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Open access journal: http://periodicos.uefs.br/index.php/sociobiology
ISSN: 0361-6525
DOI: 10.13102/sociobiology.v70i3.8701
Sociobiology 70(3): e8701 (September, 2023)
Introduction
Leaf-cutting ants (LCA) (Hymenoptera: Formicidae,
Attini) (genera Atta and Acromyrmex) cause signicant
economic damage and are, therefore, considered an important
agricultural pest in their areas of occurrence (Holldöbler &
Wilson, 1990; Urbas et al., 2007). Several methods for the
management of LCA have been used, such as insecticide
application, fungicides, and the use of entomopathogenic fungi.
However, some of these methods have high costs and adverse
effects on the environment and human health (Montoya-Lerma
et al., 2012; Della Lucia et al., 2013, Zanetti et al., 2014).
An alternative and less environmentally toxic method is
the use of substances with a deterrent effect. These substances
Abstract
Acromyrmex balzani Aa opaciceps
Ac. balzan
A. opaciceps
Ac.
balzaniA. opaciceps
versus
Sociobiology
An international journal on social insects
Arcle History
Edited by
Keywords
Aa opacicepsAcromyrmex balzani
Corresponding author
act on the insect’s behavior and prevent or inhibit foraging
and are generally associated with secondary metabolites
produced by plants (Dethier et al., 1960; Lara, 1991).
LCA efciently manage their waste (here referred
to as nest refuse, NR) produced in the colony, since such
behavior guarantees the health of individuals and the integrity
of their symbiotic fungus (Bot et al., 2001). Previous studies
showed that ants avoid cutting tissues from plants that are in
contact with dry NR mounds (Zeh et al., 1999; Farji-Brener &
Sasal, 2003; Ballari & Farji-Brener, 2006) and, more recently,
Sousa-Souto et al (2022) observed that the liquid NR extract
also proved to be deterrent against ant attack to the plants.
Thus, odors associated with NR may be the main mechanism
of deterrent action.
ID
Leandro Sousa-Souto, Bianca G. Ambrogi, Rafaella S. Santos – Nest refuse extract delays bait collection
LCA cultivates the same species of symbiont fungus,
Leucoagaricus gongylophorus (Pagnocca et al., 2011), and
maintains similar habits of hygiene, and communication
between nestmates. Likewise, the NR produced by different
species of these two genera probably has similar chemical
characteristics. Given these similarities, it is likely that the
deterrent effect of the NR extract is interspecic.
In the present study, we veried the interspecic
deterrent effect of NR in liquid formulation (hydroalcoholic
extract) on leaf-cutting ants of the species Acromyrmex balzani
Emery, 1890 and Atta opaciceps Borgmeier. For this, the study
started from the following hypotheses i) since NR is toxic and
avoided by leaf-cutting ants, even attractive baits prepared
with this substrate can also be avoided; ii) if substrate-treated
baits have an intraspecic deterrent effect, there is a high
probability that they also have an interspecic effect.
Material and Methods
Study site and LCA species used
The study was carried out on the campus of the
Federal University of Sergipe in São Cristóvão (10º55’33.55
“S 37º6’8.327” W) and the chosen species were the most
common and have the highest density in the area.
The species Acromyrmex balzani has a wide distribution
in disturbed environments, in addition to a high density of
colonies in these environments, reaching 900 colonies/ha
(Sousa-Souto et al., 2013). This species has the characteristic
of discarding NR outside the nest, in piles on the soil surface,
adjacent to the nest entrance. Likewise, the Atta opaciceps
species was chosen because it is easily found in the study
area, forming mounds up to 2.5 m in diameter with numerous
entrances (Delabie et al., 1997).
Nest refuse (NR) collection
The nest refuse produced by A. balzani was collected
directly in the eld, at the study site, and in the mounds
discarded by the colonies. The NR from A. balzani colonies
is characterized as a light-colored residue in front of the nest
entrance. The material was collected with a spoon, deposited
in paper bags, taken to the Laboratory, and placed in an oven
at 60 °C for 48 hours in order to eliminate possible pathogens
or contaminants.
