on the underlying soil. Appl Soil Ecol 29(2):165–171. https://doi.org/10.1016/j.apsoil.2004.11.001 Bang H-S, Na Y-E, Kim M-H, Roh K-A, Lee J-T (2007) Effect of Temperature on the Development of Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae). Korean J Appl Entomol 46(3):357–362. https://doi.org/10.5656/KSAE.2007.46.3.357 Bang H-S, Wardhaugh KG, Hwang S-J, Kwon OS (2003) Development of Copris tripartitus (Coleoptera: Scarabaeidae) in Two Different Rearing Media. Entomol Res 33(3):201–204. https://doi.org/10.1111/j.1748-5967.2003.tb00070.x Byrne M, Dacke M, Nordström P, Scholtz C, Warrant E (2003) Visual cues used by ball-rolling dung beetles for orientation. J Comp Physiol A: Sens Neural Behav Physiol 189(6):411–418. https://doi.org/10.1007/s00359-003-0415-1 Caveney S, Scholtz CH, Mcintyre P (1995) Patterns of daily flight activity in onitine dung beetles (Scarabaeinae: Onitini). Oecologia 103(4):444–452. https://doi.org/10.1007/bf00328682 Champely S (2020) pwr: Basic Functions for Power Analysis. In https://CRAN.R-project.org/package=pwr De Cáceres M, Legendre P (2009) Associations between species and groups of sites: indices and statistical inference. Ecology 90(12):3566–3574. https://doi.org/10.1890/08-1823.1 deCastro-Arrazola I, Hortal J, Noriega JA, Sánchez‐Piñero F (2020) Assessing the functional relationship between dung beetle traits and dung removal, burial, and seedling emergence. Ecology 101(10). https://doi.org/10.1002/ecy.3138 Dormont L, Epinat G, Lumaret J-P (2004) Trophic Preferences Mediated by Olfactory Cues in Dung Beetles Colonizing Cattle and Horse Dung. Environ Entomol 33(2):370–377. https://doi.org/10.1603/0046-225x-33.2.370 Dormont L, Jay-Robert P, Bessière J-M, Rapior S, Lumaret J-P (2010) Innate olfactory preferences in dung beetles. J Exp Biol 213(18):3177–3186. https://doi.org/10.1242/jeb.040964 Dormont L, Rapior S, Mckey DB, Lumaret J-P (2007) Influence of dung volatiles on the process of resource selection by coprophagous beetles. Chemoecology 17(1):23–30. https://doi.org/10.1007/s00049-006-0355-7 el Jundi B, Smolka J, Baird E, Byrne MJ, Dacke M (2014) Diurnal dung beetles use the intensity gradient and the polarization pattern of the sky for orientation. J Exp Biol 217(Pt 13):2422–2429. https://doi.org/10.1242/jeb.101154 Errouissi F, Haloti S, Jay-Robert P, Janati-Idrissi A, Lumaret J-P (2004) Effects of the Attractiveness for Dung Beetles of Dung Pat Origin and Size Along a Climatic Gradient. Environ Entomol 33(1):45–53. https://doi.org/10.1603/0046-225x-33.1.45 Feer F, Pincebourde S (2005) Diel flight activity and ecological segregation within an assemblage of tropical forest dung and carrion beetles. J Trop Ecol 21(1):21–30. https://doi.org/10.1017/s0266467404002056 Finn JA, Giller PS (2002) Experimental investigations of colonisation by north temperate dung beetles of different types of domestic herbivore dung. Appl Soil Ecol 20(1):1–13. https://doi.org/10.1016/S0929-1393(02)00011-2 Frank K, Brückner A, Blüthgen N, Schmitt T (2018) In search of cues: dung beetle attraction and the significance of volatile composition of dung. Chemoecology 28(4):145–152. https://doi.org/10.1007/s00049-018-0266-4 Giménez Gómez VC, Verdú JR, Velazco SJE, Zurita GA (2021) Dung beetle trophic ecology: are we misunderstanding resources attraction? Ecol Entomol 46(3):552–561. https://doi.org/10.1111/een.13001 Hanski I, Cambefort Y (1991) Dung Beetle Ecology. Princeton University Press. http://www.jstor.org/stable/j.ctt7zv085 Hartig F (2024) DHARMa: Residual Diagnostics for Hierarchical (Multi-Level / Mixed) Regression Models. In https://CRAN.R-project.org/package=DHARMa Houston WWK, McIntyre P (1985) The daily onset of flight in the crepuscular dung beetle Onitis alexis. Entomol Exp Appl 39(3):223–232. https://doi.org/https://doi.org/10.1111/j.1570-7458.1985.tb00463.x Inouchi J, Shibuya T, Hatanaka T (1988) Food Odor Responses of Single Antennal Olfactory Cells in the Japanese Dung Beetle, Geotrupes auratus (Coleoptera: Geotrupidae). Appl Entomol Zool 23(2):167–174. https://doi.org/10.1303/aez.23.167 Kamiński MJ, Byk A, Tykarski P (2015) Seasonal and Diel Activity of Dung Beetles (Coleoptera: Scarabaeoidea) Attracted to European Bison Dung in Białowieża Primeval Forest, Poland. Coleopterists Bull 69(1):83–90. https://doi.org/10.1649/0010-065x-69.1.83 Krell-Westerwalbesloh S, Krell F-T, Eduard Linsenmair K (2004) Diel separation of Afrotropical dung beetle guilds—avoiding competition and neglecting resources (Coleoptera: Scarabaeoidea). J Nat Hist 38(17):2225–2249. https://doi.org/10.1080/00222930310001618921 Lenth RV (2024) emmeans: Estimated Marginal Means, aka Least-Squares Means. In https://CRAN.R-project.org/package=emmeans Lobo JM, Cuesta E (2021) Seasonal variation in the diel activity of a dung beetle assemblage. PeerJ 9:e11786. https://doi.org/10.7717/peerj.11786 Martín-Piera F, Lobo JM (1996) A comparative discussion of trophic preferences in dung beetle communities. Miscel·lània Zoològica, 19(1) Nervo B, Tocco C, Caprio E, Palestrini C, Rolando A (2014) The Effects of Body Mass on Dung Removal Efficiency in Dung Beetles. PLoS ONE 9(9):e107699. https://doi.org/10.1371/journal.pone.0107699 NIE (2023) Endangered species. National Institute of Ecology Perera NN, Weston PA, Barrow RA, Weston LA, Gurr GM (2022) Contrasting Volatilomes of Livestock Dung Drive Preference of the Dung Beetle Bubas bison (Coleoptera: Scarabaeidae). Molecules 27(13):4152. https://doi.org/10.3390/molecules27134152 R Core Team (2023) R: A Language and Environment for Statistical Computing. In R Foundation for Statistical Computing. https://www.R-project.org/ Ribeiro PHO, Frizzas MR, Vaz-De-Mello FZ, Gawryszewski FM (2024) The evolution of body coloration in dung beetles: diel activity and sexual dimorphism. Evol Ecol 38(4):449–460. https://doi.org/10.1007/s10682-024-10300-9 Roberts JM, Clunie BJ, Leather SR, Harris WE, Pope TW (2023) Scents and sensibility: Best practice in insect olfactometer bioassays. Entomol Exp Appl 171(11):808–820. https://doi.org/10.1111/eea.13351 Salomão RP, Maia ACD, Bezerra BM, Iannuzzi L (2018) Attractiveness of Different Food Resources to Dung Beetles (Coleoptera: Scarabaeidae) of a Dry Tropical Area. Neotrop Entomol 47(1):69–78. https://doi.org/10.1007/s13744-017-0515-1 Tocco C, Dacke M, Byrne M (2021) The finely defined shift work schedule of dung beetles and their eye morphology. Ecol Evol 11(22):15947–15960. https://doi.org/10.1002/ece3.8264 Urrutia MA, Cortez V, Verdú JR (2022) Links Between Feeding Preferences and Electroantennogram Response Profiles in Dung Beetles: The Importance of Dung Odor Bouquets. J Chem Ecol 48(9–10):690–703. https://doi.org/10.1007/s10886-022-01383-1 Venables WN, Ripley BD (2002) Modern Applied Statistics with S (Fourth ed.). Springer. https://www.stats.ox.ac.uk/pub/MASS4/ Verdú JR, Lobo JM, Numa C, Pérez-Ramos IM, Galante E, Marañón T (2007) Acorn preference by the dung beetle, Thorectes lusitanicus, under laboratory and field conditions. Anim Behav 74(6):1697–1704. https://doi.org/10.1016/j.anbehav.2007.03.016 