DOI: 10.5061/dryad.9cnp5hqfk
title: Data from: Nitrogen fertilizer decreases survival and reproduction of female locusts by increasing plant protein to carbohydrate ratio

description: 1. Nitrogen limitation theory predicts that terrestrial plants should
 benefit from nitrogen inputs and that herbivores should benefit from
 subsequent higher plant protein contents. While this pattern has generally
 been supported, some herbivorous insects have shown preference and higher
 performance on low protein (p), high carbohydrate (c) diets as juveniles.
 2. However, little is known about the effects on reproduction in adults.
 Using nitrogen fertilizer, we demonstrate that high plant p:c has negative
 effects on Senegalese locust (Orthoptera: Oedaeleus senegalensis)
 reproduction and survival in an agroecological setting. 3. For this, we
 measured p:c in millet plants (Pennisetum glaucum) that received two
 levels of fertilizer (high and moderate) and a control, then we caged
 locusts on these plants for two weeks. In the laboratory, we gave locusts
 the choice between untreated millet leaves and leaves that received one of
 the two fertilization treatment. 4. We found that fertilization increased
 p:c ratio in a concentration dependent fashion. We counted the number of
 locusts alive over the course of two weeks and showed that fewer females
 survived on fertilized plants than on control plants. Females that ate
 plants from the high fertilization treatment laid lighter eggs. Finally,
 we showed that female locusts prefer unfertilized plants to plants with a
 high p:c. 5. We hypothesize that this pattern will apply broadly to
 species that have extensive carbohydrate needs, such as long-distance
 migrators. Because many ecological studies focus primarily on nitrogen or
 protein, and fail to consider carbohydrates, this study has important
 implications for how ecologists consider nutrient limitation of primary
 consumers in ecosystems globally. 
description: <b>Material and
 Methods</b> <b>A Sahel
 migrant: <i>Oedaleus senegalensis</i></b>
 Locusts
 are grasshoppers that, in response to certain environmental cues including
 high population density, will shift from solitarious to gregarious
 phenotypes, which can subsequently lead to swarms. This phenomenon is
 termed locust phase polyphenism (Cullen et al., 2017; Pener &
 Simpson, 2009) and is poorly understood in non-model locust species like
 <i>Oedaleus senegalensis</i> (Song, 2011). <i>O. senegalensis</i> is a grass-feeder and a major pest of millet and other cereal crops of subsistence agriculture in the Sahel zone of West Africa. Eggs start hatching with the first precipitation events of the rainy season (Maiga et al., 2008), which historically occur from June through September. <i>O. senegalensis</i> typically produces three generations that all migrate following the Intertropical Convergence Zone (Maiga et al., 2008). We conducted our experiments in August 2017 to coincide with the development of the first generation which is responsible for critical damages to seedling millet. The movement of local populations have yet to be formally established, therefore the migratory status of the locusts we caught is difficult to assign, however given that our location is at the southern range of the migration area, it is likely that locusts had not migrated yet and were local adults. <b>Experimental design</b> Our field site was the village of Nganda, in the West Central Agricultural Region of Senegal where most of millet and groundnut production takes place. The two crops are typically rotated from one year to the next. Pearl millet (<i>Pennisetum glaucum</i>) is a rain-fed crop with excellent tolerance to drought, sandy soil, low nutrient availability, and high temperatures.  On August 2<sup>nd</sup> 2017 (day 0), we applied two levels of N to a millet field using urea (N:P:K 46:0:0), for a total of 3 treatments: 1) control; 2) medium N (87.5kg N. Ha<sup>-1</sup>); 3) high N (175kg N.Ha<sup>-1</sup>). We selected these amounts because 175kg N.Ha<sup>-1 </sup>is similar to the fertilization rates of most crops (<i>Fertilizer use by crop</i>, 2006). For each treatment we had 4 plots of 400 m<sup>2</sup> each.  