The NR produced by A. opaciceps was collected from
10 colonies maintained at the Laboratório de Entomologia
Florestal - UFS. This substrate is discarded in plastic chambers
arranged for that purpose and has a dark brown color. The
material was collected and deposited in paper bags and later
taken to the oven at 48 °C for 48 hours for drying.
Hydroalcoholic extracts preparation
Hydroalcoholic extracts (HE) were prepared following
the methodology of Barbosa et al. (2006) with adaptations of
Sousa-Souto et al. (2022). The HE were prepared with 80mL
of 100% ethyl alcohol, 80mL of distilled water, and 40 mL
of crushed NR of A. balzani or A. opaciceps. The extracts
were strained and mixed twice a day, placed at rest at room
temperature for 48h when they were then vacuum ltered
through a Büchner funnel and lter paper (90 mm diameter),
and stored in a refrigerator at 8 °C before eld tests.
Preparation of attractive baits
The attractive baits were composed of colored straws
impregnated with citrus pulp, allowed to dry and sprayed with
a 1:1 control solution (ethyl alcohol and distilled water) or
with hydroalcoholic extract of the substrate produced by both
species, differentiated by color between treatments: white –
control (Cont); pink - Acromyrmex extract (ExtAc); blue -
Atta extract (ExtAt).
The citrus pulp was used in this study because it is
reported as a substance with high attractive power for ants
and for that reason widely used in ant killer baits, so that ants
disseminate the toxic substance inside the colony (Boaretto &
Forti, 1997; Carlos et al., 2009).
Field tests
For the tests, 30 A. balzani and 36 A. opaciceps eld
colonies at the study site were systematically selected. The
colonies were marked with wooden sticks and their location
was georeferenced with GPS. We identied the foraging areas
of each colony and, following a methodology modied from
Moreira & Forti (1999), with 10 attractive articial baits per
treatment offered, totaling 30 baits per colony.
In our study area, A. balzani forages without the
formation of permanent foraging trails. As a result, ants return
to the nest in a scattered manner. For this reason, baits were
offered in two different ways: for A. balzani colonies, the
baits were arranged like a half moon, at 25 cm of distance
from the nest entrance (Figure 1A). For A. opaciceps, the
baits were arranged in a line, parallel to the foraging trail,
at 10 cm of distance from the trail and 1 m from the nest
entrance (Figure 1B).
After the rst contact of a worker with any of the
baits, we started the period of 30 minutes of observation
for colonies of A. balzani and 20 minutes for colonies of A.
opaciceps and the number of baits collected by leaf-cutting
ants was recorded. The difference in the observation time
between species was due to the size of the colonies and the
form of recruitment (smaller nests and slower recruitment in
A. balzani). During the observation period, we consider as
collected any bait that has been removed from the pile, even
if the bait has subsequently been abandoned.
The number of baits remaining between treatments was
compared using a Generalized Linear Mixed Model (GLMM)
with a Poisson distribution and a logarithmic link function.
This modeling approach offers more exibility for variables
that do not follow a normal distribution (Bolker et al., 2009). We
used the “lme4” package in R to adjust the GLMM to our data.
Sociobiology 70(3): e8701 (September, 2023)
The model consisted of the response variable ‘number
of baits remaining’, the xed effect, represented by the
variable ‘treatment’, with three levels (ExtAc, ExtAt, and
Control), and the random effect ‘nests’, allowing to evaluate
the variation in the number of baits remaining among the
different observed colonies, even if the treatment effect is
the same for all colonies. In another approach, the “survival”
time of the baits (i.e., the time until they were removed)
between the different treatments was tested using survival
analysis (Kaplan-Meier), followed by pairwise comparisons
using log-rank tests, to verify if there was a preference for
the collection by a certain treatment level, at a signicance
level of 5% (Crawley, 2007). The Kaplan-Meier method is
widely used to estimate the survival function when dealing
with censored data, that is, where the event of interest (bait
withdrawal) is not observed for all baits in the group or
in all treatments, during the observation period (Barber &
Jennison, 1999). Data were analyzed using R software (R
Development Core Team, 2023).
