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).
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 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 L. neoniger , but not L. niger , with the
highest protein (L) diet may be due to different overwintering
conditions for each species. L. neoniger was overwintered at a
higher temperature compared to 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 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 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 Rhytidoponera sp., L. nigerand 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.
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 et al. 2007; Toth 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.
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 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 L.
niger – higher protein diets increased growth, but higher carbohydrate
diets were preferred. In 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 withL. 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 L. neonigeris 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;
Sorenşen, Busch, & Vinson, 1985; E. O. Wilson, 1976), or to
stores/trash enriched with protein (Dussutour & Simpson, 2009).