El Colegio de la Frontera Sur
Espionaje por estímulos feromonales en la abeja sin aguijón Scaptotrigona mexicana Guérin (Apidae, Meliponini)
TESIS presentada como requisito parcial para optar al grado de Maestría en Ciencias en Recursos Naturales y Desarrollo Rural
por
Erik de Jesús Solórzano Gordillo
2015
DEDICATORIA
A Dios
Por permitirme tiempo y salud para cumplir mis metas, por indicarme el camino cuando me sentía perdido
A mi padre
Alberto de Jesús Solórzano Magaña por el apoyo incondicional, por el cariño y por enseñarme que la perseverancia y el esfuerzo son el mejor camino para lograr todo aquello que quieres
AGRADECIMIENTOS
A El Colegio de la Frontera Sur Unidad Tapachula, por permitirme realizar mis estudios de posgrado.
Al Consejo Nacional de Ciencia y Tecnología (CONACYT) por la beca de maestría otorgada.
Al Dr. Daniel Sánchez Guillén por guiarme, por el apoyo incondicional y la sabiduría brindada durante mi estancia en el proyecto Abejas de Chiapas.
A los Dres. Julio Cesar Rojas León y Leopoldo C. Cruz López por el apoyo otorgado en la realización de este proyecto.
A los Dres. José Pablo Liedo Fernández, Ariane Liliane Jeanne Dor Roques y M. en C.
Miguel Ángel Guzmán Díaz por sus valiosos comentarios y observaciones al escrito.
A la Dra. Lislie por los consejos brindados durante el proceso de la realización de este proyecto.
A mis compañeros y amigos del proyecto de abejas Leonardo, Andy, Ricardo, Jovani y
Augusto por su apoyo en la parte experimental.
A mis compañeros de la maestría generación 2014-2015, por ser mi familia durante esta etapa de mi vida.
Al M. en C. Oscar Fernando Mikery Pacheco por su amistad y apoyo incondicional.
A Hernán Villatoro Moreno y Ana Karen Serrano Domínguez por haberme brindado su amistad durante todo este tiempo y apoyarme en todo momento incondicionalmente.
Esta investigación fue financiada por los proyectos: “Evolución de la cleptobiosis en
Lestrimelitta” Consejo de Ciencia y Tecnología (No.128702). Responsable Dr. Daniel
Sánchez Guillén; “Efecto del uso del suelo en la conservación de la biodiversidad de las abejas” Consejo de Ciencia y Tecnología (No.106043); “Espionaje olfativo en contra de un cleptoparitismo, Lestrimelitta niitkib” UC-MEXUS.
ÍNDICE
ÍNDICE DE FIGURAS ...... I
I. INTRODUCCIÓN ...... 1
II. CAPITULO Eavesdropping between colonies of the stingless bee Scaptotrigona mexicana Guérin (Apidae, Meliponini) during foraging ...... 7
III. CONCLUSIONES ...... 25
IV. REFERENCIAS CITADAS ...... 26
V. Anexo 1. Distribución geográfica de S. mexicana en la República Mexicana.
(fuente: Discover life, Ascher et al., 2007)...... 31
VI. Anexo 2. Dispositivo de marcaje de abejas...... 32
VII. Anexo 3. Experimento 1, marcas de olor en condiciones de campo...... 33
VIII. Anexo 4. Experimento 2, bioensayo glándulas labiales y mandibulares.. .. 34
ÍNDICE DE FIGURAS
1. Proporción de abejas forrajeras (± error estándar) colectadas en cada alimentador
en experimento 1. Letras diferentes muestran diferencia significativa (Prueba de
comparaciones múltiples de Tukey, α =
0.05).…………...……………..…………………………………...………………….....…23
2. Proporción de abejas forrajeras (± error estándar) visitando los alimentadores con
extractos de glándulas de obreras del mismo nido, diferente nido o alimentador
limpio (control) en experimento 2. Letras diferentes muestran diferencia significativa
(Prueba de comparaciones múltiples de Nemenyi, α =
0.05)..…………….……………………………………………………………………....…. 24
I
I. INTRODUCCIÓN
La mayoría de las especies de abejas sin aguijón (Hymenoptera, Apidae,
Meliponini) utilizan señales químicas, auditivas, visuales, táctiles y térmicas para reclutar a otras abejas de su misma colonia a fuentes de alimento, proceso denominado reclutamiento (Nieh, 2004). Este es un mecanismo que permite a las abejas sin aguijón encontrar la ubicación específica del alimento con una alta precisión, incluso más eficiente que la danza realizada por Apis mellifera (Sánchez et al., 2004). De todos los canales sensoriales usados durante el reclutamiento, se ha planteado que es la señalización química, por ejemplo el uso de feromonas, la información más importante utilizada por las abejas reclutadas (Nieh, 2004; Sánchez et al., 2011).
