DOI: 10.5061/dryad.547d7wmg4
title: Investment in regeneration versus asexual reproduction is resource-dependent in a freshwater annelid

description: The post-embryonic developmental processes of regeneration and asexual
 agametic reproduction are widespread and often co-occur in animals. These
 traits are of great ecological significance, but their physiological
 dynamics within species are not well understood. In naid annelids,
 regeneration and asexual reproduction via fission are evolutionarily
 related and mechanistically similar yet distinct, making these animals
 useful systems in which to study resource allocation strategies between
 the two processes. How asexual reproductive investment varies as a
 function of somatic investment demands was tested in the naid Pristina
 leidyi by repeatedly amputating the heads of individual worms, allowing
 regeneration to proceed, and measuring reproductive output over time.
 Treatments were replicated under high and low food levels to determine to
 what extent the investment dynamic between regeneration and fission is
 affected by the resource pool. Reproductive output was affected by injury
 and regeneration frequency in a resource-dependent manner, such that only
 worms with less food availability exhibited reproductive deficits; injury
 and regeneration did not affect reproductive output of worms under the
 high food condition. When reproductive output was decreased, this occurred
 not through a reduction in offspring quantity but a reduction in offspring
 quality. In the offspring of experimental animals, body size and fission
 speed were dependent on parental feeding level and to a lesser and
 inconsistent extent on parental injury history, but regeneration speed was
 unaffected by parental treatment. These findings suggest that in a species
 capable of both regeneration and asexual reproduction: 1) the resource
 pool is a key factor mediating the resource investment pattern between
 regeneration and fission; 2) sacrificing per-offspring investment rather
 than fecundity may be an optimal strategy if resources are limiting; 3)
 regeneration and fission have evolved distinct resource allocation
 pathways. This work prompts further questions about the physiological
 dynamics between regeneration and asexual reproduction in animals, such as
 whether and to what extent these have evolved adaptively, including in
 response to injury and resource pressures.
description: Animal culture and material The use of
 animals in this study did not require approval from an ethical committee.
 Established cultures of <em>P. leidyi</em> (see Bely &
 Wray, 2001)<em> </em>were cultured at room temperature (~23
 ⁰C) in glass bowls (12 cm diameter) filled with ~150 ml of artificial
 spring water (1% artificial seawater) (ASpW). Strips of brown paper towels
 were provided as substrate. Cultures were fed once weekly with 10 mg
 powdered Spirulina. Half-volume water changes were administered
 weekly. To generate experimental animals, 109
 healthy-looking worms of similar size were pulled from a culture that had
 been established 3 weeks prior, to ensure that the culture was undergoing
 active growth. These worms were moved to individual wells of 24-well
 plates filled with 1.5 mL ASpW. Each individual worm was fed once weekly
 with 0.15 mg Spirulina. Individuals were monitored daily for fission
 (zooid release). <em>P. leidyi</em> typically forms fission
 zones (FZ) at approximately two-thirds the length of the body, within
 segments 14–16. Because it possesses the original head and represents the
 larger fission product, the anterior zooid is considered the “parent” and
 the posterior zooid, once released, is considered the “offspring”, by
 convention. The first posterior zooid released by each worm was moved to a
 new 24-well plate, and these 109 young worms were used as experimental
 animals (F<sub>0</sub>) as described below and illustrated in
 Fig. 1. All F<sub>0 </sub>worms were produced within 5 days of
 each other (Fig. 1a). Individual <em>P. leidyi</em> are
 estimated to have a lifespan of approximately 1 year (Bely, AE,
 unpublished data) and F<sub>0</sub> worms born within days of
 each other were thus considered of approximately equivalent age.
 Experiment One: Effect of injury and feeding on survival and
 reproduction  An experiment with a 2×4 factor design,
 including two food levels and four injury levels, was conducted to test
 the effects of anterior amputation injury (and regeneration) frequency on
 reproductive output and how these are modulated by feeding.
