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The developmental high-wire: Balancing resource investment in immunity and reproduction


Worthington, Amy (2022), The developmental high-wire: Balancing resource investment in immunity and reproduction, Dryad, Dataset,


The strategic allocation of resources into immunity poses a unique challenge for individuals, where infection at different stages of development may result in unique trade-offs with concurrent physiological processes or future fitness-enhancing traits. Here, we experimentally induced an immune challenge in female Gryllus firmus crickets to test whether illness at discrete life stages differentially impacts fitness. We injected heat-killed Serratia marcescens bacteria into antepenultimate juveniles, penultimate juveniles, sexually-immature adults, and sexually-mature adults, and then measured body growth, instar duration, mating rate, viability of stored sperm, egg production, oviposition rate, and egg viability. Immune activation significantly impacted reproductive traits, where females that were immune-challenged as adults had decreased mating success and decreased egg viability compared to healthy individuals or females that were immune-challenged as juveniles. Although there was no effect of an immune challenge on the other traits measured, the stress of handling resulted in reduced mass gain and smaller adult body size in females from the juvenile treatments, and females in the adult treatments suffered from reduced viability of sperm stored within their spermatheca. In summary, we found that an immune challenge does have negative impacts on reproduction, but also that even minor acute stressors can have significant impacts on fitness-enhancing traits. These findings highlight that the factors affecting fitness can be complex and at times unpredictable, and that the consequences of illness are specific to when during an individual’s life an immune-challenge is induced.


Experimental Design

We randomly divided antepenultimate females into either the control or immune-challenged treatment group, and each female was assigned one of four timepoints to receive their treatment: 1) antepenultimate, 2) penultimate, 3) sexually-immature, or 4) sexually-mature. Antepenultimate females received their treatment on the first day of the experiment when they were two molts away from adulthood, as indicated by small wing buds and an ovipositor extending only 1-2 mm past the end of the abdomen. Penultimate females received their treatment five days after molting into their final juvenile instar, and were identified by their large wing buds and an ovipositor approximately 5-6 mm in length. Sexually-immature females received treatment two days after eclosing into adults once their exoskeleton had fully hardened to prevent excess injury while receiving their treatment. At this early adult stage, a females’ eggs are only just beginning to develop within the ovaries and females have low receptivity to mating (Solymar & Cade 1990). Finally, sexually-mature females received treatment seven days after eclosing into adults – a time when ovaries are full of developed eggs and females become receptive to mating (Worthington & Kelly 2016a).

At their designated treatment time, crickets were cold-anesthetized for 3:15, 3:30, or 4:00 minutes for antepenultimates, penultimates, and adults, respectively. To elicit a non-lethal yet robust immune response, we sterilized the abdomens of immune-challenged crickets with 70% ethanol and inserted a sterile glass microcapillary needle between the second and third abdominal sclerites to inject 1.0 × 104 cells/5 μL of the heat-killed bacterium Serratia marcescens (obtained as a live Microkwik culture from Carolina Biological Supply #155450A and diluted to concentration with phosphate-buffered saline). This number of cells is equivalent to an LD50 dose of live S. marcescens (Worthington & Kelly 2016b), however we used heat-killed S. marcescens to avoid the pathogenic effects of live bacteria while still effectively activating the immune response (Adamo 2004; Stahlschmidt et al. 2015) and inducing sickness behavior (Adamo et al. 2010). We plated heat-killed S. marcescens to test for viability, and no live colonies were ever observed after exposure to heat. Control females were anesthetized, sterilized, and handled underneath the stereoscope, but did not receive any injection. After receiving their treatment, females were returned to their original containers and monitored until they recovered.

All females were reared individually until they reached their 9th day of adulthood, at which time each female was placed into a 1.2-L container with a randomly assigned healthy adult male to mate with and provisioned with a cardboard shelter, a cotton-plugged water vial, and a piece of cat food. Mated adult males 2-3 weeks post-eclosion were randomly chosen from our breeding population. Only males of average size were paired with females, as obviously small and large males were avoided during selection. Each male was used only once. After 24 hours, the male was removed and a small cup of moistened fine sand (Reptilite, Ft. Pierce, FL, USA) was added for the female to oviposit into. Females were given 48 hours to oviposit before the trial was ended on the 12th day of adulthood and females were processed for the presence and viability of sperm stored in their spermatheca, as well as the number of eggs present in their ovaries.

Growth & Development

Mass (to the nearest 0.01 mg) and pronotum length (the distance between the anterior and posterior edges at the midline) were measured for the antepenultimate crickets on the first day of the experiment, and then again on the first day of the penultimate and adult instars. Each cricket was photographed at 0.75× magnification using a Leica IC90-E camera mounted on a Leica M80 stereoscope, then pronotum was digitally measured to the nearest 0.001 mm using LAS Core Software (Version 4.9). Crickets were monitored daily for molting or death, and the number of days that each individual spent in the penultimate instar was calculated from the dates we recorded. Food and water were replaced only as needed to minimize disturbance.

Sperm Viability within Spermatheca

On day 12 of adulthood, females were cold-anesthetized for 5 min so we could dissect their spermatheca to perform a sperm viability assay. The LIVE/DEADTM assay (Molecular Probes, Eugene, OR, USA) stains live sperm green using SYBR®-14 and stains dead sperm red using propidium iodide, and has been effectively used to quantify viability on sperm recovered from spermatheca (McNamara et al. 2014b). After dissection, we placed each spermatheca in 20 µL of Beadle’s saline (128.3 mM NaCl, 4.7 mM KCl, and 23 mM CaCl2) and gently ruptured with fine forceps. Following 10 minutes of incubation, we added 5 µL of 1:50 SYBR®-14 solution (1.25µL SYBR®-14 in 50 µL Beadle’s Saline), then incubated the solution in the dark for 5 minutes before adding 2.5 µL of propidium iodide and incubating in the dark for an additional 5 minutes. Following this final incubation, we pipetted 10 µL of the solution into each well of a disposable hemocytometer (INCYTO C-Chip, Covington, GA, USA). Sperm were visualized at 400× magnification on a fluorescent microscope (Leica DM2000 LED, Leica Microsystems GMBH, Wetzlar, Germany). Sperm located within five predetermined squares of the grid were counted as living (fluoresced green) or dead (fluoresced red). All sperm counts were made by D.J.B., who was blind to experimental treatment at time of assay. Sperm viabilities are reported as the percentage of viable sperm within the spermatheca (i.e. the number of live sperm divided by the total number of sperm).

Fecundity & Egg Viability

At the same time that the spermatheca was dissected, the total number of eggs contained within the ovaries was quantified. To approximate maternal investment egg size, five fully-developed eggs (i.e. those most posterior) from the right ovary were imaged and their length recorded. Upon dissecting the female on day 12, the oviposition egg cup was maintained at 27 ºC for a further nine days to allow the oviposited eggs to develop. The moist sand was then air dried for 24 hours so the eggs could be collected using a fine mesh sieve. We quantified both the total number of eggs laid and the proportion of those eggs that were viable. Eggs were only considered viable if they had eye spots after 10 days of development. Fecundity was calculated as a sum of the eggs found within the lateral oviducts and the total number of eggs that were oviposited.

Usage Notes

Missing data is indicated by "NA", all "0" values are true zeroes.