DOI: 10.5061/dryad.rjdfn2zmp
title: Investigating the role of non-helpers in group living thrips

description: Behavioural variation among individuals is a hallmark of cooperative
 societies, which commonly contain breeders and non-breeders, helpers and
 non-helpers. In some cases, labour is divided, with non-breeders
 “helping”. Conversely, in some societies, subordinate non-breeders may not
 help. These individuals may be (i) an insurance workforce ensuring
 continuity of help for breeders when other helpers are lost, (ii)
 conserving energy while waiting to breed themselves, or (iii) simply of
 too poor physiological quality either to help or breed. In the Australian
 Outback, Acacia thrips Dunatothrips aneurae (Thysanoptera) glue
 Acacia phyllodes into “domiciles” using silk-like secretions, either alone
 or cooperatively. Domicile maintenance is important for humidity, so
 repair can be interpreted as helping. I found that not all females helped
 to repair damage; some repaired partially or not at all
 ("non-helpers"). At the same time, some co-foundresses are non-
 or partially reproductive ("non-breeders”), and their role is
 currently unknown. I first tested the possibility that helping
 and breeding are divided, with non-helping females breeding, and
 non-breeders helping. In a lab experiment, I rejected this idea.
 Experimentally damaged domiciles were typically repaired by reproductive
 females, and less so by non- or partially reproductive individuals. To
 test whether non-helpers are an insurance workforce, I successively
 removed repairing females and found that non-helping females from the same
 domicile did not increase effort as a result, rejecting this hypothesis.
 Then I tested whether non-helpers were conserving energy waiting to breed.
 In a field experiment, I removed all other females, allowing either a
 helpful female or a non-helper to “inherit” her domicile. Isolated like
 this, non-helpers laid very few eggs compared to helpers or naturally
 occurring single foundresses, despite similar ovarian
 development. My findings show that labour was not divided:
 reproduction and helping covaried positively, probably depending on
 individual variation in female quality and intra-domicile competition.
 Non-helping females were neither an insurance workforce nor conserving
 energy waiting to breed. They are likely simply of poor quality,
 freeloading by benefiting from domicile maintenance by others. I
 hypothesize they are tolerated because of selection for indiscriminate
 communal brood care in the form of domicile repair.
description: Lab and field studies were conducted between January and October
 2013, at a large patch of <em>Acacia aneura,</em> the host
 plant for <em>D. aneurae</em>, at the Bald Hills paddock (S
 30° 57′ 39′; E 141° 42′ 18′′) near the UNSW Arid Zone Research Station,
 Fowlers Gap near Broken Hill, NSW, Australia (see Gilbert and Simpson
 2013, Biol. J. Linn. Soc.). <em>D. aneurae</em> domiciles were
 identified and either collected or marked using coloured tape. I collected
 domiciles by hand and placed them into ziplock bags inside a cool box,
 moving them to a laboratory at room temperature within 12 h, and
 maintained them using protocols described in Gilbert & Simpson
 (2013). I examined the domiciles under a dissecting microscope. 
 <em>Are reproduction and domicile repair divided among
 foundresses? </em>In domiciles containing two or more females, I
 made a small tear in the wall (roughly 20% of the silk area), folded back
 the silk, and observed repair behaviour (defined as stretching strands of
 "silk" singly between phyllode surfaces) by the foundresses.
 After scoring in this way, all group members were removed and dissected
 under a microscope (see protocol below) for assessment of ovarian status.
 I also measured female body size to the nearest 20 µm (pronotum width at
 50⨉ magnification). <em>Are non-helping
 foundresses an insurance workforce for breeders? </em>In a subset of
 19 domiciles, I counted the individual silk strands applied by each female
 while repairing the experimental damage. After 1 h I removed the first
 repairer to respond and observed the behaviour of the remaining female(s)
 over the subsequent 1 h period. Over successive 1 h periods, I removed
 one-by-one the repairers in the sequence that I saw them begin repairing
 and counted silk strands laid down by the remaining females. Removed
 individuals were dissected and scored for ovarian development as described
 below, and their pronotum width was measured.
 <em>Are non-helping foundresses waiting to breed
 themselves?</em> In a field removal experiment using domiciles
 containing 2 or 3 females, I first categorised each female as “repairer”
 or "nonrepairer", as above: I slightly damaged the domicile
 wall, then observed the behaviour of the inhabitants for 3 h. I used a
 longer observation period of 3 h under field conditions where observation
 was more difficult, to be sure to identify nonrepairing females. After
 classifying, I removed all individuals from all domiciles. I then assigned
 domiciles randomly to treatment groups in which I reinstated to her
 original domicile either a repairer (R group, n=11) or a nonrepairer (NR
 group, n=8). In this way, I allowed (or forced) this female to “inherit”
 her domicile. Replacement was completed within 30 minutes of removal. All
 other females I dissected to assess reproductive status as described
 below. The remaining female I left in the domicile. I also created two
 control groups (CS, control singletons, and CP, control pairs). The CS
 group consisted of naturally occurring singly built domiciles (n=46),
 while the CP group consisted of domiciles naturally built by pairs of
 females (n=9). In the CP and CS groups, I performed the same experimental
 domicile damage before removing and immediately replacing all domicile
 inhabitants. After manipulation, I monitored domiciles for 21 days to
 assess (1) the time to repair the domicile, and (2) the number of
 offspring produced.  <em>Time to repair domicile:
 </em>Each day I visually categorised repair as “no repair”,
 “incomplete repair” or “complete repair”. “No repair” was where a
 negligible number of silk strands had been added and the damaged area
 remained substantially open to the environment. “Incomplete repair” was
 where repair had begun, but strands were sparse and perturbation of the
 phyllodes (e.g. by mild wind) would likely reopen the hole in the silk.
