! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!!!!!!!!!!!!!80+.) 9 Effectiveness of the minima caste of the Leafcutter Ant ( Atta cephalotes ) on cleaning harvested leaves Tori Braun University of California, Santa Barbara Department of Ecology, Evolution, and Marine Biology EAP Tropical Biology and Conservation, Fall 201 6 16 December 2016 ABSTRACT Leafc utter ants have been practicing agriculture longer than humans themselves. Leafcutter ant species, Atta cephalotes common to Central and South America, cultivate a unique fungus, Leucocoprinus gonog yl ophora, that sus tains the lives of millions of leafcutter ants within a single colony. In order to be efficient farmers, A. cephalotes have developed a caste system consisting of a queen, soldiers, workers, and minima. Each caste has a specific role within the colony, how ever, the role of the minima is still highly debated among scientists It is hypothesized that the minima clean the surface of the leaves collected from nearby plants on the trail from the plant source to the entrance of the colony. Using two different met hods, the cleanliness of the leaves at the leaf source and the entrance to the colony were observed. The first method consisted of placing the leaf sample onto an agar plate, while the second method consisted of swabbing the leaf surface of the samples ont o an agar plate and observing whether the samples presented fungal growth or not. Overall, it was shown that there was little difference between leaf cleanliness as both the leaf source and the colony entrance had 60% of the samples grow fungus, however, other patterns such as fungal diversity and presence of mites were observed and analyzed. On average, there was a higher diversity of fungi at the leaf source compared to the entrance of the colony which may suggest that the minima may only be cleaning fo r certain types of fungi. A presence of mites found exclusively on samples taken from the leaf source could suggest that minima also clean off pests from the leaves. T hese results seem to indicate that the role of the minima is crucial to the colon y, as th ey perform multiple unique tasks in order to protect their precious food source. Efectividad en limpiar hojas de la casta mÂ’nima de la hormiga cortadora de hojas ( Atta cephalotes ) RESUMEN Las hormigas cortadoras de hojas han estado practicando agric ultura inclusive por mÂ‡s tiempo que los seres humanos. La especie Atta cephalotes nativa de Centro y SuramÂŽrica, cultiva al hongo Leuco co prinus gonogylophora el cual sirve de alimento para millones de
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) : hormigas dentro de una misma colonia. Para que el cu ltivo del hongo sea existoso, A. cephalotes ha desarrollado un sistema de castas conformado por una reina, soldados, obreras y mÂ’nimas. Cada una de estas castas juega un rol especÂ’fico dentro de la colonia, sin embargo el papel de las mÂ’nimas es motivo de debate en la comunidad cientÂ’fica. Una hipÂ—tesis es que estas hormigas limpian la superficie de las hojas colectadas a lo largo del camino entre la planta y la entrada de la colonia. Utilizando dos mÂŽtodos, determinÂŽ la presencia de hongos en hojas reciÂŽn cortadas de la planta y hojas antes de ingresar a la colonia. El primer mÂŽtodo consistiÂ— en poner la hoja sobre una placa de agar, y el segundo en frotar la superficie de la hoja sobre la placa de agar. Posteriormente determinÂŽ la presencia o ausencia de crecimiento de hongos y el nÂœmero de morfoespecies de los mismos. Se encontrÂ— poca diferencia en cuanto al crecimiento de hongos en muestras provenientes de la planta y muestras provenientes de la entrada de la colonia. Ambos grupos presentaron crecimient o de hongos en un 60% de las muestras, sin embargo las hojas reciÂŽn cortadas presentaron ligeramente mayor riqueza de especies de hongos asÂ’ como la presencia de Â‡caros Esto podrÂ’a indicar que las mÂ’nimas solamente limpian las hojas de ciertos hongos que potencialmente puedan llegar a ser perjudiciales para L. gonogylophora La presencia de Â‡caros exclusivamente en las hojas reciÂŽn cortadas sugiere que las mÂ’nimas tambiÂŽn limpian las hojas de estos organismos. En conjunto, estos resultados parecen indicar que el rol de la mÂ’nima es crucial, al realizar mÂœltiples tareas que ayudan a proteger su recurso alimenticio. Atta cephalotes, commonly known as the leafcutter ant, is unique to the Neotropics and can be found throughout Costa Rica and Central and South America Leafcutter ants are recognized for their mutual symbiosis with a fungus Leucocoprinus gonogylophora which they grow with the leaf fragments they harvest (Stevens, 1983 ). This fungus is essential for Atta cephalotes survival and the fungu s also solely r elies on the ants. L. gonogylophora has lost certain enzymes to digest woody plant matter, therefore it relies on the leaves the ants feed it while the ants rely on the fruiting bodies of the fungus that are essential for their growth (Smith sonian Tropical Research Institute, 2016). Along with the fungus