Wensler RJ (1974) Crepuscular activity of adult Sericesthis geminata (Coleoptera: Scarabaeidae): Influence of circadian rhythmicity and light intensity. New Z J Zool 1(2):197–204. https://doi.org/10.1080/03014223.1974.95178 Supplementary Files ESM1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6232839","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433999438,"identity":"e2cb3316-5ee8-416f-88a0-025c21d1f6d3","order_by":0,"name":"Suk Young Hong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYPCCA0DM2PjwQwWyAGEtzIeNJc6QpoUtTYK3jQgtBsd7zB5+YbgjZ86/xkBCcl5dnsEB5ocfGM7cw63lzBlzYxmGZ8aWM94YGBRuO1xscIDNWILhRjFuLTdyzKQlGA4nbrhxxiBBctuBxA0HGMwYGD4kEKflAO+cOqAW9m8EtUh+AGk535bYwNvADNTCA7TlBm4tkmeOlUkzGBw2NrjBfJhZ4tjhxJmHeYolEs7g1sJ3vHmb5I+Kw3IG5w+2//xQU5fYd7x944cPx3BrUTgAjEMeAyBLAqaIGYhxa2BgkG8AppQfIBb/ATzKRsEoGAWjYEQDAFunYBiq2GTsAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5556-504X","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Suk","middleName":"Young","lastName":"Hong","suffix":""},{"id":433999439,"identity":"9b8f0756-5967-40f4-82a4-fb6c28beb532","order_by":1,"name":"Minwoo Oh","email":"","orcid":"","institution":"NIE: National Institute of Ecology","correspondingAuthor":false,"prefix":"","firstName":"Minwoo","middleName":"","lastName":"Oh","suffix":""},{"id":433999440,"identity":"4868d612-0e81-4162-bf1b-243228a299ec","order_by":2,"name":"Eun Ju Lee","email":"","orcid":"https://orcid.org/0000-0003-3669-9004","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Eun","middleName":"Ju","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-03-15 12:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6232839/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6232839/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79909783,"identity":"2ac9bb8c-2ce6-4bed-8158-da72d149fd9a","added_by":"auto","created_at":"2025-04-04 11:25:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261022,"visible":true,"origin":"","legend":"\u003cp\u003eSection and plan view of the bioassay arena.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6232839/v1/305c6831d027f37091f8b170.png"},{"id":79909782,"identity":"decc1ec6-8037-41ac-bb70-6d81022ec1ef","added_by":"auto","created_at":"2025-04-04 11:25:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75224,"visible":true,"origin":"","legend":"\u003cp\u003eResults of trophic preference of dung beetles. The bars indicate the mean number of dung beetles at traps for each mammal trophic guild. The significance levels were based on pairwise comparisons. The asterisks refer to statistical significance: \u0026lt;0.001 ‘***,’ \u0026lt;0.01 ‘**,’ \u0026lt;0.05 ‘*,’ \u0026lt;0.1 ‘.’.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6232839/v1/2ae782ebe2987009c910cf98.png"},{"id":79910725,"identity":"4b629c33-6e7b-4c25-8273-729dfcfbaea2","added_by":"auto","created_at":"2025-04-04 11:33:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68150,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the preference of dung beetles between single trophic guilds, i.e., omnivore and herbivore. The bars indicate the number of dung beetles at each mammal. The significance levels were based on pairwise comparisons. The asterisks refer to statistical significance: \u0026lt;0.001 ‘***,’ \u0026lt;0.01 ‘**,’ \u0026lt;0.05 ‘*,’ \u0026lt;0.1 ‘.’.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6232839/v1/03de943c4f5c8b22207ac104.png"},{"id":79909785,"identity":"6dfef595-4494-4145-ad27-2cabcf23f08b","added_by":"auto","created_at":"2025-04-04 11:25:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90915,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the diel activity of dung beetles. For each species, the bar chart on the left shows the day/night comparison, and the bar chart on the right shows the daytime comparison. Note the difference in y-axis limits between the two graphs. The significance levels for the diurnal/nocturnal comparison were based on ANOVA, and the daytime activity was based on the pairwise comparisons. The asterisks refer to statistical significance: \u0026lt;0.001 ‘***,’ \u0026lt;0.01 ‘**,’ \u0026lt;0.05 ‘*,’ \u0026lt;0.1 ‘.’.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6232839/v1/9cb19d6d54ea0bcbf3aef7e9.png"},{"id":82233092,"identity":"90cd94c3-3ae1-4a50-a0b0-51d3eb0a989c","added_by":"auto","created_at":"2025-05-08 06:31:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1003801,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6232839/v1/6e01d765-e7ba-4776-879e-f1e69a76fa74.pdf"},{"id":79909787,"identity":"5b489734-6885-4c63-904e-d9765fdf7037","added_by":"auto","created_at":"2025-04-04 11:25:32","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":344068,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6232839/v1/a02bd9b1dc7118f33a701a9e.pdf"}],"financialInterests":"","formattedTitle":"Olfactometers can test dung beetle olfactory response and diel activity: a case study in South Korea.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTo understand insect ecology and behavior, controlled experiments in laboratory conditions have been widely used. There are many advantages of conducting experiments in laboratory settings to natural settings. First and foremost, it is possible to control for desired conditions while excluding unwanted ones. Plus, replicates of the same conditions can be made to increase the sample size. Second, experiments can be conducted regardless of weather conditions so that experiments can be done on schedule. Also, the results are less likely to be influenced by the weather. The downside is that there is a possibility of introducing unexpected biases, due to apparatus design, experiment design, or insect behavior (Roberts et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nevertheless, laboratory experiments have allowed us to gain valuable insights into the world of insects.\u003c/p\u003e \u003cp\u003eOne group of insects studied extensively in laboratory settings are dung beetles (Geotrupidae, Scarabaeinae, and Aphodiinae). They have been tested to check the olfactory response to different types of dung (Dormont et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dormont et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Verd\u0026uacute; et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gim\u0026eacute;nez G\u0026oacute;mez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Perera et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), olfactory responses to different single or multiple volatile organic compounds (VOCs) (Inouchi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Frank et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), ecosystem services (Bang et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Nervo et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; deCastro-Arrazola et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and reproduction and development (Finn \u0026amp; Giller, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Bang et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These experiments were often conducted in tandem with field experiments to ascertain findings made in the field.