Dependiendo de la especie, las abejas sin aguijón pueden utilizar una o varias estrategias para depositar feromonas: (1) a través de rastros de olor depositados sobre diferentes sustratos (por ejemplo piedras y hojas) de regreso a su nido, (2) del marcaje de la propia fuente de alimentación y, (3) la liberación de feromonas en el aire durante su camino de regreso a la colonia (Lindauer y Kerr, 1960; Schmidt et al., 2003; Nieh,
2004; Sánchez y Vandame, 2013). Sin embargo, estas señales pueden ser interceptadas por otros individuos a los que no se tenía la intención de comunicar; este fenómeno es conocido como espionaje (Slaa y Hughes, 2009).
Se pueden distinguir dos formas de espionaje, el social y el interceptivo. El espionaje social ocurre cuando se recopila información sobre otros individuos con la finalidad de establecer un rango de dominancia relativa; este tipo de espionaje se presenta en algunas especies de peces y aves territoriales (Peake, 2005).
1
El espionaje social podria ser importante en la organización colonial de algunos insectos sociales (en hormigas y avispas) con determinada dominancia jerárquica (Slaa y Hughes, 2009). Por ejemplo, la hormiga Cephalotes specularis “hormiga-tortuga- espejo” espía y copia los movimientos de la hormiga Crematogaster ampla con mayor dominancia jerárquica por ser hiper-agresiva y pasa desapercibida evitando el ataque y aprovechando los recursos defendidos por C. ampla (Powell et al., 2014). Por lo tanto, es poco probable que el espionaje social sea relevante en el forrajeo de insectos sociales debido a que éstos ocupan preferentemente otros canales de comunicación
(Slaa y Hughes, 2009).
El espionaje interceptivo se refiere a la intercepción de señales destinadas a otros receptores (Peake, 2005), y se piensa que es más probable que ocurra en insectos sociales (Slaa y Hughes, 2009). Por ejemplo, es común que varias colonias de insectos sociales colecten recursos en un área común, surgiendo entonces la posibilidad que las forrajeras de estas colonias se encuentren con las señales dejadas por individuos de la misma especie pero de diferente colonia (intercepción conespecífica) o de colonias de diferente especie (intercepción heterospecífica), usando esta información para decidir su patrón de forrajeo (Slaa y Hughes, 2009).
El espionaje recopila información a un costo relativamente bajo comparado con los costos asociados a la comunicación de manera directa, ya que no se invierte ni tiempo ni energía en la búsqueda de recursos, además que el espionaje brinda información de mejor calidad que la señalización absoluta entre los individuos, debido a que este permite la comparación simultánea de al menos dos individuos en un contexto competitivo, ya que estas señales reflejan fielmente recursos rentables a pesar de la
2 competencia (McGregor, 1993). La competencia por recursos es muy frecuente entre colonias de insectos sociales por lo que el espionaje es considerado una estrategia ventajosa para encontrar nuevas fuentes de alimento ya que el esfuerzo de búsqueda se incrementa sustancialmente (Slaa y Hughes, 2009).
El espionaje juega un papel importante en la evolución de las complejas y sofisticadas redes de comunicación en los animales (Peake et al., 2002), y es un elemento clave en la comunicación de las abejas (Nieh, 1999; Nieh, 2004). Mientras algunas especies de abejas sin aguijón utilizan el espionaje olfativo interceptivo (Peake,
2005) para detectar fuentes de alimento descubiertos por otras abejas (Kerr et al., 1963; kerr et al., 1981; Kerr, 1994), otras especies pueden detectar las feromonas de especies agresivas para evitar conflictos costosos con sus competidores (Lichtenberg et al., 2011).
