DOI: 10.5061/dryad.g79cnp5n1
title: Data from: Growth and survival of the superorganism: ant colony macronutrient intake and investment

description: In this study, we used two common ant species (Lasius niger and L.
 neoniger) to assay how they translate variation in the diet (both in
 composition and frequency) into growth. We measured colony development for
 over 8 months and measured several phenotypic traits of the worker caste,
 and examined whether forager preference corresponded with diet quality.
 Optimal colony growth was a balance between survival and growth, and each
 of these were maximized with different nutrient regimes. Interestingly,
 forager preference was not totally aligned with the diet that maximized
 colony growth. Our results highlight that: 1) organism and superorganism
 size are controlled by the same nutrients, and this may reflect a common
 molecular basis for size across life’s organizational levels, 2) there are
 nutrient trade-offs that are associated with life-history trade-offs,
 likely leading to selection for a balanced diet, and 3) the connection
 between the preference of foragers for different nutrients and how
 nutrient combinations affect colony success and demographics are complex
 and only beginning to be understood.
description: Lasius species colony growth on experimental diets
 The experimental procedure was carried out simultaneously for two
 ant species: <i>Lasius niger</i> (L., 1758) (Fig. 2) commonly
 termed as the black garden ant, widespread in Europe and northern America
 (Klotz, 2008); and <i>Lasius neoniger</i> Emery, 1893 known as
 the turfgrass ant, distributed in northern and Midwestern North America
 (Wilson, 1955). Both species are highly abundant in urban/suburban lawns
 and gardens and have similar diets. Natural diets are hemipteran honeydew
 and living and dead insects (Klotz, 2008; Wilson, 1955).
 <i>L. niger</i> (Fig. 2) queens were collected in
 Münster (NRW, Germany) during a single nuptial flight on July 5, 2017.
 <i>L. neoniger</i> queens were collected in Richmond (IN,
 U.S.A) during two nuptial flights on June 30 and July 12, 2017. Queens
 were housed in 18x150 mm test tubes half-filled with water and plugged
 with cotton. Most females initiated egg-laying after the first week. No
 food was provided until the emergence of the first workers’ cohort. Once
 workers were present, colonies were housed in 500 ml plastic boxes
 containing cotton-stoppered test tubes with water. Box walls were covered
 with fluon (polytetrafluoroethylene) to prevent escape. Colonies were
 housed in incubators at 25°C for <i>L. niger</i> and to 30°C
 for <i>L. neoniger</i>. Colonies were overwintered at 10°C
 (<i>L. niger</i>) and 15°C (<i>L. neoniger</i>)
 for two and one months, respectively, between January and February 2018.
 An acclimatization phase of 15 days prior and post overwintering period
 was allowed for gradual temperature transition. The higher temperature
 used in <i>L. neoniger</i> was due to concurrent experiments
 carried out with multiple, ecologically different, species. Differences in
 rearing and overwintering temperatures were due to differences in lab
 constraints. Colonies in the present study were
 randomly assigned to treatments after the first census (approximately one
 month of growth). The treatments were feeding frequency and diet
 composition (P:C) (Fig. 3). Feeding frequency had three levels: a 125 µl
 aliquot every other week (0.5), every week (1), or twice per week (2). The
 P:C had four levels representing a linear change in P:C from approximately
 1:2 up to 1:16 with increments of rough doubling (see Fig. 3 for details).
 The exact ratio used differed slightly between species, as follows (L.
