A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (π), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

The opposed forces of differentiation and admixture across glacial cycles in the butterfly Aglais urticae.

Glacial cycles lead to periodic population interbreeding and isolation in warm-adapted species, which impact genetic structure and evolution. However, the effects of these processes on highly mobile and more cold-tolerant species are not well understood. This study aims to shed light on the phylogeographic history of Aglais urticae, a butterfly species with considerable dispersal ability, and a wide Palearctic distribution reaching the Arctic. Through the analysis of genomic data, four main genetic lineages are identified: European, Sierra Nevada, Sicily/Calabria/Peloponnese, and Eastern. The results indicate that the Sardo-Corsican endemic taxon ichnusa is a distinct species. The split between the relict lineages in southern Europe and the main European lineage is estimated to have happened 400-450 thousand years ago, with admixture observed during the Quaternary glacial cycles, and still ongoing, albeit to a much smaller extent. These results suggest that these lineages may be better treated as subspecific parapatric taxa. Ecological niche modelling supported the existence of both Mediterranean and extra-Mediterranean refugia during the glacial periods, with the main one located on the Atlantic coast. Nevertheless, gene flow between populations was possible, indicating that both differentiation and admixture have acted continuously across glacial cycles in this cold-tolerant butterfly, generally balancing each other but producing differentiated lineages in the southern peninsulas. We conclude that the population dynamics and the processes shaping the population genetic structure of cold-adapted species during the Quaternary ice ages may be different than those classically accepted for warm-adapted species.

Assessing the aesthetic attractivity of European butterflies: A web-based survey protocol.

Aesthetic attractivity stands as an underestimated yet fundamental feature of species in conservation biology, significantly driving disproportionate protection efforts towards charismatic species. Despite the evidence, few attempts sought to precisely quantify the impact of aesthetic attractivity in defining priority of species for conservation actions (e.g. inclusion in International Union for Conservation of Nature red lists and protection lists). This study protocol describes the setting of an online test (available from April 2022 to April 2023 at www.unveiling.eu) designed to i) quantify the aesthetic attractivity to humans of the 496 European butterfly species and ii) identify which features (both in the perceived animal and in the perceiver) influence the aesthetic attractivity of a given butterfly species. The test is divided in 5 sections (personal data, ranking, single morphological features, emotional engagement, dispositional variables) aimed at profiling the relation each participant has with the species examined. In the long-term, evaluating butterflies' aesthetic attractivity could facilitate the critical assessment of current conservation strategies, such as the process of selection of flag and umbrella species by research institutions, environmental associations and Non Governative Organizations. This is expected to provide the much-needed evidence to set up unbiased biodiversity conservation strategies and counteract the selective anthropogenic pressure which favours the extinction of unattractive species, being no or less protected compared to charismatic species.

Hybridization fuelled diversification in Spialia butterflies.

The importance of hybridization and introgression is well documented in the evolution of plants but, in insects, their role is not fully understood. Given the fact that insects are the most diverse group of organisms, assessing the impact of reticulation events on their evolution may be key to comprehend the emergence of such remarkable diversity. Here, we used an insect model, the Spialia butterflies, to gather genomic evidence of hybridization as a promoter of novel diversity. By using double-digest RADseq (ddRADseq), we explored the phylogenetic relationships between Spialia orbifer, S. rosae and S. sertorius, and documented two independent events of interspecific gene flow. Our data support that the Iberian endemism S. rosae probably received genetic material from S. orbifer in both mitochondrial and nuclear DNA, which could have contributed to a shift in the ecological preferences of S. rosae. We also show that admixture between S. sertorius and S. orbifer probably occurred in Italy. As a result, the admixed Sicilian populations of S. orbifer are differentiated from the rest of populations both genetically and morphologically, and display signatures of reproductive character displacement in the male genitalia. Additionally, our analyses indicated that genetic material from S. orbifer is present in S. sertorius along the Italian Peninsula. Our findings add to the view that hybridization is a pervasive phenomenon in nature and in butterflies in particular, with important consequences for evolution due to the emergence of novel phenotypes.

Genomics Reveal Admixture and Unexpected Patterns of Diversity in a Parapatric Pair of Butterflies.

