Insect Science (2018) 25, 75–86, DOI 10.1111/1744-7917.12408 ORIGINAL ARTICLE The cephalic labial gland secretions of two socially parasitic bumblebees Bombus hyperboreus (Alpinobombus) and Bombus inexspectatus (Thoracobombus) question their inquiline strategy Nicolas Brasero1, Baptiste Martinet1, Thomas Lecocq1,2, Patrick Lhomme3, Paolo Biella4,5, Irena Valterova´ 6,Klara´ Urbanova´ 6,7, Maurizio Cornalba8, Heather Hines3 and Pierre Rasmont1 1Laboratory of Zoology, Research institute of Biosciences, University of Mons, Mons, Belgium; 2Research Unit Animal and Functionalities of Animal Products (URAFPA), University of Lorraine—INRA, Vandoeuvre-les-Nancy,` France; 3Department of Biology, the Pennsylvania State University, University Park, Pennsylvania, USA; 4Faculty of Science, Department of Zoology, University of South Bohemia, Ceskˇ e´ Budejovice,ˇ Czech Republic; 5Biology Centre of the Academy of Sciences of the Czech Republic, v.v.i., Institute of Entomology, Ceskˇ e´ Budejovice,ˇ Czech Republic; 6Academy of Sciences of the Czech Republic, Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic; 7Faculty of Tropical AgriSciences, Department of Sustainable Technologies, Czech University of Life Sciences, Prague, Czech Republic and 8Department of Mathematics, University of Pavia, Pavia, Italy Abstract Social parasitic Hymenopterans have evolved morphological, chemical, and behavioral adaptations to overcome the sophisticated recognition and defense systems of their social host to invade host nests and exploit their worker force. In bumblebees, so- cial parasitism appeared in at least 3 subgenera independently: in the subgenus Psithyrus consisting entirely of parasitic species, in the subgenus Alpinobombus with Bombus hy- perboreus, and in the subgenus Thoracobombus with B. inexspectatus. Cuckoo bumblebee males utilize species-specific cephalic labial gland secretions for mating purposes that can impact their inquiline strategy. We performed cephalic labial gland secretions in B. hyperboreus, B. inexspectatus and their hosts. Males of both parasitic species exhibited high species specific levels of cephalic gland secretions, including different main com- pounds. Our results showed no chemical mimicry in the cephalic gland secretions between inquilines and their host and we did not identify the repellent compounds already known in other cuckoo bumblebees. Key words bumblebees; Bombus hyperboreus; Bombus inexspectatus; cephalic labial gland secretions; inquiline strategy; social parasitism Introduction Brood care is an energy-consuming activity for social insects ranging from nest construction to offspring breed- ing (Smisteh et al., 2012). This high energetic cost has Correspondence: Nicolas Brasero, Laboratory of Zoology, favored the evolution of cheating species that exploit Research Institute of Biosciences, University of Mons, Place du brood care behavior of heterospecifics for their own Parc 20, 7000 Mons, Belgium. Tel: 0032(0)65373435; email: offspring rearing (Roldan & Soler, 2011). This adap- [email protected] tive strategy involves the exploitation of host social Nicolas Brasero and Baptiste Martinet are co-first authors and behavior by specialized species (so called social para- have contributed equally to this work. sites) (Lenoir et al., 2001). Social parasites have evolved 75 C 2016 Institute of Zoology, Chinese Academy of Sciences 76 N. Brasero et al. morphological, chemical, and behavioral adaptations the geographical distribution of B. inexspectatus and (usurpation behaviors) to overcome the sophisti- B. hyperboreus and their ecological niches are restricted cated recognition system (based on species-specific (Rasmont & Iserbyt, 2014). pheromonal and cuticular signatures) and defense sys- The infiltration strategies of parasitic females have been tems of their social host (Fisher & Sampson, 1992; Martin the focus of abundant research (Nash & Boomsma, 2008): et al., 2010). some bumblebee species produce very low amounts of Among bumblebees (Hymenoptera: Apidae: Bombus), cuticular hydrocarbons (chemical insignificance) and/or the cuckoo species exemplify such a social parasitic strat- acquire their host’s chemical profile (chemical mimicry) egy. Their life cycle relies on a nonmutual symbiotic re- (Dronnet et al., 2005; Martin et al., 2010), whereas lationship (called inquilinism) with their hosts (Lhomme others use repellents (allomones) to defend themselves et al., 2012). This interaction leads to a decrease or, in against the attacks of host workers (Zimma et al., 2003; some cases, to a complete loss of host reproductive suc- Martin et al., 2010). More recently, a few studies have cess (Lhomme et al., 2013). These “cheaters” infiltrate started to investigate the strategies used by