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\begin{document}

\title{Growth and survival of the superorganism: ant colony macronutrient
intake and investment}



\author[1]{Yeisson Gutiérrez}%
\author[2]{Tung Phung}%
\author[2]{Harald Mumma}%
\author[2]{Arden Ambrose-Winters}%
\author[3]{Christoph Scherber}%
\author[2]{Chris Smith}%
\affil[1]{University of Münster}%
\affil[2]{Earlham College}%
\affil[3]{Westfälische Wilhelms-Universität Münster}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
In this study, we used two common ant species (\emph{Lasius niger} and
\emph{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. Individuals (workers) and colonies (superorganisms)
increased in size with increasing amounts of protein in the diet, and as
a function of how much food was available. 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.%
\end{abstract}%



\sloppy


\section*{INTRODUCTION~}

{\label{introduction}}

Nutrition is an upstream organizer of most organismal processes, and
thus dealing with variation in the availability of nutrients is among
the most consequential challenges, and selective agents, faced by all
organisms (Stearns, 1992; Zera \& Harshman, 2001). While many nutrients
are necessary for maintenance and growth, the majority consumed and used
are macronutrients ingested in high quantity, such as proteins,
carbohydrates, and fats (Raubenheimer, Simpson, \& Mayntz, 2009). The
molecular mechanisms that translate incoming macronutrients into
maintenance, new tissue, or storage, are well understood, and tend to be
highly conserved (Conlon \& Raff, 1999).

Superorganisms, like eusocial insects, are groups of individuals of the
same species operating in a synergistic way (D. S. Wilson \& Sober,
1989). Superorganisms are characterized by the~reproductive~division of
labour, such that some individuals do not reproduce~(somatic~-- germ
line division) (H\selectlanguage{ngerman}ölldobler \& Wilson, 2009). The nutritional challenges
of any cell or organism are also faced by~superorganisms, but are
compounded by the additional level of complexity (cooperating organisms
on top of cooperating cells, etc.) -- the labour that is divided amongst
cells in `unitary' organisms is divided amongst individuals in
the~superorganism. How, then, do nutritional challenges scale from
organism to~superorganism?~Do organism and superorganism have different
nutritional optima and/or face the same nutrition-mediated trade-offs?
If individual and superorganism optima are not aligned, then processes
occurring at the individual level may limit those at the superorganismal
level.

Social insect colonies undergo a coordinated development that is
responsive to the environment and also lineage/species specific, termed
`sociogenesis' (E. O. Wilson, 1985). `Sociometry' is a general term
applied to the metrics associated with superorganisms, such as colony
size, individual size (and size distributions), the numbers and ratios
of different castes, among others. As a general pattern in studies of
sociometry and sociogenesis, worker size increases with the number of
workers in the colony (Smith \& Tschinkel, 2006; Tschinkel, 1987, 1993,
1998). This relationship is at least in part due to increasing resource
availability to developing individuals in larger colonies - increased
nutrient availability does increase worker production and worker size
(Goetsch, 1937; Metzl, Wheeler, \& Abouheif, 2018; Passera, 1974; Smith,
2007; Wills et al., 2015). Increased protein/nitrogen content has also
been associated with the production of larger workers (majors/soldiers)
and sexual castes~(Bono \& Heithaus, 2002; Goetsch, 1937; Hunt \&
Nalepa, 1994; Passera, 1974; Schmidt, Hunt, \& Smith, 2012; Smith,
Anderson, Tillberg, Gadau, \& Suarez, 2008; Smith \& Suarez, 2010;
Smith, 1942). However, previous studies have been largely observational
or used natural foods~(e.g., increased insect prey) that confound
protein/nitrogen content with many other nutrient
differences.~Furthermore, many previous studies focus on short-term
colony dynamics, documenting adult worker survival rather than
longer-term growth (e.g., Dussutour \& Simpson, 2009, 2012; Wills et
al., 2015).

The nutritional basis of individual and superorganism size is a major
unresolved issue in the understanding of social insect growth and
development. In the present study, we used ants as a model superorganism
and employed a nutritional geometry framework (Simpson \& Raubenheimer,
1995) that allowed for the manipulation of both the ratio and amounts of
protein and carbohydrate available to colonies. We hypothesized
that~worker size and colony size would both increase with increasing
availability of protein in the diet -- that is, worker number and size
are both protein-limited. This hypothesis is consistent with sociometric
observations. An important alternative hypothesis is that worker and
colony development are limited by different nutrients (i.e., worker and
colony optima differ). For example, worker size may indeed be
protein-limited, but colonies may be more carbohydrate limited due to
the metabolic demands of the standing crop of adult workers. This
alternative hypothesis is premised on there being a nutritionally
mediated life-history trade-off between growth (new tissue)
and~maintenance (current tissue, Fig. 1).