Results
Deterrence was observed for extracts from colonies
of LCA and this effect differed signicantly from control
extracts. Both foragers of A. balzani and A. opaciceps avoided
the baits treated with NR extracts, regardless of their origin
(intraspecic or interspecic). Ants avoided treated baits even
with the attractive effect of citrus pulp. In general, 85% of
the baits with NR were rejected, while approximately 80%
of the control baits were carried to the nests during the
observation period.
For A. balzani colonies, the average number of remaining
baits (unloaded) was 2.93 ± 0.61 (mean ± standard error) for
the control (Cont), 9.27 ± 0.36 for the intraspecic extract
(ExtAc ) and 9.6 ± 0.28 for the interspecic extract (ExtAt).
Thus, there was a signicant difference between the number
of remaining baits of Cont x ExtAc (p = 0.001) and Cont. x
ExtAt (p < 0.001), but there was no signicant difference
between ExtAc versus ExtAt treatments (p = 0.906) (Figure 2A).
Fig 1. Experimental design indicating the arrangement of the baits near the nests of leaf-cutting ants: For Acromyrmex
balzani the baits were arranged radially (A). For Atta opaciceps nests the baits were placed parallel to the foraging trail (B).
Fig 2. Results of the bioassay with eld colonies of Acromyrmex balzani. A - Boxplot showing the remaining baits among the three
treatments. Horizontal lines indicate median values, the top and bottom box indicate the upper and the lower quartiles, respectively, and
dots are outliers. In B, bait survival probability over the observation time of 30 minutes (1800 seconds).
Leandro Sousa-Souto, Bianca G. Ambrogi, Rafaella S. Santos – Nest refuse extract delays bait collection
Regarding the “bait survival”, there was a signicant
difference in the probability of bait survival of the ExtAc
versus Control (p = 0.018) and ExtAt versus Control (p <
0.001) but there was no difference between ExtAc versus
ExtAt (p = 0.508) (Figure 2B).
Similar results were observed for colonies of A.
opaciceps. The average number of remaining (unloaded)
baits was 1.47 ± 0.36 (mean ± SE) for control, 6.44 ± 0.57
observed in ExtAc, and 8.19 ± 0.43 for the (ExtAt). There
was a signicant difference between the NR extracts and
the control (p < 0.001), and betwwen ExtAc versus ExtAt
(p = 0.015) (Figure 3A). As observed in A. balzani, there
was a signicant difference in the “bait survival” among the
extracts (p < 0.001), but no difference between the ExtAc
versus ExtAt treatments (p = 0.52) (Figure 3B).
Discussion
The nest refuse from LCA is being better studied and
most results have shown multiple uses, either as an additional
source of nutrients and organic material to the soil (Cerda et
al., 2012; Santos et al., 2018; 2019), or through its deterrent
effect against herbivory of the ants themselves (Sousa-Souto
et al., 2022) and, more recently, against aphids (Mecenas
et al., 2023). Our results show that extracts in liquid form,
prepared with the disposal substrate (NR) have both intra and
interspecic deterrent effects on the foraging behavior of ants,
corroborating similar results using dry waste (Zeh et al., 1999;
Farji-Brener & Sasal, 2003; Ballari & Farji-Brener, 2006).
In the present study, hydroalcoholic extracts from A.
balzani and A. opaciceps colonies were efcient in reducing
bait collection by ants when compared to the control extract.
Almost all control baits were collected by the different
sampled colonies and there was little collection of treated baits
(less than 40% in the most active colonies). In addition, some
behavioral changes were noted, although not measured, as for
example, the processing time (touch of antennae and jaws on
the bait) and the start of loading the baits were signicantly
shorter for control baits when compared to the baits of the
ExtAt and ExtAc treatments. Besides the longer processing
time of baits with NR by workers, few baits were actually
loaded, even after the observation time had ended.