\u003c/p\u003e \u003cp\u003eOlfactory responses of dung beetles are tested using olfactometers, an apparatus used to study how insects respond to volatile chemical compounds. In the case of dung beetles, their ability to differentiate between dung types (Dormont et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dormont et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and their preference (Dormont et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Salom\u0026atilde;o et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gim\u0026eacute;nez G\u0026oacute;mez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Perera et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have been investigated. Other experiments have sought to \u0026ldquo;look outside the dung\u0026rdquo; and explored dung beetle preference for acorns (Verd\u0026uacute; et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and rotting meat, arthropod carcasses, fungi (Gim\u0026eacute;nez G\u0026oacute;mez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The results of these studies were similar to field experiments, in some cases with higher accuracy (Gim\u0026eacute;nez G\u0026oacute;mez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This shows that olfactometers are versatile and accurate tools for testing dung beetle preference.\u003c/p\u003e \u003cp\u003eIn addition to olfactory responses, it may be possible to test dung beetle diel activity using olfactometers. Many dung beetles burrow into the ground when they are not searching for dung (Houston \u0026amp; McIntyre, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Caveney et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Depending on the time of day when they surface and become active, they are categorized into diurnal, nocturnal, and crepuscular species. While many species were found to be active around noon (Krell-Westerwalbesloh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), interspecific differences exist. The differences may stem from physiological factors such as body temperature requirements, competition in the form of temporal resource partitioning, or both (Krell-Westerwalbesloh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Feer \u0026amp; Pincebourde, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Information on diel activity and its cause can give us clues about dung beetle morphology, evolutionary history, and life cycle (Tocco et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ribeiro et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which can help us speculate the effects that climate change will have on dung beetles.\u003c/p\u003e \u003cp\u003eBut how do dung beetles know when to search for resources? Flight chambers were used in laboratory experiments to understand the mechanism behind dung beetle flight behavior (Houston \u0026amp; McIntyre, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Caveney et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In these experiments, dung beetles were placed in a shallow basin with loam, where they would bury themselves. Afterward, the basin was placed inside the flight chamber, and the dung beetles would emerge and eventually fly when conditions were right.\u003c/p\u003e \u003cp\u003eHowever, most experiments on dung beetle diel activity have been conducted in the field. Several pitfall traps or dung pats are deployed in the field, and they are checked at regular intervals (e.g., four hours) or at different times of the day (e.g., dawn, morning, noon, afternoon, dusk, night, etc.) depending on the research question (Krell-Westerwalbesloh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Feer \u0026amp; Pincebourde, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lobo \u0026amp; Cuesta, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, this is often very resource-intensive. More dung is required to attract dung beetles in outside settings, where 250 g to 1 kg of dung was used per trap/pat (Krell-Westerwalbesloh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kamiński et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lobo \u0026amp; Cuesta, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) compared to 15\u0026ndash;50 g of dung in indoor settings (Dormont et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Salom\u0026atilde;o et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Plus, it is very labor-intensive as many traps/pats must be deployed to obtain a sufficient sample size. Then, they need to be revisited at each interval to collect the dung beetles (Krell-Westerwalbesloh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lobo \u0026amp; Cuesta, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, bad weather conditions, such as rain, can make it difficult to conduct the experiment on schedule and possibly affect the results.\u003c/p\u003e \u003cp\u003eOlfactometers could combine the functions of the traps used in outdoor settings and flight chambers. This way, both olfactory response and diel activity can be measured simultaneously. It has the advantages of being less resource- and labor-intensive while allowing the experiment to be conducted regardless of the weather. In this study, such an olfactometer was designed and built to assess the olfactory responses and the diel activity of dung beetles in South Korea. These species\u0026rsquo; olfactory responses have only been tested in the field (unpublished data; Table A1), while there are no records of their diel activity.\u003c/p\u003e \u003cp\u003eTo ascertain that olfactometers can be used to serve dual purposes, the accuracy of the olfactometer in measuring olfactory response and diel activity will be assessed in this study. To do this, three hypotheses were tested. First, dung beetles will exhibit similar olfactory responses to the field results. Second, differences in dung preference will exist within the same mammal feeding guild. Third, dung beetles will be active at specific times of the day, with many species active at noon.