A division of labour inherent in social insect colonies is between
growing individuals (larvae) and those that maintain the colony
(workers). To further explore possible trade-offs between growth and
maintenance, we examined the flow of nutrients through these two types
of individual, larva and worker, using a stable isotope tracer
experiment. We hypothesized that protein would be more rapidly and
completely transferred to larvae while carbohydrates would persist
longer in workers.

Lastly, we examined natural (field) colony preference for macronutrient
ratios to test whether preference matched the nutrient ratios that
maximized growth in the lab. We hypothesized that colonies would prefer
what maximized lab growth. However, an alternative hypothesis is that
worker preference is not aligned with colony growth (under lab
conditions), but instead is more tuned to the adult worker optimum of
high carbohydrates -- this may be selectively advantageous by favouring
colony maintenance over growth. Furthermore, workers are the collectors
of the food and preferences based on their own physiology and current
state build from the ancestral condition of solitary insects. Other
alternative hypotheses include that colonies respond based on experience
or environmental variation, among other factors.

\section*{MATERIALS AND METHODS}

{\label{materials-and-methods}}

\subsection*{\texorpdfstring{\emph{Study
species}}{Study species}}

{\label{study-species}}

The experimental procedure was carried out simultaneously for two ant
species: \emph{Lasius niger} (L., 1758) (Fig. 1) commonly termed as the
black garden ant, widespread in Europe and northern America (Klotz,
2008); and \emph{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).

\emph{L. niger} (Fig. 2) queens were collected in Münster (NRW, Germany)
during a single nuptial flight on July 5, 2017. \emph{L. neoniger}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 \emph{L. niger} and to 30°C for \emph{L.
neoniger} . Colonies were overwintered at 10°C (\emph{L. niger} ) and
15°C (\emph{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 \emph{L. neoniger} was due to
concurrent experiments carried out with multiple, ecologically
different, species.

\subsection*{\texorpdfstring{\emph{General Data
Analysis}}{General Data Analysis}}

{\label{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).

\subsection*{\texorpdfstring{\emph{Colony
Growth}}{Colony Growth}}

{\label{colony-growth}}

A pilot experiment was conducted in 2015/16 with \emph{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 \textasciitilde{}125 \selectlanguage{greek}µ\selectlanguage{english}l 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 \selectlanguage{greek}µ\selectlanguage{english}l 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 \emph{L.
neoniger} (8 colonies per treatment combination), and 86 for \emph{L.
niger} (7-8 colonies per combination).

Portions were 125 \selectlanguage{greek}µ\selectlanguage{english}l (127.33 \selectlanguage{ngerman}± 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 \emph{L. neoniger} and 86 for \emph{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.

\subsection*{\texorpdfstring{\emph{Worker
Phenotype}}{Worker Phenotype}}

{\label{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
\textgreater{} 48h at 60°C. Due to the low mass of \emph{Lasius}workers,
we extracted a sample of ants, \emph{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.

\subsection*{\texorpdfstring{\emph{Stable Isotopes Pulse
Experiment}}{Stable Isotopes Pulse Experiment}}

{\label{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\textsuperscript{15}N and \textsuperscript{13}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 \emph{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 \textsuperscript{15}N labelled or unlabelled ammonium
nitrate (labelled: Ammonium nitrate-15N, EB0056; unlabelled: Ammonium
Nitrate SHBJ1050, Sigma-Aldrich, USA) and a small quantity
of\textsuperscript{13}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 \selectlanguage{greek}µ\selectlanguage{english}L 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\selectlanguage{ngerman}°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 \textsuperscript{13}C and 71\%
more \textsuperscript{15}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.

\subsection*{\texorpdfstring{\emph{Diet preference under field and lab
conditions}}{Diet preference under field and lab conditions}}

{\label{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 \emph{L. niger} at the
Botanical Garden of Münster (51°57'55.9''N 7°36'22.9''E), and for
\emph{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 \selectlanguage{greek}µ\selectlanguage{english}l
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 \emph{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 \emph{L. neoniger} and 23 for \emph{L. niger} ). A
first-order autocorrelation structure (corAR1) was specified as ``Time
\textbar{} 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\emph{L. 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).