 F<sub>0</sub> worms were randomly assigned to one of
 two feeding treatments: low food (LF), which received 0.15 mg Spirulina on
 the first day of each week (<em>n </em>= 55); and high food
 (HF), which received 0.15 mg Spirulina on the first and fourth days of
 each week (<em>n</em> = 54). Full water changes were
 administered for all worms on the first and fourth day of each
 week. Worms in each feeding group were randomly
 assigned to one of four injury treatments: uninjured controls (0X) and
 worms injured once (1X), twice (2X), or three times (3X) during the
 experiment (Fig. 1a). Worms were divided approximately evenly among these
 four treatments (low food: <em>n</em> = 14, 14, 12, 14; high
 food: <em>n</em> = 13, 14, 12, 16, respectively). Injuries
 were all anterior amputations, inflicted by cutting at a consistent
 position at the anterior end of F<sub>0</sub> worms.
 Specifically, at each amputation time point, all worms (regardless of
 treatment) were anesthetized in 0.05 mM nicotine and worms designated for
 injury were amputated at the junction of segments six and seven using a
 scalpel (Fig. 1b). The excised anterior portion was discarded and the
 remainder of the worm, representing the posterior-most ~3/4 of the worm
 body, was retained for the experiment.  Note that worms can continue
 fissioning even after amputation of the anterior part of the body. Three
 rounds of amputation occurred across the experiment: at week 3 (day 19 of
 the experiment, beginning with the birth of the first
 F<sub>0</sub> worm), week 6 (day 40 of the experiment), and
 week 9 (day 61 of the experiment). At each of these time points, all worms
 were anesthetized and worms designated for injury at that time were
 amputated. All worms were then transferred to clean 24-well plates in
 fresh ASpW and all worms (including those that were not amputated) were
 not fed for one week, while injured animals regenerated. Designated
 feeding schedules resumed after that week. At the first amputation time
 point (week 3 of the experiment, ~2 weeks following the “birth” (release)
 of the last F<sub>0</sub> worm), worms in the 1X, 2X, and 3X
 treatments were amputated. At the second amputation time point (week 6 of
 the experiment), worms in the 2X and 3X treatments were amputated. At the
 third amputation time point (week 9 of the experiment), only worms in the
 3X treatment were amputated. The experiment was concluded on day 100 of
 the experiment. Each F<sub>0 </sub>worm was
 scored daily for survival and fission (release of a posterior zooid,
 designated F<sub>1</sub>) across the duration of the
 experiment. The first F<sub>1</sub> produced by each
 F<sub>0</sub> worm, and the first F<sub>1</sub>
 produced following each round of amputation (regardless of whether the
 individual was in an injury treatment or not), was removed, imaged, and
 assigned to experimental treatments as described below (Fig. 1c). All
 other offspring were discarded upon discovery. Throughout this paper, the
 following shorthand is used to refer to time intervals during which
 offspring production was recorded: t<sub>0</sub> - from
 F<sub>0</sub> birth until the day of first injury;
 t<sub>1</sub> - from 1 week after first injury until the day
 of second injury; t<sub>2</sub> - from 1 week after second
 injury until the day of third injury; t<sub>3</sub> - from 1
 week after third injury until the conclusion of the experiment. These
 intervals are collectively referred to as the F<sub>1</sub>
 “birth periods”. Experiment One: Offspring quality
 assessments  During Experiment One, the first
 F<sub>1</sub> worm produced by each F<sub>0</sub>
 worm at the beginning of the experiment and following each regeneration
 period, or at each equivalent time point for uninjured worms, was
 collected. ­Offspring quality was assessed by measuring offspring volume,
 reproductive speed, and regeneration speed, as described further
 below. Offspring volume was assessed on the day of
 discovery. On that day, offspring were anesthetized and imaged under a
 2.5x objective using a Zeiss Axioplan 2 microscope running AxioVision 4.8
