DOI: 10.5061/dryad.vdncjsz48
title: Data for: Maternal provisioning of offspring with defence chemicals in a facultatively parthenogenetic stick insect

description: Parents can invest in offspring by transferring environmental factors,
 such as nutrients or diet-derived defence chemicals, into eggs or embryos.
 However, in systems where females can reproduce facultatively without a
 male (facultative parthenogenesis), it is not known how reproductive mode
 and maternal environment affect offspring provisioning. The facultatively
 parthenogenetic stick insect Megacrania batesii sprays a defensive fluid
 from paired prothoracic glands. Here, we report that some hatchlings of M.
 batesii can spray even prior to their first feeding, and provide evidence
 that both eggs and hatchlings contain the same diet-derived chemical (the
 alkaloid actinidine) that is present in adult defensive spray. We also
 explored potential causes of variation among hatchlings in the capacity to
 spray, using a fully crossed experiment to investigate how offspring
 provisioning is affected by sexual vs. parthenogenetic reproduction and
 high vs. low maternal diet. We found that high maternal diet resulted in
 increased egg size but slower egg development, and maternal diet
 interacted with genotype to affect hatchling body size. Eggs laid by
 male-paired females were larger, developed more quickly, and had higher
 hatching success by comparison with eggs laid by unpaired females,
 suggesting that mating and fertilization enhance some aspects of offspring
 performance. However, hatchlings produced by unpaired females had larger
 prothoracic glands relative to body size than did hatchlings produced by
 male-paired females, suggesting that mating is associated with reduced
 provisioning of offspring with defensive chemicals. Our results reveal a
 novel example of maternal transfer of food-derived defence chemicals to
 offspring, and suggest that offspring provisioning with defence chemicals
 is affected by female reproductive mode.
description: MATERIALS AND METHODS Animal
 maintenance Eggs of <em>M. batesii
 </em>were collected from mixed-sex populations of the Northern
 genotype and all-female populations from the Southern genotype between Cow
 Bay and Cape Tribulation, Queensland (Miller<em> et al.</em>
 2024a), in February and March of 2020 and raised in the lab at UNSW
 Sydney. Eggs were maintained in 125 mL plastic containers with moist
 cocopeat in a room with controlled temperature of 27°C and watered
 periodically to prevent desiccation. Hatchlings (F0) were housed in
 full-sib same-sex pairs in cylindric plastic cages (20 × 40 cm) and fed
 leaves of <em>Pandanus tectorius </em>until they become
 adults. The F1 descendants of those individuals were used in the
 experiment described below, including 20 females produced by Northern
 genotype mothers and 24 females produced by Southern genotype mothers.
 Reproductive mode was manipulated for F1 females descended from both
 Northern and Southern populations. Twenty days after the females started
 to lay eggs, 22 females (14 Northern genotype, 8 Southern genotype) were
 housed with males in separate cages (sexual treatment), while 22 females
 (6 Northern genotype, 16 Southern genotype) were kept alone
 (parthenogenetic treatment). The reproductive treatments were set up in
 the most ecologically relevant way for <em>M. batesii</em>,
 since adult females in all-female populations are rarely found in close
 proximity with other adult females, but many adult females in mixed-sex
 populations are guarded continuously by males (Boldbaatar<em> et
 al.</em> 2024). Consistent with previous reproductive mode
 manipulations on this species (Wilner<em> et al.</em> 2025),
 females in the sexual treatment laid mostly fertilized eggs as indicated
 by the near-even sex ratio of their offspring (34 males, 48 females, 41%
 male offspring), whereas females in the parthenogenetic treatment laid
 exclusively unfertilized eggs and produced exclusively female offspring
 (66 females).   Chemical
 analyses To investigate whether mothers of <em>M.