\section*{RESULTS}

{\label{results}}

\subsection*{\texorpdfstring{\emph{Colony Survival and
Growth}}{Colony Survival and Growth}}

{\label{colony-survival-and-growth}}

The first census of colony growth was in August 2017 and the last in
April 2018 (months spent overwintered were not included in the
analysis). Colony survival was affected only by diet treatment
in\emph{L. neoniger} (G = 9.19, df = 3, \emph{P} = 0.03, Fig. 4a), but
neither species showed an effect of feeding frequency on survival. Only
the diet low in carbohydrates (L) showed decreased survival relative to
all other treatments (\emph{P} \textless{} 0.05 in all comparisons after
FDR correction). For both species, each fixed effect (diet P:C, feeding
frequency and time) in the mixed-effects model was statistically
significant, as were the interactions Time*Diet and Time*Frequency (Fig.
5, Table S2). The latter two-way significant interactions suggest that
the dynamics of diet P:C and feeding frequency changed with time, some
of which were likely due to differences becoming more pronounced over
time. Additionally, species-specific effects on growth were apparent
from the non-recovery of \emph{L. neoniger} colonies post-winter,
especially due to differential survival (above).

In the analysis of overall growth, diet P:C and feeding frequency were
statistically significant for both species, but there were no
significant interactions (Fig. 4b, Table S3). The high carbohydrate diet
(H) produced the lowest growth, differing from the low carbohydrate
(L,\emph{P} \textless{} 0.05) and medium-high carbohydrate (MH,
\emph{P}\textless{} 0.005) diets in post-hoc comparisons. The frequency
that colonies were fed was highly significant, with only colonies fed
every other week suffering reduced growth relative to the other two
treatment levels (\emph{P} \textless{} 0.001 in both post-hoc
comparisons). Colonies of \emph{L. niger} grew larger than colonies of
\emph{L. neoniger} , seemingly owing to differences in overwintering and
recovery from winter

\subsection*{\texorpdfstring{\emph{Worker
Phenotypes}}{Worker Phenotypes}}

{\label{worker-phenotypes}}

Differences among treatments in worker phenotypes are summarized in Fig.
6 for both species and full results are given in Table S4. All measured
traits were significantly affected by diet P:C and feeding frequency
separately in \emph{L. niger} , but only head width exhibited a
significant diet P:C x Frequency interaction. In \emph{L. neoniger} ,
worker lean mass and lipid content were affected by diet P:C only,
whereas head width was affected by feeding frequency only. \emph{L.
neoniger} worker dry mass was the only trait affected by both diet P:C
and feeding frequency, and no interactions were significant for all the
measured traits of this particular species.

Overall, increasing protein content in diet increased head width, lean
and dry mass (Fig. 6). But as noted above, the effect was the opposite
for worker lipid content. Although \emph{L. neoniger} head width was not
affected by diet P:C, the directional difference in means is consistent
with the results obtained for lean and dry mass (i.e., more
proteinaceous diets correspond to larger workers).

In addition, while feeding frequency only had a statistically
significant effect on \emph{L. neoniger} worker dry mass (both higher
frequency treatments were marginally different than the lower
frequency,\emph{P} = 0.06 and \emph{P} = 0.08), the direction of the
effect was consistent across all dependent variables assessed for both
species (Fig. 6) - more frequent feedings (i.e., greater food
availability) led to larger and fatter individuals.

\subsection*{\texorpdfstring{\emph{Stable Isotope Pulse
Experiment}}{Stable Isotope Pulse Experiment}}