that they cultivate, their eusocial nature also makes them interesting study subjects as each caste has a different role within the colony. The queen's purpose is to begin a colony, rear her young until they are old enough to sustain themselves and the colony, and then lay eggs for the remainder of her life. Soldiers can be up to 20 millimeters in length and have a specific job of d efending the nest Worker ants cut leaves and carry them back to the colony in order to sustain the fungus they cultivate and can reach up to ten millimeters in length. Lastly, there are minima ants that are around two millimeters in length and are mostly found "riding" the leaves from the source of the leaf fragmen ts to the entrance of the colony (Stevens, 1983) The role of the minima ant, however, is still not specifically known. One hypothesis is that the minima clean the surface of the leaves from fungi that could be detrimental to the fungi they cultivate It i s hypothesized that cleaning done by the minima may speed fungal growth and even remove foreign fungi (Stevens, 1983). Another hypothesis states the minima exist to defend the harvested leaf fragments from predators, such as parasitic flies that attack wor ker ants (Holbrook, 2009). Due to the uncertainty of the role of the minima caste, I asked the question: Do minima leafcutter ants clean the harvested leaf fragments as they are being transported from the source to the colony, and if so, how effective is t he cleaning done by the minima ants? I
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) ; hypothesize that minima clean the leaves, therefore leaf samples that contain minima will ultimately be "cleaner" than leaves collected from the source. Within my hypothesis, I predict first that there will be a lower rate of fungal presence on leaves with minima, and second, that there will be a lower rate of fungal diversity on leaves with minima. MATERIALS AND METHODS Field Methods In order to observe if leafcutter ants clean the surface of the leaves on the jour ney from the plant source to the colony I took samples from two different leafcutter ant colonies on November 26 and the November 29 of this year The first leafcutter ant colony is located on the Magic Path in Bajo del Tigre and will be referred to as Si te 1 The second leafcutter ant colony is also located in Bajo del Tigre near the path to Frank Joyce's house and will be referred to as Site 2. At both sites the source of the leaves being collected wa s located and I took samples At the leaf source I f irst observed the behavior of the ants and found no minima to be on the su rface of the leaves the workers were carrying towards the colony Thus, I collected ten leaf samples without minima at each site from the leaf source by using sterile tweezers and t aking the leaf from the ant and placing in into a sterile Ziploc bag. Next, I took samples from the entrance to the colony. First I took observations and noticed that there was a mix between leaves that had zero, one, or two minima on them. However, becaus e I wanted to test the effect of the minima, samples were only collected that had one minima on them. Samples at the colony were also collected by taking ten leaf samples at each site swiping off the minima with a sterile swab, and putting the leaf sample into a sterile Ziploc bag. Overall, 40 leaf fragments ranging from 20 25 millimeters in length, were collected between the two sites. Lab Methods T he samples were taken to the lab at the Monteverde Institute. In the lab two different methods were used to observe leaf cleanliness. For the first method, five samples from the leaf source were placed on to potato dextrose a gar plate s by removing the leaf sample from the Zi ploc bag with sterile tweezers and placing only one leaf fragment on each agar pla te. Five samples collected by the colony entrance were also placed onto agar plates. Then, the ten agar plates were place d in the incubator at 37 Â¡ C Leaf samples were observed under a dissecting microscope after one, two, and three days of incubation The same method was then performed with the samples from Site 2, therefore there were 20 agar plates in total. When observing the sample s on the agar plates presence of fungi was noted, the different types of fungi were noted and photographed and anything else that stood out, such as a presence of mites was recorded The second method I used consisted of swabbing each individual leaf sam ple. The other five samples from the leaf source and colony from each site were swabbed with a sterile cotton swab and then the swab was placed onto a potato dextrose agar plate. Each leaf sample swabbed was placed onto its own agar plate, thus there were 20 plates in total used in this method. These plates were also placed into an incubator at 37 degrees Celsius and were observed after one, two, and three days of incubation under the dissecting microscope. During each observation the presence of fungi was noted, along with how many fungi spots and the number of different types that were growing.