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Arena design\u003c/h2\u003e \u003cp\u003eThe olfactometer designs were adapted from Perera et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) in the use of the square shape and pitfall traps, and Verd\u0026uacute; et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) in the use of a control trap with no dung and the use of a buffer in the center. This study used soil instead of leaf litter as a buffer, as used in Houston and McIntyre (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), so that dung beetles can bury themselves outside their activity period. The olfactometer was made of a 5 mm acrylic sheet, assembled in a cube shape with dimensions of 600 mm \u0026times; 600 mm \u0026times; 400 mm (width, length, height) with the top and bottom open. A \u0026ldquo;field\u0026rdquo; for dung beetles to search for dung was made with a 5 mm acrylic sheet, placed 120 mm above the ground. A 10 mm foam board was placed above the acrylic sheet to prevent dung beetles from slipping. The floor had five holes where five \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varnothing\\:\\)\u003c/span\u003e\u003c/span\u003e100 mm beakers were placed. The beaker in the center was the \u0026ldquo;starting point\u0026rdquo; for the dung beetles and was filled with soil. The four beakers in each corner had funnels and roof structures that acted as pitfall traps. The roof structures held broth bags filled with dung baits. A mesh cover was placed on the top of the arena to prevent dung beetles from escaping. The sides of the arena were bordered with cardboard, and blinds in the laboratory were lowered so that direct sunlight could not enter the arena. Artificial light was used to supplement natural light in the relatively dark indoor setting during the day (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). An \u0026ldquo;IKEA TR\u0026Aring;DFRI LED bulb E26 1055 lumen\u0026rdquo; was used for lighting. The color temperature was adjusted to 2700K from 07:30\u0026thinsp;~\u0026thinsp;10:30 and 16:30\u0026thinsp;~\u0026thinsp;19:30, and 4000 K from 10:30\u0026thinsp;~\u0026thinsp;16:30. Airflow within the arena was checked with a smoke test.\u003c/p\u003e \u003cp\u003eBecause the roof structure was made of foam boards that may absorb odors, one type of dung was assigned for each roof structure. Each corner of the arena was assigned a mammal feeding guild (carnivore, herbivore, omnivore) and control (no dung) to minimize the possibility of mixing up different odors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experiment\u003c/h2\u003e \u003cp\u003eSix experiments were conducted with six dung beetle species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Experiments one through four compared dung from mammals of three trophic guilds: omnivore, herbivore, and carnivore. It was based on the assumption that dung from mammals of the same trophic guilds has a similar attraction to dung beetles (Hanski \u0026amp; Cambefort, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Mart\u0026iacute;n-Piera \u0026amp; Lobo, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). However, experiments five and six were carried out to verify this assumption, which is why comparisons between two omnivores and two herbivores were made, respectively. A control trap without any bait was also used in all experiments to test if dung beetles are trapped by factors other than olfactory response. Before the main experiments, three pilot studies were conducted with \u003cem\u003eCopris tripartitus\u003c/em\u003e using boar, cattle, and leopard cat dung.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e(a) List of species used for this experiment and (b) combinations of mammal dung used for the lab experiment. Six experiments were carried out using dung from different mammals. All six species were used for all six experiments. Hyphens (-) indicate no dung for the respective dung type.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e(a) Species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c8\" namest=\"c4\"\u003e \u003cp\u003e(b) Experiment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e#\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies name\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e#\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHerbivore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOmnivore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarnivore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePhelotrupes auratus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecattle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eboar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eleopard cat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSisyphus schaefferi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ewater deer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eboar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eleopard cat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCopris tripartitus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecattle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eraccoon dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eleopard cat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eOnthophagus fodiens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ewater deer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eraccoon dog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eleopard cat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eOnthophagus rugulosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eraccoon dog / boar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAphodius rectus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecattle / water deer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Test subjects and materials\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe six dung beetles chosen for the experiment were \u003cem\u003ePhelotrupes auratus\u003c/em\u003e, \u003cem\u003eSisyphus schaefferi\u003c/em\u003e, \u003cem\u003eC. tripartitus\u003c/em\u003e, \u003cem\u003eOnthophagus fodiens\u003c/em\u003e, \u003cem\u003eOnthophagus rugulosus\u003c/em\u003e and \u003cem\u003eAphodius rectus\u003c/em\u003e. The omnivore dung was from boar (\u003cem\u003eSus scrofa\u003c/em\u003e) and raccoon dog (\u003cem\u003eNyctereutes procyonoides\u003c/em\u003e), the herbivore dung was from cattle (\u003cem\u003eBos taurus\u003c/em\u003e) and water deer (\u003cem\u003eHydropotes inermis\u003c/em\u003e), and the carnivore dung was from Leopard cat (\u003cem\u003ePrionailurus bengalensis\u003c/em\u003e). All the dung beetles and mammals were native to South Korea.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eDung beetles with varying dung preferences were chosen, which were identified with the indicator species analysis (De C\u0026aacute;ceres \u0026amp; Legendre, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) with data from field experiments (unpublished data; Table A1). \u003cem\u003eA. rectus\u003c/em\u003e and \u003cem\u003eO. rugulosus\u003c/em\u003e were attracted to boar dung, \u003cem\u003eC. tripartitus\u003c/em\u003e to boar and cattle dung, \u003cem\u003eS. schaefferi\u003c/em\u003e to boar and leopard cat dung, and \u003cem\u003eO. fodiens\u003c/em\u003e and \u003cem\u003eP. auratus\u003c/em\u003e to all types of dung. All species were collected at Yeoncheon-gun, Gyeonggi-do province, except for \u003cem\u003eC. tripartitus\u003c/em\u003e, which could not be captured in the wild due to its status as an endangered species (NIE, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As such, they were brought from Jeju Technopark Biodiversity Research Institute via a research loan for the period of the experiment. All dung beetles were kept in cages half-filled with soil when not used for the experiment.