CRISPR/Cas9 mediated mutations as a new tool for studying taste in honeybees

Abstract

8Background: Honeybees rely on nectar as their main source of carbohydrates [1]. Sucrose, glucose 9 and fructose are the main components of plant nectars [2] [3]. Intriguingly, honeybees express only three 10 putative sugar receptors (AmGr1, AmGr2 and AmGr3) [4] , which is in stark contrast to many other 11 insects and vertebrates. The sugar receptors are only partially characterized [5] [6]. AmGr1 detects 12 different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while 13 AmGr3 is assumedly a fructose receptor. 14 Results: We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception 15 when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 16 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete 17 loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations 18 introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioural change and serve 19 to characterize the function of taste receptors in vivo.20 Conclusion: CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in 21 vivo. Biophysical receptor characterisation in Xenopus oocytes and nonsense mutation of AmGr3 in 22 honeybees unequivocally demonstrate that this receptor is highly specific for fructose. Graphical Abstract 24 See Fig. 0. 25 Background 29 Honeybees (Apis mellifera) are not only important pollinators world-wide. The highly social insects 30 perform an intricate division of labour and are well-known for their astonishing skills in learning and 31 communication. When it comes to taste, however, honeybees display a rather poor set of receptors. 32Because plant-derived nectar is their sole source of carbohydrates, sugar perception is naturally of 33 utmost importance for honeybees. The bees sense the sugar composition of a food source with only a 34 few fine contact chemoreceptors on their antennal tip [7]. In contrast to many other insects such as the 35 fruit fly (Drosophila melanogaster) with 68 genes and mosquitoes (Anopheles gambiae) with 75 genes, 36 the genome of the honeybee comprises only ten genes coding for gustatory receptors (Grs) [4]. Among 37 these only three code for sugar receptors: AmGr1, AmGr2 and AmGr3 [4] [8]. The taste receptors are 38 expressed in the brain, the antennae, mouthparts, tarsi and the gut of the honeybee. With this small set 39 of receptors honeybees evaluate a diverse set of sugars like sucrose, fructose, maltose and melicitose 40 in nectar in varying composition and in amounts ranging from 5% to 80%. In flowers of mint plants 41 (Laminacea), buttercups and clematis (Ranunculaceae), for example, sucrose is the main sugar, 42whereas other flowers such as those of oilseed rape contain relatively more glucose and fructose [3] [9]. 43The sugar trehalose, in contrast, acts as blood sugar [10] [2]. 44How honeybees recognize the different sugars in nectar and in the inner organs ...

Peripheral taste detection in honey bees: What do taste receptors respond to?

“…The development of new methods such as CRISPR/Cas9 or RNAi, which can be applied to the honey bee (Değirmenci et al., 2020; Guo et al., 2018; Wang et al., 2013), could provide valuable ways for addressing taste receptor function. Combining these strategies of receptor knock‐down with behavioral and/or electrophysiological analyses requires time and considerable efforts but appears at the present time as a privileged choice for studying GR genes in the honey bee.…”

“…For instance, expressing GRs in Xenopus oocytes and coupling this expression with electrophysiological recordings (e.g., patch clamp recordings) enable the characterization of AmGr tuning (Değirmenci et al., 2018; Jung et al., 2015; Lim et al., 2019; Takada et al., 2018). Alternatively, the development of RNAi or CRISPR/Cas9 methods allows knocking out a GR gene and determining the consequences of its loss via electrophysiological and/or behavioral analyses (Değirmenci et al., 2020).…”

“…In the silk moth, BmGr9 may have a similar role as the receptor it encodes specifically binds to fructose and is expressed in the larval gut (Sato et al., 2011). Recent studies in which AmGr3 was transiently expressed in Xenopus oocytes have shown that in honey bees AmGr3 is specialized in fructose detection (Değirmenci et al., 2020; Takada et al., 2018). When double nonsense mutations were introduced into AmGr3 using a CRISPR/Cas9 approach, the mutants exhibited a very low fructose responsiveness compared to control bees but responded normally to sucrose (Değirmenci et al., 2020; Figure 6a,b).…”

“…Recent studies in which AmGr3 was transiently expressed in Xenopus oocytes have shown that in honey bees AmGr3 is specialized in fructose detection (Değirmenci et al., 2020; Takada et al., 2018). When double nonsense mutations were introduced into AmGr3 using a CRISPR/Cas9 approach, the mutants exhibited a very low fructose responsiveness compared to control bees but responded normally to sucrose (Değirmenci et al., 2020; Figure 6a,b). Some mutant bees still responded to fructose in these experiments, thus leading to the suggestion that perception of fructose could occur, though in a reduced manner, via AmGr1 and its co‐receptor AmGr2 , when co‐expressed in the same gustatory neuron.…”

Opposing Actions of Octopamine and Tyramine on Honeybee Vision

“…Yet, our current experiments do not allow us to specify which receptors are activated by OA or TA. Here we suggest targeted knockout of individual receptor genes using CRISPR/Cas9 [ 45 ] or RNA interference [ 46 ] in future studies.…”