Experimental design
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 (Pennisetum glaucum ) is a rain-fed
crop with excellent tolerance to drought, sandy soil, low nutrient
availability, and high temperatures. On August 2nd
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-1); 3) high N (175kg
N.Ha-1). For each treatment we had 4 plots of 400
m2 each.
We collected millet leaves twice during the experiment for nutrient
analysis and Specific Leaf Area (SLA), first on August
7th (day 5) for both; and then on August
14th (day 12) for SLA and August
20th (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.
(15). We recorded stand establishment for all treatments on August
15th (day 13) by counting the number of plants over 10
m transects, with 4 transects per plot (16 transects per treatment).
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 9th (day 7) and
August 18th (day 16). We collected locusts at a nearby
location on August 7th (Minna, GPS coordinate 13º 49”
33.7N; 15º25’ 00.7W). The same day, we pooled and weighed 5 females and
3 males adult O. senegalensis for each cage. Every 2-3 days we
checked the cages and recorded mortality. On the last day of the
experiment, August 21th (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 O. senegalensis preference for fertilized vs.unfertilized plants by conducting a choice experiment from August
7th (day 5 after fertilization) to August
10th (day 8 after fertilization). We had two
treatments: control vs. medium N, and control vs. 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.