DOI: 10.5061/dryad.xd2547djt
title: The developmental high-wire: Balancing resource investment in immunity and reproduction

description: 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.
description: <em>Experimental Design</em> 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 <em>Serratia marcescens</em> (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 <em>S.
 marcescens</em> (Worthington & Kelly 2016b), however we used
 heat-killed <em>S. marcescens</em> 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 <em>S.
 marcescens</em> 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. <em>Growth &
 Development</em> 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.
 <em>Sperm Viability within Spermatheca</em>
 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). <em>Fecundity & Egg
 Viability</em> 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.
description: Missing data is indicated by "NA", all "0"
 values are true zeroes.