We studied the evolutionary relationship of two widely distributed parapatric butterfly species, Melitaea athalia and Melitaea celadussa, using the ddRAD sequencing approach, as well as genital morphology and mtDNA data. M. athalia was retrieved as paraphyletic with respect to M. celadussa. Several cases of mito-nuclear discordance and morpho-genetic mismatch were found in the contact zone. A strongly diverged and marginally sympatric clade of M. athalia from the Balkans was revealed. An in-depth analysis of genomic structure detected high levels of admixture between M. athalia and M. celadussa at the contact zone, though not reaching the Balkan clade. The demographic modelling of populations supported the intermediate genetic make-up of European M. athalia populations with regards to M. celadussa and the Balkan clade. However, the dissimilarity matrix of genotype data (PCoA) suggested the Balkan lineage having a genetic component that is unrelated to the athalia-celadussa group. Although narrowly sympatric, almost no signs of gene flow were found between the main M. athalia group and the Balkan clade. We propose two possible scenarios on the historical evolution of our model taxa and the role of the last glacial maximum in shaping their current distribution. Finally, we discuss the complexities regarding the taxonomic delimitation of parapatric taxa.

Cuticular Hydrocarbon Profile of Parasitic Beetles, Aethina tumida (Coleoptera: Nitidulidae).

Cuticular hydrocarbons (CHCs) cover insects' bodies and play important roles in chemical communication, including nestmate recognition, for social insects. To enter colonies of a social host species, parasites may acquire host-specific CHCs or covertly maintain their own CHC profile by lowering its quantity. However, the chemical profile of small hive beetles (SHBs), Aethina tumida, which are parasites of honey bee, Apis mellifera, colonies, and other bee nests, is currently unknown. Here, adults of SHB and honey bee host workers were collected from the same field colonies and their CHC profiles were analysed using GC-MS. The chemical profiles of field-sampled SHBs were also compared with those of host-naive beetles reared in the laboratory. Laboratory-reared SHBs differed in their CHC profiles from field-sampled ones, which showed a more similar, but ten-fold lower, generic host CHC profile compared to host workers. While the data confirm colony-specific CHCs of honey bee workers, the profile of field-collected SHBs was not colony-specific. Adult SHBs often commute between different host colonies, thereby possibly preventing the acquisition of a colony-specific CHC profiles. An ester was exclusive to both groups of SHBs and might constitute an intraspecific recognition cue. Our data suggest that SHBs do not use any finely tuned chemical strategy to conceal their presence inside host colonies and instead probably rely on their hard exoskeleton and defence behaviours.

Evidence for multiple colonisations and Wolbachia infections shaping the genetic structure of the widespread butterfly Polyommatus icarus in the British Isles.

The paradigm of isolation in southern refugia during glacial periods followed by expansions during interglacials, producing limited genetic differentiation in northern areas, dominates European phylogeography. However, the existence of complex structured populations in formerly glaciated areas, and islands connected to mainland areas during glacial maxima, call for alternative explanations. We reconstructed the mtDNA phylogeography of the widespread Polyommatus Icarus butterfly with an emphasis on the formerly glaciated and connected British Isles. We found distinct geographical structuring of CO1 haplogroups, with an ancient lineage restricted to the marginal European areas, including Northern Scotland and Outer Hebrides. Population genomic analyses, using ddRADSeq genomic markers, also reveal substantial genetic structuring within Britain. However, there is negligble mito-nuclear concordance consistent with independent demographic histories of mitochondrial versus nuclear DNA. While mtDNA-Wolbachia associations in northern Britain could account for the geographic structuring of mtDNA across most of the British Isles, for nuclear DNA markers (derived from ddRADseq data) butterflies from France cluster between northern and southern British populations - an observation consistent with a scenario of multiple recolonisation. Taken together our results suggest that contemporary mtDNA structuring in the British Isles (and potentially elsewhere in Europe) largely results from Wolbachia infections, however, nuclear genomic structuring suggests a history of at least two distinct colonisations. This two-stage colonisation scenario has previously been put forth to explain genetic diversity and structuring in other British flora and fauna. Additionally, we also present preliminary evidence for potential Wolbachia-induced feminization in the Outer Hebrides.

