DOI: 10.5061/dryad.866t1g1sq
title: Factors driving the within-plant patterns of resource exploitation in a herbivore

description: 1. Selective pressures exerted on the feeding behavior of animals have
 been extensively studied to understand their foraging patterns. In
 herbivores, specific within-plant patterns of resource exploitation have
 been reported, but their determinants remain poorly understood. 2. Here,
 we describe and decipher the determinants of the foraging pattern of the
 pollen beetle, Brassicogethes aeneus, a pollinivorous insect that is a
 pest of oilseed rape (Brassica napus). This insect feeds from flowers for
 almost all of its life cycle, except for a couple of weeks preceding
 blossoming. During this period, only flower buds are available and the
 insect destroys them to feed from the pollen they contain, causing serious
 yield losses. 3. We found that during this critical period, pollen beetles
 exhibit a stereotypic intra-inflorescence feeding pattern that depends on
 flower bud maturity. To explain this pattern, we first deciphered the
 selective pressures driving pollen beetles’ feeding behavior. Using a set
 of manipulative laboratory experiments, including behavioral experiments
 on plant tissues and artificial substrates, chemical characterization of
 plant tissues and performance experiments, we show that the pollen
 beetles’ feeding behavior does not seem to be driven by specialized
 metabolites or an attempt to reach an optimal nutrient balance, but rather
 by a process of maximization of total macronutrient intake. Next, using
 Optimal Diet Choice models, we found that one aspect of the
 intra-inflorescence feeding pattern, the preference for young over old
 flower buds, could be well explained through the lens of total
 macronutrient intake maximization per unit of time to access the resource.
 4. Our study provides new insights into small-scale foraging patterns, and
 highlights the need to characterize and assess the relative influence of
 several components of diet quality when deciphering selective pressures
 driving foraging patterns.
description: <strong>Feeding
 tests</strong>
 <strong>2.1. Tests on entire
 plants</strong> To describe the feeding pattern of the pollen
 beetle, one individual was placed on the main inflorescence of an entire
 plant at the green-yellow bud stage as described in Hervé et al. (2014a).
 After three days, the
 number of buds damaged by
 feeding was counted. Buds
 damaged by feeding are recognizable as they show irregular holes of
 usually large sizes and located anywhere on the buds, while buds damaged
 by oviposition show stereotypic small holes at their basis. The length
 (i.e. maturity) of damaged buds was measured under a binocular microscope
 (0.1 mm precision). Ten replicates were performed.
 <em>Data analysis</em> – All statistical analyses were performed with
 the R software v. 4.0.2 (R Core Team, 2020). The mean number of buds
 damaged was compared between maturity classes (young, intermediate and old
 buds) using a Wald test
 applied on a Generalized Linear Mixed Model (GLMM) including the
 individual plant as random factor (distribution: Poisson, link function:
 log) (R package ‘lme4’ (Bates et al., 2015) and ‘car’ (Fox &
 Weisberg., 2019)). Here and in the following experiments, pairwise
 comparisons of Estimated Marginal Means (EMMeans) were systematically
 performed using the 'emmeans' package (Lenth, 2019) and
 p-values adjusted using the False Discovery Rate
 correction (Benjamini & Hochberg 1995). 
 <strong>2.2. Tests on plant organs
 </strong> To confirm the preference for flowers over
 flower buds and to assess the contribution of resource accessibility and
 resource chemical composition to this preference, dual-choice tests were
 performed. One individual was placed in a Petri dish (Ø = 55 mm) for 2 h
 with two different food sources, and it was then recorded whether each
 food source had been damaged or not. Three choice tests were conducted:
 one flower vs. one old bud (to confirm preference for flowers), one old
 bud vs. six anthers just excised from one old bud (to assess influence of
 resource accessibility), and six anthers just excised from one flower vs.
 six anthers just excised from one old bud (to assess influence of resource
 chemical composition). Old buds rather than young buds were chosen to
 decipher the factors responsible for the preference for flowers for
 practical aspects and to avoid any bias related to a physical factor, the
 anthers of old buds and flowers having the same size. Twenty to 27
 replicates were performed per choice test. 
 <em>Data
 analysis</em> – For
 each choice test, the probability of being damaged was compared between
 the two food sources using a Cochran’s Q test, which considered the
 replicate as pairing factor (R package ‘RVAideMemoire’, Hervé 2021).
 <strong>2.3. Tests on artificial substrates
 </strong> An experimental setup based on agar disks was designed to assess the respective and
 relative contributions of macronutrients (either their quantity and ratio)
 and defense metabolites in the preference of pollen beetles for flowers.