 imaging software. Body dimensions were measured with ImageJ using the Fiji
 package (v1.47f ) (Schindelin et al., 2012) and volume was approximated by
 using the formula for a cylinder
 (π<em>W</em><sup>2</sup><em>L</em>)
 (Fig. 1d). To calculate total body volume, worm length and width were
 measured. Whole worm length (<em>L</em>) was measured by
 tracing from the base of the proboscis to the tip of the pygidium along
 the anteroposterior axis. Whole worm width (<em>W</em>) was
 averaged across three roughly equally spaced positions along the body, as
 worms vary slightly in diameter along the body length. Especially under
 higher food conditions, <em>P. leidyi</em> can develop
 multiple FZs along the anteroposterior body axis simultaneously, such that
 new offspring sometimes already possess one or occasionally even two FZs
 when they are released from the parent. The total volume contained within
 all FZs (if present) in an offspring was separately calculated (if more
 than one FZ was present in an individual, the volumes of all its FZs were
 pooled to produce a single measure of FZ volume). Note that FZs, even
 early stage ones, are easily identified by differences in opacity and
 slight narrowing of body width relative to older tissues. Total FZ volume
 was calculated by measuring, for each FZ, the length and width of the FZ,
 with width measured around the center of the FZ. Note that imaging for
 body size measurements did not begin until partway through
 t<sub>0</sub>, leading the sample size for F<sub>0
 </sub>body volume to be slightly smaller than the sample size for
 F<sub>0</sub> fission speed. Following
 imaging, worms were randomly assigned to one of two assay groups: an
 “offspring fission speed” assay and an “offspring regeneration speed”
 assay (Fig. 1c). For the fission speed assay,
 F<sub>1</sub> worms were placed individually in fresh 24-well
 plates filled with ASpW and maintained on the same feeding regimen as
 their respective F<sub>0</sub> worm. F<sub>1</sub>
 worms were scored daily to determine both the date of a first FZ
 appearance and the completion of the first fission (release of a
 “F<sub>2</sub>” offspring). Time (# days) from “birth” to
 formation of a first FZ and from formation of the first FZ to zooid
 release (fission speed) were recorded and their sum calculated (Fig. 1c).
 After fission, the F<sub>1</sub> worm and all of its offspring
 were discarded. For the regeneration speed assay,
 within an hour following imaging, F<sub>1</sub> worms were
 amputated in the manner described previously and transferred individually
 to fresh 24-well plates filled with spring water. Worms were not fed
 during this experiment. Worms were checked daily for two indicators of
 full regeneration: the emergence of visible chaetae in the new anterior
 segments (four in total) and the emergence of the proboscis. Time from
 injury to full regeneration (# days) (regeneration speed) was recorded
 (Fig. 1c). At the completion of regeneration, worms were discarded. If
 amputated F<sub>1</sub> worms produced offspring, which was
 common within 1-2 days following amputation if a fission zone was present
 at birth (itself common in F<sub>1</sub> derived from HF
 F<sub>0</sub>), the date of that offspring release was noted,
 and such offspring were discarded. Experiment Two:
 Direct effect of feeding on regeneration speed To
 provide additional context for interpreting offspring regeneration speed,
 a follow-up experiment was run to assess how feeding level directly
 affects regeneration speed of individual worms (rather than that of their
 offspring). For this experiment, 24 worms were pulled from a culture
 (maintained as above), and 12 worms were each randomly assigned to LF and
 HF levels, as described above. Worms were maintained individually and fed
 under LF and HF conditions for two weeks and then amputated in the same
 manner as described above. Worms were scored daily for regeneration
 completion, as described above. Three LF worms and one HF worm died
 following amputation and were excluded from analysis.
