In that same study, Huang and Yang report anecdotal evidence that suggests equal yields in orchards that use chemicals vs. orchards that use ant bridges to control for pests. Similarly, Peng and Christian , report lower levels of fruit damage in cashew with the presence of weaver ants. However, as vegetation complexity declines in agroecosystems, tree density and diversity may also decrease , as well as the possibility to generate connections between the arboreal vegetation, which might impact arthropod populations . The lack of connectivity between trees in managed systems can have a significant impact on the mobility of worker ants and their ability to control resources. This impact may be particularly marked at greater distances from the nest, where ant dominance may be lower . This in turn may influence the ecosystem services provided by ants, particularly the suppression of pest outbreaks . Shaded coffee plantations, which maintain high levels of shade and structural complexity , can sustain complex networks of organisms, which can result in biological pest control . In coffee systems, ants are a functionally diverse and abundant group of ground and arboreal-nesting arthropods and are considered important biological control agents . Ants are predators of the most devastating coffee pest, plastic garden container the coffee berry borer , a beetle that drills cavities in coffee berries and severely damages the seed .

Several species of arboreal ants, with nests attached to or inside tree trunks, branches, or twigs, control adult and immature stages of this pest either through direct predation or deterrence . Ants of the genus Azteca are numerically dominant in shaded coffee plantations. These ants forage intensively on coffee plants , and deter CBB adults by removing them from the coffee plant, therefore lowering fruit damage . In shaded coffee plantations, Azteca sericeasur ants nest on shade trees and access adjacent coffee plants through the leaf litter or available pathways, such as fallen branches, vines, and other vegetation , matching the description by Longino for this species in forest habitats. In more intensively managed coffee systems, with fewer and more distant nesting trees, connectivity may be sparse or absent and artificial connections might buffer against this loss. Vegetation structure and arboreal characteristics in coffee plantations are likely to be important factors influencing ant foraging behavior and nesting in arboreal ants . However, the influence of vegetation connectivity on the foraging of this dominant arboreal ant and its effect on pest removal in coffee plantations has not yet been studied. Previous work has documented the importance of arboreal connections for ants and biological control in agricultural systems. For example, various studies and farmers’ manuals suggest that connecting nests to adjacent trees using bamboo strips enables weaver ants to colonize new trees, which increases ants’ efficiency in removing pests, including the pentatomid insect Tesserarotoma papillosa .

However, there is little evidence about the effect of increasing arboreal connectivity on biological control using experimental data. We report an experiment testing the influence of adding connections between shade trees and coffee plants and its effects on CBB removal on coffee plants. To our knowledge, this is the first study providing experimental data on the effect of adding connectivity on ant activity and pest removal in coffee agroecosystems. Specifically, we tested one hypothesis: Connectivity affects CBB removal in this system by increasing recruitment rates of A. sericeasur ants to prey items; we predicted that A. sericeasur ants use artificial connections between nesting trees and coffee plants; plants with connectivity have higher ant activity than isolated plants; plants with connections have grater removal rates of CBB by A. sericeasur ants; and A. sericeasur activity and CBB removal rates by A. sericeasur ants decrease with increased distance from A. sericeasur nests.We conducted the study in a 300 ha shaded coffee plantation in the Soconusco region of Chiapas, Mexico. The coffee plantation is located at 1,100 m a.s.l. in the Sierra Madre de Chiapas Mountains. The natural vegetation types are high and mid-elevation perennial forest and the climate is semitropical with rainfall typically occurring between May and October . The coffee plantation can be characterized as a commercial polyculture, where coffee plants grow under the canopy of shade of trees, mostly in the genus Inga , providing an average canopy cover of 75% .Within the farm, we haphazardly selected 20 non-overlapping sites located at least 10 m away from each other with one Inga micheliana tree containing an A. sericeasur carton nest on the tree trunk . Azteca sericeasur is a polydomous, arboreal ant species , which occurs in ~13% of trees at our study site , and forages on coffee plants . Trees were selected only if ant nests were noticeably active. In each site, we quantified ant activity on the nest tree as the number of ants crossing a single point on the main trunk during 1 min. This methodology has been used in previous studies to measure overall ant activity of a nest . We then selected the six coffee plants nearest to the nesting tree, making sure they were not directly touching each other or the tree by removing branches and vines . We then randomly assigned three of the coffee plants at each site to a connection treatment and three as controls without connections, then measured ant activity on the plants by counting the number of ants passing a point on the central trunk for 1 min. We connected treatment coffee plants to the nesting tree using jute string . Strings remained in the field for 3 days to allow for ant acclimation to disturbance and for ants to establish new foraging pathways. After 3 days, we returned to the sites and re-measured ant activity on the nesting tree and coffee plants. Observations took place between 10 a.m. and 1 p.m. and were immediately stopped as soon as it started raining, as this drastically decreases ant activity. To test how connectivity impacts potential biological control provided by ants, we added dead adult CBB onto connected and control coffee plants to directly assess ant removal rates. We collected CBB-infested coffee berries from the field, dissected them,extracted female adult CBB individuals , and placed them in the freezer for up to 24 hr, after which beetles were dead. Three days after placing strings and after re-assessing ant activity, we placed 10 dead CBB adults on a small piece of white card on each coffee plant near the center of the trunk, left cards for 30 min, plastic pot and then counted the number of CBB remaining. Cards were balanced on coffee branches and were bent slightly to keep the CBB from falling. Restricting movement of sentinel prey, either by gluing them to observation sites or by freezing them, is a common technique for assessing predator behavior .

