Study animals
Male G. sigillatus used in this study were randomly selected from
a large outbred colony, initiated with crickets collected from a wild
population from Riverside, CA in 2014. All crickets used in this study
were of the same generation. Newly hatched, experimental crickets were
reared in 55 L plastic storage bins packed with egg carton to increase
rearing surface area and provisioned with a standard diet (roughly equal
parts Envigo© 2018CM Teklad Certified Global 18%
protein rodent diet and Purina® Cat Chow Complete
pellets) and water (glass vials plugged with moist cotton) ad
libitum . The nutritional content for this standard diet is 18.6%
protein, 6.2% fat, 44.2% carbohydrate (rodent diet) and 32.0%
protein, 12.0% fat, and 37.6% carbohydrate (Purina®Cat Chow). When sex differences became apparent (4thor 5th instar), juvenile males were removed from stock
colonies and housed individually in small (450 mL) plastic containers,
ensuring virgin status and age control for all experimental crickets,
and provisioned with food (roughly equal parts rodent diet and cat food)
and water (glass vials plugged with cotton) ad libitum . A small
section of egg carton was also provided as a refuge. All individuals
were housed in an environmental chamber at 32°C on a 16 h:8 h light:dark
cycle. Males were checked daily to determine the date of final molt to
adulthood and then randomly assigned to a diet treatment.
Artificial diets and measuring dietary intake
Isocaloric diets (which differed in P:C but not total calories) were
created using a protocol from an earlier study that quantified the
amount of carbohydrates and protein maximizing calling effort (P:C 1:8)
and immune function (i.e., encapsulation ability) (P:C 5:1) in maleG. sigillatus (Rapkin et al., 2018). Briefly, proteins
consisted of a 3∶1∶1 mixture of casein, albumen, and peptone, with
digestible carbohydrates consisting of a 1∶1 mixture of sucrose and
dextrin. All diets contained Wesson’s salts (2.5%), ascorbic acid
(0.28%), cholesterol (0.55%), and a vitamin mix (0.18%). After the
appropriate dry weight of protein and carbohydrate had been added to the
mixture, the remainder of the mixture was adjusted to the appropriate
composition with crystalline cellulose.
Upon adult eclosion, each cricket was given one dish of his assigned
diet (high protein diet: n = 249; high carbohydrate diet: n = 245),
which was changed every 3 days over a period of 21 days (7 trays total
for each male). Diet was provided in feeding platforms created by gluing
the upturned plastic lid of a 2 mL microcentrifuge tube in the center of
a plastic petri dish (50 mm diameter, 9 mm deep). The materials and
design of the feeding platforms ensured that food could be collected in
the petri dish if any spilled while subjects were feeding. Diet
consumption was calculated as the difference in dry weight before and
after feeding. Prior to weighing, any feces were removed from the
feeding platform using fine forceps. The dry weight of all
food-containing trays, obtained by holding them in drying oven at 30°C
for at least 72 h, was measured both before and after feeding to
quantify how much of each diet was consumed by an individual throughout
the feeding trials, as this alters the total amount of nutrients
available. This is an especially important facet of evaluating the
effects of macronutrients, but one not often considered in such
assessments.