{\label{stable-isotope-pulse-experiment}}

There was a statistically significant effect of caste by sample time for
atomic enrichment of both \textsuperscript{15}N and\textsuperscript{13}C
(K-W test, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} \textgreater{} 12, d.f. = 3, \emph{P}
\textless{} 0.005 for both \textsuperscript{15}N
and\textsuperscript{13}C). It can be noticed that incoming nutrients are
first ingested by workers, with worker atomic per cent
of\textsuperscript{15}N and \textsuperscript{13}C peaking at 24h. The
24h workers had nearly double the atomic enrichment of
\textsuperscript{13}C compared to all other treatments and had five-fold
higher enrichment of\textsuperscript{15}N (Fig. 7). Workers were
statistically different (\emph{P} \textless{} 0.05) from larvae at both
24 and 96-hour collections for \textsuperscript{15}N, but the difference
was not statistically significant in the 24-96 hour comparison
for\textsuperscript{13}C. No other pairwise comparisons were
statistically significant. We had predicted that we would see a movement
of more nitrogen to larvae relative to carbon, but this was not evident
in the data. However, if anything the opposite appears more likely.
Larval atomic per cent \textsuperscript{15}N remained constant over both
sample times while the mean for atomic per cent \textsuperscript{13}C
increased over sampling points (this result is trending toward being
statistically significant, \emph{P} = 0.11). Larval C:N was much higher
than that of adult workers, 7.24 \selectlanguage{ngerman}± 0.18 vs. 5.00 ± 0.11 (mean ± SE,
F\textsubscript{1,39} = 108.9, \emph{P} \textless{} 0.001), likely due
to the high lipid content of larvae. Thus, despite a high protein need
relative to workers for growth, the carbohydrate needs of larvae were
substantial.

\subsection*{\texorpdfstring{\emph{Diet
preference}}{Diet preference}}

{\label{diet-preference}}

About 20 baiting trials were conducted for each species in the field,
but several time intervals and bait stations had to be discarded due to
intrusion by other species or a lack of \emph{Lasius} presence. \emph{L.
niger} clearly tended to avoid the diet with the highest protein content
(termed ``L'' in this study because of the low carbohydrate content) but
did not differentiate among the remaining diets (Fig. 8), although
pair-wise comparisons revealed that ML and MH were significantly
preferred over L (P \textless{} 0.05). On the other hand, \emph{L.
neoniger} exhibited an overwhelming preference for the ML diet, with
diet being highly significant in the mixed-effects model
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 52.01, \emph{P} \textless{} 0.001). This trial
was also replicated with two \emph{L. neoniger} (ca. 2 years) lab
colonies, and the result was qualitatively similar (data not shown).

In order to understand whether the preference for ML was driven by the
numerator (carbohydrates) or the denominator (protein) in the ratio, we
offered new baits with both macronutrients to \emph{L. neoniger} field
colonies, yet in series where only one varied. We hypothesized that the
preference for ML was a composite of a combined preference for
carbohydrates and proteins, especially because their response to the
gradient in ratios was non-linear. When carbohydrates were held constant
the ants had a strong preference for lower levels of protein; the number
of ants present with the least amount of protein was double that
compared to the most protein. The intermediate level of protein received
an intermediate level of visitation. The lab results were consistent
with the field data. The effect of diet in these trials was again highly
statistically significant (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 29.09,
\emph{P}\textless{} 0.001). When protein was held constant, the ants had
an increasing preference for more carbohydrates, with the preference
increasing with increasing levels of carbohydrates
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}= 13.47, \emph{P} = 0.001) (Fig. S1).

\section*{DISCUSSION}

{\label{discussion}}

We describe a robust pattern where individual and superorganism (i.e.
ant colony) growth increased with dietary protein, both as a function of
the amount provided and its quantity relative to carbohydrates. This
pattern was supported across two independent experiments on two common
ant species in central Europe and the midwestern United States. These
results support our hypothesis that colony size and the size of
individuals within colonies are protein limited. Availability of protein
likely constrains colony growth and is also a possible regulator of size
and caste determination within colonies.

We also show evidence of nutrient trade-offs for colony growth and
survival, as well as for individual size and lipid storage. A visual
schematic of nutrient trade-offs is depicted in Fig. 1, such that for a
particular trait there is a single optimal P:C. Trade-offs exist when
different traits have different optima, namely, when the lines
representing trait values over differing P:C values intersect. The lines
for both colony (growth vs. survival) and individual traits (size vs.
lipid content) intersect in our data (Fig. 9). The area of intersection
represents the optimal investment, assuming that each of the traits has
equal fitness effect. For both individual and colony-level traits, for
both species, the intersection is at the level of the intermediate P:C
used in this study. Interestingly, we may have detected the optimum P:C
for both colony survival and growth; that is, intermediate P:C levels
have the highest value of both survival and growth. On the other hand,
the maximum values for the individual-level traits of size and lipid
content are at the extreme P:C levels used in this study -- the largest
workers were produced at the highest relative P and the fattest workers
at the highest relative C. The existence of trade-offs at the individual
level suggest that colonies can manipulate the nutrients available to
individuals within a nest in response to a changing external
environment. For example, in response to environmental stress, colonies
may modify the nutrients available to individuals within the nest,
biasing provisioning toward carbohydrates over protein. At the colony
level, a nutritionally mediated trade-off between growth and survival
implies that selection may optimize nutrient collection to benefit the
life-history trait most beneficial at a given time in the life cycle
(including having variable strategies for nutrient collection).