 Statistical analysis All statistical
 analyses were performed in the R computing environment (R Development Core
 Team, 2019). Most analyses were performed using generalized linear (mixed)
 models (GLMMs) via the lme4 package (Bates et al., 2015), and the 95%
 confidence intervals (CIs) for fitted values were estimated via the
 ciTools package (v0.6.1; Haman & Avery, 2020). The significance of
 explanatory variables in all models was determined with a Wald test.
 Pairwise comparisons were accomplished using the emmeans package (v1.8.9;
 Lenth, 2023), when appropriate. Analyses of F<sub>0</sub> worm
 data included tests of the effects of feeding and injury on survival and
 fecundity. A GLMM with a binomial error distribution (logit link) was
 constructed to test differences in F<sub>0</sub> survival over
 the course of Experiment One between feeding and injury treatments, with
 the response variable being whether or not a worm survived following a
 particular injury (and lived to the next injury, if applicable). GLMMs
 with a Poisson error distribution (log link) were constructed to test
 differences in fecundity (# of offspring produced) between feeding and
 injury treatments for different periods of time:
 t<sub>0</sub>, t<sub>1</sub>,
 t<sub>2</sub>, t<sub>3</sub>, and total duration
 of the experiment. Worms were randomly assigned to plates based on injury
 treatments but not feeding, such that some plates only held high-food
 worms and others only low-food worms, and worms were maintained in the
 same relative well position across the experiment. Therefore, inclusion of
 plate as a random effect in a mixed model created a singularity with
 feeding. A nested model using plate as a fixed effect to substitute for
 feeding was constructed to test whether there were significant differences
 between plates of the same feeding group. As no significant contribution
 of plate distinguishable from a feeding effect was apparent, and identical
 results were obtained between the singular mixed-effects models and
 reduced GLMs without the plate variable, these latter models were used for
 the final analyses. Analyses of
 F<sub>1</sub> worm (offspring) data included tests on the
 effect of parent feeding and injury treatments on offspring body volume,
 fission speed and associated measures, and regeneration speed. Offspring
 birth period (t<sub>0</sub>, t<sub>1</sub>,
 t<sub>2</sub>, or t<sub>3</sub>) was additionally
 modeled as a fixed effect in order to facilitate comparisons between
 offspring from recently- and not recently-injured
 F<sub>0</sub> worms of the same approximate age as a control.
 A GLMM with a gamma error distribution (log link) was constructed to test
 differences in body volume, with the volume of any FZs present included as
 a fixed effect. A binomial GLMM was used to test the probability of
 F<sub>1</sub> being born with a FZ already present. Time from
 birth to FZ detection in the fission speed assay (fission latency period),
 time from FZ detection to fission completion in the fission speed assay
 (fission speed), and time from injury to regeneration completion in the
 regeneration speed assay (regeneration speed), were each analyzed via the
 construction of GLMMs with a Poisson error distribution (log link).
 Inclusion of the identity of the F<sub>0</sub> worm that a
 F<sub>1</sub> worm was born from as a random effect in these
 models, with the exception of those for body volume, led to singular fits,
 as worm identity was not sufficiently replicated across levels of feeding.
 Therefore, F<sub>0</sub> identity was modeled as a fixed
 effect to check for outlier contributions of individual worms to these
 various responses. As no outliers were detected through this method, and
 identical results were obtained between the singular mixed-effects models
 and reduced GLMs without F<sub>0</sub> identity, these latter
 models were used for the final analyses with the exception of those for
 body volume, which was analyzed via a GLMM as described above.
 In Experiment One, although each worm was assigned to a
 particular injury treatment at the start of the experiment, amputations
 were performed sequentially over time such that at any particular time
 point in the experiment, a worm may have only experienced a subset of its
 planned amputations (Fig. 1). For example, at the end of
 t<sub>1</sub>, the maximum number of anterior amputations that
 a worm could have experienced during the experiment is one; therefore, the
 real injury history of all worms assigned to the 1X, 2X, and 3X injury
 treatment groups at this point is effectively identical. Given this
 experimental design, data were analyzed in two different ways: 1) with
 worms strictly assigned to their initial injury treatment group, and 2)
 with worms pooled based on their actual injury history at the given birth
 period in the experiment (thus maximizing effective sample sizes). For the
 latter approach, for each birth period, the actual injury history of an
 F<sub>0 </sub>worm was considered to be its injury treatment.
