DENTINE ENAMEL enamel prisms ameloblasts at EFF striae of Retzius (old EFFs) Havers - Halberg Rhythms in Canis familiaris . Russell T. Hogg 1 , Timothy G. Bromage 2 , Bin Hu 2 1 Rehabilitation Sciences, FGCU; 2 Basic Sciences and Biomaterials & Biomimetics, NYU College of Dentistry Figure 2. Variation of RP (i.e., HHO) in mammals. The left image shows a histological section of an eastern wooly lemur molar (Avahi laniger), whose RP = 3 days (yellow arrows = striae of Retzius, with 3 cross-striations visible between them, red arrows demarcate transition between striations). The right image shows a section from an elephant molar (Elephas recki), where RP = 14 days (black arrows = cross striations, blue lines = striae of Retzius). Acknowledgments & References HHO was quantified as the periodicity, in days, between successive striae of Retzius (Retzius periodicity, RP) in 11 domestic dogs of different breeds (see Table 1). To obtain this periodicity, 24-hour growth increments (cross-striations) were counted between successive striae of Retzius on histological specimens. Data were gathered from embedded thin sections of teeth, imaged using polarized light microscopy. For information on the interpretation of RP from histological sections, see Figs. 1 and 2. Natural logarithms (ln) of RP data were regressed against ln body mass and compared to prior results obtained for primates (Hogg et al. 2015). r 2 = 0.8 p < 0.01 n = 48 r 2 = 0.05 p = .3 n = 22 100 μm Discussion Introduction Results Materials & Methods We aim to assess variation in Havers-Halberg oscillation (HHO) periodicities among domestic dogs (Canis familiaris). The HHO is hypothesized to be a hypothalamus- generated biorhythm that coordinates multiple life history variables including body size, metabolic rate, and reproductive output (Bromage et al. 2009). The periodicity of this clock varies among and within species, but not within individuals. In monkeys and apes, HHO periodicity is strongly correlated with body mass, but in lemurs this is not the case. Lemurs also have a very restricted range of HHO variation (Hogg et al. 2015). This raises the question of how HHO rhythms and body size evolution can be decoupled. Domestic dogs have high body mass variation but low variation in life history variables such as lifespan, linked to intense artificial selection for IGF1 gene sequence variants (Sutter et al. 2007) rather than neuroendocrine mechanisms such as the HHO. Based on this, we hypothesize that HHO variation in dogs 1) will be narrow considering their body mass range, and 2) will not be correlated with body mass; they will therefore resemble lemurs, providing potential insight into lemur evolution. Specimen Breed Sex RP ln RP Body Mass (kg) ln Body Mass 2599 ? f 5 1.609 3.345 1.208 3410 Spaniel m 5 1.609 11.350 2.429 3474 ? f 5 1.609 31.780 3.459 3475 ? m 5 1.609 15.770 2.758 3515 St. Bernard m 5 1.609 90.710 4.508 3532 ? ? 4 1.386 6.894 1.931 3535 French Poodle f 6 1.792 20.259 3.009 3544 Doberman m 6 1.792 17.187 2.844 3545 Collie m 5 1.609 20.259 3.009 3546 German Shepherd m 4 1.386 16.826 2.823 "Harold" English Bulldog ? 4 1.386 27.240 3.305 Figure 1. Formation of striae of Retzius,. An ameloblast epithelium deposits enamel matrix appositionally, moving from the dentine toward the future enamel surface (curved black line to right). Over a predetermined rhythmic interval, a visible line forms in enamel at successive positions of the epithelium (enamel formation front, or EFF). the striae of Retzius. 24-hour growth lines (cross-striations) can between striae can be summed to determine the RP (see Fig. 2 & 3). Similar growth lines appear in dentine. The hypotheses are corroborated, in that 1) dogs have a narrow range of HHO variation, and 2) dog HHO period is not significantly correlated with body mass. HHO periodicities in dogs range between 4-6 days even though our sample spans nearly 2 orders of magnitude in body mass. This may explain why dogs can have such a large size variation, yet have a fairly consistent life history package in terms of lifespan and reproductive output. I.e., IGF1 mutations explain the body size variation, but do not have an impact on HHO biology and life history, which seems not to have been as affected by artificial selection. Based on our results, we predict that similar physiological mechanisms may be at play in lemurs. Figure 3. HHO periodicities in sections from Canis familiaris. The left image is taken from dentine, the right image from enamel. Large white arrows signify long-period growth lines (Andresen lines in dentine, striae of Retzius in enamel), whereas small white arrows indicate one group of 24-hour growth increments between two long period lines. In both cases, the RP = 4 days. Table 1. Raw data included in study. Body mass data are taken from records for the individuals included. RP (HHO) Group ln body mass ln RP (HHO) ln RP (HHO) ln body mass r 2 = 0.12 p = 0.74 n = 11 Group ln body mass Fig. 4. RP variation for the same body mass range among all three groups. Dogs and lemurs (purple and green) both exhibit much less variation than monkeys and apes (blue). Fig. 5. ln body mass across RP = 4, 5, and 6 (the dog range). Both dogs have an overall larger body mass for the same RP as monkeys and apes, implying a faster life overall life history (t test p = .007). For lemurs, n = 2 so no analysis was run. This research funded by the 2010 Max Planck Research Award which is endowed by the German Federal Ministry of Education and Research to the Max Planck Society and the Alexander von Humboldt Foundation in respect of the Hard Tissue Research Program in Human Paleobiomics. Bromage TG, Lacruz RS, Hogg RT, Goldmann HM, McFarlin SC, et al. (2009) Lamellar bone reconciles enamel rhythms, body size, and organismal life history. Calcif Tissue Int 84: 388-404. Hogg RT, Godfrey LR, Schwartz GT, et al..(2015) Lemur biorhythms and life history evolution. PLOS One 10: e0134210. doi:10.1371/journal.pone.0134210. Sutter NB, Bustamante CD, Chase K, Gray MM, Zhao K, et al. (2007) A single IGF1 allele is a major determinant of small size in dogs. Science 316: 112-115. Fig. 6. Regression of ln RP against ln body mass among domestic dog sample. Results corroborate the hypothesis that body mass and HHO are not significantly correlated in dogs. Fig. 7. Regression of ln RP against ln body mass among primates (from Hogg et al. 2015). Lemurs have a poor correlation and narrow range of variation in RP relative to body size, similar to dogs. D = Daubentonia.