Carbohydrate or sugar resources, predominantly from extrafloral nectaries – vegetative structures that secrete nectar– and hemipteran honeydew, are one of the most widely used food resources by ants and parasitoid wasps . These stable and highquality plant and animal exudates represent an important component of ant diets . Sugar resources can drive the formation of ant mosaics during colonization and establishment , influence ant abundance and diversity, and determine foraging decisions . Nectar and honeydew resources also provide an important source of nutrition for parasitoid wasps and benefit parasitoid egg production, egg viability, growth, and increase lifespan . For example, parasitoid wasps often use EFNs to find their hosts, or oviposit near EFNs to increase the probability of encountering hosts that visit EFNs . Parasitoid wasps in the Eucharitidae family have developed a set of morphological, chemical and behavioral adaptations that allow them to access ant brood through the use of nectar resources . Adult female wasps in this family oviposit near EFNs where ants forage for food . When planidias emerge, they find their way to ant mandibles as ants feed on EF nectar, hitchhike on ant bodies, and are thus accidentally transported to the nest. Once in the nest, adult ants transfer parasitoid planidias onto their brood during feeding . Despite the importance of nectar resources for ants and parasitoids, research on nectar-mediated interactions has focused primarily on the benefits of mutualistic interactions to the plant rather than focusing on consumers . For instance, planting blueberries in a pot few studies have provided robust evidence about the benefits of nectar resources to ant colony reproduction , or the mechanisms involved in nectar-mediated multitrophic interactions, such as parasitoid-host interactions .
Parasitoids of ants have been previously described as “the missing link” in ant community dynamics because we know very little about how and in which contexts they shape ant communities . Diversity and availability of nesting resources determine ant community structure and species co-occurrence. Nesting resources are a spatially-segregated microhabitat usually taking the form of cavities located in arboreal substrates such as twigs, branches, tree trunks and bark. These cavities can occur naturally when wood-boring beetles build galleries that are then abandoned, leaving vacant cavities suitable for ant colonization . In natural forests, the diversity of nest cavities available for twig-nesting ants – ants that nest in hollow twigs– influences colonization, the diversity of species, and colony growth . Similarly, in tropical agroforests a greater diversity of cavity sizes promotes niche differentiation in a community of TNAs, therefore explaining higher species diversity and species co-occurrence . In a similar study, Armbrecht et al. found that an array of twigs belonging to different tree species increases ant species richness. Furthermore, the close relationship between ants and their nesting resources has evolutionary implications for certain ant species. For instance, workers of Cephalotes persimilis choose cavities that match the size of their heads to increase individual nest survival, which has led to the evolution of a specialized head-disk that allows workers to protect their nest from other ant intruders . Although it has long been established that microhabitat characteristics affect parasitism, primarily through host concealment , less is known about the specific role of nesting resources used by hosts on parasitism, particularly in social insects.
It is possible that nests provide protection for parasitoids and thus constitute an attractive habitat for parasitoid wasps , leading to the development of sophisticated mechanisms that allow parasitoids to be integrated into the social life of antcolonies, as in the case of the chemical camouflage displayed by Eucharitidae wasps . Alternatively, characteristics of nesting resources, such as the entrance size, could determine the ability of parasitoids to access the nest. For example, a smaller nest entrance could allow ants to prevent parasitoids from getting in, if large enough to be detected. Ants and parasitoid wasps respond differently to changes in habitat complexity . Natural enemies are generally more abundant when habitat complexity increases, possibly because they are able to find refuge from predators and alternative food sources . Habitat complexity enhances predator mobility and colonization , and therefore may increase host mortality by providing adequate refugia for parasitoids, as has been shown for predator-prey systems . Furthermore, parasitoid-host interactions can depend on environmental contexts, as has been already suggested for predator-prey systems . For instance, in aquatic systems, diverse predator guilds are less effective when plant structures are more diverse , but also the availability of refuges can reduce intraguild predation . Various scenarios are possible with parasitoid-host interactions: on the one hand, environmental context could determine the success of parasitoids to find their hosts ; on the other hand, more complex habitats could provide moreresources to feed when hosts are not abundant enough, allowing their persistence in the environment. In the specific case of ants and their parasitoids, Wilkinson and Feener demonstrated that habitat complexity such as the diversity of plant architectures, allows ant hosts to escape parasitism from parasitoid flies. Although there has been considerable progress in our understanding of the role of habitat complexity on parasitoids in general, the interaction between parasitoid wasps and their ant hosts remains poorly understood.