The Pleistocene species pump past its prime: Evidence from European butterfly sister species.

The Pleistocene glacial cycles had a profound impact on the ranges and genetic make-up of organisms. While it is clear that the contact zones that have been described for many sister taxa are secondary and have formed in the current interglacial, it is unclear when the taxa involved began to diverge. Previous estimates based on small numbers of loci are unreliable given the stochasticity of genetic drift and the contrasting effects of incomplete lineage sorting and gene flow on gene divergence. Here, we use genome-wide transcriptome data to estimate divergence for 18 sister species pairs of European butterflies showing either sympatric or contact zone distributions. We find that in most cases, species divergence predates the mid-Pleistocene transition or even the entire Pleistocene period. We also show that although post-divergence gene flow is restricted to contact zone pairs, they are not systematically younger than sympatric pairs. This suggests that contact zones are not limited to the initial stages of the speciation process, but can involve notably old taxa. Finally, we show that mitochondrial divergence and nuclear divergence are only weakly correlated and mitochondrial divergence is higher for contact zone pairs.

High resolution DNA barcode library for European butterflies reveals continental patterns of mitochondrial genetic diversity.

The study of global biodiversity will greatly benefit from access to comprehensive DNA barcode libraries at continental scale, but such datasets are still very rare. Here, we assemble the first high-resolution reference library for European butterflies that provides 97% taxon coverage (459 species) and 22,306 COI sequences. We estimate that we captured 62% of the total haplotype diversity and show that most species possess a few very common haplotypes and many rare ones. Specimens in the dataset have an average 95.3% probability of being correctly identified. Mitochondrial diversity displayed elevated haplotype richness in southern European refugia, establishing the generality of this key biogeographic pattern for an entire taxonomic group. Fifteen percent of the species are involved in barcode sharing, but two thirds of these cases may reflect the need for further taxonomic research. This dataset provides a unique resource for conservation and for studying evolutionary processes, cryptic species, phylogeography, and ecology.

Two ways to be endemic. Alps and Apennines are different functional refugia during climatic cycles.

Endemics co-occur because they evolved in situ and persist regionally or because they evolved ex situ and later dispersed to shared habitats, generating evolutionary or ecological endemicity centres, respectively. We investigate whether different endemicity centres can intertwine in the region ranging from Alps to Sicily, by studying their butterfly fauna. We gathered an extensive occurrence data set for butterflies of the study area (27,123 records, 269 species, in cells of 0.5 × 0.5 degrees of latitude-longitude). We applied molecular-based delimitation methods (GMYC model) to 26,557 cytochrome c oxidase subunit 1 (COI) sequences of Western Palearctic butterflies. We identified entities based on molecular delimitations and/or the checklist of European butterflies and objectively attributed occurrences to their most probable entity. We obtained a zoogeographic regionalisation based on the 69 endemics of the area. Using phylogenetic ANOVA we tested if endemics from different centres differ from each other and from nonendemics for key ecological traits and divergence time. Endemicity showed high incidence in the Alps and Southern Italy. The regionalisation separated the Alps from the Italian Peninsula and Sicily. The endemics of different centres showed a high turnover and differed in phylogenetic distances, phenology and distribution traits. Endemics are on average younger than nonendemics and the Peninsula-Sicily endemics also have lower variance in divergence than those from the Alps. The observed variation identifies Alpine endemics as paleoendemics, now occupying an ecological centre, and the Peninsula-Sicily ones as neoendemics, that diverged in the region since the Pleistocene. The results challenge the common view of the Alpine-Apennine area as a single "Italian refugium".

A new comprehensive trait database of European and Maghreb butterflies, Papilionoidea.

Trait-based analyses explaining the different responses of species and communities to environmental changes are increasing in frequency. European butterflies are an indicator group that responds rapidly to environmental changes with extensive citizen science contributions to documenting changes of abundance and distribution. Species traits have been used to explain long- and short-term responses to climate, land-use and vegetation changes. Studies are often characterised by limited trait sets being used, with risks that the relative roles of different traits are not fully explored. Butterfly trait information is dispersed amongst various sources and descriptions sometimes differ between sources. We have therefore drawn together multiple information sets to provide a comprehensive trait database covering 542 taxa and 25 traits described by 217 variables and sub-states of the butterflies of Europe and Maghreb (northwest Africa) which should serve for improved trait-based ecological, conservation-related, phylogeographic and evolutionary studies of this group of insects. We provide this data in two forms; the basic data and as processed continuous and multinomial data, to enhance its potential usage.