 niger/L. neoniger): 1:2/1:1.7; 1:4/1:3, 1:8/1:5, 1:16/1:14.5 (see Table S1
 for details). For simplicity, the ratios for <i>L. niger
 </i>are those used in figure and table labels. The difference in
 diets between species was an unintentional artefact of differing lab
 protocols – though the diets of both species are roughly linear and
 represent similar increments. The starting number of colonies was 96 for
 <i>L. neoniger</i> (8 colonies per treatment combination), and
 86 for <i>L. niger</i> (7-8 colonies per
 combination). Portions were 125 µl (127.33 ± 0.38 mg,
 mean ± SD) of isocaloric chemically defined diets prepared using casein,
 whey protein, egg powder and sucrose in agar solution (Dussutour &
 Simpson, 2008). Insect vitamins (Vandersant, MP Biomedicals, Germany) and
 a preservative (Methylparaben, Dephyte, Germany) were also added equally
 to the diets. For details on the diet constitution and recipes see Table
 S1. Food leftovers were removed after three days and new batches of food
 were prepared every three weeks. The development of the colonies was
 monitored over eight monthly censuses (disregarding the months spent
 overwintering) by assessing the worker number. Every colony census was
 done in a non-invasive way by visually inspecting the test tubes under a
 stereomicroscope (SZX7, Olympus, USA) at 40X magnification. Colonies were
 considered ‘dead’ only if the queen died. Several
 different worker traits were measured: head width, dry mass, lean mass and
 lipids content. These characteristics, individually and in aggregate, are
 typically responsive to nutrition and change with colony-level development
 and health (Smith & Tschinkel, 2009; Tschinkel, 1993). Head width
 of young workers (recognized by their lighter colouration, Fig. 2) was
 used as a proxy of body size (Schwander, Rosset, & Chapuisat,
 2005). Head width images were taken at 56X magnification using a
 stereomicroscope (SZX7, Olympus, USA) with an attached camera (Retiga
 2000R, Q-Imagine, Canada) and measured at the maximal distance across the
 eyes using iSolutions-Lite software (iMT Technology, USA). The number of
 ants measured per colony varied due to availability (8 ± 2.6, mean ± SD).
 Lipid extractions were done following the protocol of protocol of
 Tschinkel and Smith (2009). In brief, samples were first dried for
 &gt; 48h at 60°C and weighed. Subsequently, we repeatedly washed the
 samples with diethyl ether (Merck KGaA, Darmstadt, Germany) in a Soxhlet
 extractor during 24h. Finally, the ants were dried again after for
 &gt; 48h at 60°C and weighed. This process yielded three measures,
 namely, dry mass, lean mass, and the proportion of dry mass that was
 lipids (i.e., the portion extracted, calculated as (dry mass – lean
 mass)/dry mass). Due to the low mass of <i>Lasius</i> workers,
 we extracted a sample of ants, <i>en masse</i>, for each
 colony. The number of ants per colony was 8.8 ± 1.8 (mean ± SD). Both dry
 mass and lean mass were normalized as the mass of the sample divided by
 the number of ants in the sample. Linear models ('lm' function)
 were used to analyse dry mass, lean mass and lipid content; head width was
 analysed using generalized linear models ('glm' function) with
 Gamma distribution. Stable Isotopes Pulse
 Experiment In order to examine the flow of nutrients
 into a colony, we used a stable isotope pulse. Diets enriched with the
 heavy stable isotopes <sup>15</sup>N and
 <sup>13</sup>C (when abbreviating carbon with C we use the
 superscript for atomic mass in order to differentiate from C for
 carbohydrates) were fed once and the flow of the isotopes to different
 castes was assayed over time. After the final census of <i>L.
 neoniger</i>, a subset of eight colonies in the ML treatment were
 randomly assigned to either of two treatment groups, those receiving the
 heavy isotope enriched diet (Table S1) or those receiving an unenriched
 (but otherwise identical) control diet. Five of the eight colonies were
 provided with the enriched diet, and the remaining three were fed the
 unenriched diet in order to control for background isotopic levels. The
 1:4 diet used in the previous experiment was modified so that isotopically
 labelled materials could be easily integrated. The basic methods of Shik
 et al. (2018) were followed. A small quantity of
 <sup>15</sup>N labelled or unlabelled ammonium nitrate
 (labelled: Ammonium nitrate-15N, EB0056; unlabelled: Ammonium Nitrate
 SHBJ1050, Sigma-Aldrich, USA) and a small quantity of
 <sup>13</sup>C labelled or unlabelled glucose (labelled:
 D-Glucose-1-13C, MBBC0227V; unlabelled: D-(+)-Glucose, SLBJ0583V,
 Sigma-Aldrich, USA) were added so that the P:C ratio of the diet was not
 changed. The supplemental ammonium nitrate amounted to 0.9% (by dry mass)
 of the protein sources in the diet, and the supplemental glucose was
 approximately 4% of the carbohydrate sources in the diet. Colonies were
 offered 125 µL of the diet for 24 h. After this period, the food was
 removed and a random sample of approximately five workers and five (mostly
 last-instar) larvae was withdrawn, frozen for 24 h, and then stored in a
 drying oven at 60°C (following the methods of Smith & Tillberg
 (2009)). Colonies were then sampled again after 96 h following the same
 procedure. Dried samples were homogenized in sterile aluminium capsules
 and elemental analysis was carried out at the University of California at
 Davis Stable Isotope Facility using a PDZ Europa ANCA-GSL elemental
 analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer
 (Sercon Ltd., Cheshire, UK). The atomic per cent enrichment was calculated
 as the atomic per cent of the heavy isotope (relative to the lighter
 isotope) divided by the same quantity for the same sampling time and caste
 in the unlabelled colonies. The individuals in colonies fed enriched
 diets, overall, had 9% more <sup>13</sup>C and 71% more
 <sup>15</sup>N compared to individuals in colonies fed
 unlabelled diets. Each element was analysed separately using the atomic
 per cent excess as the response variable. Time (24 vs. 96 h) and
 developmental caste (worker vs. larva) were the categorical predictors.
