DOI: 10.5061/dryad.v15dv41xp
title: Generational variation in nutrient regulation for an outbreaking herbivore

description: Multivoltine insects can produce multiple generations in one year.
 Favorable conditions support more generations, leading to serious
 outbreaks. For herbivores, plant nutrient availability is a major
 environmental factor affecting fitness and it can shift substantially
 throughout seasons. In a stochastic environment, organisms can adopt
 several strategies to regulate their nutrient intake and maximize
 performance. However, data regarding nutrient regulation of wild
 herbivores are scarce, and even more so regarding potential
 intergenerational plasticity. To bridge this gap, we measured nutritional
 regulation and performance of an outbreaking multivoltine herbivore – one
 of the most serious agricultural pests in the Sahel: Oedaleus
 senegalensis. We surveyed a field population in Senegal and measured its
 nutritional preference and regulation across two generations (G1 and G3)
 using artificial diets and plant choice experiments. In the field, G1
 locusts were five to ten times more abundant than G3 locusts. We found
 that G1 and G3 locusts selected different protein: carbohydrate ratios but
 also that the strength of regulation was different. G1 locusts regulated
 their nutrient target more tightly than G3 locusts. In contrast, studies
 with laboratory populations demonstrate strong regulation for
 grasshoppers, appearing less plastic than field populations. Both
 generations selected a carbohydrate-biased nutrient ratio, although it was
 more carbohydrate-biased for G3 locusts. In both cases, plant nutrient
 contents in the field were more protein-biased than their preferred diet.
 Therefore, choices by locusts were likely influenced by other ecological
 variables such as leaf toughness or plant defenses. G1 females were
 heavier and laid more eggs than G3 females. However, G3 locusts survived
 longer during the experiment than G1 locusts, suggesting a potential
 generational trade-off between reproduction and survival. Our data
 highlight the importance of studying nutritional regulation in situ and
 incorporating field and lab data to better understand foraging decisions
 and nutritional trade-offs.
description: <strong>I Oedaleus senegalensis: a multivoltine and
 migratory species</strong> The Senegalese
 grasshopper <em>O. senegalensis</em> is a grass‐feeder and a
 major pest of millet and other cereal crops of subsistence agriculture in
 the Sahel zone of West Africa. <em>O. senegalensis</em>
 typically produces three generations throughout the rainy season, although
 when conditions are unfavorable, only two generations are produced (Batten
 1969). This species is considered a non-model locust (Song 2011) and
 locust phase polyphenism (Pener and Simpson 2009, Cullen et al. 2017) is
 typically poorly understood in non‐model species (Song 2011). However,
 Senegalese grasshoppers, like other locust species, are migratory. They
 are nocturnal fliers that can travel hundreds of kilometers in one night
 (Riley and Reynolds 1983, Cheke 1990, Maiga et al. 2008). G1 and G2 travel
 from South to North along the Intertropical Convergence Front, ahead of
 the heavy rains. G3 migrates back South at the end of the rainy season
 (Maiga et al. 2008). <strong>II Field site, host
 plants, and locust abundance</strong> We
 conducted these studies in the summer of 2018 in the Kaffrine region of
 Senegal. The regional woody shrubland savanna landscape is topographically
 flat and marked by agricultural expansion that has replaced native dry
 forests (Mbow et al. 2008). Precipitation ranges from an average of 2 mm
 in the dry season (November–April) to an average of 737 mm during the
 rainy season (May–October). Dry season temperatures average ~ 27 °C
 compared to rainy season average of ~29 °C (D’Alessandro et al. 2015). The
 early rainy season is marked by quick onset of sprouting vegetation and
 concomitant emergence or increased activity of associated animals, fungi,
 and bacteria. By the end of the rainy season, annual plants including
 crops are reaching maturity with thick leaves and seeds (Fig. 1), and
 natural enemies of grasshoppers are well established and
 ubiquitous. The Kaffrine region, along with Kaolack and
 Fatick regions, is known as the ‘West Central Agricultural Region’, or
 Peanut Basin (Tappan et al. 2004) and produces most of the country’s
 millet and peanut. The two crops are typically grown in rotation. Pearl
 millet (<em>Pennisetum glaucum</em>) is a rain‐fed crop with
 excellent tolerance to drought, sandy soil, low nutrient availability and
 high temperatures. While <em>O. senegalensis</em> is
 considered the main pest of millet, these grasshoppers are typically found
 to be more abundant in fallow fields containing weed species (Toure et al. 2013, Word et al. 2019, Le Gall et
 al. 2020a). We
 decided to contrast millet with one of those weed grasses. We selected
 <em>Paspalum scrobiculatum</em>, also called kodo millet or
 dugubupicc locally (“dugub” is millet and “picc” is bird in Wolof), a wild
 native grass because it is readily eaten by <em>O.