One point to be reported is that many baits with NR
registered as “counted” were abandoned by the workers after
two or three minutes that were removed from the point of
origin. This behavior was not observed in ants that collected
the control baits. In addition, the behavior of attempting to
remove the citrus pulp inside of the plastic straw was also
observed. In fact, many abandoned straws were cut or bitten
by ants. This behavior is further evidence that baits with NR
showed characteristics that were recognized by the ants as
harmful to them, corroborating the hypothesis that odor is the
mechanism behind deterrence (Sousa-Souto et al., 2022). In
fact, a preliminary analysis through liquid chromatography
(HPLC-DAD) of the NR extracts suggests that the substances
present may be secondary metabolites from plants foraged by
the colonies, such as gallic and p-coumaric acids, two simple
phenolic acids (Mecenas et al., 2023).Thus, although the strong
Fig 3. Results of the bioassay with eld colonies of Atta opaciceps. A - Boxplot showing the remaining baits among three treatments.
Horizontal lines indicate median values, the top and bottom box indicate the upper and the lower quartiles, respectively, and dots are outliers.
In B, bait survival probability over the observation time of 30 minutes (1800 seconds).
Sociobiology 70(3): e8701 (September, 2023) 5
attractiveness of the citrus pulp in the baits treated with NR,
the deterrent effect was effective, causing rejection or later
abandonment of the treated baits.
Ants select the material to be harvested from the
physical or chemical characteristics of the host plant, such as
the absence of trichomes in the leaves, nutritional value of the
material, levels of secondary metabolites, energy expenditure
to incorporate this material into the symbiotic fungus garden
(Garcia et al., 2005; Verza et al., 2007). Vegetable materials
considered unsuitable for the cultivation of the fungus or
which may be toxic are generally rejected at the time of
cutting by foragers, or before incorporation into the fungus,
by garden workers (Verza et al., 2007). Thus, it is possible
that the rejection of baits with NR at the time of foraging
shows that the workers were able to associate the odor of
the baits with those phenolic acids mentioned above or other
potential risks for the colony.
Although LCA shares the same species of symbiont
fungus and has similarities in communication between
nestmates, the colonies differ through their highly specic
chemical prole (Viana-Bailez et al., 2011). The experimental
design of the present study, however, does not allow us to
conclude that the low collection of baits treated with the
interspecic extract was due to the recognition of the baits as
material from a rival colony (territorial behavior) or simply
due to the inherent odor of the disposal substrate.
Considering the composition of nest refuse between
the two species, we can highlight some striking differences
between these two substrates (Della Lucia et al., 2013).
First, there are differences in the origin of the plant material
of both NRs, for A. balzani (ExtAc) the NR composition is
of herbaceous plants (with a predominance of grasses). The
material is totally discarded outside the colony, making it drier
and more fragmented. The disposal material of A. opaciceps
is done mainly through underground chambers, although
some colonies deposit their NR in external piles adjacent to
the nest (Sousa-Souto et al., 2022). The incorporation of nest
waste in the inner chambers makes the substrate texture more
compact, with high moisture content and a more pronounced
odor. It is reported that residues deposited internally are more
susceptible to the proliferation of pathogens, compared to
waste discarded outside the nests (Farji-Brener et al., 2016).
Even with all these differences, however, the observed
interspecic effect of the extracts indicates that the deterrence
can be triggered simply by the characteristic odor of the
refuse substrate.
Despite advances in knowledge of the potential
deterrent effect of nest refuse on ant foraging, it is still
unclear whether such effects are long-term and whether its
use would be viable on a large scale. Thus, new studies must
be conducted to ll in the gaps that still exist, conrming (or
not) the use of NR extracts as a new perspective in integrated
pest management.
Acknowledgments
We would like to thank Hosana H. Mecenas, for the
support of data collection, to Dr. Genésio Tâmara Ribeiro,
from the Laboratório de Entomologia Florestal (LEFLO) da
Universidade Federal de Sergipe (UFS) and Dr. Yana Teixeira
dos Reis, from the Laboratório de Entomologia (UFS) for
their help in acquiring the substrate. To Dr. Paulo Cesar de
Lima Nogueira, from the laboratório de Química Orgânica
(UFS) for his assistance in conducting the chromatographic
analyses. To Dr. Sinara M. Moreira for suggestions for
improving the methodology. To the Conselho Nacional de
Desenvolvimento Cientíco e Tecnológico (CNPq), CAPES
(PROAP), and CAPES/FAPITEC (88881.157451/2017–01)
for funding.
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