\subsection*{\texorpdfstring{\emph{Development of ant
colonies}}{Development of ant colonies}}

{\label{development-of-ant-colonies}}

Individual workers and colonies (superorganisms) increased in size with
increasing amounts of protein in the diet, and as a function of how much
food was available, and yet there was no interaction between the two
(Figs. 4-7, 9). However, the high mortality exhibited by \emph{L.
neoniger} colonies under one of the experimental diets (L) is consistent
with other studies that find either lesser stress resistance or nitrogen
toxicity in high protein/low carbohydrate diets (Fig. 4)(Cook, Eubanks,
Gold, \& Behmer, 2010; Dussutour \& Simpson, 2009, 2012). We hypothesize
that the high colony mortality was due to a decrease in lipid stores, as
individual lipid content is a buffer for colony survival (Dussutour,
Poissonnier, Buhl, \& Simpson, 2016; Christopher R Smith, 2007). The
lower survival in \emph{L. neoniger} , but not \emph{L. niger} , with
the highest protein (L) diet may be due to different overwintering
conditions for each species. \emph{L. neoniger} was overwintered at a
higher temperature compared to \emph{L. niger} (although the temperature
transition was the same for both species) and thus workers may have had
higher metabolic rates and depleted greater amounts of their lipid
reserves, leading to higher worker and colony mortality. In agreement
with our data, lipid storage in other insects is increased with
increased carbohydrate content of diet (Dussutour et al., 2016;
Warbrick-Smith, Behmer, Lee, Raubenheimer, \& Simpson, 2006). Note, the
higher temperature used in \emph{L. neoniger} was due to the concurrent
overwintering of multiple, ecologically different, species.

Other studies have demonstrated that a change in the macronutrient
ratios, toward a higher protein content, has toxic effects (Dussutour \&
Simpson, 2009, 2012; Harrison, Woods, \& Roberts, 2012; Simpson \&
Raubenheimer, 2009). However, the P:C ratios in our study were all
relatively high compared to studies documenting toxicity -- increased
protein content in our study was generally correlated with both
increased worker number and worker size. A decrease in colony growth
with even higher levels of protein was found in our preliminary study,
which may have been due to a toxicity effect. Together, our preliminary
data and the data presented in this paper suggest that optimal growth is
achieved through a trade-off between sufficient carbohydrates for lipid
storage on the one hand, and enough protein for growth on the other (but
less than will cause toxicity). In both \emph{Lasius} species, this
optimum appears to be at a P:C of 1:4 to 1:8. These results are
consistent with previous studies using similar diets, though those
studies measured mortality rather than reproduction - mortality was
lowest between 1:3 and 1:5 in \emph{Rhytidoponera} sp., \emph{L.
niger}and \emph{Solenipsis invicta} (Cook et al., 2010; Dussutour \&
Simpson, 2009, 2012).

Colony growth was non-linear with respect to the amount of food
provided. Colonies fed once per week did not grow any better than those
fed twice weekly, suggesting that we found a maximum growth rate for
colonies on the provided diets. Doubling food availability essentially
saturated their ability to turn those nutrients into new ants. Colonies
fed only once every two weeks, however, had decreased growth. Therefore,
our levels of nutrient provisioning were appropriate to assess nutrient
limitation as a function of both the amount and P:C ratio of provided
diets.

\subsection*{\texorpdfstring{\emph{Colony and Individual
Phenotypes}}{Colony and Individual Phenotypes}}

{\label{colony-and-individual-phenotypes}}

Our study demonstrated that average ant size increased with increasing
protein (relative to carbohydrates) and the total amount of diet
provided. While we did not examine gene expression in this study, it has
been established that caste determination in some social insects is
regulated behaviourally and morphologically by nutrient-sensing genes,
including those involved in insulin and Tor signalling (Wheeler, Buck \&
Evans 2006; Patel \emph{et al.} 2007; Toth \emph{et al.} 2007; and
reviewed in Chandra et al., 2018; Smith, Toth, Suarez, \& Robinson,
2008; Toth \& Robinson, 2007), and caste determination is largely
explained by size variation (Trible \& Kronauer, 2017). Thus, a
universal feature of `organismality' is that size scales with the
nutritional environment during development because the translation of
nutrients into growth is achieved through conserved processes at the
cellular level. If there existed a super-superorganism then we would
also expect that its size, along with the size of its constituent parts,
is nutritionally regulated; note that supercolonies, as seen in some ant
species (Holway, Lach, Suarez, Tsutsui, \& Case, 2002), do not likely
fit the evolutionary definition of `organism'.