Algunas abejas sin aguijón viven en ambientes donde los recursos alimenticios son escasos, por lo tanto es imprescindible reclutar otros individuos de la misma colonia para explotar estos recursos (Roubik y Aluja, 1993; Liow et al., 2001). Muchas especies de abejas sin aguijón extienden su zona de alimentación cientos de metros, por ejemplo
Trigona amalthea deja un rastro de olor que se extiende aproximadamente 900 m
(Roubik y Aluja, 1993; Van Nieuwstadt y Ruano, 1996). Estos rastros de olor crean una larga ruta pero con un alcance relativamente corto en el canal de comunicación que puede ser detectado por otras abejas forrajeras que entrelazan sus rutas de búsqueda de alimento (Nieh, 2004).
Varios estudios han mostrado que las forrajeras de los géneros Trigona y
Scaptotrigona secretan feromonas de reclutamiento almacenadas en glándulas labiales
3 y mandibulares (Lindauer y Kerr, 1960; Stangler et al., 2009). Sin embargo, se ha demostrado que las glándulas mandibulares poseen un efecto de alarma o repelencia en la especie de abeja sin aguijón Trigona recursa (Jarau et al., 2006), por lo tanto su papel durante el reclutamiento es controversial (Barth et al., 2008), por lo que es importante evaluar el comportamiento de espionaje de abejas con ambos tipos de glándulas por separado.
Aunque se han realizado diversos estudios enfocados en la comunicación de las abejas sin aguijón, aún se desconocen muchos aspectos relacionados con el espionaje.
Estudios realizados en Scaptotrigona mexicana muestran una alta precisión para indicar un lugar específico donde se encuentra el recurso (Sánchez et al., 2004), y que las marcas de olor y las señales visuales son fuentes de información eficientes para el reclutamiento de forrajeras (Sánchez et al., 2007; Sánchez et al., 2008). Sin embargo, la comunicación multimodal juega un papel importante en el reclutamiento de forrajeras en esta especie (Sánchez et al., 2011), ya que los diferentes canales sensoriales empleados por esta especie contribuyen a una mayor precisión, aunque algunos podrían dar información redundante. Estos mecanismos de comunicación varían entre especies de abejas, tanto en la presencia y formas que puedan adoptar, lo que determina que la precisión espacial del reclutamiento sea diferente entre las especies de meliponinos (Sánchez et al., 2004).
La mayoría de los estudios de espionaje en meliponinos se ha enfocado en evaluar el espionaje interespecífico, espionaje entre distintas especies (Nieh et al.,
2004; Lichtenberg et al., 2011), sin embargo, no se han realizados estudios que evalúen el espionaje intraespecífico (espionaje entre colonias de la misma especie). El
4 espionaje intraespecífico puede estar involucrado de manera importante en algunos procesos ecológicos, por ejemplo en la variación intraespecifica de rasgos que afectan la dinámica poblacional y competencia (Bijlsma y Loeschcke, 2005; Bolnick et al.,
2011). Estos procesos pueden contribuir a la formación de conductas novedosas como la cleptobiosis (el robo de recursos entre individuos de la misma o de diferente especie;
Hölldobler y Wilson, 1990) e incluso a la aparición de subespecies (Montresor et al.,
2003; Jacobson et al., 2015; Luo y Wei, 2015). Por lo tanto, el objetivo principal de esta investigación fue evaluar el comportamiento de espionaje intraespecífico de
Scaptotrigona mexicana. Se eligió a esta especie porque se conoce con profundidad su comportamiento de reclutamiento para colectar recursos (Sánchez et al., 2008;
Sánchez et al., 2009; Sánchez et al., 2011) y por ser exitosamente utilizada para la polinización de cultivos en la región del sureste mexicano. Esta abeja sin aguijón se distribuye de manera natural desde Chiapas y por la costa del Golfo de México hasta
Tamaulipas, tanto por tierras bajas con bosque tropical perennifolio como en las laderas de las montañas con bosques de pino y bosques mesófilos de montaña, a una altitud que oscila alrededor de los 1000 m. Se presenta también en forma discontinua al sur del Estado de México y en el extremo más oeste de la Sierra Madre del Sur en
Guerrero (Ayala, 1999) ver anexo 1. Se propusieron los siguientes objetivos específicos: (1) Evaluar la preferencia de forrajeras de Scaptotrigona mexicana hacia marcas de olor del mismo nido o de diferente y (2) evaluar la preferencia de individuos recolectores (forrajeras) de S. mexicana hacia las feromonas labiales y mandibulares de individuos del mismo o de diferente nido. La hipótesis que se plantea es que las forrajeras de una colonia de Scaptotrigona mexicana prefieren las feromonas depositadas por las forrajeras de una segunda colonia conespecífica cuando son
5 presentadas con un recurso altamente rentable debido a un proceso de asociación de las feromonas con el recurso.
