MATERIALS AND METHODS
Study
species
The experimental procedure was carried out simultaneously for two ant
species: Lasius niger (L., 1758) (Fig. 1) commonly termed as the
black garden ant, widespread in Europe and northern America (Klotz,
2008); and Lasius neoniger Emery, 1893 known as the turfgrass
ant, distributed in northern and Midwestern North America (E. O. 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; E. O. Wilson, 1955).
L. niger (Fig. 2) queens were collected in Münster (NRW, Germany)
during a single nuptial flight on July 5, 2017. L. neonigerqueens 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 L. niger and to 30°C for L.
neoniger . Colonies were overwintered at 10°C (L. niger ) and 15°C
(L. neoniger ) 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 L. neoniger was due to
concurrent experiments carried out with multiple, ecologically
different, species.
General Data
Analysis
All analysis and modelling were conducted for each species separately as
the aim of this research was not to compare the species but to study the
effects of the treatments simultaneously for both of them.
All data analysis was done in R version 3.6.2 (R Core Team, 2019).
Type-II analysis of variance tables based on 1-d.f. chi square tests
were used to assess the significance of terms in all models (e.g. lm,
glm, lme) using the ’Anova’ function in the “car” library (Fox &
Weisberg, 2018); these tests are marginal, i.e. each term is tested in
presence of all other terms in the model and tests are not
order-dependent. Data were summarized using libraries plyr (Hadley
Wickham, 2010), dplyr (H Wickham, 2018) and Rmisc (Hope, 2013). Figures
were made after the means and confidence intervals calculated from the
raw data using ggplot2 (Hadley Wickham, 2016) with RColorBrewer
(Neuwirth & Brewer, 2014).
Colony
Growth
A pilot experiment was conducted in 2015/16 with L. neoniger .
Twenty-four Colonies were monitored over six months; colonies were
distributed among three treatments varying in the protein to
carbohydrate content of their diet. Colony growth (the number of
workers) increased near linearly with increasing parts of carbohydrates,
where colonies fed a diet with a P:C ratio of 1:1 produced fewer workers
than those fed a diet of 1:2, and those fed a diet of 1:3 produced the
greatest number of workers. The experiment also documented that a weekly
provision of ~125 µl of artificial diet (see below) was
sufficient to maintain colony growth and survival. These pilot data
informed our choice of the quantity of diet to provision and the P:C
range.
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 (protein to carbohydrate
ratios, abbreviated as P:C henceforth) (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), hereafter referred to as low
carbohydrate (L), medium-low (ML), medium-high (MH), and high
carbohydrate (H). The starting number of colonies was 96 for L.
neoniger (8 colonies per treatment combination), and 86 for L.
niger (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 10 months by counting larvae and workers on a monthly
basis. 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.
Data were analysed to answer three related questions: 1) Did colony
survival vary depending on treatments? 2) Did the dynamics of growth
vary with treatments? and 3) Did overall growth vary with treatments?
G-tests (function ’G.test’) were used to assess differences in colony
survival as related to treatments using the library RVAideMemoire
(Hervé, 2014). The ’G.multicomp’ function was used for post-hoc
comparisons - p-values were corrected for false discovery rate (FDR).
A linear mixed-effects model for each species, function ’lme’ from
library nlme (Pinheiro, Bates, DebRoy, & Sarkar, 2014) was used to
assess growth dynamics. Growth was approximated as the number of workers
in each monthly census, excluding colonies that died. Feeding frequency,
diet P:C and census number (the latter as a third-order polynomial,
identified after initial visual inspection of the data) were the fixed
effects (factorial), and colony ID was expressed as a random factor (96
colonies for L. neoniger and 86 for L. niger ).
Additionally, census number was added as a first-order autocorrelation
structure (corAR1) grouped by colonies.
To assess the effects of treatments on overall growth we used a two-way
analysis of variance with the frequency of feeding and diet P:C as fixed
effects and the difference in numbers of workers between the last and
second census (i.e. the first census that had experienced the
treatments) as the response variable.
Worker
Phenotype
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 using
gravimetry after repeatedly washing the samples with ether; we used the
protocol of Tschinkel and Smith (2009). Samples were first dried for
> 48h at 60°C. Due to the low mass of Lasiusworkers, we extracted a sample of ants, en masse , for each
colony. The number of ants per colony was 8.8 ± 1.8 (mean ± SD). The
lipid extraction gave three measures, dry mass, lean mass, and the
proportion of dry mass that was lipids (i.e., the portion extracted).
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 isotopes15N and 13C (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 L. neoniger , 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 ML 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 15N labelled or unlabelled ammonium
nitrate (labelled: Ammonium nitrate-15N, EB0056; unlabelled: Ammonium
Nitrate SHBJ1050, Sigma-Aldrich, USA) and a small quantity of13C 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
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 13C and 71%
more 15N 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 L. niger at the
Botanical Garden of Münster (51°57’55.9”N 7°36’22.9”E), and for L.
neoniger 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 125 µl
portions of all four P:C ratios (as described above) in random order,
was placed near an active nest entrance. Baited nest entrances were at
least 1.5 m apart and each was used only once. 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.
After food preference for the four P:C levels had been established, we
performed an additional diet choice test with the species L.
neoniger 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 ’ML’ 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 L. neoniger and 23 for L. niger ). 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-rearedL. neoniger 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 iPhones and the
Lapse-It 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. S1).