bioRxiv preprint doi: https://doi.org/10.1101/2021.01.09.426073; this version posted January 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Poor adult nutrition impairs learning and memory in a parasitoid 2 wasp 3 4 Hossein Kishani Farahani1*, Yasaman Moghadassi2, Jean-Sebastien Pierre3, Stéphane Kraus4, 5 Mathieu Lihoreau4* 6 7 1. Equipe Recherches Agronomiques, Agronutrition, Carbonne, France 8 2. Department of Plant Protection, Faculty of Agriculture and Natural Resources, 9 University of Tehran, Karaj, Iran 10 3. University of Rennes 1, UMR-CNRS 6553 EcoBio, Avenue du Général Leclerc, Campus 11 de Beaulieu, 35042 Rennes Cedex, France. 12 4. Research Center on Animal Cognition (CRCA), Center for Integrative Biology (CBI); 13 CNRS, University Paul Sabatier – Toulouse III, France 14 * Corresponding authors: 15 [email protected] 16 Equipe Recherches Agronomiques, Agronutrition Co., Carbonne, France 17 Phone : +33781944086 18 [email protected] 19 Research Centre on Animal Cognition, UMR 5169 CNRS, University of Toulouse III, France, 20 phone: +33 6 33 43 57 06 21 Running Title: nutrition quality and adults memory 22 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.09.426073; this version posted January 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 Abstract: 24 Animals have evolved cognitive abilities whose impairment can incur dramatic fitness costs. 25 While malnutrition is known to impact brain development and cognitive functions in vertebrates, 26 little is known in insects, whose small brain appears particularly vulnerable to environmental 27 stressors. Here, we investigated the influence of diet quality on learning and memory in the 28 parasitoid wasp Venturia canescens. Newly emerged adults were exposed for 24h to either 29 honey, sucrose solution 20%, sucrose solution 10%, or no food, before being conditioned in an 30 olfactory associative learning task in which an odor (orange) was associated to a reward (host 31 larvae). Wasps fed honey showed 3.5 times higher learning performances and 1.5 times longer 32 memory retention times than wasps fed sucrose solutions and starved wasps. Poor diets also 33 reduced longevity and fecundity. Our results demonstrate the importance of early adult nutrition 34 for optimal cognitive function in these parasitoid wasps that must quickly develop olfactory 35 memories for choosing high quality hosts for their progeny. 36 37 Keywords: Venturia canescens; Ephestia kuehniella; olfactory learning; memory retention; 38 nutrition 39 40 41 42 43 44 45 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.09.426073; this version posted January 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 46 Introduction 47 Animals rely on various forms of learning and memories to exploit resources in their 48 environment and adapt to changing conditions [1, 2, 3]. These cognitive abilities are sustained by 49 brains that require large amounts of proteins to grow [4], but also lipids and carbohydrates for 50 maintenance [5, 6, 7]. The process of learning, itself, imposes important energetic costs [8], and 51 the formation of persistent (long-term) memories involves protein synthesis [9, 10]. Therefore, 52 the ability of animals to acquire key nutrients in food is expected to directly impact their 53 cognitive performances [11]. 54 Malnutrition is known to affect cognitive functions in vertebrates (e.g. pigeons: [12], 55 mice: [13], cats and dogs: [14]). In humans, for instance, high fat and caloric diets have been 56 associated with hippocampal-dependent memory loss [15, 16]. By contrast, little is known about 57 the cognitive effects of malnutrition in invertebrates. Insects, in particular, rely on an impressive 58 range of learning and memory forms to interact socially and forage, and these cognitive abilities 59 are implemented by only few neurons [17, 18]. The miniature brain of insects is thus particularly 60 vulnerable to a range of environmental stressors, including poor nutrition [19]. For instance, in 61 the fruit fly Drosophila melanogaster, larvae fed diets with unbalanced protein to carbohydrate 62 ratios showed reduced learning performances in an aversive olfactory differential learning task 63 [20]. In the Western honey bee Apis mellifera, adults fed pollen with a deficit in specific fatty 64 acids (i.e. Omega-3, Omega-6) had impaired learning and memory performances in an appetitive 65 olfactory differential learning task [21, 22]. These cognitive effects of malnutrition may incur 66 particularly strong fitness costs in many solitary species where adults rely on learning and 67 memory to find food and nourish their progeny by themselves. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.09.426073; this version posted January 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 68 Solitary parasitoid wasps, such as Venturia canescens, learn to associate an odor with a 69 high-quality host to select nutritionally rich environments for the development of their offspring 70 [23, 24, 25]. Before engaging in oviposition, adults encompass a critical period soon after 71 emergence, where they need to find food [26, 27, 28]. Females typically acquire carbohydrates 72 from nectar and honeydew [29, 30, 31]. Other key nutrients such as proteins, minerals and fat, 73 are occasionally obtained from pollen [32]. Since olfactory memory formation is nutritionally 74 demanding, we hypothesized that wasps fed highest quality diets would show the best cognitive 75 performances. 76 Here, we tested whether diet quality during early adulthood affect the cognitive 77 performances of V. canescens wasps. We experimentally exposed emerging females to one of 78 four nutritional conditions of decreasing quality in terms of nutrient concentration, nutrient 79 diversity, and energy content (honey – see composition in Table 1, sucrose 20%, sucrose 10%, 80 no food). We then tested the impact of diet on cognition in a conditioning assay in which wasps 81 had to associate an odor to a reward (host) in a flight tunnel. We further tested the influence of 82 diet on fitness by monitoring the longevity and the reproductive success of these wasps. 83 Results 84 Wasps did not show innate odor preference 85 We first tested the influence of the nutritional condition on innate attraction to odors, by giving 86 individual wasps a simultaneous choice between two odor sources (orange and vanilla) in a flight 87 tunnel with two decisions chambers for 15 min (see details in Figure 1). The wasps did not 88 display any preference for either odor, irrespective of their nutritional condition (χ2 2 = 0.13, P 89 = 0.93, N = 50). The proportion of wasps that made no choice (i.e. when the wasps did not fly 90 after 5 mins in the tunnel) remained stable (30±2%) and was similar across nutritional conditions 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.09.426073; this version posted January 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 91 (χ2 2 = 0.9, P = 0.69, N = 50). This indicates that the nutritional condition did not affect the 92 motivation nor the motor activity of wasps in response to odorants. We therefore arbitrarily 93 chose the orange odor as the conditioned odor (CS+) and the vanilla odor as the new odor (NOd) 94 in all the subsequent behavioral tests. 95 Wasps fed honey showed highest learning performance 96 We then tested the effect of the nutritional condition on olfactory learning. To do so, we first 97 conditioned each wasp in the presence of 30 host larvae and the orange odor (CS+) for 2h. We 98 then tested the conditioned wasps for odor preference by giving them a choice between the CS 99 (orange) and the NOd (vanilla) in the flight tunnel for 15 min. Learning was observed in wasps 100 exposed to each of the four nutritional conditions but at different magnitudes (Figure 2; Binomial 101 GLM Feeding; χ2=87.33, P<.0001; Conditioning: χ2=14.48, P=0.0001; Feeding× conditioning: 102 χ2=7.7, P=0.005). The highest proportion of correct choices for the CS (85±3%, N =50) was 103 observed in wasps fed honey. This proportion decreased with diet quality, reaching intermediate 104 levels in wasps fed sucrose diets (72±2%, N= 50), and a minimum level in starved wasps 105 (62±2%, N = 50). The proportion of wasps that did not make a choice remained low across 106 nutritional conditions (12 ± 2%, N=50; Figure 2), but increased with decreasing diet quality, 107 reaching a maximum in starved wasps (29±3%, N =50; Binomial GLM Feeding; χ2=12.3, 108 P=0.006; Conditioning: χ2=8.93, P=0.002; Feeding× conditioning: χ2=7.61, P=0.005; Figure 2). 109 Therefore, wasps fed highest diet quality showed highest learning performances. 110 Wasps fed honey showed longest memory retention 111 We further tested the effect of the nutritional condition on olfactory memory retention by testing 112 wasps in the flight tunnel at different time periods, between 2h and 30h post conditioning (Figure 113 3). Memory retention was significantly longer in wasps fed honey (27±3h, N= 600) than in 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.09.426073; this version posted January 10, 2021.