6
II. CAPITULO Eavesdropping between colonies of the stingless bee Scaptotrigona mexicana Guérin (Apidae, Meliponini) during foraging
------Mensaje reenviado ------
Asunto: NAWI - Submission Confirmation
Fecha: 11 Nov 2015 13:32:04 -0500
De: Naturwissenschaften (NAWI)
Responder a: Naturwissenschaften (NAWI)
Para: Daniel Sánchez-Guillén
Dear Dr Sánchez-Guillén, Thank you for submitting your manuscript, Eavesdropping between colonies of the stingless bee Scaptotrigona mexicana Guérin (Apidae, Meliponini) during foraging, to The Science of Nature. During the review process, you can keep track of the status of your manuscript by accessing the following web site: http://nawi.edmgr.com/ Your username is: DSanchez Guillén-298 Your password is: available at this link http://nawi.edmgr.com/Default.aspx?pg=accountFinder.aspx&firstname=Daniel&last name=S%c3%a1nchez-Guill%c3%[email protected] Should you require any further assistance please feel free to e-mail the Editorial Office by clicking on "Contact Us" in the menu bar at the top of the screen. With kind regards, Springer Journals Editorial Office The Science of Nature Now that your article will undergo the editorial and peer review process, it is the right time to think about publishing your article as open access. With open access your article will become freely available to anyone worldwide and you will easily comply with open access mandates. Springer's open access offering for this journal is called Open Choice (find more information on www.springer.com/openchoice). Once your article is accepted, you will be offered the option to publish through open access. So you might want to talk to your institution and funder now to see how payment could be organized; for an overview of available open access funding please go to www.springer.com/oafunding. Although for now you don't have to do anything, we would like to let you know about your upcoming options.
7
1 Naturwissenschaften - Original paper
2
3
4 Eavesdropping between colonies of the stingless bee Scaptotrigona mexicana Guérin
5 (Apidae, Meliponini) during foraging
6
7 Erik Solórzano-Gordillo . Julio C. Rojas . Leopoldo Cruz-López . Daniel Sánchez*
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9 El Colegio de la Frontera Sur, Carretera Antiguo Aeropuerto km 2.5, Tapachula, Chiapas,
10 México. C.P. 30700
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13 * Author for correspondence: Daniel Sánchez, e-mail: [email protected]. Tel.: +52 01 962 62
14 89800-Ext. 5400.
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19
8
20 Abstract
21 Stingless bees use chemical signals to inform nestmates the location of rich food sources.
22 However, such information can be intercepted by unintended conspecifics from another colony.
23 In this paper, we investigated if interception of chemical information occurs in the stingless bee
24 Scaptotrigona mexicana by performing two experiments. In the first experiment, foragers were
25 exposed to odor-marked feeders by nestmates and by non-nestmates and their preferences were
26 recorded. In second experiment, we marked feeders with mandibular and labial gland extracts of
27 nestmates and of non-nestmates. Our results indicate that eavesdropping is present at the
28 intraspecific level. However, we found that eavesdroppers oriented better towards non-nestmate
29 odors using odor marks than using gland extracts, due presumably to the more complex
30 composition of odor marks, which include chemicals from other parts of the bees. Thus foragers
31 exposed to pheromones from non-nestmates are able to orientate towards food sources marked
32 with these compounds. We discuss this finding within an ecological and behavioral framework.
33
34 Keywords: pheromone, recruitment, foraging, meliponine, espionage
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40
9
41 Introduction
42 Most species of stingless bees (Apidae, Meliponini) are known to recruit nestmates to food
43 sources through spatial information encoded in chemical, sound, visual, tactile, and presumably
44 temperature related signals (Nieh 2004). Recruitment communication in stingless bees allows
45 them to find a specific food location with high precision even more efficiently than the dance of
46 the honey bee, Apis mellifera (Sánchez et al. 2004). Chemical signaling is perhaps the most
47 important information used by recruits, though visual cues can also be meaningful (Nieh 2004;
48 Sánchez et al. 2011).
49 Recent studies have shown that stingless bee species use similar hydrocarbon-based
50 compounds to recruit nestmates to food sources (Jarau et al. 2004a; Jarau et al. 2004b; Schorkopf
51 et al. 2007), so detection of these chemicals by unintended receivers can occur, leading to
52 espionage-like behavior during foraging (Goodale et al. 2010).
