Does hot spring bathing behavior affect the parasites and gut microbiome of Japanese macaques?

Methods

Summary

The field study will be carried out over two winter seasons (January – March 2020 and December 2020 - January 2021) and one summer season (June – September 2020) at Jigokudani Monkey Park (JMP), Shiga Heights, Nagano Prefecture, Japan (36^o43′58″N, 138^o27′46″E). The monkeys do not perform HSBB in summer, so this season will be used as a comparison. There are more than 160 Japanese macaques in the study group, but not all individuals perform hot spring bathing behavior (HSBB). We will select a stratified random subset of ca. 16 adult females, eight bathers and eight non-bathers (our natural ‘treatment’ groups in the experiment), determined based on ease of observation during the first winter season. Lower-ranking individuals do not typically have access to the hot spring, but we will try to select individuals of varying rank classes nonetheless for a more even sample.

Instantaneous sampling will be used to record behavior of focal individuals every minute during 90-minute focal-animal sessions (ca. 2 sessions/week/subject) to assess: (1) HSBB (duration and frequency), (2) nit-picking (active louse egg removal; see below) rates, and (3) grooming sites (during both self- and allo-grooming). Grooming sites of subjects will be divided into 9 body regions following Zamma (2002). To estimate louse abundance, the rate of nit-picking behavior per grooming-minute will be recorded. A previous study showed that the conspicuous behavior performed by macaques during grooming, in which they pick and remove an object from the base of the hair and subsequently ingest it (literally, nit-picking), is directed at louse eggs 99% of the time. Agonistic and other affiliative behaviors will be recorded ad libitum to assess relative dominance rank and social relationships.

Fecal samples will be collected from focal individuals immediately after defecation and placed into sealable plastic bags. At the earliest opportunity, samples will be partitioned and stored in: (1) sodium acetate-acetic acid-formalin (SAF) fixative for protozoa screening and (2) lysis buffer for bacterial analysis (Hayakawa et al., 2018). Samples will be stored in the dark at room temperature until processing.

Parasites will be identified using standard protocols. We will test for Giardia using sedimentation with trichrome staining for microscopy as well as direct immunoflourescence (DIF), for Entamoeba spp. and Balantidium coli using standard sedimentation protocols for microscopy, and for Cryptosporidium spp. using acid-fast and methyl-violet staining for microscopy as well as DIF assays. These methods also allow for general quantification of infection intensity. Molecular characterization will also be used in screening for Giardia, Cryptosporidium, and Entamoeba species, e.g. using methods developed by Itagaki et al., (2005) for Giardia and Li et al., (2017) for Cryptosporidium and Entamoeba.

Lastly, to characterize the gut microbiome, we will follow the methods developed by Hayakawa et al., (2018). DNA from feces will be extracted and purified, and then the V3-V4 region of 16S rRNA gene will be amplified. Raw sequences will be processed with a 97% similarity cutoff to classify operational taxonomic units (OTUs). For taxonomic identification, OTUs are assigned through the ribosomal database project (RDP) classifier at 50% confidence. Using a similar framework, and related to the parasitological work noted above, we will also attempt to classify the gut eukaryome (helminths and protozoa only) – i.e. the community of eukaryotic organisms inhabiting the gut of each macaque. Like the gut microbiome, the gut eukaryome also has implications for the health and fitness of free-living organisms.

Challenges

Although we will only follow sixteen individuals, working with social animals living in a group of 160 and in a popular tourist destination imposes its own set of challenges. The work requires good concentration and focus, as well as the ability to avoid disturbance of and by tourists. Selecting the most appropriate set of subjects is also not a straightforward task.

Moreover, as the field site lacks appropriate equipment and facilities, we will have to exercise due diligence in transferring samples to the Kyoto University Primate Research Institute (KUPRI) in Inuyama and conduct molecular analyses there in a timely fashion to minimize issues related to sample degradation.  

Lastly, it may not be immediately clear as to what mechanism might underlie variation in parasitism and gut microbiome, if observed, as this study is correlational in nature. However, observing variation is the beginning of any rigorous ecological study, and we believe now is the time to begin such an endeavor with these unique hot-spring bathing monkeys

Pre Analysis Plan

Our research aims to investigate the effects of HSBB on parasitism and gut microbiota in Japanese macaques inhabiting JMP, Shiga Heights, Nagano, Japan.

Several questions guide this research: (1) Does HSBB have effects on the risk of protozoan infection in Japanese macaques, i.e. does the diversity and intensity of infection vary between bathing and non-bathing individuals? (2) Does HSBB impact the gut microbiome of Japanese macaques, i.e. does its alpha, beta and gamma diversity differ across bathing and non-bathing individuals, and are the patterns similar for the gut eukaryome as well? (3) Does HSBB impact the abundance and distribution of lice on the body of Japanese macaques?

The following hypotheses frame this research toward testing the above questions. HSBB occurs primarily or only in winter, so individuals that bathe (bathers) are predicted to have higher risk of intestinal protozoan infection during this season than non-bathers if hot springs are a source of infection. Simultaneously, or perhaps consequently, bathing will significantly alter gut bacterial composition, either directly through differential exposure to bacterial types or indirectly through mediation of the gut eukaryome or other aspects of macaque physiology (e.g. stress physiology, immunology, etc.). In contrast, we predict ectoparasites to be less common or distributed differentially on the body in bathers due to heat exposure; the heat should restrict louse activity (e.g. egg-laying) to parts of the body that are rarely immersed (e.g. head, shoulders). In summer, we predict no significant differences in protozoan infection, gut microbiome/eukaryome or ectoparasite load between bathers and non-bathers, as bathing is restricted to winter.