 senegalensis</em> and is preferred over other known host grass
 species like <em>Cenchrus biflorus</em> (Maiga et al. 2008) as
 shown by a cafeteria experiment that we ran in 2016 (SI, Table
 1). We measured locust abundance and collected locusts
 for our experiments in the village of Gniby in fallow and millet fields.
 We selected Gniby because it consistently supports high density
 <em>O. senegalensis</em> populations (Word et al. 2019, Le
 Gall et al. 2020a). We collected adult G1 on August 4th and 5th 2018 at
 three locations 1) 14°43N, 15°67W; 2) 14°42N, 15°67W; 3) 14°25N, 15°40W.
 On September 20th and 22nd we recorded abundance and collected adult G3 at
 two locations: 1) 14°41N,15°66W and 2) 14°41N,15°67W. To measure
 abundance, for G1 we counted locusts in twenty plots of one square meter
 each. Each plot was separated by at least ten meters. For G3, the millet
 reached above eight feet high, so we used a transect method and recorded
 abundance along ten transects that were ten meters long, one meters wide,
 and separated by five meters each. Results were standardized to locusts
 per square meter. At both time points, locusts were transported to the
 Direction de la Protection des Végétaux (DPV) field station in Nganda and
 kept in wire mesh cages with ambient local vegetation before being used
 for subsequent experiments. <strong>III
 Artificial diet choice experiment</strong> The
 aim of this experiment was to compare the protein:carbohydrate ratios
 selected by G1 and G3 adults when provided with artificial diets. We
 initiated the choice experiments in August after the start of the rainy
 season (G1) and in September (G3) towards the end of the rainy season
 within a day or two of locust collection. For this, we weighed and put
 locusts in individual aerated plastic containers (15 x 10 cm). Each cage
 contained a water tube, a perch for roosting, and two dishes containing
 artificial diets. We prepared and dried the food in our laboratory at
 Arizona State University (United States) following the method developed by
 Simpson and Abisgold (1985). In total, we made three diets varying in
 protein to carbohydrate ratios but otherwise isocaloric: p35:c7, p28:c14,
 and p35:c7. For each diet, “p” stands for percent of protein in the diet,
 and “c” stands for percent of carbohydrates in the diet, by dry mass. The
 protein component of all foods was a 3:1:1 mix of casein, peptone and
 albumen, while the digestible carbohydrate (henceforth carbohydrates)
 component was a 1:1 mix of sucrose and dextrin. All foods contained
 similar amounts of Wesson’s salt (2.4%), cholesterol (0.5%), linoleic acid
 (0.5%), ascorbic acid (0.3%) and vitamin mix (0.2%) (Dadd, 1961). The
 remainder of the diet was cellulose, a non-nutritive bulking
 agent. We gave the locusts one of two treatments of
 pre-weighed diet pairings: p7:c35 & p35:c7 or p7:c35 &
 p28:c14 and used 20-25 locusts per treatment (approximately half males and
 half females). After three days, the diets were removed and dried for
 24-36 h at 60ºC (drying oven Memmert and Kowell C1-1) and then re-weighed
 to record consumption at the nearest 0.1 mg. Locusts were weighed at the
 beginning and the end of the experiment to calculate mass gain.