\u003c/p\u003e \u003cp\u003eFor each experiment, 20\u0026ndash;30 individuals of dung beetles were used, which was determined with the power test (Table A2). The power test was done using the \u0026ldquo;pwr\u0026rdquo; package (Champely, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although the use of different individuals per test is suggested (Roberts et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), same individuals were used multiple times for the experiment because of the difficulty of collecting endangered species \u003cem\u003eC. tripartitus, O. rugulosus\u003c/em\u003e, and \u003cem\u003eS. schaefferi\u003c/em\u003e (Bae et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDung from mammals was collected and portioned into bags of 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e3 grams. Boar dung was collected at a farm in Taebaek-si, Gangwon-do province. Cattle dung was collected at Seoul National University Animal Farm in Pyeongchang-gun, Gangwon-do province. Leopard cat dung was collected at Seoul Zoo. Raccoon dog feces were collected from Seoul Wildlife Center when raccoon dogs were rescued and in rehabilitation. Water deer dung was collected near the field experiment sites in Yeoncheon-gun, Gyeonggi-do province. They were frozen until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Procedure\u003c/h2\u003e \u003cp\u003eBefore the experiment, the beaker in the center was half-filled with soil, and the four beakers on the sides were filled to 5\u0026ndash;10 mm, depending on the size of the species. Then, frozen dung was put into broth bags and tied underneath the roof structure. 24\u0026ndash;30 individuals of dung beetles were placed in the center beaker and covered up with soil to the top. Only one species of dung beetle was used per experiment, and in the case of dung beetles whose sex could be distinguished easily (\u003cem\u003eC. tripartitus, O. rugulosus, O. fodiens\u003c/em\u003e), the sex ratio was matched to approximately 1:1. In between the experiments, the roof structure, plastic funnels, beakers, and the floor board were washed to get rid of the odors from the previous experiment. The roof structure and floor boards were washed with tap water, while the plastic funnels and beakers were washed thoroughly with detergent.\u003c/p\u003e \u003cp\u003eEach experiment started at noon or after 19:30, depending on the activity pattern of the species. The beaker in the center was covered with an object above the soil for approximately 30 minutes so that dung beetles could get accustomed to the environment. The traps were checked every three hours from 07:30 to 19:30. The experiment was carried out for 48 hours, but when less than 20 individuals fell into the traps within this time, the experiment continued until 20 individuals made their choice. The experiments for \u003cem\u003eC. tripartitus, O. rugulosus, O. fodiens, P. auratus\u003c/em\u003e, and \u003cem\u003eA. rectus\u003c/em\u003e were conducted from 24 September to 5 November in 2023, while the experiments for \u003cem\u003eS. schaefferi\u003c/em\u003e were conducted from 8 July to 26 July in 2024. Due to the seasonality of dung beetles, experiment six for \u003cem\u003eO. fodiens\u003c/em\u003e and experiments five and six for \u003cem\u003eA. rectus\u003c/em\u003e could not be conducted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Data analysis\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Dung preference\u003c/h2\u003e \u003cp\u003eThe results of experiments one through four were used to find the trophic preference of dung beetles. The results of the four experiments were used to fit generalized linear models (GLM) for the number of individuals that fell into each trap. A Poisson distribution with link function log was used, and the mammal trophic guild was used as the fixed variable. For each model, residual fit was assessed with the DHARMa package (Hartig, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and the Analysis of Variance (ANOVA) was performed to test for statistical differences. Then, pairwise comparisons between trophic guilds were conducted using the emmeans package (Lenth, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The p-values of the pairwise comparisons were adjusted using the Tukey method.\u003c/p\u003e \u003cp\u003eThe results of experiments five and six were used to find the preference of dung beetles within the same mammal trophic guild, i.e., herbivore and omnivore. A GLM was fitted for each experiment, using the same methods employed for experiments one through four, except that mammal species were used as the fixed variable. All statistical analyses were conducted using R (R Core Team, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Diel activity\u003c/h2\u003e \u003cp\u003eThe results of all six experiments were pooled to analyze the diel activity of each dung beetle species. First, whether dung beetles were nocturnal or diurnal (day/night) was assessed. This was done by comparing the number of dung beetles caught in the traps at 07:30 (night) to the sum of individuals caught at other times, i.e., 10:30, 13:30, 16:30, and 19:30 (day). Second, dung beetle activity during different hours of the day (daytime activity) was compared to find out when dung beetles were most active. The comparison was made between the number of individuals caught at 10:30, 13:30, 16:30, and 19:30. For both cases, GLMs were fitted using Poisson distribution with link function log, using the activity pattern or activity time as fixed variables. However, the negative binomial distribution was employed when residuals were poorly fit, using the MASS package (Venables \u0026amp; Ripley, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The consecutive methods were identical to that used for dung trophic preference.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Pilot experiment\u003c/h2\u003e \u003cp\u003eThe three pilot studies conducted with \u003cem\u003eC. tripartitus\u003c/em\u003e using boar, cattle, and leopard cat dung exhibited precise results. Preference for cattle dung was pronounced, while close to zero individuals were found in control traps (Table A3). As such, the main experiment was initiated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Dung preference\u003c/h2\u003e \u003cp\u003eThe first hypothesis that dung beetles exhibit similar olfactory response to the field experiment was not true for most dung beetles in this experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The second hypothesis that dung beetles will have preference for different mammals of the same trophic guild was valid only for \u003cem\u003eP. auratus\u003c/em\u003e, while it was only marginally true for \u003cem\u003eC. tripartitus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. auratus\u003c/em\u003e was a generalist in the field experiment. However, in the olfactometer bioassay, it displayed significant differences in preference for dung from different feeding guilds (Table A4a). Pairwise comparisons showed significant differences between omnivore dung and control, omnivore dung and carnivore dung, and