While worker size (such as head width or lean mass) was regulated by the
quantity of protein in the environment, individual lipid content was
regulated by the amount of carbohydrates (Fig. 6). As noted above, there
was thus a trade-off faced by organisms with regard to their nutritional
choices for these two major macronutrients (Fig. 9). Solitary insects,
including caterpillars and last instar grasshoppers, tend to maximize
growth with P:C near 1:1 (reviewed in Behmer 2009), whereas flies
performed better with a more carbohydrate bias (Lee et al., 2008; Young,
Buckiewicz, \& Long, 2018). It is clear that in these studies there are
life-history trade-offs inherent in different P:C ratios, such as
longevity (maximized with increasing decreasing protein) and egg-laying
(higher at increased protein).

While data are currently limited across insect taxa, we hypothesize that
superorganism growth is maximized at a higher carbohydrate diet compared
to most solitary insects. This prediction is premised on the majority of
the standing biomass of a superorganism being non-growing,
non-reproducing workers. While larvae have a higher carbon to nitrogen
(C:N) ratio than workers, this is largely due to their high lipid
content. The colony's germline, reproductive gynes and males, have a
higher nitrogen content (i.e., much lower C:N ratio) (Schmidt et al.,
2012; C R Smith et al., 2008; Chris R Smith \& Suarez, 2010). A logical
extension of this prediction is that social insect growth and
development is less protein limited than solitary insects and this may
have been an inherent benefit to social living. This hypothesis is in
line with how metabolic rate scales with body size in insect organisms
and superorganisms. On a per mass basis, metabolism scales constantly
across solitary and social insects (Hou, Kaspari, Vander Zanden, \&
Gillooly, 2010). Thus, eusociality and increased colony size are
selectively advantageous with regard to increased metabolic efficiency.

\subsection*{\texorpdfstring{\emph{Nutrient flows within colonies and
colony-level
preferences}}{Nutrient flows within colonies and colony-level preferences}}

{\label{nutrient-flows-within-colonies-and-colony-level-preferences}}

We predicted that larvae would receive incoming protein,
disproportionately, compared to carbohydrates and that the opposite
would be true for workers. Using stable isotope labelling of the most
preferred and optimal diet of \emph{L. neoniger} , fed as a pulse, we
traced nitrogen (as a proxy for protein) and carbon (as a proxy for
carbohydrates) through colonies over four days. As is necessarily the
case, nutrients were transferred from workers to larvae, but the
signature of excess isotope enrichment in workers was gone in four days.
Contrary to our expectation, after four days there were no differences
between workers and larvae for either nutrient, and there, if anything
(the result is marginally statistically significant), was the opposite
pattern of nutrient transfer than predicted with larvae having slightly
more of an excess of carbon than workers, but not nitrogen. Both workers
and larvae at four days still showed atomic excess of labelled isotopes
of both elements compared to colonies fed unenriched diets (i.e., they
still had labelled nitrogen in/on their body). It is possible that
proteins were being stored in workers until larvae were hungry - the
timeframe of our study was insufficient to examine differences between
developmental castes in diet assimilation. Additional studies using the
basic isotope pulse strategy employed here, but with more time-points
and a higher resolution of sampling, will help disentangle some of the
complex interactions within colonies that are difficult to infer from
behaviour, such as the relative distribution of nutrients to different
sub-groups within a nest, and how rapidly they are distributed and
assimilated (Shik et al., 2018).