Stabilisation of some names of European butterflies (Lepidoptera: Pieridae) in their prevailing usage.

To stabilise nomenclature in its prevailing usage, the authors designate three lectotypes, respectively for the nominal taxa Pontia daplidice race nitida Verity, 1908 (Museo di Storia naturale della Università di Firenze, sezione di Zoologia, 'La Specola' MZUF), for Euchloe crameri Butler, 1869 (Natural History Museum UK, NHMUK) and for Euchloe ausonia var. esperi Kirby, 1871 (National Museum of Ireland, NMI). They also designate two neotypes, respectively for [Papilio] ausonia Hübner, [1804] (MZUF) and for Pontia simplonia Freyer, 1829 (Jura Museum, Eichstätt, JME).

How long is 3 km for a butterfly? Ecological constraints and functional traits explain high mitochondrial genetic diversity between Sicily and the Italian Peninsula.

Populations inhabiting Mediterranean islands often show contrasting genetic lineages, even on islands that were connected to the mainland during glacial maxima. This pattern is generated by forces acting in historical and contemporary times. Understanding these phenomena requires comparative studies integrating genetic structure, functional traits and dispersal constraints. Using as a model the butterfly species living across the Messina strait (3 km wide) separating Sicily from the Italian Peninsula, we aimed to unravel the mechanisms limiting the dispersal of matrilines and generating genetic differentiation across a narrow sea strait. We analysed the mitochondrial COI gene of 84 butterfly species out of 90 documented in Sicily and compared them with populations from the neighbouring southern Italian Peninsula (1,398 sequences) and from the entire Palaearctic region (8,093 sequences). For each species, we regressed 13 functional traits and 2 ecological constraints to dispersal (winds experienced at the strait and climatic suitability) against genetic differentiation between Sicily and Italian Peninsula to understand the factors limiting dispersal. More than a third of the species showed different haplogroups across the strait and most of them also represented endemic haplogroups for this island. One fifth of Sicilian populations (and 32.3% of endemic lineages) had their closest relatives in distant areas, instead of the neighbouring Italian Peninsula, which suggests high relictuality. Haplotype diversity was significantly explained by the length of the flight period, an intrinsic phenology trait, while genetic differentiation was explained by both intrinsic traits (wingspan and degree of generalism) and contemporary local constraints (winds experienced at the strait and climatic suitability). A relatively narrow sea strait can produce considerable differentiation among butterfly matrilines and this phenomenon showed a largely deterministic fingerprint. Because of unfavourable winds, populations of the less dispersive Sicilian butterflies tended to differentiate into endemic variants or to maintain relict populations. Understanding these phenomena required the integration of DNA sequences, species traits and physical constraints for a large taxon at continental scale. Future studies may reveal if the patterns here shown for mitochondrial DNA are also reflected in the nuclear genome or, alternatively, are the product of limited female dispersal.

No evidence of queen immunisation despite transgenerational immunisation in Crematogaster scutellaris ants.

Like vertebrates, invertebrates evolved acquired immunity based on memory-like mechanisms, known as immunisation. Immunisation and its transmission among individuals are phylogenetically ancestral and conserved characters that have been reported in different insect orders. Physiological mechanisms are still largely unknown, and the high variability in responses in different host-parasite systems led to different conclusions. In social insect species, the complex organisation of colonies further complicates the interpretation of the immune responses. In ants, it has been shown that the expression of immunisation depends on species, caste and physiological status of individuals. In this study, we investigate the occurrence of immunisation in queens of Crematogaster scutellaris ants using the fungus Metarhizium anisopliae as elicitor. Foundation in C. scutellaris is claustral and monogynic, allowing us to test the existence of the phenomenon in two distinct physiological conditions, corresponding to the claustral and colonial phases of queens. Queens and foundresses challenged with heavy doses of the pathogen showed higher mortality if previously exposed to light doses, indicating the absence of immunisation in our experimental settings. On the other hand, evidence of the trans-generational immunisation in the same host-parasite system has been recently found, where workers produced by queens exposed to light doses of M. anisopliae survived longer than those belonging to the control group. These results indicate that foundresses exposed to M. anisopliae can elicit an increased resistance in the offspring without providing themselves with a similar increased immune response and that immunisation and trans-generational immunisation are uncoupled phenomena in this host-parasite system.

