DOI: 10.5061/dryad.7d7wm385d
title: Sex-dependent effects of infection on guppy reproductive fitness and offspring parasite resistance

description: Infection imposes energetic costs on hosts. Hosts typically respond by
 shifting resources, potentially affecting the quantity and quality of
 offspring they produce. As the sexes differ in their optimal reproductive
 strategies, infection of mothers versus fathers may affect offspring
 quantity and quality in different ways. Here, we test how experimental
 infection of guppies Poecilia reticulata with the ectoparasite
 Gyrodactylus turnbulli affects parental reproductive fitness and offspring
 parasite resistance. We compared breeding pairs in which one or neither
 parent had previously been infected. In terms of reproductive fitness,
 parental infection experience did not affect the size, body condition, or
 number of offspring produced, but fathers who experienced the heaviest
 infections produced offspring ~55 days sooner than average. This result
 may represent terminal investment by the males most affected by infection,
 or may indicate that these males have a faster pace of life, investing in
 reproduction at the expense of parasite defence. We found that offspring
 age, parental infection experience, and parental infection severity
 together strongly predicted offspring parasite resistance. Only among
 pairs in which one parent had been infected, older offspring, which were
 those born soonest after the parent’s infection, tended to experience
 heavier infections. This result may therefore reflect temporary
 infection-induced reductions in parental investment in offspring quality.
 Beyond this effect of offspring age, offspring of infected mothers
 experienced 105 fewer worm days than those of infected fathers: fathers,
 but not mothers, that experienced heavy infections themselves produced
 offspring that also experienced heavy infections. The parent-offspring
 regression for infected fathers is therefore consistent with previous
 evidence that parasite resistance is heritable in this system, and yields
 a narrow sense heritability estimate of 0.69±0.13. By contrast, the
 mother-offspring regression (slope: -0.13±0.17) provides novel insight
 that mothers may engage in transgenerational immune priming. Overall, our
 results suggest that the sexes strike a different balance between
 offspring quantity and quality when faced with infection, with potentially
 broad implications for disease and host-parasite coevolutionary dynamics
 in nature.
description: <em>(a) Fish Origin and Maintenance</em>
 We used laboratory-bred descendants of guppies from the Caura
 River in Trinidad. This laboratory population was founded by fish
 collected in 2012 from a high-predation population located at an
 undeveloped, rural site on the Caura River, Trinidad (UTM: 20 P 679527.7 m
 E, 1180376.4 m N based on WGS84 Datum; elevation 112m). Since 2012, the
 population has been maintained in large numbers, and we continue to see
 substantial variation in disease resistance, as is clear from our results.
 Fish were maintained and bred in 20 L tanks of mixed sexes under standard
 conditions, as follows. Tanks were on a recirculating system (Aquaneering)
 with approximately 20% daily water changes, a lighting schedule of
 12L:12D, and an average temperature of 25±1°C. In these systems,
 wastewater from tanks through foam, sand, and ultraviolet filters before
 re-entering other tanks, preventing unintentional parasite transmission.
 Fish were fed flake and Artemia daily. <em>(b)
 Parasite origin and maintenance</em>  Our
 parasite line was established by transferring one worm from a commercially
 obtained guppy to an uninfected host from a mixed laboratory-bred stock, a
 ‘culture fish’. This parasite line was maintained on groups of three to
 six culture fish per 1.8 L tank under standard conditions. Twice per week,
 all culture fish were anaesthetized (tricaine methanesulfonate ‘MS222’; 4g
 per L), and parasites were counted using a dissecting microscope.
 Uninfected fish were added to replace recovered or dead fish.
 <em>(c) Parental infection</em> <em>and
 breeding design</em> Parental fish were
 anaesthetized (using MS222 as above) and placed in close proximity to a
 dead, highly infected culture fish until at least two worms transmitted
 (mean±SEM=2.8±0.27), as observed under a dissecting microscope. Once
 infected, parental fish were isolated in 1.8 L tanks maintained under
 standard conditions for the duration of their infection. We counted worms
 every three days for 15 days to record individual infection trajectory
 over time. After 15 days, we treated fish with levamisole (0.002g per L)
 to clear infection. We verified that this treatment had cleared infection
 by screening the fish for parasites under anaesthetic, three times with a
 minimum of four days between screens. We recorded the length and weight of
 the parents before and after infection. Parasite naïve parents were
 exposed to the same conditions and manipulations, with the exception of
 parasites. We set up 38 total breeding pairs of one
 male and one female using the recovered fish and parasite naïve fish in
 three different treatments: recovered mother, naïve father (13 pairs);
 recovered father, naïve mother (11 pairs); and both parents naïve (14
 pairs). We did not set up pairs in which both parents were recovered
 because previous experience suggested this treatment may not have yielded
 sufficient offspring, and our primary objective was to examine the
 independent effects of maternal and paternal infection. These breeding
 pairs were housed in 1.8 L tanks under standard conditions, including fry
 mesh structures and gravel, to reduce possible fry cannibalism by the
 parents. Between May and December 2021, these breeding tanks were checked
 daily for the presence of offspring, which were moved to a separate 1.8 L
 tank. Each brood produced from a pair was moved to a separate 1.8 L
 tank. <em>(d) Offspring
 infection</em>  Once they reached adulthood,
 offspring were separated into individual 1.8 L tanks. A total of 108
 offspring were collected: 32 from recovered fathers and naïve mothers; 38
 from recovered mothers and naïve fathers; and 38 from naïve parents. We
 used two batches of fish, one in August 2021 (52 offspring from 33
 breeding pairs, with an average age at infection of approximately 4.4
 months) and one in May 2022 (56 offspring from 32 breeding pairs, with an
 average age at infection of approximately 7.8 months). According to the
 needs of other concurrent experiments, 94 fish were infected manually, as
 described under parental infection, and 14 were infected via “co-housing”:
 they were housed in 1.8 L tanks with an infected conspecific and screened
 for infection every 24 hours. Once infected, these fish were isolated in a
 1.8 L tank and treated the same as the manually infected offspring and
 parents from that point forward. As noted below, we included infection
 type as a fixed effect in the statistical models and found that it did not
 explain significant variation in our response variables. On all infected
 offspring, we counted the worms every three days for 12 days and then
 killed the host. 