 Artificial substrates consisted of 3 % agar disks (Ø = 5 mm, thickness = 2 mm) supplemented with
 macronutrients (casein:whey 80:20 as protein source and sucrose as
 carbohydrate source) and/or pure standards of defense metabolites,
 depending on the experiment. Macronutrient and defense metabolite
 concentrations used in experiments varied depending on the hypothesis
 tested (see Results and Table S1). In all experiments on artificial
 substrates, two individuals were placed in a Petri dish (Ø = 35 mm) for 3
 h, with two to three disks depending on the experiment. Insects were
 filmed during the experiment and their movements tracked with the
 Ethovision XT software v. 15 (Noldus, Wageningen, Netherlands). The
 feeding behavior was estimated as the cumulative duration spent on each
 disk. Following preliminary observations, it was assumed that individuals
 were feeding when on disks and that the feeding speed was constant. Thirty
 replicates were performed per experiment. 
 <em>Data
 analysis</em> – For
 each experiment, the total
 time spent on disks was compared between treatments using a Wald test
 applied on a Linear Mixed Model (LMM) that included the replicate as
 random factor (R packages ‘lme4’ and ‘car’). The response was
 systematically square-root transformed to ensure model
 fitting. <strong>3. Chemical characterization of plant
 organs</strong>
 <strong>3.1.
 Macronutrients</strong> Macronutrient quantification was performed on
 five samples of three different plant organs: anthers from flowers, old
 buds and young buds. Each sample comprised organs collected from 14-16
 plants (four organs per plant), immediately frozen into liquid nitrogen
 then freeze-dried. Plant organs were sampled from the same plants to
 ensure unbiased comparisons. Total soluble proteins (P) were extracted
 from 10 mg of dried powder that was agitated for 15 min at room
 temperature in 1 mL of acidified phosphate buffer (0.2 M, pH = 6.8), then
 centrifuged at 12,000 g for 30 min at 4 °C. Quantification was performed
 using the Bradford’s method (Bradford, 1976) and standard solutions of
 bovine serum albumin as references. Total digestible carbohydrates were
 extracted from 10 mg of dried powder in 2 mL of phosphate buffer (0.2 M,
 pH = 6.5) for 20 min at 95 °C. After centrifugation at 14,000 g for 5 min
 at 4 °C, the supernatant was collected, the extraction steps were repeated
 and both supernatants were pooled. Total digestible carbohydrates (C) were
 quantified using the hot anthrone test (van Handel, 1967) and standard
 solutions of glucose as references. 
 <em>Data analysis
 </em>– Macronutrient
 composition was compared between pairs of plant organs (flower vs. old
 buds and old buds vs.
 young buds) using Welch
 t-tests. Tests were performed for P, C and P+C content as well as for the
 P:C ratio. <strong>3.2. Defense metabolites
 </strong> Compounds were chosen according to previous studies
 (Hervé et al. 2014a,b; Seimandi-Corda et al. 2019): S-methylcysteine sulfoxide (SMCSO) and flavonols
 that were suspected to be phagodeterrent for the pollen beetle (Hervé et
 al. 2014b), and glucosinolates that are known defense compounds of
 Brassicaceae (Hopkins, van
 Dam & van Loon, 2009) although their effects on the pollen beetle
 was not clear (Hervé et al., 2014a,b). Material (i.e. anthers from flowers and old
 buds) was collected as described for macronutrients, and
 metabolites were extracted
 and quantified as described in Hervé et al. (2014a) and Seimandi-Corda et
 al. (2019).  <em>Data analysis</em> – Metabolic profiles of plant organs
 were compared
 multivariately using a redundancy analysis (RDA) on centred and scaled
 data, and an associated permutation test with 9 999 permutations (R
 packages ‘vegan’; Oksanen et al., 2018). Univariate Welch t-tests with
 FDR-adjusted p-values were also performed to compare plant organs
 separately for each compound. 
 <strong>4.
 Performance
 experiments</strong>
 <strong>4.1. Performance on plant
 organs</strong> Here and in the following experiments,
 performance was assessed as the survival time following a defined feeding
 period. Survival was chosen over fecundity as performance estimator since
 pollen beetles produce eggs continuously as they feed (Hervé et al.,
 2014a; Ekbom & Ferdinand, 2003) and lay eggs only in flower buds.