Saccharomyces cerevisiae Induces Immune Enhancing and Shapes Gut Microbiota in Social Wasps.

Trained immunity is the enhanced response of the innate immune system to a secondary infection after an initial encounter with a microorganism. This non-specific response to reinfection is a primitive form of adaptation that has been shown to be conserved from plants to mammals. Insects lack an acquired immune component and rely solely on an innate one, and they have expanded it upon traits of plasticity and adaptation against pathogens in the form of immune priming. The recent discoveries of the role of Saccharomyces cerevisiae in the insect's ecology and the ability of this yeast to induce trained immunity in different organisms suggest that insects could have developed mechanisms of adaptation and immune enhancing. Here, we report that two yeast strains of S. cerevisiae, previously shown to induce trained immunity in mammals, enhance resistance to Escherichia coli infection in the paper wasp Polistes dominula. The reduction of injected E. coli load by S. cerevisiae strains was statistically significant in future foundresses but not in workers, and this occurs before and after hibernation. We thus investigated if the effect on E. coli was mirrored by variation in the gut microbiota composition. Foundresses, showing immune enhancing, had statistically significant changes in composition and diversity of gut bacterial communities but not in the fungal communities. Our results demonstrate that S. cerevisiae can prime insect responses against bacterial infections, providing an advantage to future foundress wasps to carry these microorganisms. Understanding the mechanisms involved in the generation of specific and long-lasting immune response against pathogenic infections in insects and the influence of the induction of trained immunity on the commensal gut microbiota could have a major impact on modern immunology.

Home economics in an oak gall: behavioural and chemical immune strategies against a fungal pathogen in Temnothorax ant nests.

Nest architecture is a fundamental character shaping immune strategies of social insects. The arboreal ant Temnothorax unifasciatus nests in cavities such as oak galls where the entire colony lives in a unique small chamber. In these conditions, physiological and behavioural strategies likely prevail over compartmentalisation and are presumably tuned with colony size. We designed two experiments to study chemical and behavioural immune strategies against the entomopathogenic fungus Metarhizium anisopliae in colonies of different sizes. First, we compared spore germination and length of germinal tubes inside artificial nests, designed to impede the contact between the ants and the fungus, in colonies of different size. In the absence of direct contact, Temnothorax unifasciatus colonies inhibit fungal growth inside their nests, presumably through volatile compounds. The analysis revealed a positive correlation between fungistatic activity and colony size, indicating that workers of smaller colonies do not invest a higher per capita effort in producing such substances compared to larger colonies. Second, we performed a removal experiment of contaminated and non-contaminated items introduced inside the nests of colonies of different size. Small colonies challenged with contaminated fibres showed an increased removal of all the items (both contaminated and non-contaminated) compared to small colonies challenged with non-contaminated fibres only. Conversely, larger colonies moved items regardless of the presence of the spores inside the nest. Colony size qualitatively affected removal of waste items showing a pathogen elicited reaction in small colonies to optimise the reduced workforce, while the removal behaviour in larger colonies revealed to be expressed constitutively.

Integrating three comprehensive data sets shows that mitochondrial DNA variation is linked to species traits and paleogeographic events in European butterflies.