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) < RESULTS I found that 60% of the leaf samples from the leaf source grew fungi after one day of incubation and 60% of the leaf samples from the entrance to the col ony grew fungi after one day of incubation (Figure 1). O n average the leaf samples from the colony grew less different types of fungi compared to the samples collected from the leaf source (Table 1 ). From the leaf on agar method, at the source, there was an average of 2.2 different typ es of fungi, while at the colony there was an average of two different types of fungi. In the swab method, the source showed an average of 3.4 different types of fungi, while the colony showed an average of three different ty pes of fungi. Dif ferent and unique morphospecies were also found between the leaf source and the entrance to the colony. For example, one morphospecies I called "white jelly" was found in every sample from both the source and the colony. However, another m orphospecies of fungi that I called "green hairy" was only found in samples at the leaf source. Lastly, when observing the leaf samples under the dissecting microscope, I noticed a prese nce of mites on the surface of the leaves Nine mites in total were fo und on four different samples solely from the leaf source (Figure 2). Figure 1. Presence of Fungi Between Source and Colony Samples. This chart demonstrates that 60% of samples from the source grew fungi after 1 day and 60% of the samples from the colony grew fungi after 1 day.
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) = DISCUS SION Ultimately, the first prediction of my hypothesis was not supported. I had expected to see a higher rate of cleanliness in sampl es with minima than without. However, I observed an even rate of cleanliness between samples from the leaf source, without minima, and samples from the entrance to the colony, with one minima My second prediction, on the other hand, was supported by my results. I had expected to see a lower rate of fungal diversity in samples taken from the entrance to the colony than in samp les taken from the leaf source. In both methods, the colony showed lower rates of fungal diversity than the leaf source. Both the rejection of the first prediction and the acceptance of the second prediction may suggest the m inima clean specific morphospec ies of fungi that may harm L. gonogylophora Leafcutter ants have been subjected to thousands of years of evolution and thus have developed job "roles based on sustaining a balance between fuel supplies and energy consumption in the colony" (Newitz, 2016). It is probable that !"#$%&%'$#()* % + % % % % / % 01$234$ % > #.0-%!?'%/@#7!9A ; ; : : 9 :B: 2 #$#)C!?'%/@#7!9A < : D 9 ; : > #.0-%!?'%/@#7!:A ; < < < : ;B< 2 #$#)C!?'%/@#7!:A ; < 9 ; < ; Figure 2. Mite presence on leaf samples. Nine mites were found within 10 samples from the leaf source. No mites were found on any of the 10 samples from the colony. Table 1. Number of fungal morphosepecies within each sample (1 5). Fungal diversity, on average, is higher at the leaf source in both Method 1 (leaf on agar) and Method 2 (swab me thod).
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) E it is not cost efficient for the minima to clean off fungi that would not threaten their food source. This finding could also suggest that the leaves are not cleaned until a later stage of the journey and could support the claim that t he "fungus garden is tended by certain workers, which weed out any foreign fungi that may be broug ht in with the leaves" (Borror et al. 1989). Differences in fungi found at the leaf source and the entrance to the colony may also tell us about the vulnera bility of L. gonogylophora Because the morphospecies of fungi, "white jelly" was found in all samples in both the leaf source and the entrance to the colony, this may suggest that this specific morphospecies is not harmful to the leafcutter ants' food su pply. On the other end of the spectrum, the morphospecies, "green hairy" was only found in samples taken from the leaf source. This finding would lead me to predict that this specific morphospecies of fungi could potentially threaten L. gonogylophora Acco rding to recent research, l epiotaceous fungi can be vulnerable to changes in the environment (Vellinga, 2004). This supports that the fungus the leafcutter ants grow may be highly susceptible to other fungi, diseases, and pests that could ultimately be det rimental to its health. With minima there to monitor and clean the fungi, however, their role becomes vital within the colony. Lastly, the presence of mites may suggest that the minima have multiple roles within the colony. Because several mites were foun d on the surface of the leaves without minima, this could suggest that the minima are also concerned with ridding