Workers are non-growing, and thus feed primarily on carbohydrates to
fuel their metabolism. As discussed above, we show that colony growth is
maximized at intermediate levels of protein in the diet. When given
choices of the same diets used to assay colony growth, colony preference
was not in alignment with the optimum for colony growth in \emph{L.
niger} -- higher protein diets increased growth, but higher carbohydrate
diets were preferred. In \emph{L. neoniger} , on the other hand, colony
preference was uncannily aligned with optimal colony growth (data were
consistent between field and lab colonies). Further work done only
with\emph{L. neoniger} found that preference for protein and
carbohydrates in the diet is a result of clear preferences for each of
the varying macronutrients, and thus the ants are judging the ratio.
When each protein and carbohydrates were manipulated individually, the
ants had a clear preference for increasing carbohydrates and for
decreasing amounts of protein. What caused a decrease in preference at
the higher levels of carbohydrates in the first preference experiment on
\emph{L. neoniger}is unclear, but the effect persisted across multiple
preparations of the diets and was consistent in the lab and field (Fig.
8, Fig. S1).

It is difficult to draw generalizable conclusions from a single
(one-season) diet/bait preference assay, and it is also difficult to
judge optimality in diet from growth only under laboratory conditions.
That being said, increased carbohydrate preference by foragers may be an
adaptive strategy because this prioritizes survival over growth, and ant
colonies tend to be long-lived. For example, when starved, colonies will
prioritize investment in growth over investment in reproduction (Smith,
2007), presumably because they will have future attempts at reproduction
should they survive the current period of low resource levels.
Preference/foraging, though, may not be optimal due to many types of
constraints (Pyke, 1984), and studies on some ants have failed to
tightly link forager preference with productivity/fitness (Seal \&
Tschinkel, 2007). Foragers are responsive to the presence of larvae in
the nest and adjust the collection of resources to more protein in their
presence (Cassill \& Tschinkel, 1999; Dussutour \& Simpson, 2009).
Therefore, it is not as though overall colony preference, as expressed
through foragers, is not regulated by feedback. Furthermore, ants are
capable of filtering nutrients once foragers return to the colony.
Although in non-social organisms this mechanism would be composed mainly
by selective absorption of nutrients and excretion of excess, ants use
the larvae as a protein stomach and the nutrients are distributed across
nest members (Deby L Cassill, Butler, Vinson, \& Wheeler, 2005;
Soren\selectlanguage{polish}ş\selectlanguage{english}en, Busch, \& Vinson, 1985; E. O. Wilson, 1976), or to
stores/trash enriched with protein (Dussutour \& Simpson, 2009).

\textbf{Conclusion}

We have shown that protein limitation scales up to limit the size of
both organisms and superorganisms. The selective benefits of size, such
as task efficiency, longevity, and metabolic efficiency, also trade-off
with physiological resource storage. These factors are predicted to
select for tuning of dietary preferences, and we found evidence of
dietary preference -- and ants appear to make decisions on the diet by
assessing multiple ingredients independently. The mechanisms by which
superorganisms integrate nutritional information and translate nutrition
into growth decisions, including feedbacks, are only beginning to be
elucidated.

\section*{ACKNOWLEDGEMENTS}

{\label{acknowledgements}}

We would like to thank D. Ott for fruitful discussions on insect
nutrition and stoichiometry, and T. Czaczkes for valuable insights on
colony housing and maintenance. We are grateful to J. Gadau, K-H. Knorr
and T. Schmidt for kindly providing materials to YG. Preliminary colony
growth data for \emph{L. neoniger} was collected by B. Crawley and M.
Saleh. Additionally, M. Bailey, I. Barrera Lopez, D. Neumeister, E.
Nicholson, S. Pant, E. Perkins, S. Slavinskas, A. Serna and M. Zenker
all helped with this study through ant care and data collection.

\textbf{Author contributions:} CRS and YG designed the experiment. YG,
TP, AAW and HM collected the data. YG, CRS and CS analysed the data. YG
and CRS wrote the manuscript, which was revised and approved by all of
the authors.

\textbf{Funding}

Financial support to YG was provided by Colciencias -- Colombia
(doctoral studies abroad program, 679-2014). Funding to CRS was provided
by Earlham College through the Ford-Knight Student-Faculty Collaborative
Research Grant, and the Summer Science Research Fund.

\textbf{Competing Interests:} The authors declare that they have no
competing interests.

\textbf{Ethical approval:} All applicable national guidelines for the
care and use of animals were followed. Permission for \emph{L.
niger}collection was granted by the city of M\selectlanguage{ngerman}ünster (67-20-0032, 2017).