In coffee agroecosystems, ants are considered one of the most abundant and diverse groups of natural enemies . Besides their relevance for agricultural management, ants are a model system to study questions in community ecology. Ants participate in a variety of species interactions , their abundance and diversity is often explained by the availability of food and nesting resources , they are sensitive to habitat disturbance , and are potentially regulated by top down forces such as parasitoids . Resource mediated interactions, in combination with the influence of habitat complexity on both ants and parasitoids, pose an exciting opportunity to investigate whether nectar and nesting resources, and habitat complexity together, mediate important processes in the ant colony life . In this study, we examined whether colonization by TNA’s, colony size, and parasitoid-host interactions between parasitic wasps and their ant hosts are influenced by the addition of sugar resources, the size of nest entrances, and habitat complexity in a coffee agroecosystem. Specifically, we addressed the following questions: Does the addition of nectar and nesting resources of different entrance sizes influence colonization and colony size of TNAs? Does the addition of nectar resources and nest entrance size influence parasitism rates of TNAs? and Is the effect of nectar resources on ant colonization, growth and parasitism context dependent?. We hypothesized that adding nectar resources would result in a higher proportion of colonized nests, larger colony size, raspberries in pots and a “preference” for certain ant species to colonize nests of specific sizes. Further, we expected to find a higher proportion of parasitized ant pupae with nectar addition, and lower parasitism rates in nests with a smaller entrance size. Finally, we hypothesized that ant colonization, colony size, and parasitism would be higher in more complex habitats. This study took place within a 300 hectare organic, shaded coffee agroecosystem located at 1000 m a.s.l. in the Sierra Madre mountains of the Soconusco region in Chiapas, Mexico. The climate is semitropical with rains between May and October. The annual rainfall varies between 4000-5000 mm. The agricultural management is categorized under “commercial polyculture” with a percent canopy cover that varies from 50% to 91% , where trees of the genus Inga dominate the coffee landscape. Within the agroecosystem, we established 20 sites, each separated from each other by 15 m. Each site consisted of two Inga micheliana trees that were randomly selected from a map. I. micheliana trees were selected making sure there were no Azteca sericeasur ants nesting on the selected trees or trees within 10 m. We characterized habitat complexity in each site. We measured percent of canopy cover 2 m to the N, S, E and W of each tree with a densitometer. Within 5 m of each tree, we counted the number of coffee plants, measured weed height, estimated the percent of ground covered with weeds, and measured height and circumference of all trees. We constructed a Vegetation Complexity Index using the vegetation data. We calculated index values for vegetation surrounding each tree by dividing the values of each variable by the highest observed value across all trees for each variable. Then we took the average across the two trees within one site to obtain a single value between 0 for low vegetation complexity and 1 for high vegetation complexity.
To evaluate the effect of nectar and nesting resources on colonization and colony size of TNAs, we added artificial nectaries and artificial nests made of bamboo to each of the two I. micheliana trees in each plot. The artificial nectaries consisted of 2 bottles affixed with 4 2ml Eppendorf tubes filled with either nectar or water, and emerging cotton strings to deliver the liquid to insect visitors . The nectar solution consisted of one part of nectar and three parts of water. We randomly assigned one tree in each site to either the sugar treatment and the other to the control treatment. We added one artificial nectary device per tree at a manageable height of 3 m approximately, reachable with a standard aluminum ladder. We replenished both sugar and water solutions to 1.5 ml every two weeks and monitored ant activity on the devices. Artificial nectaries remained in the field for 3 months during the rainy season. We added artificial nests made from bamboo to I. micheliana trees . On each tree, we added six artificial bamboo twigs of two different entrance sizes to increase resource variability and therefore diversity of colonizing ants . Artificial nests remained in the field for three months. To assess ant colonization and colony size, we collected artificial nests after three months. We transported artificial nests to the lab, froze them for 10 min. to decrease ant mobility, and then opened each nest to assess whether they were occupied, to identify ant occupants, and to count adults and brood in each nest. We assessed ant parasitism by rearing all ant brood. We placed brood in plastic containers covered with a fine mesh to allow air flow. In the case of ant species without cocooned pupae , we placed brood in containers with adults, to allow for sanitary brood care to prevent fungal infections during rearing . Thus, for those species, we placed the entire nest in the plastic container, along with water and honey to allow for colony maintenance. We maintained brood for 10 days, during which time we recorded emergence of parasitoids and collected all emerged adults in plastic vials containing alcohol. After 10 days, we collected all pupae and larvae and dissected them under the microscope to account for parasitoids that did not fully develop during the rearing time. Parasitoid identification was done using the annotated keys to the genera of nearctic Chalcidoidea and Wheeler and Wheeler key to hymenopterous parasites of ants. To determine if the addition of nectar resources, nest entrance size and VCI influence ant colonization , we performed generalized linear mixed models with “glmer”, using the “lme4” package in R . We used the “cbind” function to create the dependent variable using the number of nests that were occupied and the number of nests that were not occupied, included site as a random factor, and used a Binomial error distribution. We compared two models. In the first, nest occupation was tested as a function of both nectar and artificial nest size treatments and the interaction between the two as fixed factors, with the VCI included as an additional predictor. In the second, VCI was removed. We selected the best model using the Akaike’s Information Criterion calculated with the “MASS” package . To determine if the addition of nectar resources, nest entrance size and VCI influence colony size , we performed GLMM as above for each of the three most common ant species. We used site as a random factor and a Poisson error distribution. We used three dependent variables for analysis – workers and brood . To determine the effect of both treatments, we compared two models for each dependent variable. In the first, the number of workers, larvae or pupae were tested as a function of both nectar and size treatments and the interaction between the two as fixed factors, and the VCI. In the second, VCI was removed.