Effects of age and nutritional state on the expression of gustatory receptors in the honeybee (Apis mellifera)

Abstract

Gustatory receptors (Grs) expressed in insect taste neurons signal the presence of carbohydrates, sugar alcohols, CO2, bitter compounds and oviposition stimulants. The honeybee (Apis mellifera) has one of the smallest Gr gene sets (12 Gr genes) of any insect whose genome has been sequenced. Honeybees live in eusocial colonies with a division of labour and perform age-dependent behavioural tasks, primarily food collection. Here, we used RT-qPCR to quantify Gr mRNA in honeybees at two ages (newly-emerged and foraging-age adults) to examine the relationship between age-related physiology and expression of Gr genes. We measured the Gr mRNAs in the taste organs and also the brain and gut. The mRNA of all Gr genes was detected in all tissues analysed but showed plasticity in relative expression across tissues and in relation to age. Overall, Gr gene expression was higher in the taste organs than in the internal tissues but did not show an overall age-dependent difference. In contrast Gr gene expression in brain was generally higher in foragers, which may indicate greater reliance on internal nutrient sensing. Expression of the candidate sugar receptors AmGr1, AmGr2 and AmGr3 in forager brain was affected by the types of sugars bees fed on. The levels of expression in the brain were greater for AmGr1 but lower for AmGr2 and AmGr3 when bees were fed with glucose and fructose compared with sucrose. Additionally, AmGr3 mRNA was increased in starved bees compared to bees provided ad libitum sucrose. Thus, expression of these Grs in forager brain reflects both the satiety state of the bee (AmGr3) and the type of sugar on which the bee has fed.

Fig 1. Gustatory receptor mRNA levels are not equal in every body part in both newly-emerged and forager honeybees.

All expression levels are relative to the expression of the reference gene RP49 (RPL32) and are normalised to AmGr1 in the forager brain. A. Expression levels of the candidate sugar receptors (AmGr1 and AmGr2) and fructose receptor (AmGr3) genes across the un-manipulated forager (≈2–3 wk old) and newly emerged honeybee (≈24 h old) anatomy. B. Expression levels of the candidate bitter receptors in forager and newly emerged bees (NA represents unavailable data.). C. Expression levels of the unknown and potentially Apis-specific receptor genes in forager and newly-emerged bees. Note: AmGr9 mRNA expression levels were detected in all tissue types in both groups however levels were too low to include reliable expression values. Cells are shaded to indicate level of expression. N^{soft tissue}: 20 individual tissues. N^{hard tissue}: 75–150 individual tissues.

Fig 2. mRNA expression of the three candidate sugar receptors, AmGr1, AmGr2 and AmGr3 in the brains of ‘starved’ forager honeybees (provided with 10μl of 0.7M sucrose then held for 24 h without food).

All expression levels are relative to mRNA expression of the reference gene RP49 (RPL32) in the brain. The expression of each gene has been normalised to the mRNA expression of that same gene under 0.7M sucrose ad libitum feeding conditions over 96h, ‘fed’ condition, set at a value of 1.0 (represented by the hashed line). Expression levels are not comparable between genes. *: P < 0.05 One-Sample Wilcoxon Signed Rank test. N = 3–4 biological replicates (15–20 whole brains measured as mRNA pooled from groups of 5 brains).

Fig 3

mRNA expression of the three candidate sugar receptors, A. AmGr1, B. AmGr2 and C. AmGr3 in the brains of forager honeybees following 96 h ad libitum feeding on one of three 0.7 M carbohydrate diets (disaccharide sucrose or either monosaccharide glucose or fructose). All expression levels are relative to mRNA expression of the reference gene RP49 (RPL32) in the brain. The expression of each gene has been normalised to the mRNA expression of that same gene under 0.7M sucrose ad libitum (first bar in each graph), set at a value of 1.0. Expression levels are not comparable between genes. *: P < 0.05 One-Sample Wilcoxon Signed Rank test. N = 2–4 biological replicates (10–20 whole brains, measured as mRNA pooled from groups of 5 brains).