DOI: 10.5061/dryad.7wm37pw0w
title: The significance of genetic distance and nest occupation on the worker-worker similarity of gut bacterial microbiome and cuticular hydrocarbon profile in a sweat bee

description: The cuticular hydrocarbon (CHC) profile and the gut microbiome (GM) are
 crucial traits which have a significant impact on the life of bees. In
 honey bees, the CHC profile and the GM interact finely through
 trophallaxis, such that the characteristics of the GM are partially
 defined by the chemical recognition among sisters. However, most of the
 known primitively eusocial bees show simpler social traits, including
 moderate genetic relatedness among colony members, often due to
 workers' nest drifting or dispersal, and lack of trophallaxis. Hence,
 primitively eusocial bees offer a great opportunity to evaluate the
 respective role of worker-worker genetic relatedness and of the
 environment in which the adult lives (residency nest) on the interaction
 between CHC profile and GM. Here, we investigated such relationships in
 the primitively eusocial digger bee Halictus scabiosae (Halictidae). We
 found a high rate of nest-drifting by workers, which leads to a consequent
 highly variable intra-colonial genetic relatedness. Genetically closely
 related workers, even occupying distant nests, did possess both a more
 similar microbiome profile and a more similar CHC profile. Additionally,
 sharing the same nest seemed to account for the similarity of both CHC
 profile and GM among workers. Interestingly, differences in microbiome
 profile and in CHC profile were highly and positively correlated across
 workers, even after controlling for genetic relatedness. The results of
 our study point towards an impact of genetic relatedness on the GM and the
 CHC profile, but also suggest that microbiome and CHC profile are
 partially acquired through adult nest environment, and that microbiome
 possibly has a role in shaping the cuticular chemistry.
description: <strong><em>DNA extraction and genotype determination
 </em></strong> We studied genetic
 relatedness between workers using microsatellite markers. DNA extraction
 was performed from one leg of each individual using Chelex 100 (Walsh et
 al. 1991). The Chelex extraction method has become a staple in the field
 of honey bee research due to its rapidity and efficacy in DNA extraction
 (Evans et al. 2013). This method has been demonstrated to yield DNA of
 sufficient quality for microsatellite analysis. Consequently, it was
 selected as the primary extraction method for the samples in this study,
 and its effectiveness was subsequently validated. Eight microsatellite
 markers were used in this study, with primers reported in Table S1. Two
 different multiplex reactions were designed (Multiplex 1: LHMS10, rub73,
 rub02, rub72; Multiplex 2: rub37b, rub30, rub77, LM27) using forward
 primers fluorescent-labeled and amplified under the following PCR
 conditions: initial denaturation at 94°C for 3 min; 35 cycles of 30 s at
 94°C, 30 s at 55°C, and 45 s at 64°C; a final elongation for 10 min at
 64°C. In order to ascertain the feasibility of the multiplex reactions, a
 polymerase chain reaction (PCR) test was performed. This involved the
 amplification of five samples of <em>H. scabiosae</em>, both
 individually and in combination in the different multiplexes. The
 annealing temperature was selected based on the articles in which the
 primers were designed (Kukuk et al. 2002; Paxton et al. 2003; Soro and
 Paxton 2009). Once the efficacy of the multiplex reaction had been
 verified, the remaining samples were amplified.
 Amplified products were sent for detection to the company Secugen
 S.L (Madrid, Spain), and the identification of alleles was carried out
 using GENEMAPPER 3.7 software (Applied Biosystems) to export the data
 matrix for subsequent analysis.
 <strong><em>Statistical
 analyses</em></strong> The number of total
 and private alleles of each nest aggregation, as well as the unbiased
 expected heterozygosity (uH<sub>e</sub>) were obtained using
 GenAlEx 6.5 (Peakall and Smouse, 2006). The presence of null alleles was
 evaluated with Micro-Checker version 2.2.3 (Van Oosterhout et al., 2004),
 and the conformity of the populations with the Hardy-Weinberg equilibrium
 was checked using GENEPOP on the web version 4.1 (Rousset, 2008). Analysis
 of molecular variance (AMOVA) was carried out based on 999 replicates
 using GeneAlex 6.5 (Peakall & Smouse 2006) to assess how genetic
 diversity varied within and between nest aggregations. Genetic distances
 between individuals (as a proxy of genetic relatedness) were calculated
 and their differences between populations were evaluated by a Principal
 Coordinates Analysis (PCoA). Finally, in order to analyze the genetic
 clustering of the <em>H. scabiosae</em> aggregations, the
 STRUCTURE v2.3.4 program (Pritchard et al., 2000) was used. All possible
 existing populations (K; 1-10) (burn-in: 1 x 104; MCMC: 105) were
 simulated using ten iterations each to obtain the most probable one
 according to the ∆K value (Evanno et al., 2005) obtained with STRUCTURE
 HARVESTER web v0. 6.94 (Earl and von Holdt, 2012).
description: # The significance of genetic distance and nest occupation on the
 worker-worker similarity of gut bacterial microbiome and cuticular
 hydrocarbon profile in a sweat bee
 [https://doi.org/10.5061/dryad.7wm37pw0w](https://doi.org/10.5061/dryad.7wm37pw0w) ## Description of the data and file structure This is a dataset of the microsatellite-derived genotypes of sweat bee workers of the species Halictus scabiosae. *H. scabiosae* workers were collected from two nest aggregations near the small town of Alberese, within the Maremma Regional Park (Tuscany, Italy: 42°40′5″N, 11°6′23″E). The two nest aggregations (hereafter: aggregation 1 and aggregation 2), composed of around 100 (aggregation 1, Casa Gialla Barbicato) and 50 (aggregation 2, Sagrado Agriturismo) nests, were located 1.2 km appart. Five *H. scabiosae* workers *per* nest were collected by netting, from a total of 18 nests (7 nests from the aggregation 1, labelled A to G and 11 nests from the aggregation 2, labelled H to U). Microsatellite alleles are named according to the detected size in base pairs. The presence of a 0 (zero) means that this allele could not be detected, probably due to poor PCR amplification. Column A contains the samples (1 to 5) coded according to nest and aggregation. Column B contains the locations where the aggregations were found. Columns C to R contain the alleles of the microsatellite loci. For example, column C contains the alleles coded by their size in base pairs of the LHMS10 locus, and column D contains the other alleles of the same locus, as these are samples of diploid female bees.