Categoría 3: Contenidos

One of the most striking characteristics of eusocial insects is the partitioning of reproduction among females into reproductive and non reproductive specialists. Caste differentiation is for some taxa a function of genotype (Schwander et al. 2010), but most variation in caste is explained by environmental cues (Lattorff & Moritz 2013). For the eusocial honey bee Apis mellifera, differences in larval feeding regimes largely explains the differentiation of a genetically totipotent larvae into a reproductive queen or a functionally sterile worker (reviewed by Hartfelder et al. 2015). Within honey bee colonies, female larvae fed almost exclusively on royal jelly develop into queens, whereas, those fed less of this proteinacious substance develop into workers (Haydak 1970). These nutritional differences during early life stages elicit two distinct phenotypic responses; large queens that are long-lived and fecund, and relatively small workers that are short-lived and functionally sterile.

Adult honey bee queens signal their reproductive potential through production of queen mandibular pheromone (QMP). This multi-component pheromone encourages workers to refrain from activating their ovaries and to re-direct their reproductive energy into

helping the queen reproduce (Slessor et al. 2005). The queen’s signal is proportional to her fertility (Kocher et al. 2008) and declines in fertility are accompanied by changes in

the ratio of components within the pheromone. When queen fertility declines below a threshold level, her daughter workers can to some extent re-activate their ovaries to lay male-haploid eggs.

Queen pheromone and royal jelly therefore have, in a sense, 'opposite' effects on

reproduction. Royal jelly is produced by workers and queen caste development. QMP, by contrast, is produced by queens and suppresses reproduction in developed workers. It is not clear, however, if QMP and royal jelly are functionally associated, as regulators of antagonistic pathways or if they simply have functionally opposite effects upon the same regulatory pathway.

Evidence for the later hypothesis suggests that queen pheromone increases the production of the egg yolk precursor protein vitellogenin (Vg) (Fischer & Grozinger 2008), where Vg binds to receptors on the hyperpharengeal glands, and is subsequently incorporated into the royal jelly medium that nurses feed to developing larvae (Amdam et al. 2003). Further, royal jelly stimulates ovary development (Haydak 1970), and differences in the number of ovarioles (units that produce eggs) within ovaries have been correlated with the threshold response by workers, as workers with more ovarioles are less responsive to QMP (Kocher et al. 2010).

Gene expression studies comparing differences between queen and worker castes and between queen-less (colonies without a queen) and queen-right (colonies with a queen) workers have highlighted the degree of overlap in the genes and gene pathways that regulate caste differentiation and worker ovary activation (Grozinger et al. 2007). The insulin signaling/ IGF-1 like signaling (IIS) pathway regulates queen worker caste

differentiation (Wheeler et al. 2006), as larvae with reduced expression of an insulin receptor, transition into workers even when fed a queen diet of royal jelly (Wolschin et al. 2011). The IIS pathway may also regulate QMP responsiveness as nurses who are more responsive to QMP (Fussnecker et al. 2011), have lower levels of insulin signaling (Mutti et al. 2011b), compared to more QMP unresponsive foragers. Further, juvenile hormone (JH) a key regulator of reproductive development, is up-regulated in queen destined larvae in response to royal jelly, leading to an increased production of Vg that is incorporated into the developing ovary (Barchuk et al. 2002). In adult honey bees, differential expression of JH and Vg are also associated with transitions from nurse and foraging states, and JH specifically is thought to be differentially regulated by QMP component 9-ODA (Robinson et al. 1992).

A comparative study using Drosophila melanogaster and Apis mellifera has shown that royalactin, the main component of royal jelly, functions as a ligand for the epidermal growth factor receptor (EGFR) in both species. Royalactin-induced increases in EGFR signaling appear to encourage queen development in bees, and to promote queen bee-like characteristics in the fruit fly (Kamakura 2011) In Drosophila, royalactin up regulates JH and Vg, inducing a queen-like increase in ovarian development. More recently, it has been shown that decreasing EGFR signaling through genetic knock-down inhibits worker bees from re-activating their ovaries in queen-less environments (Formesyn et al. 2014), suggesting that this pathway may also play a key role in QMP perception. It has already been shown that Drosophila females elicit a homologous ovarian response to QMP as seen in worker bees (Camiletti et al. 2013), allowing for a unique approach to test the functional interaction between QMP and royal jelly.

Given the degree of mechanistic relatedness between royal jelly and QMP, it is puzzling why the queen remains fertile in spite of her own production of ovary suppressing queen pheromone. Although this has not been studied in Apis, queens of one termite species (Yamamoto & Matsuura 2011) and two ant species (Holman et al. 2012; Vargo 1992) show slight declines in reproduction in response to species-specific synthetic queen pheromone. These responses by queens to their own pheromonal cue are not as dramatic as those seen in workers and suggest that caste specific developmental regimes, like a larval diet of royal jelly, may confer some degree of reproductive safe-guarding to the negative effects of QMP.

In the present study I will conduct two functional tests of the relationship between ovary- inhibiting queen pheromone and ovary-stimulating royal jelly. First, I will measure the ovary phenotypes of egg number, ovary size and fecundity associated with exposing

Drosophila females to QMP, royal jelly, or both. If QMP and RJ functionally interact to regulate ovary development then I expect, that females treated with RJ and QMP will have a less severe ovary phenotype compared to females treated with QMP alone. Conversely, if ovarian effect of QMP is not dependent on treatment with RJ, then I expect that the ovarian effect associated with QMP will not be affected by RJ treatment. In a second test of functional association, I will test the ovarian phenotypes associated with QMP in flies where the effects of RJ have been genetically mimicked, either through up-regulation of the EGFR signalingpathway or ubiquitous expression of royalactin, a key component of RJ. Here again, I expect that if QMP and RJ are functionally

associated than I should see less severe ovarian phenotypes in these transgenic flies, than if QMP and RJ were working independently.