We collected millet leaves twice during the experiment for nutrient analysis and specific leaf area (SLA), first on August 7<sup>th </sup>(day 5) for both; and then on August 14<sup>th</sup> (day 12) for SLA and August 20<sup>th</sup> (day 18) for nutrient analysis. For both analyses we collected at least 30 leaves per treatment (3-5 g fresh mass) from different plants and from every plot. After collection, we kept the plants in an ice cooler until arrival at the phytosanitary base of Nganda (Direction de la Protection des Végétaux) where we photographed them over a 10 cm x 10 cm grid. We then dried them for 24-36 h in a drying oven (Kowell C1-I) at 60ºC and weighed them. We recorded SLA with ImageJ (National Institute of Health). 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 seconds 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-sulfuric acid method, following the protocol of Clissold et al. (2006). On each plot we built 3-4 cages (14-16 cages per treatment) using screening mesh (Phifer, Aluminium screen mesh). Each cage was 50 cm wide and 60 cm high, poles were 90 cm rebars, and the metallic mesh was buried underground and held by 50 cm rebars. We closed the cages with staples and binder clips. Each cage contained at least two millet plants. We calculated growth by measuring plant height in the cages or for neighboring plants on August 9<sup>th</sup> (day 7) and August 18<sup>th</sup> (day 16). We collected locusts at a nearby location on August 7<sup>th</sup> (Minna, GPS coordinate 13º 49” 33.7N; 15º25’ 00.7W). The same day, we pooled and weighed 5 females and 3 males adult <i>O. senegalensis</i> for each cage. We picked this density so that grasshoppers would not run out of food in the cages (crops often do not occur significant damages at density below 10 grasshoppers per square meter (Grasshopper Identification & Control Methods, 2008). We slightly skewed the sex ratio towards females because we were interested in collecting egg data. The grasshoppers were left in the cages and allowed to decline naturally, every 2-3 days we checked the cages to record mortality. On the last day of the experiment, August 21<sup>th </sup>(day 19), the remaining locusts were removed, and we scraped the first 10 cm of soil with a trowel and manually sieved the soil for eggs. Eggs were kept in alcohol and brought back to the lab to be counted, we then dried and weighed them. We compared <i>O. senegalensis</i> preference for fertilized <i>vs.</i> unfertilized plants by conducting a choice experiment from August 7<sup>th</sup> (day 5 after fertilization) to August 10<sup>th</sup> (day 8 after fertilization). We had two treatments: control <i>vs.</i> medium N, and control <i>vs.</i> high N. For each treatment we tested 24 grasshoppers (12 females and 12 males). Locusts were hosted in a plastic cage with two leaf blades in water tubes and a perch for roosting. Plants were collected in the same fashion as for the nutrient analysis and kept in a water bucket until the beginning of the experiment. At the end of the experiment, plant material was dried, and we calculated dry consumption using a regression equation that we obtained from drying 50 millet leaves: Dry mass millet leaf = 0.0162 + 0.16 * Wet mass millet leaf  . <b>Statistical analysis</b> Statistical analyses were carried in JMP Pro 14. Fertilization effects on plants:<b> s</b>pecific leaf area (SLA), plant protein, and plant carbohydrates were analyzed with ANOVAs followed by a Tukey HSD when we found differences among treatments. To evaluate effects on ratio, plant protein and carbohydrate content was analyzed by MANOVA, we used a Pillai’s test statistic. Choice experiment: the dry amount of plant eaten for each treatment was compared using MANCOVA techniques; we used Pillai’s test statistic. We used start mass as a covariate to correct for size differences among individuals. We included sex as an independent variable when its effect was significant. The dry amount of food eaten by males, and by females, was analyzed using ANOVAs followed by a Tukey HSD when we found differences among treatments. Locust performance in the field:<b> </b>The number of locusts alive over time was averaged by plots and analyzed by survival analysis followed by post-hoc comparison with Bonferroni correction (Tripathi & Pandey, 2017). Total egg mass and egg number, and average egg mass were averaged for each plot. We analyzed total egg mass and egg number with a Kruskall-Wallis test. We used a Welch’s anova to compare average egg mass because variances were not homogeneous and used Games-Howell HSD for posthoc comparisons (McDonald, 2009).