description: # Sex-dependent effects of infection on guppy reproductive fitness and
 offspring parasite resistance
 [https://doi.org/10.5061/dryad.7d7wm385d](https://doi.org/10.5061/dryad.7d7wm385d) ## Description of the data and file structure ### Files and variables #### File: 20241205\_TGIP\_supplement.pdf **Description:** This .pdf file is an RMarkdown file of the code and output of the analyses we present in the paper. #### File: 2024TGIPData.csv **Description:** This .csv file contains the raw data from our experiment. ##### Variables These variables all refer to the offspring: * SEX: sex of the fish * PREL: fish standard length in mm before infection. * DOB: date in YYYYMMDD the fish was born * INFTYPE: Whether the fish was infected under anaesthesia through the manual exposure to a heavily infected, euthanized donor fish (‘manual’), or through cohousing with a live infected donor (‘cohouse’) * LENGTHRESID: The residuals of length of the focal fish on sex. Females tend to be larger than males: using the residuals of this relationship allows us to test for both size and sex differences. * PREW: Fish weight in mg before infection * LASTW: Fish weight in mg after infection * preSMI: Body condition - scaled mass index - before infection * postSMI: Body condition - scaled mass index - after infection * deltaSMI: Body condition - scaled mass index - change over the course of infection * DOSE: Initial number of parasites fish was infected with * INFBIRTHDAYS: Time in days between the first day of parental infection and fish birth * LATENCY: Time in days between the day the parental pair was set up to breed and the offspring’s birth, corrected so the minimum value is 0 (as fish were not virgins, some females produced offspring before being paired with a male). * PAIRBIRTHDAYS: Time in days between the day the parental pair was set up to breed and the offspring’s birth * MAXWORMS: The maximum number of worms recorded on the fish over its infection * FULLAUC: The area under the curve of infection load (number of worms) over time - ‘infection integral’ * AGEATINF: Age in days fish was when it was infected * DATEINF: Date of infection The datasheet also contains the following variables about the infected parent. Where two variables are listed, those with an ‘M’ prefix refer to the mother, and those with an ‘F’ refer to the father: * PARINFTREAT: Which parent was infected - mother, father or neither. * PINF: Binary variable denoting that one parent was (1) or was not (0) infected. * PAUC: Parental (either the mother’s or father’s, depending on which was infected) area under the curve (AUC) of infection; NA if neither parent infected or parental AUC unknown * MOTHERID: ID of the parent * MOTHERINF: Whether (1) or not (0) the parent was infected * MMAXWORM: The maximum number of worms recorded on the fish over its infection * MAUC: The area under the curve of infection load (number of worms) over time - ‘infection integral’ * MDATEINF: The date in YYYYMMDD the parent was infected. * MPREW: Fish weight in mg before infection * MPOSTW: Fish weight in mg after infection * MPREL: Fish length in mm before infection * MPOSTL: Fish length in mm after infection * MpreSMI: Body condition - scaled mass index - before infection. * MpostSMI: Body condition - scaled mass index - after infection. * MdeltaSMI: Body condition - scaled mass index - change over the course of infection. * MDIED: Whether or not the mother or father died during the observation period * FATHERID: ID of the parent * FATHERINF: Whether (1) or not (0) the parent was infected * FMAXWORM: The maximum number of worms recorded on the fish over its infection * FAUC: The area under the curve of infection load (number of worms) over time - ‘infection integral’ * FDATEINF: The date in YYYYMMDD the parent was infected. * FPREW: Fish weight in mg before infection * FPOSTW: Fish weight in mg after infection * FPREL: Fish length in mm before infection * FPOSTL: Fish length in mm after infection * FpreSMI: Body condition - scaled mass index - before infection. * FpostSMI: Body condition - scaled mass index - after infection. * FdeltaSMI: Body condition - scaled mass index - change over the course of infection. * FDIED: Whether or not the mother or father died during the observation period * TOTBABIES: Total number of babies produced by that mother Throughout the datasheet we use "NA" to denote missing values. These values are either missing because this is what is dictated by the experimental design (e.g. some parents were not infected, so will have NAs for all the infection variables), or because fish died before these data could be recorded (e.g. some fish have NA for post infection weights because they died during infection), or because of experimenter error. ## Code/software We used R statistical software to analyse our data. The code and output are included in the pdf provided here. We used the following packages, most up to date versions as of December 2024. library(glmmTMB) #for GLMMs library(visreg) #for plotting models library(GGally) #for pairwise plotting library(ggplot2) #for general plotting library(readxl) #for reading in excel file library(dplyr) #for dataframe management library(plyr) #for dataframe management library(car) #for ANOVA function library(DHARMa) #for model validation library(lmodel2) library(lsmeans) library(multcomp) library(ggpubr) library(arm) library(BSDA) library(tidyr)