bumblebee in- host colonies to take advantage of their food resources quiline offspring to be tolerated by host workers (Lhomme and workers (Nash & Boomsma, 2008). In bumblebees, et al., 2012; Lhomme et al., 2015). obligate inquilinism evolved in at least 3 subgenera inde- For male offspring of inquiline species, avoiding host pendently (Hobbs, 1967; Wilson, 1971; Michener, 1974): recognition is especially challenging because of their in the subgenus Psithyrus consisting entirely of parasitic early production of large amounts of the species-specific species (Williams, 1998), in the subgenus Alpinobombus Cephalic Labial Gland Secretions (CLGS) involved in with B. hyperboreus (Milliron & Oliver, 1966; Richards, attracting conspecific females during the premating be- 1973) (Fig. 1A), and in the subgenus Thoracobombus with havior (Zˇ a´cekˇ et al., 2009; Lhomme et al., 2012; Ayasse B. inexspectatus (Yarrow, 1970; Williams, 1998; Muller,¨ & Jarau, 2014). Indeed, CLGS are a complex mixture 2006) (Fig. 1C). of (mainly aliphatic) compounds (Calam, 1969) derived All Psithyrus species are completely dependent on from saturated fatty acids (Luxova´ et al., 2003) involved in their host because these socially parasitic bumblebees do premating behavior (Bergstrom¨ et al., 1981). The species- not produce a worker caste, do not have pollen collec- specific CLGS are synthesized de novo by cephalic labial tion baskets and produce little to no wax to build cells glands in the head of bumblebee males (Zˇ a´cekˇ et al., (Benton, 2006; Sramkova & Ayasse, 2008). In the 2013). This highly concentrated specific signature are Alpinobombus subgenus, the circum arctic B. (Alpinobom- discriminated by host workers and potentially lead to bus) hyperboreus is mainly known as an inquiline of B. male offspring destruction (Lhomme et al., 2012). Con- (Alpinobombus) polaris (Fig. 1B) at least in some parts of sequently, a strong selective pressure (beside the sex- its range (Milliron & Oliver, 1966; Løken, 1973; Richards, ual selection) on the inquiline male CLGS should likely 1973; Pape, 1983; Stenstrom¨ & Bergmann, 1998; Rampini lead to adaptive changes. Similarly to alternative chemi- et al., 2012), but nests of B. (Alpinobombus) balteatus cal infiltration strategies, one could expect 3 alternative and B. (Pyrobombus) jonellus have been found to be in- adaptive patterns: (i) chemical insignificance meaning filtrated by B. hyperboreus in Norway (Bergwall, 1970; that males do not produce CLGS for premating behav- Gjershaug, 2009). In the Eurasian Arctic and in North ior or produce CLGS only after leaving the host nest or America, some females were reported harvesting pollen even males produce undetected compounds; (ii) chemi- (Gjershaug, 2009), suggesting a facultative inquilinism cal mimicry meaning that the inquiline actively produce for this species. In the Thoracobombus subgenus, Yarrow the same compounds as its host; or (iii) chemical repel- (1970) and Muller¨ (2006) have highlighted a number of lency meaning that specific repellent compounds are pro- peculiar biological and morphological traits of B. (Tho- duced within the CLGS and act as “chemical shield” to racobombus) inexspectatus (i.e., absence of worker caste, avoid host worker aggressiveness. A previous study on the loss of wax production and reduction of the hair-like struc- cuckoo bumblebee B. (Psithyrus) vestalis demonstrated tures of the hind leg used to gather pollen) that likely a chemical repellent effect of CLGS on host workers are adaptations to parasitic life. Bombus (Thoracobom- triggered by tetradecyl acetate and (Z)-11-hexadecenyl bus) ruderarius (Fig. 1D) was described as the only host acetate (Lhomme et al., 2015). The chemical strategies of B. inexspectatus (Muller,¨ 2006). This subalpine Euro- used by other Psithyrus or non-Psithyrus inquiline species pean species occurs in the Cantabrian Mountains and the are unknown. Here, we perform (i) the first detailed de- Alps from 1200 to 2100 m in altitude where it is uncom- scription of the 2 non-Psithyrus inquiline CLGS and (ii) mon (Tkalcu,˚ 1963, 1965; Yarrow, 1970; Amiet, 1996; comparative analyses of CLGS composition between B. Rasmont et al., 2015). Compared to their respective hosts, hyperboreus, B. inexspectatus and their hosts. We finally C 2016 Institute of Zoology, Chinese Academy of Sciences, 25, 75–86 CLGS in social parasite 77 Fig. 1 Photos of the studied bumblebee species: (A) Bombus hyperboreus male (PRAS1067) from Alaska, USA (photo: P. Rasmont), (B) B. polaris male (PRAS0936) from Norway (photo: P. Rasmont), (C) B. inexspectatus male from Alps, Italy (photo: P. Rasmont), and (D) B. ruderarius male from Pyrenees, France (photo: P. Rasmont). discuss the potential chemical strategies involved in