53 There are some studies that have shown chemical espionage between species of stingless
54 bees (Lichtenberg et al. 2011; Nieh et al. 2004). Nieh (1999) hypothesized that communication
55 inside the hive may have evolved to avoid espionage by competitors. However, all stingless bee
56 species that are known to communicate the location of food sources use in some degree chemical
57 signals in the field, so this hypothesis might not be true. These chemical signals consist of
58 pheromones secreted by either labial or mandibular glands; even though both are associated with
59 recruitment to food sources, current evidence shows that mandibular pheromones are related to
60 colony defense (Jarau et al. 2006), and to keep competitors off the food source (Jarau et al.
61 2004b; Lichtenberg et al. 2011), however, other authors still suggest that these mandibular marks
62 are indeed used to signal the location of food sources (Lindauer and Kerr 1960; Nieh et al. 2003).
10
63 It is interesting to notice that no study regarding intraspecific espionage has been
64 performed in stingless bees or any other social bee. Such interaction may have special impact in
65 ecological processes, like population dynamics and intraspecific competition (Bijlsma and
66 Loeschcke 2005; Bolnick et al. 2011), since it may contribute to the formation of new behaviors
67 or even subspecies (Jacobson et al. 2015; Luo and Wei 2015; Montresor et al. 2003).
68 Thus, the main goal of this research was to determine if Scaptotrigona mexicana is able to
69 develop espionage-like behaviors intraspecifically. Thus carried out experiments to reach the
70 following specific goals: (1) determine the preference of S. mexicana foragers to odor marks of
71 the same or different nest and, (2) if S. mexicana foragers are able to develop a higher preference
72 towards the labial and mandibular pheromones of non-nestmates than to their own.
73
74 Materials and methods
75 Study site The experiments were conducted between 9:00 to 16:00h at the campus of El Colegio
76 de la Frontera Sur (ECOSUR), Tapachula (14° 53' N, 92°17' W, 75 m altitude), Chiapas, Mexico
77 from June to September 2015. We used six healthy, queenright colonies of S. mexicana housed in
78 wood boxes (20 × 20 × 40 cm), with approximately 2,500-3,000 bees and with enough food
79 reserves (Sánchez et al. 2008).
80
81 Bee training Five to ten foragers of each colony were trained to a specific feeder located at 15 m.
82 Feeders consisted of a tripod holding a square piece of cardboard (15 X 15 cm), which offered
83 2.0M sucrose solution inside a Petri dish (10 cm diameter X 1.5 cm height; appendix 2). Foragers
84 had access to the food through several holes made to the Petri dish. Since S. mexicana foragers
11
85 do not show territorial behavior when collecting food (personal observations), more than one
86 colony could visit the same feeder. Therefore we used a device to mark the thorax of the foragers
87 with a water-based paint at the colony entrance to identify the specific colony (Mikery-Pacheco
88 et al. 2013; Sánchez et al. 2009).
89
90 Experiment 1 - Odor-marking in field conditions The objective of this experiment was to
91 determine if espionage occurs under field conditions. We performed this experiment with two
92 colonies at a time and consisted in three phases. Odor-marking phase: foragers of two colonies
93 were simultaneously trained to their own feeder (appendix 3a). On top of the Petri dish a circular
94 piece of filter paper was placed so that foragers had to walk on it to mark it (appendix 3b). After
95 80 visits of bees to these feeders, the piece of paper was removed and stored in ice (appendix 3c).
96 Any forager that did not have the color of the colony under training was captured and kept in a jar
97 until the end of the experiment. Non-nestmate odor phase: in this phase we exposed foragers
98 from colony 1 (eavesdropping colony) to odor marks of colony 2 (subordinate colony) by
99 removing the feeder of colony 1, so foragers from this colony started visiting the feeder of colony
100 2 (appendix 3d). After 2 minutes of exposure, this feeder was removed and phase 3 began.
101 Espionage phase: two new feeders were placed 20 cm away from each other in a row-wise
102 fashion, each showing the piece of filter paper marked in the odor-marking phase; an additional
103 feeder showing an untouched piece of paper worked as a control (appendix 3e). For 20 min we
104 trapped and registered any bee from the two colonies (eavesdropping and subordinate colony)
105 that landed in any of the feeders to avoid pseudo-replication. In order to avoid site learning, we
106 exchanged the locations of the feeders every 5 min. Foragers were released when the experiment
107 was over. Six replicates were performed.