herbivore dung and control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Table A5a). The difference within a single trophic guild was not statistically significant between omnivores. However, it was significant for herbivores, with water deer dung preferred over cattle dung (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Because no dung beetles fell into control traps, the comparison between control and other dung types were mostly significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. schaefferi\u003c/em\u003e was attracted to omnivore and carnivore dung in field settings. But in laboratory settings, herbivore and omnivore dung were more attractive than carnivore dung, and less individuals were found in traps with carnivore dung than control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Table A5a). This species did not exhibit preference to different mammals within a single trophic guild (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn the case of \u003cem\u003eC. tripartitus\u003c/em\u003e, the results of the field experiment and the lab experiment matched. It was attracted to herbivore and omnivore dung, with statistical significance against control and carnivore dung (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, Table A4a, A5a). Single trophic guild experiments showed that raccoon dog dung and water deer dung was preferred over control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003cem\u003eO. fodiens\u003c/em\u003e\u0026rsquo; lab experiment results also matched the field experiment, because it displayed generalist characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Table A4a, A5a). However, the comparison between control and other traps were also insignificant. This species did not display preference for different mammals within a single trophic guild (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003cem\u003eO. rugulosus\u003c/em\u003e preferred omnivore dung in the field experiment. However, while the difference between trophic guilds was significant (Table A4a), pairwise comparisons only showed a significant difference between control and omnivore (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, Table A5a). The difference within a single trophic guild did not yield significant results, but boar dung and water deer dung had more individuals than raccoon dog dung and cattle dung, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. rectus\u003c/em\u003e was attracted to omnivore dung in the field experiment. However, this was different in laboratory conditions. Preference towards dung from different trophic guilds was exhibited (Table A4a), but herbivore dung and carnivore dung were preferred (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, Table A4a, A5a).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Diel activity\u003c/h2\u003e \u003cp\u003eThe third hypothesis that dung beetles would be active at specific times of the day was true for all the species tested. Also, as expected, many diurnal species were active at noon. All dung beetles except for \u003cem\u003eC. tripartitus\u003c/em\u003e were diurnal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table A8). For daytime activity, species had distinct patterns. \u003cem\u003eP. auratus\u0026rsquo;\u003c/em\u003e activity was spread relatively evenly throughout the day (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Table A4b, A5b). For \u003cem\u003eS. schaefferi\u003c/em\u003e, activity peaked during noon, and declined sharply after 16:30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Table A4b, A5b). In comparison, \u003cem\u003eO. rugulosus\u003c/em\u003e was most active later in the day, with peak activity at 16:30 and reduced activity at 10:30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, Table A4b, A5b). The daytime activities of \u003cem\u003eO. fodiens\u003c/em\u003e and \u003cem\u003eA. rectus\u003c/em\u003e were similar, with activity peaks from noon to 16:30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, Table A4b, A5b). Because \u003cem\u003eC. tripartitus\u003c/em\u003e was a nocturnal species, no significant relationship was observed for daytime activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eContrary to the first and second hypotheses, the results obtained differed from the field experiments, and dung beetle olfactory response was not demonstrated for mammals of different trophic guilds nor within the same trophic guild. As assumed in the power test, it was expected that no dung beetles would fall into control traps. However, more dung beetles than expected were trapped in the control traps, obscuring the results.\u003c/p\u003e \u003cp\u003eThe main reason that dung beetles were found in control traps is because of flight activity, which caused them to fall into random traps. During the experiment, many dung beetles flew into the olfactometer wall or mesh cover and \u0026ldquo;accidentally\u0026rdquo; dropped into one of the traps (personal observation). This suggests that a change in olfactometer design is required to prevent these accidents. A good example is the design used by Verd\u0026uacute; et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), which had dung beetles walk through a narrow tunnel at the sides of the central chamber to get to the source of the VOCs. This would require more effort from dung beetles, and result in reduced numbers at control traps. In fact, Verd\u0026uacute; et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) demonstrated the feasibility of this design by showing that significantly more dung beetles were attracted to resources than control traps.\u003c/p\u003e \u003cp\u003eThe second reason is that the sun's direction may have presented problems with orientation. Dung beetles orient themselves with the direction of the sun, which was reported many times with \u0026ldquo;roller\u0026rdquo; dung beetles, or dung beetles that form dung balls and roll them away to nest elsewhere (Byrne et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; el Jundi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although only \u003cem\u003eS. schaefferi\u003c/em\u003e belonged to the roller group, all dung beetles used in this experiment moved towards the sun in the cages where they were kept before and in between experiments (personal observation). Despite the blinds and cardboards set up to prevent direct sunlight from entering the olfactometer, polarized light or light intensity gradients may have influenced dung beetle orientation (el Jundi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Dung beetles attempting to escape with this directional bias may randomly fall into one of the traps on the way. As such, it may be possible to address this with the design change suggested in the previous paragraph.