The Allometry of Brain Miniaturization in Ants

Extensive studies of vertebrates have shown that brain size scales to body size following power law functions. Most animals are substantially smaller than vertebrates, and extremely small animals face significant challenges relating to nervous system design and function, yet little is known about their brain allometry. Within a well-defined monophyletic taxon, Formicidae (ants), we analyzed how brain size scales to body size. An analysis of brain allometry for individuals of a highly polymorphic leaf-cutter ant, Atta colombica, shows that allometric coefficients differ significantly for small ( ! 1.4 mg body mass) versus large individuals (b = 0.6003 and 0.2919, respectively). Interspecifically, allometric patterns differ for small ( ! 0.9 mg body mass) versus large species (n = 70 species). Using mean values for species, the allometric coefficient for smaller species (b = 0.7961) is significantly greater than that for larger ones (b = 0.669). The smallest ants had brains that constitute 15% of their body mass, yet their brains were relatively smaller than predicted by an overall allometric coefficient of brain to body size. Our comparative and intraspecific studies show the extent to which nervous systems can be miniaturized in taxa exhibiting behavior that is apparently comparable to that of larger species or individuals. Copyright © 2011 S. Karger AG, Basel Received: August 27, 2010 Returned for revision: September 30, 2010 Accepted after revision: October 28, 2010 Published online: January 20, 2011 W.T. Wcislo Smithsonian Tropical Research Institute Apartado 0843-03092 Balboa, Panama (Republic of Panama) Tel. +507 212 8128, Fax +507 212 8148, E-Mail WcisloW @ si.edu © 2011 S. Karger AG, Basel 0006–8977/11/0000–0000$38.00/0 Accessible online at: www.karger.com/bbe Seid /Castillo /Wcislo Brain Behav Evol 2 mammals (b = 0.77). Yet the smallest species reported in that study was 2.5 mg, which is relatively large in comparison to many arthropods. In contrast, there are detailed volumetric brain studies of particular beetle (Coleoptera) or Strepsiptera species with extremely small body sizes [e.g. Beutel et al., 2005; Grebennikov, 2008; Polilov, 2008; Polilov and Beutel, 2010], but there are no data for closely related large-bodied forms. Here we ask whether the scaling relationships observed in other taxa hold for animals with very small body sizes compared with large-bodied relatives? We studied intraspecific brain scaling in the leaf-cutter ant, Atta colombica, in which body mass spans 3 orders of magnitude, and polymorphic workers differ in behavior and physiology [Weber, 1972; Hölldobler and Wilson, 1990]. Furthermore, Atta ants have a diphasic cephalic allometry [Wilson, 1953], so we also tested for diphasic allometry in brain volume. Interspecifically, we extended an earlier study of brain scaling in ants [Wehner et al., 2007] by substantially increasing the number of species (n = 70 vs. 10); the taxonomic coverage (31 genera from 5 subfamilies and 2 informal groupings vs. 3 genera from 1 subfamily); and we included species that are over 60 times smaller (0.039 vs. 2.5 mg body mass), spanning approximately 4 orders of magnitude among species. Materials and Methods Ant Collections For the intraspecific study, specimens were collected from a single nest of Atta colombica in Gamboa, Colon Province, Republic of Panama. There is extensive continuous size variation among Atta workers [Weber, 1972], and to sample the full range of size variation we dug into various fungus chambers and collected workers in or near the gardens, along with soldiers; newly emerged callow workers were excluded, but otherwise the ages of individuals were unknown. Individuals of 70 ant species were collected either from queen-right laboratory colonies or as individual foragers, either in the vicinity of Gamboa, or near Gainesville, Fla., USA ( table 1 ). Voucher specimens are deposited in the Dry Reference Collection of the Smithsonian Tropical Research Institute, and the Museo de Invertebrados ‘Graham Fairchild’ de la Universidad de Panamá. Interspecific Body and Brain Measurements In most ant species there is continuous variation in worker size [Hölldobler and Wilson, 1990], but some species are monomorphic (i.e. a single mode in worker body size distribution), while others are polymorphic (i.e. multiple modes in the distribution of worker body size). It may be problematic to compare workers having different social roles among different species (e.g. large soldiers vs. small workers), or monomorphic and polymorphic species. Furthermore, the sampling among taxa was uneven because we included multiple individuals for the polymorphic species in order to capture the full size range in polymorphic species ( table 1 ). To address these 2 problems, we conducted 1 set of analyses using the full data set (n = 261 ants from 70 species), and a second set of analyses using mean values for each species (n = 70); we refer to these as full and reduced data sets, respectively. Histological Brain Sectioning and Volumetric Reconstructions for Atta colombica Ants were weighed using an AND  GR-202 microbalance (accuracy to 0.01 mg). The brain of each ant was quickly removed from the head capsule and immediately placed in fixative (6% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer) in preparation for standard histological sectioning. After fixation for 12–24 h, the brains were rinsed in cacodylate buffers and postfixed in 1–1.5% osmium tetroxide for 2–3 h. The brains were then rinsed in buffer followed by H 2 O and dehydrated in DMP and acetone in preparation for embedding in Epon . Brains were infiltrated in Epon, by first placing them in a 50/50 mixture of Epon/ acetone and then transferring them to 100% Epon. They were then embedded in Epon in Beem capsules and cured at 60 ° C overnight. Embedded brains were sectioned in a microtome (Microm HM 355s) at 5 m sections using stainless steel disposable knives. Serial sections were kept in order and placed individually on glass slides and then stained with toluidine blue. Coverslips were then placed over the sections using Permamont  and the sections were photographed using a Nikon 8700 camera attached to a Nikon Eclipse E600 compound microscope. Serial digital sections were then imported into a computer, and were traced, aligned and stacked using the program Reconstruct [Fiala, 2005] to calculate the 3-D volume of each brain. Measurements of Brain Mass for Interspecific Comparisons For the interspecific study we used brain mass as a measure of size. Collected ants were weighed using a Sartorius CPA2P microbalance after their removal from laboratory colonies or usually within 24 h after collection from the field. Collected ants were dissected under cold Ringer’s solution (150 m M NaCl, 24 m M KCl, 7.0 m M CaCl2, 4.0 m M MgCl2, 5.0 m M HEPES buffer, and 131 m M sucrose, pH = 7.0). The brain, including both the supraand subesophageal ganglion and all sensory lobes, was quickly removed from the head capsule, usually in less than 1 min, and then cleaned of all tracheae and fat bodies. Each brain was then placed on a small piece of tared Parafilm within a small droplet of Ringer’s solution. The Ringer’s solution was wicked away using finely twisted pieces of Kimwipes  and the brain was weighed within 4 s. To assess weight loss due to water evaporation from exposed brains, we measured weight loss through time for 5 ants of different body sizes. The steepest rate of water loss occurred within the first 20 s following removal from Ringer’s solution (data not shown). We used data points for the first 20 s starting at the time we could detect weight loss, given the 1 g resolution of the balance, to calculate the slope of the rate of weight loss from a linear regression, and we took this to be the maximum rate loss. We used this worst-case slope to calculate the expected loss of weight over the interval needed to prepare and weigh the specimen, and then expressed this weight loss as a percentage of total brain mass ( fig. 1 ).