GoKawiilAnimals and housing
Long-term laboratory colonies of naked mole-rats (H. glaber) were maintained in Berlin, Germany and Pretoria, South Africa. Damaraland mole-rat (F. damarensis) colonies were maintained in Pretoria, and Micklem’s mole-rat (Fukomys micklemi), Mashona mole-rat (Fukomys darlingi) and Mechow’s mole-rat (Fukomys mechowii) were maintained in České Budějovice, Czech Republic. (The following species were kept in the laboratory (Pretoria, South Africa) for unrelated studies after being collected from the wild: Highveld mole-rat (Cryptomys hottentotus pretoriae), Natal mole-rat (Cryptomys hottentotus natalensis), common mole-rat (Cryptomys hottentotus hottentotus), Cape mole-rat (G. capensis), Cape dune mole-rat (B. suillus), and Emin’s mole-rat (H. emini).
In Berlin, naked mole-rats were kept under controlled environmental conditions, with ambient temperature kept at 30–32 °C and humidity at 50–70%, under dim illumination. Eighteen colonies were housed in custom-designed, interconnected plastic chamber systems (Fräntzel Kunststoffe). Animals were provided with a daily ad libitum diet of tubers (primarily sweet potatoes, celery root, cucumbers, bananas and carrots) and supplemented weekly with ProNutro (Bokomo). All husbandry and experimental procedures were approved by the local governmental authorities in Berlin (Landesamt für Gesundheit und Soziales, licenses G 0196/17 and G 0121/23). When selecting animals for experiments, a non-invasive salivary swab assay was established to determine sex by genotyping. In brief, saliva was collected from live naked mole-rats using a swab (Geyer). The extraction of genomic DNA was performed with proteinase K. Precipitation of the DNA was performed to concentrate the DNA solution used as template for the PCR. To determine whether the animal is a male, we amplified a 163-bp region of the sex-determining gene on the Y chromosome, Sry using Hg-SRY-For 5′-GAAGAACGGCCATTTTTCGG-3′ Hg-SRY-Rev 5′-GCATTCATGGTGTGGTCTCG-3′.
To check the DNA quality and PCR condition, we amplified a 446-bp region of the mitochondrial 16S rRNA gene using Hg-16S-For, 5′-TGGTGATAGCTGGTTGTCCA-3′ and Hg-16S-Rev, 5′-TAGTCTTTCCTTGCGGCACT-3′, amplicon detectable in both sexes as previously described57.
In South Africa, eight colonies of naked mole-rats were maintained in tunnel systems consisting of multiple plastic chambers (designated for food storage, toileting and nesting) connected by acrylic glass tunnels. Animals were fed a varied diet of chopped vegetables (primarily sweet potatoes, cucumbers and carrots) with weekly ProNutro supplementation. Nesting material consisted of wood shavings. The holding rooms were kept at 29–32 °C with 50–70% relative humidity.
In the Czech Republic, the animal rooms of Micklem’s mole-rat, Mashona mole-rat and Mechow’s mole-rat were kept at 25 ± 1 °C, 50 ± 10% relative humidity and photoperiod of 12 h light:12 h dark. Animals were fed ad libitum with vegetables (such as carrots, potatoes, sweet potatoes, beetroot, apple and cucumbers) and a rodent dry food mix. Animals were given the opportunity to carry out their natural digging behaviours in peat and were provided extra enrichment such as tree branches and plastic tubes for gnawing.
All procedures were approved by the relevant institutional animal care and use committees and local governmental authorities in Berlin (Max Delbrück Center; Landesamt für Gesundheit und Soziales, licenses G 0196/17 and G 0121/23) and Pretoria (University of Pretoria), in accordance with national and international guidelines for the ethical treatment of laboratory animals. Procedures in Pretoria were approved by the Animal Ethics Committee of the University of Pretoria (license no. NAS199/2020, NAS071-2023, NAS313/2022, NAS324/2022, NAS209-2021, NAS011/2025 and NAS130_2025) and received DALRRD Section 20 approval (SEpi-Bizhub24110620262, SDAH-Epi-22101309360, SDAH-Epi-23032315040, SDAH-Epi-23041710040, 12/11/1/1/8 (2002 LH), SEpi-Bizhub25021311032 and SEpi-Bizhub25061009490). All naked mole-rats used in this study were derived from colonies originally captured by J. Jarvis, primarily in Mtito Andei and Lerata, Kenya, and represent a mixed parentage1.
Behavioural assays
Social rank determination
To assess dominance hierarchies within the eight colonies from South Africa, we followed a previously established assay for naked mole-rat social rank22,58. In brief, two plastic chambers were connected by a transparent tube, with one animal placed in each chamber. When both entered the tube simultaneously, the individual climbing over the other was scored as dominant. Each dyad was tested in at least three trials, with pairings pseudo-randomized and repeated across multiple months. A ranking index (R.I.) was calculated as wins divided by total trials and normalized to the colony maximum (R.I. = 1 for the top-ranking individual). Ranks were assigned categorically (rank 1: R.I. > 0.8, rank 5: R.I. < 0.2). For experiments, we used high-ranking individuals with R.I. ≥ 0.7 and low-ranking individuals with R.I. ≤ 0.3.
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