\u003c/p\u003e \u003cp\u003eThird, the olfactometer was made of materials that could retain odors from VOCs, unlike the recommended glass or polytetrafluoroethylene (PTFE) (Roberts et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Measures were taken to prevent this from influencing the results, such as assigning a roof structure for each dung type and cleaning components of the olfactometer between experiments. However, because dung beetles possess acute olfactory senses (Dormont et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Urrutia et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), there is a possibility that residual VOCs may have influenced dung beetle activity. Yet, because larger baits attract more diversity and abundance of dung beetles (Errouissi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), it is probable that the dung baits had a far more significant influence on dung beetle response than residual VOCs.\u003c/p\u003e \u003cp\u003eAlso, experiment planning could have been improved. At the initial planning stage, it was assumed that no dung beetles would fall into the control traps. Therefore, the number of experiments and individuals used per experiment was insufficient for some species, especially ones prone to fall into control traps. In addition, dung from different mammals belonging to the same trophic guild may be different in attractiveness to dung beetles, as seen by the preference of \u003cem\u003eP. auratus\u003c/em\u003e for water deer dung over cattle dung in this experiment. Similar results were reported in the past (Frank et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Perera et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting that pooling data according to trophic guilds may not be advisable. Therefore, conducting multiple experiments with the same combination of dung is suggested.\u003c/p\u003e \u003cp\u003eOverall, the results regarding the olfactory response of dung beetles were unclear and did not align with the findings from the field experiments. However, since several potential factors were identified, new experiments with enhanced olfactometer design and improved experimental planning should yield more meaningful results.\u003c/p\u003e \u003cp\u003eIn contrast to the olfactory response, diel activity yielded clearer results. The third hypothesis that dung beetles are active at specific times of the day and that many species will be active at noon was supported. The contrast between diurnal and nocturnal activity was particularly pronounced. Like many species belonging to the tribe Coprini, \u003cem\u003eC. tripartitus\u003c/em\u003e is a nocturnal species (Hanski \u0026amp; Cambefort, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). This may be due to its large body size, which allows it to conserve heat in colder night temperatures (Lobo \u0026amp; Cuesta, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Other species were clearly diurnal. However, many individuals of \u003cem\u003eP. auratus\u003c/em\u003e and \u003cem\u003eA. rectus\u003c/em\u003e were active at night, although it was statistically insignificant. While the reason for this is unclear, covering the center beaker for 30 minutes may not have been sufficient for these species to adapt to the new environment.\u003c/p\u003e \u003cp\u003ePatterns in daytime activity were observed for most diurnal dung beetles. In line with the hypothesis, many species were active at noon, but each species had specific periods of activity. How the dung beetles could tell when to be active is not known, although it has been suggested that light intensity and temperature may be the reason (Wensler, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Houston \u0026amp; McIntyre, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Caveney et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). This experiment was conducted in an indoor setting where daytime temperatures were between 17.0\u0026deg;C and 25.5\u0026deg;C, which was higher than the threshold temperature suggested by Houston and McIntyre (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) for \u003cem\u003eOnitis alexis\u003c/em\u003e. In fact, many dung beetles were active between the lower temperature ranges between 17.0\u0026deg;C and 19.0\u0026deg;C during the experiment, likely because of the colder climates of South Korea compared to southern Africa where \u003cem\u003eO. alexis\u003c/em\u003e is from. Therefore, the largest variation in the environment was in the light conditions.\u003c/p\u003e \u003cp\u003eAlthough direct sunlight could not reach the interiors of the olfactometer, indirect sunlight was available. Dung beetles orient themselves with the position of the sun, but when it is obscured, they can use other cues, such as polarized light and changes in light intensity, to find their way (el Jundi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarly, dung beetles may be able to use these signs to discern the time of day, as polarized light changes with the sun\u0026rsquo;s position (Cronin \u0026amp; Marshall, 2011), and as light intensity changes throughout the day.\u003c/p\u003e \u003cp\u003eIn addition, although artificial light was used during the day, it does not seem to have influenced dung beetle diel activity. First, the light source was not nearly as bright as the sun. Although studies on the effect of artificial light during the day are limited, the fact that dung beetles are affected by light intensity (Wensler, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Houston \u0026amp; McIntyre, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Caveney et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; el Jundi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) suggest similar brightness to the sun is required for artificial light to influence activity. Second, the results of this experiment also suggest little influence of artificial light. The color temperature of the artificial light was maintained at 4000K from 10:30 to 16:30, but \u003cem\u003eS. schaefferi\u003c/em\u003e and \u003cem\u003eO. rugulosus\u003c/em\u003e had significantly different numbers of individuals that were active between 10:30\u0026thinsp;~\u0026thinsp;13:30 and 13:30\u0026thinsp;~\u0026thinsp;16:30. However, it cannot be dismissed that the difference in active periods may be more pronounced without artificial light, nor could it be dismissed that species such as \u003cem\u003eO. fodiens\u003c/em\u003e and \u003cem\u003eA. rectus\u003c/em\u003e were not influenced. Therefore, additional experiments are required to confirm the effects of artificial light.\u003c/p\u003e \u003cp\u003eAnother factor that may influence dung beetle diel activity is the circadian rhythm. Houston and McIntyre (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) and Wensler (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) observed that dusk-crepuscular dung beetles emerged just below the soil surface before dusk. However, when the light conditions were manipulated, they would burrow back instead of taking flight. The authors suggested that circadian rhythm directs dung beetles to surface, while light conditions dictate flight. All in all, although the actual factor for dung beetle diel activity remains uncertain, the sun's contribution appears to be considerable.