The Beginning of the Study on Urinary Markers in Bats

In 2017, I embarked on my first postdoctoral project at the National Institute of Ecology in South Korea. Initially, I struggled to kickstart a new project on gibbons. Dr. Sun-Sook Kim, the leader of my research team, at that time shared an interesting observation: Eastern Bent-winged bats (Miniopterus fuliginosus) often urinate when handled. This reminded me of my PhD course, where I studied the hormones and behaviour of wild bonobos in the DR Congo. I realized that I could initiate a similar study on bats using their urine. In July 2017, I had the opportunity to capture and handle bats for the first time in Mungyeong, South Korea (Fig. 1). As anticipated, they frequently urinated during body size measurements, enabling me to collect urine samples from over 20 individuals in two surveys in a single night. This was a stark contrast to my graduate school days, where I would follow a single female bonobo for several kilometres from dawn to dusk in the African rainforest just to collect her urine. Despite the challenge of dealing with flying insects and mosquitoes, this task was relatively easier and rewarding. The first field trip compensated for all the worry and frustration I had experienced due to my inability to initiate the primate research I desired in my new location.

Progress of the Study

Following the successful completion of my initial field trip on bats, I decided to broaden my research scope from primates to bats. After about a year of collecting urine samples, it was time to analyse them. Fortunately, I discovered that Dr. Hyeong Cheol Park’s lab at the same institute had the necessary equipment. With his assistance, I was able to complete the urine analysis. However, the process was not without its challenges. There were instances when the computer systems connected to the equipment malfunctioned, and the analysis sometimes extended into the night. One particularly stressful aspect was the distinctive odour of urine from some individuals. After processing hundreds of urine samples (Fig. 2), I would often feel nauseous and lose my appetite. While we don’t have data to draw any conclusions, I speculate that this odour could potentially be used to determine the presence or absence of Eastern Bent-winged bats in a specific habitat.

Upon completing the urine analysis, my colleague and I began exploring the pilot data. It seemed unlikely that our urine samples would show evidence of ketone bodies accumulation in urine, which would be expected if the sampled bats exhibited excessive fat metabolism. The concentration of acetoacetate detected in our diluted urine sample was notably low. However, we found expected seasonal and individual variations in urine creatine concentrations. We found no sex differences, which contrasts with findings from a study on Greater Horseshoe Bats in South Korea [1]. In that study, urinary creatinine concentrations varied with both microenvironment and sex.

In a following comprehensive analysis, we found that the concentration of creatinine, which is related to urine concentration in bats, could be well explained by their body mass and forearm length. Although we did not initially expect these two factors to interact and affect an individual’s urine concentration, our statistical model revealed such a significant interaction effect (Fig. 3). At the study design phase, we had not given much thought to the surface volume ratio (SVR), which would be a crucial factor influencing water stress in bats. However, after obtaining the results, we realised that given their large SVR compared to other small mammals without wings, the effect of SVR must be considered beforehand. It is easy to imagine that two bats with the same forearm length, but different body mass would have different SVRs. An increase in body mass probably increases body volume more than surface area, and an increase in forearm length results in an increase in surface area due to its effect on wing dimension. With this consideration, the statistically significant interaction term seems rather obvious in hindsight, but it was something my colleagues and I had overlooked at the beginning.

Submission and Acceptance of the Paper

In 2022, my colleagues and I drafted the paper and completed the first submission. We deeply considered the implications of the significant interaction term. Unlike previous papers that suggested differences in surface volume ratio (SVR) between species resulted in various adaptations, we proposed that within the same species, individuals with different SVRs would experience varying levels of water stress, affecting their behaviours. For instance, torpor bout lengths during day roosting could vary among individual bats. However, after first review, one reviewer criticised that our data did not align with our claimed. After several readings of the review, I found myself agreeing with the reviewer. Without any experimental data and based solely on the statistical significance of the correlation, it was a considerable logical leap to suggest that the difference in SVR between individual bats could affect the torpor bout length.

Our paper was finally accepted for publication after about a year of revision process. We toned down our arguments and condensed our discussion on the implications of our findings. I anticipate the day when follow-up studies, both experimental and observational, will test for differences in SVR between individuals as a function of their feeding status and degree of fat deposition, and the resulting differences in their energy expenditure and water stress management strategies. I hope that our study will serve as a starting point for further experiments and detailed observational studies. Lastly, I would like to take this opportunity to express my gratitude once again to everyone who contributed to this study.

References

1. Ryu, H., Kinoshita, K., Joo, S. & Kim, S.-S. Urinary creatinine varies with microenvironment and sex in hibernating Greater Horseshoe bats (Rhinolophus ferrumequinum) in Korea. BMC Ecol. Evol. 21, 77 (2021). https://doi.org/10.1038/s42003-023-05183-5
2. Ryu, H., Kinoshita, K., Joo, S., Choi, Y.-S. & Kim, S.-S. Increased urinary creatinine during hibernation and day roosting in the Eastern bent-winged bat (Miniopterus fuliginosus) in Korea. Commun. Biol. (2023). https://doi.org/