 Providing them with flower buds where to lay eggs would have led to an
 impossibility to prevent them from feeding, which would have biased the
 experiments. One starved individual was placed for 24 h in a Petri dish (Ø
 = 55 mm) with a non-limiting food source consisting of two flowers or two
 old buds. After the feeding period, individuals were placed in new Petri
 dishes (same size) with a moistened filter paper humidified every day and
 their survival time was recorded through daily observations. Forty-one to
 43 replicates were performed per treatment.
 <em>Data
 analysis</em> – The
 survival time was analyzed using a likelihood ratio test (LRTest) applied
 on a survival regression (distribution: Weibull, link function: log),
 which included the treatment, the sex and their interaction as explanatory
 variables (R packages ‘survival’ (Therneau, 2021) and
 ‘car’). <strong>4.2. Performance on artificial
 diets</strong> Twenty-eight artificial diets differing in their
 P+C content and P:C ratio were designed, consisting of all combinations of
 four total macronutrient concentrations (P+C 45, 90, 180 and 270 g.l-1)
 and seven macronutrient ratios (P:C 1:0.5, 1:1, 1:2, 1:3.5, 1:5, 1:8 and
 1:15). All diets were presented to insects in 3 % agar disks
 (Ø = 5 mm, thickness = 2 mm). All disks included
 casein:whey 80:20 as P source and sucrose as C source, as well as a
 constant amount of additional nutritional resources (i.e. dry yeasts and
 vitamins) and antimicrobial agents (i.e. Tegosept and propionic acid) (see
 Appendix S1). The P and C content of dry yeasts was included in the
 calculations following Lihoreau et al. (2016). After starvation, one
 individual was offered two identical disks for 48 h in a Petri dish (Ø =
 35 mm) that also contained a moistened filter paper. After the feeding
 period, disks were removed and performance was estimated as above. Fifteen
 replicates were performed per diet.
 <em>Data
 analysis</em> – The
 survival time was analyzed using a LRTest applied on a survival regression
 (distribution: Weibull, link function: log) (R packages ‘survival’ and
 ‘car’). The model included the total P+C concentration, the P:C ratio and
 their interaction as explanatory variables.
 <strong>5.
 Optimal Diet Choice
 modeling </strong> To determine whether pollen beetles’ feeding
 pattern at the inflorescence scale could be explained by a maximization of
 the total nutrient intake per unit of time, a model of Optimal Diet Choice
 (ODC) was used. As other optimal foraging models, ODC models are based on
 the profitability of each food source, which is dependent on the amount of
 nutrients acquired from the food sources and the handling time needed to
 locate, capture, manipulate and ingest these food sources. Since pollen
 beetles can be considered encountering both young and old buds
 simultaneously on a given inflorescence, the ODC model of Waddington
 & Holden (1979) was used. This model allows predicting the optimal
 proportion of each food source to be included in the diet, which maximizes
 total macronutrient intake per unit of time:    where <em>p<sub>y</sub></em> is the predicted proportion of young buds to be included in the diet, <em>M.C</em> is the ratio of total nutrient amounts ingested per bud (M: ratio of bud masses ingested, C: ratio of total nutrient concentrations per mass unit), <em>H<sub>y</sub></em> and <em>H<sub>o</sub></em> are handling times of young and old buds respectively, and <em>D<sub>y</sub></em> and 1-<em>D<sub>y</sub></em> are the relative densities of young buds and old buds on an inflorescence respectively. Distributions of the model parameters were estimated from a series of dedicated experiments described below and summarized in Table 1. Values of <em>p<sub>y</sub></em> were computed through simulations since most of the model parameters, i.e. <em>M</em>, <em>C</em> and <em>H</em>, were random variables. For that purpose, the central tendency as well as the distribution of each parameter were computed at the mean point of the two bud maturity classes, i.e. 1.75 mm long for young buds and 6 mm long for old buds (Table 1). Values of <em>p<sub>y</sub></em> were calculated for all values of <em>D<sub>y</sub></em> ranging from 0 to 100 %, by steps of 1 %. For each value of <em>D<sub>y</sub></em>, 10 000 simulations were performed in each of which parameter values were randomly sampled from their theoretical distributions. These 10 000 values allowed estimating a central tendency of <em>p<sub>y</sub></em> (considered as the median value) and an associated 95 % credibility interval. Predicted proportions of young buds to be used could then be compared with real values observed in the feeding experiment on entire plants. The real relative density of young buds at the green-yellow bud stage was estimated by measuring all buds of the main inflorescence of 15 intact plants and calculating the proportion of young buds among the total amount of young and old buds. This proportion was estimated as 0.596 using a Generalized Linear Model (GLM, distribution: binomial, link function: logit).