Figure 3 Male hops inflorescences at varying phenological stages; at stage 1, buds have
elongated into oval, but anthers remain inside of sepals; by stage 6, anthers have begun to
Figure 1 Female hops azure laying eggs on an early-stage male inflorescence (A),
panicle of male flowers of hops (B), and an ant tending a larvae while it feeds on male
flowers (C); in panel C, flowers show characteristic herbivory of pollen inside sepals.
Facultative ant mutualism in the rare hops azure butterfly,
Celastrina humulus (Lycaenidae)
Emily Mooney1, Abbey Swift1, Hailee Nolan2, Robert Schorr3,
1University of Colorado Colorado Springs, 2Colorado State University, 3Colorado Natural Heritage Program
Introduction to the Study System
Conservation of rare species relies on understanding how biotic and abiotic factors
shape abundance . The hops azure butterfly (Celastrina humulus, Family
Lycaenidae) is ranked as a G2 Imperiled species by NatureServe . Butterflies lay
eggs on male flowers of native hops, Humulus lupulus var. neomexicanus (Fig. 1A).
Larvae develop quickly feeding on the pollen-rich male inflorescences (Fig. 1B). Like
other lycaenids, hops azure larvae can form associations with several species of
ants , which walk over the caterpillars and drink nectar from specialized organs
(Fig. 1C). Kubik and Schorr (2018) found hops azure larvae tended by ants in 10 of
15 sites along Monument Creek near Colorado Springs, CO . Given the well
documented benefits of mutualism with ants , it is unclear why many larvae do
not form associations with ants. Our objective was to assess which biotic and
abiotic factors were associated with ant tending.
Photo by A. Swift
A B C
Methods In the Field
We surveyed larvae on 134 unique bines (flowering vines) along a 1.5 km section of Monument Creek in Colorado Springs, CO. Once larvae were found, we used
replicate surveys to record the number of larvae on the bine, larval developmental stage, ant attendance, ant species, and floral phenology (Fig. 2). We ranked
phenology of male flowers using a numerical score (Fig 3). To account for variation among host plants and their environments, we measured light (PAR) above each
bine using a meter and patch area. We surveyed bines within the same host plant population in 2020 and 2021.
Larvae per bine
We used a model selection approach to determine which of the
factors we measured best explained variation in likelihood of ant
tending (Y/N). We fit mixed effects models with binomially distributed
errors using the ‘glmer()’ in the package lmerTest . The model set
included univariate, additive, and 2-way interactive models (Table 1).
After initial analysis failed to support models that included an effect of
larval stage, we did not include this factor in the final model set. We
obtained AICc and ΔAIC scores from the AICcmodavg package . All
analyses were performed in R version 4.1.1 .
Model Selection and Results
Table 1 Models and fit statistics
Model Weight ΔAICc
Ants? ~ Larvae + Flowerstage + Area + (1 | Year) 0.567 0
Ants? ~ Larvae + Flowerstage + Area+ Light + (1 | Year) 0.752 0.590
Ants? ~ Larvae + Area + Light + (1 | Year) 0.884 1.644
Ants? ~ Larvae + Flowerstage * Light + Area + (1 | Year) 0.946 2.705
Ants? ~ Larvae + Flowerstage * Area + (1 | Year) 0.987 3.949
Ants? ~ Larvae + Flowerstage * Area + Light + (1 | Year) 0.998 3.965
Ants? ~ Larvae + Area * Light + (1 | Year) 0.999 4.439
Ants? ~ Larvae + Flowerstage + (1 | Year) 14.923
Ants? ~ Larvae + (1 | Year) 18.849
What’s Associated with Ant Tending?
Figure 4 Greater densities of
larvae per bine was associated
with increased likelihood of ant
tending. More larvae may
simply attract more ants. Such
density dependence can be
common in ant mutualisms 
Figure 5 Larvae on bines with
later stage flowers were more
likely to be ant tended.
Flowering phenology can
mediate associations between
herbivores and ants . Nectar
from larvae feeding on later
stage flowers may be more
attractive to ants .
Figure 6 Larvae on bines in
larger patches had increased
likelihood of ant tending. Patch
area can be a signal of site
quality perhaps due to greater
nitrogen, a key factor in these
L.R. χ2= 7.632, P= 0.006
L.R. χ2= 5.450, P= 0.005
L.R. χ2= 9.275, P= 0.010
1. Wagner DL, Grames EM, Forister ML, Berenbaum MR, Stopak D (2021) Insect decline in the Anthropocene: Death by a thousand cuts. Proceedings of the
National Academy of Sciences, 118(2): e2023989118
2. Celastrina humulus | NatureServe Explorer. https://explorer.natureserve.org/Taxon/ELEMENT_GLOBAL.2.111968/Celastrina_humulus
3. Kubik TD, Schorr RA (2018) Facultative Myrmecophily (Hymenoptera: Formicidae) in the Hops Blue Butterfly, Celastrina humulus (Lepidoptera:
Lycaenidae). Entomological News, 127(5):490–498.
4. Ness J, Mooney K, Lach L (2010) Ants As Mutualists. Ant Ecology, 9780199544:432.
5. Kuznetsova A, Brockhoff PB, Christensen RHB (2017) lmerTest Package: Tests in Linear Mixed Effects Models. Journal of Statistical Software, 82(13):1–26.
6. Mazerolle, MJ (2020) AICcmodavg: Model selection and multimodel inference based on (Q)AIC(c). R package version 2.3-1. https://cran.r-
7. R Foundation for Statistical Computing (2021) R: A language and environment for statistical computing. :R version 4.1.1 (2021-08-10)-- “Kick Things.”
8. Peterson MA (1995) Unpredictability in the facultative association between larvae of Euphilotes enoptes (Lepidoptera: Lycaenidae) and ants. Biological
Journal of the Linnean Society, 55(3): 209–223.
9. Patricelli D, Barbero F, Morgia V La, Casacci L P, Witek M, Balletto E, Bonelli S (2011) To lay or not to lay: Oviposition of Maculinea arion in relation to
Myrmica ant presence and host plant phenology. Animal Behaviour, 82(4):791–799.
10. Pierce NE, Nash DR, Baylis M, Carper ER (1991) Variation in the attractiveness of Iycaenid butterfly larvae to ants. Ant-Plant Interactions, :131–142.
11. Billick I, Brown R, Reithel JS (2005) Importance of fertilization of host plants to ant tending and growth rates in Glaucopysche lygdamus (Lepidoptera:
Lycaenidae). Annals of the Entomological Society of America, 98(4):491–495.
This project received support from the Colorado Native Plants Society Marr Grant. Funding to A. Swift was provided by
the UCCS Undergraduate Research Academy (2020) and the Department of Biology (2021). Funding to H. Nolan was
provided by the Linda Hamilton Conserving Colorado Award at CSU Fort Collins. We also thank C. Hamilton, E.
Callahan, and L. Callahan for additional help with field surveys.
Stage 1 Stage 6Stage 2 Stage 4