 Due to heterogeneity of variances, these data were analysed using a
 Kruskal-Wallis test followed by a Dunn test ('dunnTest' function
 in the FSA library (Ogle, 2018)) for pairwise differences using the
 Benjamini-Hochberg method. Diet preference under field
 and lab conditions Field trials were conducted between
 May and June 2018 in grassy lawns for both naturally occurring colonies of
 <i>L. niger</i> (N = 23) at the Botanical Garden of Münster
 (51°57'55.9"N 7°36'22.9"E), and for <i>L.
 neoniger</i> (N = 30) on the campus of Earlham College in Richmond,
 Indiana (39°49'15.2"N 84°54'47.0"W). A 7.5 cm
 microscope slide with four 125 µl portions of every P:C ratio (1:2, 1:4,
 1:8 and 1:16) in random order, was placed near an active nest entrance.
 After placement, the number of ants at each food portion was counted every
 five minutes during 40 minutes. If other ant species were seen at any food
 portion at any location, then that time point and all subsequent time
 points at that location were not included in the analysis. We assumed that
 all food portions had an equal probability of discovery. Baited nest
 entrances were at least 1.5 m apart and each was used only once. Previous
 studies have shown that <i>Lasius</i> colonies usually have
 one or few open nest entrances (Traniello & Levings, 1986), and
 the foraging activity of the workers is concentrated between 10-20 cm from
 the nest entrance (Traniello, 1983). After food
 preference for the four P:C levels had been established, we performed an
 additional diet choice test with the species <i>L.
 neoniger</i> only. The diet was modified to ascertain whether P:C
 preference was driven by P content and/or C content. Levels of P were
 varied (holding C constant), and then separately, levels of C were varied
 (holding P constant). We used the midpoint for these diets to be the most
 preferred 1:4 diet (see Fig. 3) and then halved and doubled either P or C.
 We then presented three sets of diets to field and lab colonies (as above)
 - all paired diets had either constant C or constant P (i.e., they were
 either on the vertical or the horizontal black line in Fig. 3).
 Diet preference data collected from field colonies were
 square-root transformed, in order to homogenize variances, and analysed
 using a mixed-effect model to take into account the non-independence of
 repeated counts at each colony. The model included the "bait"
 type (i.e., the diet P:C) as a fixed factor, and colony ID as a random
 factor (18 colonies for <i>L. neoniger</i> and 23 for
 <i>L. niger</i>). A first-order autocorrelation structure
 (corAR1) was specified as “Time | colony ID / bait type”. As above, the
 'lme' function from the nlme library (Pinheiro et al., 2014) was
 used to analyse data. Food preference trials were also
 conducted on two laboratory-reared <i>L. neoniger</i> colonies
 (c.a. two years old with over one hundred workers). Food portions were
 placed in foraging arenas attached to colony containers and then foraging
 was filmed using an Apple iPhone 4 with the Lapse-It (2017 free version,
 http://www.lapseit.com/) application for time-lapse. Pictures were taken
 every 5 minutes for at least 3.5 hours and all ants at each bait were
 counted for every time period. Laboratory data were not statistically
 analysed due to low sample size. Both colonies showed the same preference
 to the four diet ratios, but only one colony of the pair responded with
 high recruitment to the variable P and C diets (Fig. S2).
description: Datasets are named descriptively and are either about colony
 growth or diet. NAs indicate no data (i.e., when a
 colony was dead) while 0s indicate a lack of a measured item. neoniger or
 Lneo = Lasius neoniger, niger or Lnig = Lasius niger. "diet" and
 "freq" refer to the two treatments as described in the methods -
 diet is the amount of carbs relative to one part protein and
 'freq' is the feeding frequency. WeeklyP and WeeklyC refer to
 the actual amounts of each protein and carbs provided.