Understanding the dynamics of biodiversity, including the spatial distribution of genetic diversity, is critical for predicting responses to environmental changes, as well as for effective conservation measures. This task requires tracking changes in biodiversity at large spatial scales and correlating with species functional traits. We provide three comprehensive resources to understand the determinants for mitochondrial DNA differentiation represented by (a) 15,609 COI sequences and (b) 14 traits belonging to 307 butterfly species occurring in Western-Central Europe and (c) the first multi-locus phylogenetic tree of all European butterfly species. By applying phylogenetic regressions we show that mitochondrial DNA spatial differentiation (as measured with GST , G'ST , D and DST ) is negatively correlated with species traits determining dispersal capability and colonization ability. Thanks to the high spatial resolution of the COI data, we also provide the first zoogeographic regionalization maps based on intraspecific genetic variation. The overall pattern obtained by averaging the spatial differentiation of all Western-Central European butterflies shows that the paradigm of long-term glacial isolation followed by rapid pulses of post-glacial expansion has been a pervasive phenomenon in European butterflies. The results and the extensive data sets we provide here constitute the basis for genetically-informed conservation plans for a charismatic group in a continent where flying insects are under alarming decline.

Natural biocide disrupts nestmate recognition in honeybees.

Honeybee colonies are under the threat of many stressors, biotic and abiotic factors that strongly affect their survival. Recently, great attention has been directed at chemical pesticides, including their effects at sub-lethal doses on bee behaviour and colony success; whereas the potential side effects of natural biocides largely used in agriculture, such as entomopathogenic fungi, have received only marginal attention. Here, we report the impact of the fungus Beauveria bassiana on honeybee nestmate recognition ability, a crucial feature at the basis of colony integrity. We performed both behavioural assays by recording bee guards' response towards foragers (nestmate or non-nestmate) either exposed to B. bassiana or unexposed presented at the hive entrance, and GC-MS analyses of the cuticular hydrocarbons (CHCs) of fungus-exposed versus unexposed bees. Our results demonstrated that exposed bees have altered cuticular hydrocarbons and are more easily accepted into foreign colonies than controls. Since CHCs are the main recognition cues in social insects, changes in their composition appear to affect nestmate recognition ability at the colony level. The acceptance of chemically unrecognizable fungus-exposed foragers could therefore favour forager drift and disease spread across colonies.

Population genomics reveals evolution and variation of Saccharomyces cerevisiae in the human and insects gut.

The quest to discover the variety of ecological niches inhabited by Saccharomyces cerevisiae has led to research in areas as diverse as wineries, oak trees and insect guts. The discovery of fungal communities in the human gastrointestinal tract suggested the host's gut as a potential reservoir for yeast adaptation. Here, we report the existence of yeast populations associated with the human gut (HG) that differ from those isolated from other human body sites. Phylogenetic analysis on 12 microsatellite loci and 1715 combined CDSs from whole-genome sequencing revealed three subclusters of HG strains with further evidence of clonal colonization within the host's gut. The presence of such subclusters was supported by other genomic features, such as copy number variation, absence/introgressions of CDSs and relative polymorphism frequency. Functional analysis of CDSs specific of the different subclusters suggested possible alterations in cell wall composition and sporulation features. The phenotypic analysis combined with immunological profiling of these strains further showed that sporulation was related with strain-specific genomic characteristics in the immune recognition pattern. We conclude that both genetic and environmental factors involved in cell wall remodelling and sporulation are the main drivers of adaptation in S. cerevisiae populations in the human gut.

Trans-generational immunization in the acrobat ant Crematogaster scutellaris.

Trans-generational immunization is defined as the transmission of an enhanced resistance to a pathogen from parents to offspring. By using the host-parasite system of the ant Crematogaster scutellaris and the entomopathogenic fungus Metarhizium anisopliae, we describe this phenomenon for the first time in ants. We exposed four groups of hibernating queens to different treatments (i) a non-lethal dose of live conidiospores in Triton, (ii) a dose of heat-killed conidiospores in Triton, (iii) a control Triton solution, and (iv) a naive control. We exposed their first workers to a high dose of conidiospores and measured mortality rates. Workers produced by queens exposed to live conidiospores survived longer than those belonging to the other groups, while exposure to Triton and dead spores had no effect. Starved workers showed a significantly higher mortality. The treatments did not influence queen mortality, nor the number of offspring they produced at the emergence of the first worker, showing no evidence of immunization costs-at least for these parameters in the first year of colony development. We propose that trans-generational immunization represents an important component of social immunity that could affect colony success, particularly during the critical phase of claustral foundation.