 batesii </em>were transferring their defensive chemicals to
 offspring, we carried out gas-chromatography mass spectrometry analyses
 (GC-MS). We compared the chemical composition of egg yolk and hatchlings’
 prothoracic glands (offspring of experimental adults chosen randomly and
 frozen at -80°C until dissection) with the defence fluid sprayed by
 adults. We extracted chemicals from 18 eggs, 20 hatchlings and 2 adults
 (one of each sex) to analyse their chemical composition. The eggs were
 washed with ethanol and had their shell removed to expose the yolk. We
 dissected the prothoracic glands of hatchlings by cutting the ventral
 upper portion of the prothorax and removing the glands with a pair of fine
 forceps. Defence fluid was collected from live adults by tapping the
 prothorax with an insertion glass vial until the adults sprayed their
 defence fluid inside the vial. Yolk, prothoracic glands, and defence fluid
 were collected in insertion glass vials (300 μL). The amount of yolk,
 prothoracic glands, and defence fluid were not consistent among samples,
 precluding quantitative comparisons of chemical concentrations.
 Extractions were performed on ice to avoid loss of volatile compounds.
 Each sample was transferred to a 1.5 mL glass vial, and 220 μL of
 chloroform was added and allowed to evaporate overnight at room
 temperature, leaving a small quantity of precipitate on the bottom of the
 vial. We then added 100 μL of acetonitrile to each sample and vortexed
 each vial. The vials with the extracts were placed immediately into a
 GC-MS instrument for the analyses of the chemical composition of the
 samples.   Chemical analyses of all
 extracts were carried out on a Focus DSQ GC-MS equipped with a Triplus
 autosampler (Thermo Fisher Scientific, Germany). Separations were carried
 out using a HP-5MS capillary column with 30 m x 0.25 mm of internal
 diameter and 0.25 mm of film thickness (19091S-433, J&W
 Scientific, USA). A spitless injection with a 4 μl  injection volume was
 used, with the injector temperature set to 230°C. The source temperature
 was set at 200°C, and the start time (solvent delay) was set at 5 min.
 Oven temperature was as follows:  initial temperature 60°C (held for 2
 min), then from 60°C to 115°C (held for 2 min) with a heating rate of
 6.5°C/min, then from 115°C to 125°C (held for 1 min) with a heating rate
 of 1°C/min, and from 125°C to 325°C (held for 9 min) with a heating rate
 of 7.2°C/min. GC-MS data were processed on the qual browser in Xcalibur
 software (version 2.1, Thermo Scientific), and spectra matched against the
 Wiley NIST library (NIST wiley 9 NIST 11) using the NIST Search 2.0
 programme for identifications.  
 Diet manipulation We implemented two feeding
 treatments (high food and low food) and two reproduction treatments
 (sexual and parthenogenetic) in a full factorial design (Figure 2). Each
 female or pair experienced both feeding treatments sequentially, with each
 treatment lasting ten days. Half of the experimental adults from each
 reproduction treatment were assigned initially to the high food treatment
 and half to the low food treatment (first diet treatment). After the first
 diet treatment was completed, adults that were in the high food treatment
 were switched to the low food treatment and vice versa (second diet
 treatment). In the high food treatment, female-male pairs or single
 females received two pieces of leaf (~10 cm in length) every second day
 (five feedings over 10 days) (Figure 2). At each feeding, the sizes of
 remaining leaves from the previous feeding were recorded and the old
 leaves were then discarded. The high food treatment can be considered as
 <em>ad libitum</em> feeding (75% of the total leaf area was
 eaten). Leaves were eaten mostly by the females as adult males eat much
 less than females (Boldbaatar 2022). In the low food treatment,
 female-male pairs or single females received one piece of leaf (~ 10 cm in
 length) on the third and seventh days of treatment (two feedings over 10
 days) (Figure 2). The size of the leaf was recorded, and the leaf was
 discarded after two days from the day of feeding. Insects in the low food
 treatment ate 88% of total leaf area, with the unconsumed part of the leaf
 often dry or covered by the clip used to hang the leaf.