We used frozen sentinel prey to increase the availability and similarity of beetles on cards and to reduce the potential for live prey to escape from the arena. To assess whether CBB removal was due to ant activity, we monitored cards across the plot over a period of 30 min and recorded any arthropods present. Only ants were observed on the cards, indicating that these were responsible for removing the CBB. Although we acknowledge that the use of dead prey may alter ant behavior, it is already well established that A. sericeasur both antagonizes and predates live CBB in the field, and reduces CBB infestation on plants . We used dead prey in this experiment to more readily assess ant removal rates and infer that these changes translate to changes in the bio-control efficiency of this ant on live prey. Immediately following each experiment, we characterized the vegetation in each site because several different environmental factors are known to influence ant foraging in coffee systems . We measured the percentage of canopy cover , coffee plant height, and distance from each coffee plant to the central Inga nest tree.To test for statistical differences in ant activity on nest trees before and after connecting trees to coffee plants, we fit our data to a generalized linear mixed model . We included time , canopy cover, and their interaction as fixed effects . We also modeled nest tree identity as a random effect. To assess count data , we originally fit our model to a Poisson distribution with a log link function. However, to correct for observed over-dispersion, we modified our model to a Poisson-lognormal distribution by adding a per-observation random effect term .To test for statistical differences in ant activity on coffee plants before and after establishing connections, we used a GLMM. We included time , treatment , coffee plant distance to nest tree, the interaction between time and treatment, and the interaction between time and distance as fixed effects . Random effects were modeled with plant identity nested within site to account for the block design of the experiment and to control forvariation between our sites. To model count data and to correct for overdispersion, we used a Poisson-lognormal model with a log link function by including a per-observation random effect as described above .We modeled CBB removal by ants using a GLMM. We included treatment , coffee plant distance to nest tree, ant activity on coffee plants after string placement, the interaction between treatment and distance, and the interaction between treatment and ant activity on plants as fixed effects . We also included ant activity on nest tree and coffee plant height as covariates. Random effects were modeled with plant identity nested within site to account for the block design of the experiment and to control for variation between our sites. To model count data in our response variable, we used a Poisson distribution with a log link function.We constrained model selection to include biologically pertinent terms for inference and to aid in model interpretation. A full model of these terms was tested, along with subsequent models of different covariate combinations and a null intercept-only model of random effects . The best fit model was determined via backwards model selection compared to the full model, where the model that resulted in the lowest AIC was selected, if ∆AIC > +2 when the best fitted model was not the full model. Overall significance in models was assessed using Wald type II Chi-squared tests. Statistical differences among treatments were compared by Wald Z tests . In all cases, fixed effect parameters and the variance of random effects were estimated by maximum likelihood with Laplace approximation using the “glmer” function in the “lme4” package in R . To aid in data interpretation, we removed one coffee plant replicate from our analysis where measured ant activity was more than double that of any other plant measured and may have resulted from an unusually high buildup of scale insects which are tended by A. sericeasur on coffee. Additionally, one nest tree replicate was not included in the tree activity analysis because the data were not collected at that site. Finally, coffee plant height and distance to nest tree were centered and scaled to aid model interpretation. All analyses were performed in R .Our experiment demonstrates that the addition of string to connect shade trees and coffee plants in coffee agroecosystems facilitates movement for A. sericeasur and potentially increases ant recruitment rates. Studies in natural systems have reported increases in ant activity with arboreal connections across the arboreal stratum , possibly driven by the easy access these pathways provide to resources . Other ants, such as Pogonomyrmex spp., prefer linear arboreal substrates and switch to cleared routes as a mechanism to reduce the energetic costs of ant foraging , and in some cases to decrease the risk of encountering predators . The observed increase in ant activity on connected coffee plants after the placement of strings suggests that structural connectivity can increase ant recruitment rates to foraging areas in coffee and may enhance the efficiency of movement for A. sericeasur. This may lead to increased foraging efficiency for ants and enhanced resource capture rates on coffee. However, this could also reflect other benefits associated with using linear arboreal substrates, such as avoiding predators, a behavior that is known to occur in A. sericeasur . Using more efficient foraging pathways and thereby avoiding the leaf litter as a primary foraging substrate may potentially protect A. sericeasur workers from the attack of the phorid fly parasitoid Pseudacteon spp. .