RESULTS
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 inL. neoniger (G = 9.19, df = 3, 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 (P < 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 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,P < 0.05) and medium-high carbohydrate (MH, P< 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 (P < 0.001 in both post-hoc comparisons).
Colonies of L. niger grew larger than colonies of L.
neoniger , seemingly owing to differences in overwintering and recovery
from winter
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 L. niger , but only head width exhibited a
significant diet P:C x Frequency interaction. In L. neoniger ,
worker lean mass and lipid content were affected by diet P:C only,
whereas head width was affected by feeding frequency only. 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 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 L. neoniger worker dry mass (both higher
frequency treatments were marginally different than the lower frequency,P = 0.06 and 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.
Stable Isotope Pulse
Experiment
There was a statistically significant effect of caste by sample time for
atomic enrichment of both 15N and13C (K-W test, χ2 > 12,
d.f. = 3, P < 0.005 for both 15N and13C). It can be noticed that incoming nutrients are
first ingested by workers, with worker atomic per cent of15N and 13C peaking at 24h. The 24h
workers had nearly double the atomic enrichment of 13C
compared to all other treatments and had five-fold higher enrichment of15N (Fig. 7). Workers were statistically different
(P < 0.05) from larvae at both 24 and 96-hour
collections for 15N, but the difference was not
statistically significant in the 24-96 hour comparison for13C. 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 15N remained constant over both sample
times while the mean for atomic per cent 13C increased
over sampling points (this result is trending toward being statistically
significant, P = 0.11). Larval C:N was much higher than that of
adult workers, 7.24 ± 0.18 vs. 5.00 ± 0.11 (mean ± SE,
F1,39 = 108.9, P < 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.
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 Lasius presence. 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 < 0.05). On the other hand, L.
neoniger exhibited an overwhelming preference for the ML diet, with
diet being highly significant in the mixed-effects model
(χ2 = 52.01, P < 0.001). This trial
was also replicated with two 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 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 (χ2 = 29.09, P< 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 (χ2= 13.47, P = 0.001) (Fig. S1).