 The data and results of these pooled analyses are presented in the main
 text and figures. The data and results of unpooled analyses, in which
 worms are analyzed at each time point based on the initially assigned
 injury treatment, are presented in the Supporting Information and
 mentioned in the main text when conclusions differ from those of the
 pooled analyses. The effect of food level on
 regeneration speed in Experiment Two was tested using a Welch’s
 t-test.
description: # **Investment in regeneration versus asexual reproduction is
 resource-dependent in a freshwater annelid**
 This dataset contains all
 of the data and code used to perform the analyses in the present article.
 These were used collectively to determine how food availability and the
 frequency of anterior amputation and regeneration affect reproductive
 investment in the worm *Pristina leidyi*. There are four spreadsheets and
 eight R scripts contained in this dataset. **Description of the data and
 file structure** There are four spreadsheets containing data: 1.    
 Injury-Data-Primary.xlsx 2.     Mortality.csv 3.     Offspring-Sizes.xlsx
 4.     Extra-Regen-Test.xlsx (1) contains all of the data used in
 calculations of fecundity for F0 worms and consists of several sheets. The
 first sheet contains data related to F0 worm experimental treatments and
 offspring production. Every row represents one worm. Reported for each
 worm in the first several columns is a unique identifier code (A or B,
 indicating low or high food treatment respectively, followed by a number),
 the plate in which the worm was maintained during the experiment, its date
 of birth, the date and note indicating whether the worm died or was lost
 during the experiment, the date the worm was found dead if so, a binary
 code indicating whether a live posterior zooid was discovered in the same
 well at the time of death (1 being positive), the number of days between
 the most recent amputation and the discovery of death, which amputation
 number most recently preceded death, the assigned feeding treatment, and
 the assigned injury treatment. The remaining columns in the sheet largely
 concern offspring production. Columns K and L report the dates of
 offspring born prior to the first amputation date and the sum total of
 these respectively. Col. M reports the date of the first amputation and is
 the same for all rows regardless of whether the worm was actually injured
 or not. Col. N reports whether a fission zone was detected at the time of
 the first amputation with a binary code (1 for positive). Cols. O and P
 report the dates of offspring born during the first regeneration period
 and the sum total of these offspring respectively. Cols. Q and R report
 the date of the first offspring (F1) born after the completion of the
 first regeneration period (i.e., during t1) and a binary code (1 for
 positive) indicating whether at least one offspring was born during t1.
 Col. S reports the dates of any further offspring born during t1, and Col.
 T gives the sum total of offspring born during t1. Col. U indicates
 whether the parent worm survived to the date of the second amputation. The
 remaining columns repeat the information given from Cols. K through U for
 the second and third amputations with the exception of the final two
 columns, which report the total number of offspring produced during the
 experiment and a T/F for whether that worm survived to the conclusion of
 the experiment. Cells in some columns (e.g.: "Offspring
 During...", "Post-C* Offspring...") may appear blank, which
 simply indicates an absence of any offspring born during these intervals
 for those F0 worms. The second sheet of (1) contains data concerning the
 offspring quality experiments. Each row is one F1 worm. The first several
 columns report the parent ID code, the parental feeding treatment, the
 parental injury treatment (“CutTrt”), the parental number (part of the
 ID), the birth period of the F1 worm (“Round”), its birth date, and the
 offspring quality experiment that worm was randomly assigned to (either
 fission speed or regeneration speed). For worms assigned to the fission
 speed experiment, columns H and I report the date that a fission zone was
 first detected and the date that fission was completed, respectively.