12
108
109 Experiment 2 - Bioassays with mandibular and labial glands In this experiment, we evaluated if
110 mandibular and labial glands are involved in recruitment, leaving aside other cues that could have
111 an effect on forager's choice behavior. We worked with one colony at a time, which was
112 simultaneously exposed to hexane extracts of labial and mandibular glands of nestmates and non-
113 nestmates (Jarau 2009; appendix 4). Gland extracts were made as described by Jarau et al.
114 (2010). In each replicate, we used 10 µl of extract, which corresponded to one-bee-equivalent
115 (Reichle et al. 2013). Five to ten foragers of one colony were trained to a feeder with 2M sucrose
116 solution at 10 m away from the colony. Once several recruits were observed (approximately 20
117 bees), we removed this feeder and placed three new ones separated 20 cm from each other: 1)
118 control feeder: a petri dish feeder with a 3 × 3 cm piece of filter paper saturated with 10 µl of
119 hexane; 2) nestmate feeder: a petri dish feeder with a piece of filter paper with 10 µl of labial
120 gland extract or mandibular gland extract from nestmates and, 3) non-nestmate feeder: a petri
121 dish feeder with a piece of filter paper with 10 µl of labial gland extract or mandibular gland
122 extract from non-nestmates. For 20 min we trapped (to avoid pseudoreplication) and recorded the
123 choices of any forager from the colony under study that landed and tried to feed. To avoid site
124 learning, we exchanged the locations of the feeders every 5 min. At the end of the experiment, all
125 captured foragers were released. In total we conducted 54 replicates.
126
127 Statistics We carried out a chi-square test to pooled data to determine if the number of foragers
128 among feeders in experiment 1 followed a uniform distribution (no preference of any cue), i.e. if
129 there were preferences for nestmate or non-nestmate odor marks. Pairwise Tukey HSD test to the
130 proportion of foragers was carried out to determine any differences in the preference by foragers
13
131 among feeders. In experiment 2 we observed several zeroes and a high variation in the number of
132 foragers recorded among replicates. Thus we analyzed the proportion of foragers that chose each
133 feeder using the Kruskal-Wallis analysis of variance to determine if foragers preferred nestmate
134 to non-nestmates gland extracts. Nemenyi's pairwise comparison test (Chi square corrected for
135 ties) was applied to the proportion of foragers to determine the source of significance of global
136 tests (Pohlert 2014). All analyses were carried out in R software (R Development Core Team
137 2012).
138
139 Results
140 In experiment 1, the choice of 328 foragers was recorded, 163 for the eavesdropping colonies and
141 165 for the subordinate colonies. One-hundred and four eavesdropping foragers chose the feeder
142 marked with non-nestmate odors, while only 14 and 45 foragers chose the control and the feeder
143 marked by nestmates, respectively. On the other hand, 116 odor-target foragers preferred the
144 feeder marked by their nestmates, while 30 and 19 foragers were captured at the control and non-
145 nestmate marked feeders, respectively. A Pearson's Chi-squared test revealed that the number of
146 eavesdropping foragers was significantly higher than expected in the non-nestmate feeder (χ2 =
147 72.92, df = 2, p < 0.001; Fig. 1).
148 In experiment 2 with labial gland extracts we recorded 565 choices in 29 replicates: 228,
149 61 and 276 choices for the non-nestmate, the control and the nestmate marked feeders,
150 respectively, showing that foragers preferred more nestmate and non-nestemate marked feeders
151 than the control feeder (F2,84 = 10.23, p < 0.001; Fig. 2). We recorded 275 choices in 25 replicates
152 for the mandibular gland extract experiment, 120 for the nestmate feeder, 129 for the non-
153 nestmate marked feeder and 26 for the control feeder. Again, foragers prefered significantly
14
154 more non-nestmate and nestmate marked feeders than control feeder (F2,72 = 14.96, p < 0.001).
155 Post hoc test revealed that the proportion of foragers that visited the marked feeders was not
156 different in both labial gland and madibular gland experiments (Fig. 2).
