DOI: 10.5061/dryad.qfttdz0t1
title: It's complicated: The role of false brood cells as an anti-parasite strategy in Hymenoptera is not simple despite their ubiquity

description: Nest collection: Nests of A. morosus were collected from naturally
 abscised fronds that skirt the base of the rough fern tree, Cyathea
 australis in Kurth Kiln Regional Park, Yarra Ranges National Park,
 Cathedral Range State Park, Marysville Bushland Reserve and Grants Picnic
 Ground in the Dandenong Ranges region in eastern Victoria, Australia.
 Nests were collected across seven separate collections from December 2017
 to January 2021. Collections were performed at dawn when temperatures were
 below 15°C to ensure all bees were in their nest. Nest entrances were
 sealed upon collection and nests placed on ice in insulated boxes and
 transported to Flinders University for nest processing. Nest processing:
 Nests were opened lengthwise until the first brood cell was reached. All
 adult female A. morosus and any adult parasites in the nest entrance were
 removed and placed in 99% ethanol for preservation and identification.
 Nests of A. morosus can be characterized as either i) social – in which at
 least two adult females occupy the nest and provision brood with no false
 cells; ii) sequential solitary – solitary female provisions brood with no
 false cells; iii) false cell solitary – solitary female provisions brood
 cells that are interspersed with false cells (Fig. 1). The brood cell
 closest to the nest entrance was checked to determine the maturity of the
 brood. The nest was fully opened if the brood had reached pupation or
 adulthood, whereas, the nest was left to develop at ambient room
 temperature if brood were still developing. Nest architecture (nest
 length, brood cell lengths and false cell lengths) and contents (number of
 brood, sex of brood, number of false cells, failed cells and parasites)
 were recorded. Rates of parasitization (number of nests containing at
 least one parasitized brood cell) were designated into suites depending on
 their respective mode of entering and parasitising brood (i.e.
 Gasteruption, Anthrax, Ephutomorpha; (Hearn et al., 2021)). We categorized
 nest productivity using three metrics: as the total number of brood cells
 produced, the number of surviving brood cells, and the proportion of
 surviving brood cells in a nest. In A. morosus, it is important to make
 this distinction between the total number of brood cells and the number of
 surviving brood cells due to high rates of brood loss that can arise from
 factors other than parasitization (e.g. microbial infection) (Spessa et
 al., 2000). Nest structure: Amphylaeus morosus were only ever
 found to construct false cells and never vestibular cells (Fig. 1).
 Solitary nests of A. morosus are arranged linearly with false cells
 separating closed brood cells. False cells are often greater in diameter
 compared to brood cells as they are further enlarged by the
 nest-constructing adult female who uses the pithy ‘excavations’ as a
 barrier in front of the next brood cell (Fig. 1). In contrast, brood cells
 are fully lined in a cellophane-like material produced by adult females
 (Almeida, 2008).  Statistical analysis: All statistical tests
 were performed using R version 4.2.2 (R Core Team, 2024). Some of the
 variables analysed were strongly zero or one-truncated, and where data
 were not normally distributed, we used non-parametric tests. Orphaned
 nests of A. morosus (nests containing brood but no adult female) are
 common and this can have implications for brood survival. For some
 analyses, we therefore separated nests based on the number of adults
 present at the time of collection. We used Kruskal-Wallis tests to compare
 the differences in nest productivity variables based on the number of
 adult females present. Since social nests never produced false cells (see
 below), we restricted analyses of the effect of false cells on nest
 productivity to nests with one or zero adult females. We used Mann-Whitney
 pairwise comparisons to test the effect of false cells on nest
 productivity (total number of brood cells provisioned and number of
 surviving brood cells) between nests with one adult female and orphaned
 nests. However, these analyses may be confounded by the fact that brood
 production in orphaned nests may have been curtailed by death of the adult
 female. We therefore also repeated the Mann-Whitney analyses using the
 proportion of surviving brood cells rather than the absolute number. To
 assess how the number of false cells influenced parasitism rates, we used
 a binary logistic regression with a logit link function. We used a
 generalized linear model with a Poisson distribution with a log link
 function to test the influence of nesting strategy on count data (total
 number of brood cells produced and total number of surviving brood cells).
 Only nests containing at least one brood cell were used in all analyses.
description: # It's complicated: The role of false brood cells as an anti-parasite
 strategy in Hymenoptera is not simple despite their ubiquity Data
 collection and processing: Nest collections In total, 299 Amphylaeus
 morosus stem nests were collected from abscised fronds of the rough fern
 tree, Cyathea australis. Colony data Nests were processed within 24 hours
 after being returned to Flinders University, and opened longitudinally up
 to the first brood cell, with all adult A. morosus and additional contents
 within the nest entrance removed and placed in 99% ethanol. The maturity
 of the brood cell closest to nest entrance was examined to determine if
 the brood were at a pupal stage where they could be visually sexed.
 Because A. morosus construct brood cells in a linear array, starting from
 “cell 1” (first brood laid – furthest from the nest entrance) to a maximum
 of “cell 16” (last brood laid – closest to the nest entrance) in the
 largest of nests, the cell closest to the nest entrance is expected to be
 the least mature brood. In this species, brood laid earlier are generally
 larger and take longer to develop compared to recently laid brood (Spessa
 1999). The cell closest to the entrance will therefore give an indication
 of brood maturity for the nest. Immature brood for a nest were left in
 their cells in a sealed container and reared to pupation at ambient room
 temperature (~22 °C). The variables in these datasets were coded as
 follows for analysis: Nest_data_FC_clean.csv (individual nest data) * Nest
 -(arbitrary ordering of nests as they were processed) * Date - date nest
 was collected * Location - location nest was collected * Adult - number of
 adult females present in the nest at the time of collection * Nestlengthmm
 - total nest length * Totalbroodcelllength - nest space used for brood
 cells (and false cells) * Totalno.broodcells - total number of brood cells
 in the nest * no.surviving_broodcells - total number of surviving brood in
 the nest * No.falsecells - number of false cells * no.female_offspring -
 total number of female offspring * no.male_offspring - total number of
 male offspring * Parasitisedcellsbombylidfly - number of cells parasitized
 by bombyliid flies * Parasitisedcellsgasteruptionwasp - number of cells
 parasitized by gasteruption wasps * Parasitised_mutillid - number of cells
 parasitized by mutillid wasps 'n/a' is indicated for any nest
 length measurements that were not recorded. These nests were excluded from
 our nest measurement analyses