 “Complete repair” was where silk completely covered the damaged area,
 perturbation of the phyllodes would be unlikely to reopen the hole in the
 domicile wall, and/or the repaired wall resembled the original silk. I
 also recorded any instances of naturally occurring domicile damage (and
 its repair), destruction, or abandonment. 
 <em>Offspring in domicile: </em>After 21 days, I
 counted the number of offspring (eggs and larvae, live or dead) in each
 domicile, and dissected the female to examine her ovaries (see below).
  All individuals remaining in domiciles after the 21 d experimental period
 were also dissected as described below to assess reproductive
 status.  <em>Assessing ovary status: </em>I
 assessed ovarian development in all dissected individuals following
 Gilbert et al (2018, Scientific Reports). Foundresses were killed by
 immersing in 100% ethanol for 1 m and were dissected immediately in water.
 I recorded the number and volume to the nearest 20 µm at 50⨉ (π × length ×
 (width/2)<sup>2</sup>), assuming eggs are an ellipsoid) of any
 developing oocytes, and also of any mature, chorionated eggs that were
 ready to lay, and summed the resulting volumes to give a total volume
 within each ovary. For analysis, oocyte and mature egg volumes were
 log-transformed after adding the minimum value in the dataset to all
 values.
description: # Investigating the role of non-helpers in group living thrips
 [https://doi.org/10.5061/dryad.rjdfn2zmp](https://doi.org/10.5061/dryad.rjdfn2zmp) This dataset contains 4 data files in CSV format and 1 file of R code in Rmarkdown format.  ### Data Note that in some cases these data files include individuals involved in studies unrelated to the current paper. These individuals are excluded from the code, with appropriate explanation. Each data file corresponds to a different part of the linked paper, as follows: 1. Are reproduction and domicile repair divided among foundresses? * gilbert_2024_jae_dissection_data - fig 3 - dis.csv 2. Are non-helping foundresses an insurance workforce for breeders? * gilbert_2024_jae_sequential_removal_lab - figs 4 and 5 - xlab.csv 3. Are non-helping foundresses waiting to breed themselves? * gilbert_2024_jae_raw_data_removal_repair_tr.csv * gilbert_2024_jae_removal_ovaries.csv * Also uses some data from gilbert_2024_jae_dissection_data - fig 3 - dis.csv (see R code) ### Code All R code, and a reproduction of the results section of the paper in markdown format, are contained in the file entitled * 2024-05-09 JAE Nonhelpers R code.Rmd ## Description of the data and file structure Field codes for all data files: ##### gilbert\_2024\_jae\_1\_dissection\_data - fig 3 - dis.csv In this dataset, each line represents one female, so multi-female domiciles appear on multiple lines. Some females/domiciles from unrelated experimental studies have been excluded. Other irrelevant cases are excluded in the R code. * DATECOLL: Date domicile collected * DATEDISS: Date female dissected * ID.NEST: Domicile ID * NEST.STAGE: Max stage of nest development (Building, No Eggs, Eggs, Active, Dispersed, Old)  * F.STATE: Female developmental stage (Teneral, Alate, Dealate) - all here are dealate, this is a subset of a larger dataset * F.EXPT: Which experiment, if any, the female is assigned to. XLAB = sequential removal experiment; XR = removal experiment * TREAT: treatment group to which the focal female is assigned, within the experiment described in F.EXPT; see main text and other datasets for context * REPAIR: Repair latency coded as 3 level factor: 2 = repair; 1 = repair only after others removed; 0 = no repair * PRONOT.50: Pronotum width in um, measured at 50x * nestvol: Domicile volume (mm^3) * chorion: Chorionated (i.e., mature) oocytes present in the ovary, true or false * oocyte: Volume of developing oocytes in um3 (see Gilbert et al 2018, Scientific Reports, for the description of dissection and measurement protocols) * chorvol: Volume of chorionated oocytes in mm^3 * oovol: Volume of developing oocytes in mm^3 * repro: Whether or not female is reproductive (i.e., has developing oocytes): true or false * reprostate: Female reproductive state coded as a 3-level factor: Undeveloped, Developing oocytes, Mature oocytes * repair: Whether or not the female engaged in repair: true or false ##### gilbert\_2024\_jae\_2\_sequential\_removal\_lab - figs 4 and 5 - xlab.csv In this dataset, each line represents one female, so domiciles appear on multiple lines.  * NEST: Domicile ID * ID: Female ID * strands: Total number of strands laid by the female during the experimental observation period * timein: Duration of the experimental observation period for that female (HH:MM) * strandshour: Rate of strand deposition per hour (=strands/timein) * timetop: Time spent as a primary repairer in the group during the experimental period * timenottop: Time spent as subordinate (i.e., not primary) repairer in the group during the experimental period * strandstop: Strands laid while primary repairer in the group during the experimental period * strandsnottop: Strands laid while subordinate (i.e., not primary) repairer in the group during the experimental period * strandshourtop: Rate of strand deposition per hour while primary repairer in the group during the experimental period * strandshournottop: Rate of strand deposition per hour while subordinate (i.e., not primary) repairer in the group during the experimental period * rank: repair rank within the group, on the basis of latency to begin repairing: 1 = top repairer, 2 = second repairer, etc. * stage: Stage of domicile development (see Gilbert et al. 2013, *Biol J Linn Soc*) * choro: Whether or not a female has mature oocytes (0 = no, 1 = yes) * oocyte: Volume of developing oocytes, in um3 (this is converted to mm3 in the R script) * chorvol: Volume of mature oocytes, in mm^3 * size: Pronotum width in um * nestvol: Domicile volume in mm^3 * FOUNDRESSES: Number of foundresses in the nest * relrank: Relative rank, i.e., repair rank (see "rank" column) scaled by the number of foundresses such that first repairer = relrank 0 and last repairer = relrank 1. ##### gilbert\_2024\_jae\_3\_raw\_data\_removal\_repair\_tr.csv Each line of this dataset represents a domicile/nest, which received one of the treatments detailed in the accompanying paper (R, NR, CP, CS). * IND: number of replicates * ID: Domicile ID * FNUMBER: Number of females initially present in the nest * FBEHAVIOUR: Repair behaviour of females initially in the nest: H = helpful, U = unhelpful, M = male (never help), X = unknown * TR.INITIAL: My initial division of nests into treatment groups (RH = remove helper, RNH = remove nonhelper, C = Control) * tr: Final treatment group coding for statistical model (H = "helpful" = R group; U = "unhelpful" = NR group; C = control pairs (CP); AH = control singletons (CS). * REMAINING: Behaviour of females in nest following treatment * REPAIRTIME: Time to repair nest (days) * REPCENS: Whether or not the nest was gone/destroyed before completely repaired * GONE: Domicile disappeared during the experiment * DESTROYED: Domicile was destroyed during the experiment * STAGEAFTER: Nest stage 21 days after treatment (Building, No Eggs, Eggs, Active, Dispersed, Old) * OFFSPRINGAFTER: Number of offspring in the nest 21 days after treatment * NOTES: Notes * X27.08.2013 (and columns thereafter): Action or observation on date. MA = Manipulation; NR = Not repairing; RE = Repairing; CO = Repair complete; GO = Gone; DE = Destroyed; BD = Blown down; COLL = Collected; X = post-collection. ##### gilbert\_2024\_jae\_4\_removal\_ovaries.csv In this dataset, each line represents one female, so domiciles appear on multiple lines. Data on the volume of developing/mature oocytes is attached to this dataset from the dissection data (File 1) in the R code. * NEST: Domicile ID * FF: Number of foundresses * COMP: Repair behaviour of of females initially in domicile: H = helpful, U = unhelpful, M = male (who never help), X = unknown * TREAT: My initial division of nests into treatment groups (RH = remove helper, RNH = remove nonhelper, C = Control, C1 = control singletons) * REMOVE: Behavioural classification of individual(s) removed from nest: H = helpful, U = unhelpful, O = none removed, M = Male  * LEAVE: Behavioural classification of individual(s) left in nest: H = helpful, U = unhelpful, M = Male  * TR: Treatment applied to focal individual (R = removed at beginning of the experiment; L = left until the end of the experiment), but coding Control groups uniformly as C * ST: Behavioural classification of the focal individual: H = helpful, U = unhelpful, C = control (i.e., unknown, could be either/any of the remaining individuals) * PRESENT: Number of individuals present in the nest at the time of removal of the focal individual * TR.INDIV: Treatment applied to focal individuals (R = removed at the beginning of the experiment; L = left until the end of the experiment), coding Control groups according to the actual treatment they received (i.e., control individuals were all L). * ID.INDIV: An arbitrary ID for the individual within the nest, e.g. R1 is the first female to be removed, R2 is the second, and L1 is the first "left" female I dissected. * STATUS.INDIV: Behavioural classification of a focal individual as for ST, but coding C groups according to the possible states (i.e., "HU" could be a helper or a non-helper). * NOTES: Notes