   Prior to the experiment, several females
 were dissected to estimate the length of time required for eggs to develop
 fully in the ovaries. We observed that eggs in different stages of
 development, including five to six eggs almost fully yolked, were present
 in the females’ ovaries and oviduct. Therefore, we opted for discarding
 the eggs laid over the first five days after the start of the feeding
 manipulation. Females of <em>M. batesii</em> lay approximately
 one egg per day, and those first five laid eggs would have been fully or
 almost fully yolked prior to the diet manipulation and therefore unlikely
 to be affected by the treatments. Eggs laid in the second half of the
 treatment (last five days) were collected, photographed, and placed into
 separate containers with a layer of moist cocopeat at the bottom. The eggs
 were separated into individual containers so that hatchlings could be
 frozen without provoking the defence spray behaviour. We recorded the
 number of eggs laid, eggs size, egg development time (days from laying to
 hatching), and egg hatching success (proportion of eggs that hatched). The
 eggs, hatchling thorax and hatchlings’ dissected prothoracic glands were
 photographed using a camera (Leica MC170HD) mounted on a stereoscope
 (Leica MZ16A, Wetzlar, Germany) with lighting standardized by using a
 consistent light source and blocking out ambient light. Traits were then
 measured from the images using ImageJ (Schneider, Rasband &
 Eliceiri 2012). We measured the length and width of the eggs (mm), the
 length of the thorax (cm), and area of the prothoracic glands (calculated
 as the product of length and maximum width in pixels) as a proxy of the
 amount of defence fluid present in the glands.
   Statistical analyses
 Feeding treatment (high vs. low food), reproductive mode (pairing
 with a male vs. no pairing), maternal genotype (Northern vs. Southern
 genetic cluster), order of feeding treatment application (low→high vs.
 high→low), and all interactions among diet, reproduction and genotype,
 were modelled as fixed effects using the R package glmmTMB
 (Brooks<em> et al.</em> 2017). Gaussian error distribution was
 used to model egg development time, egg size (arbitrary units), hatchling
 size, and prothoracic gland size (log-transformed, arbitrary units),
 poisson error distribution was used to model the number of eggs laid, and
 binomial error distribution was used to model egg hatching success
 (hatched vs. unhatched outcome for each egg). Mother identity was modelled
 as a random effect in all models to account for repeated measures on
 individual focal females, but paternal identity was not modelled because
 parthenogenetically produced females had no father. We modelled
 prothoracic gland size with hatchling body size (thorax length) included
 as an additional fixed covariate to account for variation in body size
 among hatchlings. Because female and male hatchlings are very similar in
 body size and hatchling sex did not explain variation in any measures of
 hatchling performance, we did not include hatchling sex in the
 models. Akaike’s Information Criterion corrected for
 small samples (AICc) was used to determine support for covariates and
 interactions by comparing models containing all possible subsets of the
 fixed effects from the above glmmTMB models (but retaining maternal
 identity as a random effect in all models) using the “dredge” function in
 the MuMIn package (Bartoń 2024). The null (intercept-only) model was
 included among the models compared for each response variable. The top
 model and other models with ΔAICc &lt; 2 were considered to have
 statistical support (Burnham & Anderson 2002; Johnson &
 Omland 2004; Burnham, Anderson & Huyvaert 2011; Symonds &
 Moussalli 2011), and model-averaged effect coefficients were calculated
 based on all supported models using the “model.avg” function. As a measure
 of model fit, the marginal R<sup>2</sup> (proportion of total
 variance explained by the fixed effects) and condition
 R<sup>2</sup> (proportion of total variance explained by both
 fixed and random effects) were calculated for the full model using the
 “r.squaredGLMM” function. All statistical analyses were conducted using R
 version 4.1.0 (R_Core_Team 2021).