157
158 Discussion
159 Olfactory signaling has been described in many species of social bees. This is used, among many
160 other things, to communicate nestmates the location of feeding sites, either newly discovered,
161 under current exploitation or reopened (Seeley 1995; Waddington and Holden 1979). To our
162 knowledge, this is the first study that shows that olfactory signaling in a highly social stingless
163 bee species, S. mexicana, provides an opportunity for intraspecific eavesdropping. Heterospecific
164 eavesdropping has been shown in stingless bee species, in which the decodification of
165 communication signals from other species provides the opportunity to locate food sources already
166 under exploitation by heterospecifics (Lichtenberg et al. 2011; Nieh et al. 2004). The first
167 experiment in our work clearly shows that foragers that were exposed to non-nestmate odors
168 preferred the feeder marked with such odors over the one marked by nestmates. Subordinate
169 foragers, on the other hand, preferred their own odors. The question here is under what
170 circumstances foragers would find non-nestmate odors. Part of the answer can be found in the
171 mode of reproduction of stingless bees. Stingless bee reproduction promotes the creation of
172 clusters of colonies since daughter colonies require the help (food reserves) of the mother colony
173 for its formation (Roubik 1989). Short spatial distance among colonies results in a high genetic
174 similitude, possibly making the colonies to have a highly similar chemical profile, which allows
175 them to share resources, since it is known that in stingless bees chemical cues allow non-
176 nestmates to be readily accepted (Nunes et al. 2008) and that chemical mismatching can take
15
177 foragers into fight from different colonies of the same specie (Buchwald and Breed 2005).
178 However it is worth mentioning that genetics seemingly plays a secondary role in nestmate
179 recognition: Katserke et al. (2006) found that nestmate recognition, measured at different levels
180 of aggressive behavior, in the ant Formica exsecta correlated significantly with the spatial
181 distance between colonies, but not with genetic distance. So it seems that exchange of cuticle
182 hydrocarbons is very important at decreasing the rejection rate of non-nestmates. Moreover,
183 during gathering of resources foragers recognize and learn locations where nestmates are
184 normally found, so it is not strange to find non-nestmate conspecifics during foraging, if colonies
185 are located nearby, ready to spy and to steal food resources (Reeve 1989).
186 In the second experiment, when S. mexicana foragers were exposed to gland extracts, we
187 found that our eavesdroppers did not prefer the feeders marked with non-nestmate labial and
188 mandibular gland extracts, which is in contrast with the results of the first experiment; a similar
189 behavior was observed with normal foragers (Fig. 2). Jarau et al. (2006) found that labial gland
190 extracts were more attractive than both mandibular extracts and non-marked feeders. Scientific
191 community has debated the role of mandibular gland scents in food location communication.
192 While some authors mention that mandibular scents are used as alarm marks to avoid competitors
193 (Jarau et al. 2004b; Lichtenberg et al. 2011), other authors suggest that these mandibular marks
194 are used to signal the location of food sources (Lindauer and Kerr 1960; Nieh et al. 2003).
195 However, these studies focused on studying the odor of nestmates or only of gland extracts,
196 limiting their relevance to our study. We think that since stingless bee foragers use multiple
197 sensory modalities (e.g., touch, vision, olfaction, and audition) to transfer information concerning
198 resource location (Nieh 2004; Sánchez et al. 2011), it was easier to the foragers in experiment
199 one to discern between own odors and that of non-nestmates, i.e. in experiment 2 we only
16
200 manipulated the olfaction signals, but it is probably that other sensory channels were participating
201 in experiment one (Nieh et al. 2003; Nieh and Roubik 1995). Additionally, Jarau et al. (2010) and
202 John (2008) found significant differences in the proportion of the compounds in the glands of
203 different colonies of stingless bee species, supposedly to decrease competition at food sources.
204 This was not the case in our study, though, since we observed foragers choosing in equal
205 proportion on both nestmate marked feeder and non-nestmate marked feeder.
206 Previous work described S. mexicana as an organism showing a highly precise food
207 communication system (Sánchez et al. 2004; Sánchez et al. 2008). Further research found that
208 some situations might yield a lower precision (Sánchez et al. 2008; Sánchez et al. 2011). In this
209 work, we found that learning of non-nestmate odors probably influences choice behavior, and
210 that preference towards own odor marks was very low in eavesdroppers. With these results we
211 are starting to better understand the dynamics of foraging in stingless bees, and that flexibility
212 seems to be the rule.