 Other worms are left 'na'. For worms assigned to the
 regeneration speed experiment, columns J and K report the date that
 amputation was inflicted and the date that regeneration was complete,
 respectively, with other worms left 'na'. Column L calculates
 the number of days between the two dates appropriate for that given worm.
 For fission speed-assigned F1, Column M reports the number of days between
 the date of birth and the date of first fission zone appearance
 ("FALSE" for regeneration speed-assigned F1; a value of 0
 indicates that the offspring was born already containing a visible fission
 zone), and Column N is simply the sum of Cols. L and M and is meaningless
 for regeneration speed-assigned F1. The remaining sheets in (1) were used
 to record the birth dates of additional offspring during various intervals
 of the experiment for loading into the first sheet. These sheets are
 included simply for user reference and may be safely ignored or deleted.
 (2) contains data used for the analysis of mortality in F0 worms. Each row
 represents one worm. Reported are its feeding group and the most recent
 amputation the worm experienced prior to either the conclusion of the
 experiment or its death. Therefore, these do not necessarily correspond
 exactly to the originally assigned injury treatments. The final two
 columns are a binary record of survival and death following that
 amputation, with “1” indicating a positive event for that column. (3)
 contains data related to calculations of body volume in F1 worms. The
 first several columns report the F1 ID code (the parent ID followed by a
 dash and a number 1-4), the parent worm feeding treatment, the F1 birth
 period (“Round”), the offspring number (1-4), and the parent worm injury
 treatment. Cols. G-J report the estimated length and three width
 measurements (in mm), and Column K calculates the average width. Column L
 calculates body volume (in cubic mm) based on the formula for a cylinder.
 Column M is a binary reporting a “1” if a fission zone was detected in the
 offspring at the time of birth. The remaining columns give the estimated
 total length, width, and volume of any fission zone(s) detected. (4) is a
 short spreadsheet used for Experiment 2. Each row is a worm, and both its
 feeding treatment and the number of days between amputation and the
 completion of regeneration are reported. **Code/Software** There are eight
 R scripts used to conduct the analyses presented in this paper: 1.    
 mortality.R 2.     cut_to_death.R 3.     fecundity.R 4.     bodysize.R
 5.     bodysize_log.R 6.     offspring_analyses.R 7.    
 offspring_analyses_unpooled.R 8.     exp2_ttest.R All analyses were
 conducted with R v4.3.1. (1) was used to calculate mortality frequency in
 F0 worms and calls upon data files (1) and (2). Packages used for this
 analysis include: ·       multcomp v1.4-25 ·       ggplot2 v3.4.3 ·      
 readxl v1.4.3 ·       dplyr v1.1.2 ·       HH v3.1-49 ·       car v3.1-2
 ·       compute.es v0.2-5 ·       effects v4.2-2 ·       pastecs v1.3.21
 ·       graphics v4.3.1 ·       lme4 v1.1-34 ·       performance v0.10.4
 ·       ciTools v0.6.1 ·       emmeans v1.8.8 (2) was used to analyze how
 experimental treatments affected the amount of time between amputation and
 any mortality that occurred and calls upon data file (1). (3) was used for
 analyses of fecundity in F0 worms and calls upon data file (1). Additional
 packages used in this script include: ·       MASS v7.3-60.0.1 ·      
 cowplot v1.1.2 (4) was used for analysis of body size in F1 worms and
 calls upon data file (3). (5) is identical to (4) except with some slight
 code modifications to accommodate the presentation of log-transformed body
 size values. (6) was used for analyses of fission speed and regeneration
 speed in F1 worms and calls upon data file (1). Additional packages used
 in this script include: ·       FSA v0.9.5 (7) is identical to (6) except
 with some slight code modifications to accommodate the presentation of
 unpooled data. (8) was used for the analysis of Experiment 2 and calls
 upon data file (4). Additional packages used in this script include:
 ·       rstatix v0.7.2