description: # Data for: Maternal provisioning of offspring with defence chemicals in a
 facultatively parthenogenetic stick insect
 [https://doi.org/10.5061/dryad.vdncjsz48](https://doi.org/10.5061/dryad.vdncjsz48) ## Description of the data and file structure The data were collected from a fully crossed experiment in which the reproductive model (sexual / parthenogenetic) and adult diet (High / Low) were manipulated for each of 44 *Megacrania batesii* females.  Each female experienced a single reproductive treatment (either paired with a male throughout life or never paired with a male), but was subjected to both High and Low diet treatments successively. Each diet treatment was applied for 10 days, but eggs were collected from the final 5 days. The response variables included the number of eggs laid by the focal females, egg size, egg development time, egg hatching success, hatchling body size, and the size of hatchlings' prothoracic glands.  The data are divided into 4 data files for ease of analysis and plotting, as described in the README file. ### Files and variables #### **File: eggnumberdata.csv** This file contains data on the number of eggs laid by each focal female over the last 5 days of the 10-day diet-treatment application period (see Methods). Because each focal female was subjected sequentially to both High and Low diet treatments, there are two rows for each female ("motherID"), a row for the number of eggs laid while on the High diet and a row for the number of eggs laid while on the Low diet.  Columns: **motherID**: The individual identity code for each focal female. **Reproduction**: The reproduction treatment to which the focal female was subjected. Females in the "Paired" group were paired with a male and therefore mated and laid fertilized eggs (probably along with some unfertilized eggs). Females in the "Unpaired" group were housed without males and therefore laid exclusively unfertilized eggs. **Diet**: Each focal female was subjected successively to both a High diet treatment (i.e., provided with abundant leaves as food) and a Low diet treatment (provided with a limited number of leaves as food). The treatments lasted 10 days, and eggs were collected over the last 5 days (hence there are two rows for each female).  **Order**: The order in which the diet treatments were applied for each focal female (1 = first, 2 = second).  **Genotype**: The genetic origin of each focal female. "Northern" females were descended from the northern genetic cluster, while "Southern" females were descended from the southern genetic cluster, as defined in Miller, S.M., Stuart, K.C., Burke, N.W., Rollins. L.A. and Bonduriansky, R. 2024. Genetic and phenotypic consequences of local transitions between sexual and parthenogenetic reproduction in the wild. The American Naturalist 203: 727511. **Numbereggs**: The number of eggs laid by focal females during the last 5 days of High and Low diet treatments. --- **File: eggdata.csv** This file contains data on the size of the eggs laid by focal females and whether or not each egg hatched. Each row contains data for one egg. Columns: **motherID**: as above **egg**: Numbers representing the sequence in which eggs were laid during the 5-day egg collection period for each diet treatment. **Reproduction**: as above **Diet**: as above **Order**: as above **Genotype**: as above **Treatment**: Codes representing the 4 possible Reproduction x Diet treatment groups, used only for plotting. **Egglength**: The length of each egg in mm. **Eggwidth**: The width of each egg in mm. **Eggsize**: The area of each egg in mm^2, calculated as Egglength x Eggwidth. **Hatched**: 0 = egg did not hatch; 1 = egg hatched. --- **File: hatchlingdata.csv** This file contains data on phenotypes for each egg that hatched, including egg development time, hatchling body size, hatchling sex, and the area of hatchling prothoracic glands. Columns: **motherID**: as above **egg**: Numbers representing the sequence in which eggs were laid during the 5-day egg collection period for each diet treatment. **Reproduction**: as above **Diet**: as above **Order**: as above **Genotype**: as above **Treatment**: Codes representing the 4 possible Reproduction x Diet treatment groups, used only for plotting. **Diet-Genotype**: Codes representing the 4 possible Genotype x Diet treatment combinations, used only for plotting. **daycollected**: Callendar date on which each egg was collected. **dayhatched**: Callendar date on which each egg hatched. **Timedev**: Development time calculated for each egg as the number of days between collection and hatching.  **Hatchlingthorax**: Thorax length of each hatchling in cm. **Sex**: The sex of each hatchling (determined using the method described in Miller et al. 2024 Am.Nat.). **glandareal**: Area of the left prothoracic gland in pixels. **glandarear**: Area of the right prothoracic gland in pixels. **Logglandarea**: The combined area of the left and right prothoracic glands, log-transformed and divided by 10 in order to improve the distribution of residuals and make the scale comparable to thorax length. This variable was used as the response variable in the analysis of gland size. **Relativeglandarea**: The combined area of the left and right prothoracic glands divided by 100000 and by Hatchlingthorax. This variable was used only to estimate effect size. **Logrelativeglandarea:** Log of Relativeglandarea. This variable was used only for plotting. --- **File: Prophatched.csv** This file contains the proportion of eggs hatched for each focal female, used for plotting only. Columns: **motherID**: as above **Reproduction**: as above **Count**: The total number of eggs laid by the focal female during the High and Low diet treatments.  **Prophatched**: The proportion of the eggs laid that hatched, used only for plotting. ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * All of the data were collected for the described experiment by the authors