the leaf surface of potential thre ats. It is known that minima aid in the removal of pests and prevention of infection due to fun gal pathogens inside the colony (Currie & Stewart, 2001). However, this role of the minima has only been explored from inside the colony, thus further research is needed to support this conclusion. This experiment could be improved in th e future by a djusting the method s by only collect ing leaves from the colony that were observed with a minima o n the leaf for the whole journey from the source to the entrance of the colony. Due to poor forest conditions, such as tree fall and debris, following an ant from the source to t he colony was nearly impossible. By only taking samples directly from the leaf source and the colony entrance in my experiment it cannot be confirmed how long the minima had actually been on the leaf surface, and thus if they were actually cleaning t he le af surface from the leaf source to the entrance of the colony Another source of error could be due to the sensitivity of the potato dextrose agar plates. Since these plates are highly susceptible to growing fungus, some of the fungus grown could have come from different sources not related to leafcutter ants. In the future, this study could be taken a step fu rther by examining L. gonogylophora itself I believe it would be intriguing to see how this fungus reacts to the different morphospecies of fungi th at were grown on my samples This information could provide knowledge of what specific morphospecies of fungi the minima ants clean By further investigating mutualisms and their importance in nature, humans may also learn how to form mutualisms with natur e and ultimately help put an end to climate change, in order to save these highly susceptible communities. In conclusion, the role of the minima ant within the colony may serve countless functions. It is likely that the minima clean the surface of leaves f or harmful fungi, pests, and perhaps have many more functions that have not yet been studied. This role, however, is vital to the success of the intricate empire of A. cephalotes L. gonogylophora and their farming community.
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) F ACKNOWLEDGEMENTS Thank you t o my primary advisor, Andres Camacho, for helping me work out the kinks of my project and for your help analyzing my results. Thank you to my secondary advisor, Sofia Arce Flores, for all of your feedback and helpful comments. Thank you to the landowners o f Bajo del Tigre, and especially Frank Joyce, for allowing me to study the ant colonies and collect samples here. T hank you to the Monteverde Institute for allowing me to invade the lab oratory every day for two weeks to do my research. Thank you to all the UCEAP students and staff for your continued support and encouragement. And lastly, thank you to the gracious ants who let me steal and study your hard earned leaves. LITERATURE CITED B orror, Donald J., Charles A. Triplehorn, and Noorman F. Johnson. An In troduction to the Study of Insects. 6th ed. Orlando: Saunders College, 1989. Print. Currie C R &, Stuart A E 2001. Weeding and grooming of pathogen s in agriculture by ants. Proc. R. Soc. B Biol. Sci. 268 :1033 1039. Newitz, Annalee. "Why Leafcutter Ants Evolved into Sophisticated Farmers." Ars Technica. N.p., 27 Jan. 2016. Web. 06 Dec. 2016. Smithsonian Tropical Research Institute. "After the age of dinosaurs came the age of ant farmers." ScienceDaily. ScienceDaily, 20 July 2016. www.sciencedaily.com/releases/2016/07/160720094244.htm Stevens, G. C., In Costa Rican Natural History. Chicago: U of Chicago, 1983. Print. Tat e Holbrook, Rebecca Clark, & Brian Haney. "Secrets of a Superorganism." ASU Ask A Biologist. 27 Sep 2009. ASU Ask A Biologist, Web. 6 Dec 2016. http://askabiologist.asu.edu/explore/secrets superorganism Vellinga, Else C. "Ecology and distribution of Lepiotaceous fungi (Agaricaceae) A Review ." Nova Hedwigia 78.3 4 (2004): 273 299.
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) G APPENDICES Appendix 1. A (top left). Shows a mite on the surface of a leaf sample from th e source. B (bottom left). Shows a mite on the surface of a leaf sample from the source. C (top right). Shows the morphospecies of fungi, "white jelly", grown most commonly on all samples from the leaf source and entrance to the colony. D (bottom right). S hows the morphospecies of fungi, "green hairy", grown in only samples from the leaf source.
! "#$%!#&!'()(*+!,%+&-.//%0!1)/!()!2$%+)()3!4+05%6/%7!,%+5%6!!!!!!!!!! !!!!!!!!!!! 80+.) H Appendix 2. Types of fungi found in each method and at each site. This table shows the different types of fungus and the presence of that certain fungus in each sp ecific method and site. (MP = Magic Path. FH = Frank's House. Leaf = Leaf on agar method. Swab = swab method.)