\textbf{Data availability:} all raw data would be made publicly
available in the Dryad repository.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig 1.} A conceptual schematic of the relationship between
nutrient availability/use and trait value for both social colonies (top)
and individuals within colonies (bottom). The plots on the left show
overlapping optima for different social or individual traits (no
trade-off) and the plots on the right show different optima and thus
trade-offs (intersecting lines).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2---plate-Lniger/Fig-2---plate-Lniger}

\end{center}
\end{figure}

\textbf{Fig 2.} Castes and developmental stages present in a\emph{Lasius
niger} colony. Pictures were taken using a Canon 550D camera with a 100
mm macro-lens and extension rings attached. Figures are not at scale.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-3---exp-design/Fig-3---exp-design}

\end{center}
\end{figure}

\textbf{Fig 3.} Incipient ant colonies were allocated to twelve
experimental treatments (black dots) resulting from the combination of
four diets and three feeding frequencies. Values represent weekly
available nutrients. L: Low carbohydrate content; ML: Medium-Low; MH:
Medium-High; and H: High. Black lines represent the range of protein and
carbohydrates used for diet preference assay in field colonies
of\emph{Lasius neoniger} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4.} Colony growth and survival. A) Survival of colonies
in\emph{L. neoniger} was affected by diet composition
(Protein:Carbohydrates) but not by feeding frequency. In B) Colony
growth was affected by both diet composition as well as diet frequency
in both species, but these factors did not interact - the H diet had
lower growth than others, as did the lowest feeding frequency. L: Low
carbohydrate content; ML: Medium-Low; MH: Medium-High; and H: High.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-5---development/Fig-5---development}

\end{center}
\end{figure}

\textbf{Fig. 5.} Colony development was affected by diet composition
(Protein:Carbohydrates) and feeding frequency, and the dynamics changed
over time. L: Low carbohydrate content; ML: MediumLow; MH: Medium-High;
and H: High. Variation expressed as confidence intervals.~Statistical
results available in Table S2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig 6.} Workers phenotypic traits were responsive to diet P:C
and feeding frequency. Higher dietary protein led to bigger workers and
less lipid storage, and more frequent feeding was associated with bigger
workers and increased fat storage. L: Low carbohydrate content; ML:
Medium-Low; MH: Medium-High; and H: High. Variation expressed as
confidence intervals. Statistical results are available in Table S4.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-7---Isotopes-pulse/Fig-7---Isotopes-pulse}

\end{center}
\end{figure}

\textbf{Fig 7.} Atomic per cent excess in workers and larvae after a
pulse of \textsuperscript{13}C and \textsuperscript{15}N isotopes.
Workers became highly enriched rapidly (they collect the material) and
then food was transferred to larvae by 96h. While not statistically
significant, the atomic enrichment of \textsuperscript{13}C trends
toward being higher in larvae at 96h while there is no pattern of
difference in \textsuperscript{15}N; this is opposite what was
predicted. This assay was carried out with a single diet (ML,
Medium-Low) in a subset of \emph{Lasius neoniger} colonies. Variation is
expressed as confidence intervals.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-8---Diet-preference/Fig-8---Diet-preference}

\end{center}
\end{figure}

\textbf{Fig 8.} Field colonies of both \emph{Lasius} species tended to
avoid the diet with the highest protein content, and \emph{L.
neoniger}exhibited an overwhelming preference towards MH diet. L: Low
carbohydrate content; ML: Medium-Low; MH: Medium-High; and H: High.
Means and confidence intervals were extracted from the mixed-effects
model using the effects library (Fox et al., 2018) and back-transformed.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Fig 9.} Untransformed values of colony survival and growth (A
and B) and individual worker lean mass and lipid content (C and D). The
lines intersect in all graphs, suggesting that both colony level growth
and survival, as well as individual level lean and lipid mass, trade-off
as a function of the carbohydrate and protein content in the diet
(x-axis). In all cases, the lines intersect at the intermediate level
(ML or MH) of P:C used in this experiment.

\section*{Supplementary Electronic
Material}

{\label{supplementary-electronic-material}}

\textbf{Table S1.} Recipes of diets used for the experiments

\textbf{Table S2.} Mixed-effect models results for colony development

\textbf{Table S3.} Two-way ANOVA results for overall colony growth

\textbf{Table S4.} Linear and generalized linear models results for
workers phenotypes

\textbf{Figure S1.} Diet preference (i.e. recruitment) in field and
laboratory colonies of \emph{Lasius neoniger}

\selectlanguage{english}
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