\u003c/p\u003e \u003cp\u003eComparing the results of olfactory response with diel activity, it was observed that diel activity produced much clearer results. This indicates that dung beetle diel activity can be measured indoors using olfactometers, allowing the disadvantages of field experiments to be mitigated. Moreover, in the South Korean context of this study, \u003cem\u003eS. schaefferi\u003c/em\u003e and \u003cem\u003eO. rugulosus\u003c/em\u003e are rare species not found in many places. One of the few places they could be found was in forests near the Demilitarized Zone (DMZ), where frequent activity may raise suspicion from military personnel and residents, making it difficult to conduct these experiments in the field. In such special cases, lab experiments are the only feasible option.\u003c/p\u003e \u003cp\u003eWith the appropriate olfactometer design and experiment planning, both dung beetle olfactory responses and diel activity can be measured effectively. Three key factors for this are given. First, sunlight should be available where the experiment is conducted, as dung beetles rely on it to perceive the time of day. Second, soil with sufficient depth should be available at the \u0026ldquo;starting point\u0026rdquo; so dung beetles can burrow until their activity period. Third, olfactometers should be designed to prevent dung beetles from falling into random traps, whether during an attempt to escape through flight or due to directional bias presented by the sun\u0026rsquo;s position. These principles will facilitate experiments that deepen our understanding of dung beetle morphology, evolutionary history, and life cycle (Tocco et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ribeiro et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the face of anthropogenic changes such as habitat loss, overexploitation, and climate change, this understanding will better prepare us for potential impacts on insects in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis paper could not have been written without the help of many people and organizations, to whom we owe our greatest gratitude. Jeju Technopark Research Institute provided the endangered \u003cem\u003eCopris tripartitus\u0026nbsp;\u003c/em\u003eused in this experiment, where Minhee Ko kindly helped us. The staff at Seoul National University Animal farm kindly allowed us to collection of cattle dung at their farm. Leopard dung was collected with the help of Seoul Zoo. Doctor of Veterinary Medicine (D.V.M.) PhD So-Young Jung, and D.V.M. Ain Choi facilitated the administrations for dung collection, while zookeepers Seoungmin Choi and Sangha Lee collected leopard cat dung. The staff at Seoul Wildlife Center found time to help us collect raccoon dog dung, despite their busy schedule rescuing and rehabilitating wild animals. Lastly, Minseok Cho was a great help in collecting dung beetles and conducting the experiments.\u003c/p\u003e\n\u003cp\u003eFunding:\u003c/p\u003e\n\u003cp\u003eThis research did not receive any grants or funding from external sources. Fieldwork was carried out with funds from the laboratory of plant ecology (EJ Lee’s lab at Seoul National University).\u003c/p\u003e\n\u003cp\u003eConflicts of interest:\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eEthical approval:\u003c/p\u003e\n\u003cp\u003eAll applicable institutional and/or national guidelines for the care and use of animals were followed.\u003c/p\u003e\n\u003cp\u003eConsent to participate:\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication:\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data was deposited in dryad under the reference number DOI: 10.5061/dryad.jm63xsjng.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBae YJ, Cho YB, Park JY, Oh K-S, Econature (2013) vol 2). 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Anim Behav 74(6):1697\u0026ndash;1704. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anbehav.2007.03.016\u003c/span\u003e\u003cspan address=\"10.1016/j.anbehav.2007.03.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWensler RJ (1974) Crepuscular activity of adult Sericesthis geminata (Coleoptera: Scarabaeidae): Influence of circadian rhythmicity and light intensity. New Z J Zool 1(2):197\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03014223.1974.95178\u003c/span\u003e\u003cspan address=\"10.1080/03014223.1974.95178\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Activity pattern, Behavior bioassay, Insect ecology, Laboratory experiment, Resource partitioning","lastPublishedDoi":"10.21203/rs.3.rs-6232839/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6232839/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLaboratory experiments are widely used to understand insect ecology and behavior. One of the taxa frequently studied this way is the dung beetle to test olfactory responses to resources using olfactometers. Diel activity is another frequently investigated characteristic of dung beetles, but this was usually done with field experiments. The disadvantages of this are that it is labor- and resource-intensive and that weather conditions can influence the results. To address this, we propose that diel activity can also be measured with olfactometers in addition to olfactory response. A four-trap olfactometer was designed to test for inter-trophic preference between carnivore, herbivore, omnivore dung, and control (no dung). Intra-trophic preference was also examined to check differences between mammals of a single feeding guild. The diel activity was examined by checking the traps every three hours from 07:30 to 19:30. Six experiments with different combinations of dung were conducted on six dung beetle species. The six species were chosen based on their resource preference assessed from field experiments, which were expected to be mimicked in the laboratory experiment. The results for the olfactory preference were unclear and did not resemble the results of the field experiment, possibly due to limitations in olfactometer design. However, more accurate results were produced for diel activity, suggesting that using olfactometers to measure diel activity is possible. Conducting two experiments simultaneously would save time and resources and ultimately contribute to enhancing our understanding of dung beetle morphology, evolutionary history, and life cycle.\u003c/p\u003e","manuscriptTitle":"Olfactometers can test dung beetle olfactory response and diel activity: a case study in South Korea.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 11:25:27","doi":"10.21203/rs.3.rs-6232839/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e65c4ea4-cb98-4750-8e8d-8a038cfb504e","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-08T06:23:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-04 11:25:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6232839","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6232839","identity":"rs-6232839","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}