213
214 Acknowledgements We appreciate the help of the following people during field and laboratory work: Leonardo
215 Arévalo-Monterrubio, Augusto Campollo-Ovalle, Ricardo Toledo-Hernández, Andy Villarreal Cruz and Miguel
216 Guzmán. This study was possible thanks to the support of SEP-CONACYT agreement no. 128702 "Evolución de la
217 cleptobiosis en Lestrimelitta", SEP-CONACYT agreement no. 106043 “Land use effect on the conservation of bees'
218 biodiversity” and UC-MEXUS project "Olfactory eavesdropping and against a cleptoparasite, Lestrimelitta niitkib".
219 The first autor was supported with a scholarship from CONACYT.
220
221
222
17
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297 mexicana (Apidae, Meliponini). Insec Soc 58:575-579.
21
298 Schorkopf DLP et al. (2007) Spitting out information: Trigona bees deposit saliva to signal
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304
305
306
22
307 Fig. 1 Average proportion of foragers (± EE) collected at each feeder in the six replicates of experiment 1.
308 Different letters indicate significant difference (Tukey Posthoc test, α = 0.05)
Propotion foragersof
NESTMATE-ODOR NON-NESTMATE CONTROL 309 EAVESDROPPER SUBORDINATE
310
23
311 Fig. 2 Average proportion of foragers (± SE) visiting the feeders baited with nestmate gland extract, non-
312 nestmate gland extract, or clean feeder (control) in experiment 2. Different letters indicate significant
313 difference (Nemenyi's pairwise comparison test, α = 0.05)
314
Propotion foragersof
NESTMATE-ODOR NON-NESTMATE CONTROL 315 EAVESDROPPER SUBORDINATE
316
24
III. CONCLUSIONES
Se observó que las forrajeras de S. mexicana pudieron asociar las marcas depositadas por forrajeras de otra colonia con un recurso rentable cuando estuvieron previamente en contacto con estas marcas de olor depositadas en el alimento, e incluso preferirlas sobre las marcas depositadas por abejas de su propia colonia. Sin embargo, este no fue el caso con los extractos de glándulas labiales o mandibulares, en donde las abejas
únicamente pudieron distinguir el control y no los dos tratamientos con extractos glandulares de abejas de su propia colonia o de otra. Esto puede ser debido a que en el experimento con marcas de olores tenían más compuestos presentes que facilitan diferenciar una marca de olor de otra. Es posible entonces que la aparición de espionaje químico, y el consiguiente desarrollo de la cleptobiosis tenga lugar en situaciones como las de nuestros experimentos, en donde varias colonias comparten recursos que se agotan eventualmente. El siguiente paso es determinar como la repartición de recursos ocurre en condiciones naturales, y si existe algún factor que permite que algunas colonias con mayor similitud genética sean más propensas a desarrollar espionaje que las más alejadas, o viceversa.
25
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30
V. Anexo 1. Distribución geográfica de S. mexicana en la República Mexicana. Los
puntos amarillos indican un registro (fuente: Discover life, Ascher et al., 2007).
31
VI. Anexo 2. Dispositivo de marcaje de abejas. A) Abeja de S. mexicana marcada
en el tórax de acuerdo al color de su colonia (verde). B) Abeja (sobrevolando)
con el tórax marcado de color blanco cerca del dispositivo de marcaje de las
abejas de colonia de color verde. C) abeja de color anaranjado en la entrada de
la colonia marcada con color verde.
32
VII. Anexo 3. Experimento 1, marcas de olor en condiciones de campo. A)
Entrenamiento de colonias con alimentadores colocados a 15 m de distancia (2
m de distancia entre alimentadores). B) Colocación de alimentadores limpios
para el marcaje de feromonas de las 2 colonias. C) Obtención de alimentadores
marcados por 80 abejas de una misma colonia. D) Eliminación de un
alimentador y exposición de feromonas de la otra colonia. E) Fase de espionaje
(bioensayo).
33
VIII. Anexo 4. Experimento 2, bioensayo glándulas labiales y mandibulares. A)
Entrenamiento de colonias con alimentadores colocados a 10 m de distancia. B)
Eliminación del alimentador cuando se observaron aproximadamente 20
forrajeras. C) Bioensayo, se colocaron 3 alimentadores con un algodón con
sacarosa 2M separados a 20 cm uno del otro: el alimentador 1 contenía extracto
glandular de su misma colonia, el alimentador 2 extracto glandular de la otra
colonia, y el alimentador control sin extractos.
10 m
20 cm 20 cm
Alimentador 1 Alimentador 2 Control
34
35