 <strong>IV Plant choice experiment</strong>
 The aim of this experiment was to contrast nutrient selection
 results from the artificial diet choice experiment with nutrient selection
 from a choice experiment conducted with local plants. For G1, fresh plants
 were collected in Nganda on the day the experiment started and kept
 bundled with their stems soaked in water. The millet (<em>P.
 glaucum</em>) was collected at the seedling stage (~ 25 cm tall, 1-4
 leaves sprouted) in a local collaborator farmer’s field. The weed
 (<em>P. scrobiculatum</em>) was collected on the side of the
 road, also at seedling stage (~15 cm tall). For G3, we followed the same
 protocol but this time we collected mature weed leaves, mature millet
 leaves, and millet seeds at the milky stage (Fig. 1) which are also
 consumed by locusts (Boys 1978). The first plant choice experiment was
 started on August 5th; the second one involved an additional treatment
 (millet seeds) and therefore had to be divided in two experimental blocks
 to be manageable. These experimental blocks took place on September 21st
 and September 27th. For each experiment, we put 12 male
 and 12 female locusts (n=24) in individual plastic cages (14 x 8 x 4cm)
 fitted with a perch for roosting. The leaf stems were maintained in water
 in a plastic tube sealed with cotton. We cut the millet ears in pieces of
 roughly 4-5 cm and they were given as is. The experiments each lasted 24
 h. At the end of each experiment, we dried all plant material. We
 estimated dry consumption from regression equations. For this we weighed
 20-32 fresh leaves in August, and again in September; we then dried them
 at 60ºC for 48 to 72 h and re-weighed them. Regression equations for
 transforming between plant fresh and dried mass can be found in the
 supplementary information (SI, Appendix 1). For G3
 locusts, we added millet ears. However, despite cutting the millet ears,
 each piece was several folds heavier than grasshoppers and thus
 fluctuation in water content made it impossible to collect an accurate
 estimate of consumption with the same method used for leaves. Thus, we
 used visual observation to establish preference, for this an observer
 recorded which food item (weed leaves or millet seeds) had the most
 grasshopper feeding damage after 30 minutes.
 <strong>V Plant no-choice</strong>
 For the plant no-choice experiments, we used the same setup as
 for the plant choice experiment except that we gave the grasshoppers one
 plant item only: young millet or weed leaves in August (start date August
 6th), and mature millet leaves, millet kernel, or mature weed leaves in
 September (start date September 24th). We used 24 grasshoppers per
 treatment, half males and half females for G1. We had difficulties
 collecting enough females for G3 so we used 24 grasshoppers per treatment:
 9-10 females and 14-15 males. Each time, the experiment lasted a week and
 we changed millet and weed leaves every other day and every three days for
 the kernels. For each grasshopper, we recorded consumption and egg
 production (for females). We reported consumption for day 0-2 when most
 locusts were still alive. The cages were checked daily for mortality and
 the presence of parasites at the time of death was recorded (unidentified
 dipterans made up the vast majority of parasites and about 25% of the
 grasshoppers were parasitized). <strong>VI
 Chemical analysis</strong> We ran the chemical
 assays on the dried plant material in our laboratory at Arizona State
 University (United States). For this, we ground plant samples for 30 s at
 200 rpm using a Retsch MM 400 ball mill. We measured plant protein content
 with a Bradford assay and non-structural carbohydrate content using the
 phenol–sulphuric acid method (Deans et al. 2018).
 <strong>VII Statistical analysis</strong>
 Locust abundance in the field and locust mass in the plant
 no-choice experiment were analyzed using ANOVAs. (Although the data were
 not normally distributed, ANOVA is robust against the violation of
 normality when group sizes are equal (Donaldson 1968)). Other analyses met
 the assumptions of parametric tests. The protein:carbohydrate ratios
 selected in the artificial diet and plant choice experiments were analyzed
 with MANCOVAs using start mass as a covariate to account for size
 differences. The dry amount of plant eaten in the choice experiment and
 plant no-choice experiment, the protein and carbohydrate intakes from the
 artificial diet and the plant choice experiments were analyzed using
 ANCOVAs with start mass as a covariate. Sex was included as a cofactor.
 Visual preference (locusts that did not eat after 30 minutes were not
 included in the analysis) in the plant choice experiment, egg laying and
 parasitism in the plant no-choice experiment, were analyzed by nominal
 logistic fit. Survival in the plant no-choice experiment was analyzed by
 survival analysis with Weibull distribution.