FEMS Microbiology Ecology, 95, 2019, fiy235
doi: 10.1093/femsec/fiy235 Advance Access Publication Date: 14 December 2018 Research Article Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019
RESEARCH ARTICLE Bacteriome-associated Wolbachia of the parthenogenetic termite Cavitermes tuberosus Simon Hellemans1,*,†,‡, Nicolas Kaczmarek1,*,†,§, Martyna Marynowska2,, Magdalena Calusinska2,#, Yves Roisin1,|| and Denis Fournier1,*,**
1Evolutionary Biology & Ecology, Universite´ libre de Bruxelles, Avenue F.D. Roosevelt 50, CP 160/12, B-1050 Brussels, Belgium and 2Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology, L-4422 Belvaux, Luxembourg
∗Corresponding authors: Evolutionary Biology & Ecology, Universite´ libre de Bruxelles, Avenue F.D. Roosevelt 50, CP 160/12, B-1050 Brussels, Belgium. E-mail: [email protected]; [email protected]; [email protected] One sentence summary: The parthenogenetic termite Cavitermes tuberosus and the reproductive parasite Wolbachia are partners that likely engage in an intimate insect-bacteria nutritional partnership. †These authors contributed equally to this study. ‡Simon Hellemans, http://orcid.org/0000-0003-1266-9134 §Nicolas Kaczmarek, http://orcid.org/0000-0002-2879-1575 Martyna Marynowska, http://orcid.org/0000-0003-1739-8177 #Magdalena Calusinska, http://orcid.org/0000-0003-2270-2217 ||Yves Roisin, http://orcid.org/0000-0001-6635-3552 ∗∗Denis Fournier, http://orcid.org/0000-0003-4094-0390 Editor: Rolf Kummerli¨
ABSTRACT Wolbachia has deeply shaped the ecology and evolution of many arthropods, and interactions between the two partners are a continuum ranging from parasitism to mutualism. Non-dispersing queens of the termite Cavitermes tuberosus are parthenogenetically produced through gamete duplication, a mode of ploidy restoration generally induced by Wolbachia. These queens display a bacteriome-like structure in the anterior part of the mesenteron. Our study explores the possibility of a nutritional mutualistic, rather than a parasitic, association between Wolbachia and C. tuberosus. We found a unique strain (wCtub), nested in the supergroup F, in 28 nests collected in French Guiana, the island of Trinidad and the state of Para´ıba, Brazil (over 3500 km). wCtub infects individuals regardless of caste, sex or reproductive (sexual versus parthenogenetic) origin. qPCR assays reveal that Wolbachia densities are higher in the bacteriome-like structure and in the surrounding gut compared to other somatic tissues. High-throughput 16S rRNA gene amplicon sequencing reveals that Wolbachia represents over 97% of bacterial reads present in the bacteriome structure. BLAST analyses of 16S rRNA, bioA (a gene of the biosynthetic pathway of B vitamins) and five multilocus sequence typing genes indicated that wCtub shares 99% identity with wCle, an obligate nutritional mutualist of the bedbug Cimex lectularius.
Keywords: termites; reproductive parasites; Wolbachia; symbiosis; parthenogenesis; bacteriome; gut microbiota; 16S rRNA gene; deep-sequencing; qPCR; biotin
Received: 2 August 2018; Accepted: 13 December 2018
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1 2 FEMS Microbiology Ecology, 2019, Vol. 92, No. 2
INTRODUCTION endosymbionts in the nests and favour their transmission in the hosts. From the perspective of eusocial insects, their ancient ori- Endosymbiosis is a particular case of association between gin (ant and termite fossils indicate advanced sociality 100 mil- microorganisms living within eukaryotic cells. Endosymbiotic lion years ago; Rust and Wappler 2016), the diversity of their life bacteria deeply shaped the ecology and evolution of many history strategies (Lach, Parr and Abbott 2009; Jones and Eggle- arthropods. Some of these associations are occasional and dis- ton 2011), and their high speciosity (Bolton et al. 2007; Krishna play spatial and temporal variations, others are essential and Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 et al. 2013) confer a long evolutionary time and strong opportu- integrated in the host’s core physiology (Weeks et al. 2007;Wer- nities to develop intimate relationships with endosymbionts. ren, Baldo and Clark 2008; Zug and Hammerstein 2015; Diouf In the termite Cavitermes tuberosus, parthenogenesis is an et al. 2018). The most striking examples are mitochondria and integral part of the lifecycle: the queen uses sex to produce the plastids, two essential membrane-bound organelles of eukary- altruistic castes (workers, soldiers) and the dispersers (alates), otic cells, that evolved from bacteria by endosymbiosis (Maynard and thelytokous parthenogenesis to produce female neoten- Smith and Szathmary´ 1995; Embley and Martin 2006). Accord- ics, i.e. non-dispersing queens (Fournier et al. 2016)(Fig.1). ing to the fitness costs and benefits conferred to the organ- Two peculiar traits of this species attracted our attention. isms involved in the association, interactions can be perceived First, these neotenic queens are parthenogenetically produced as a continuum ranging from parasitism to mutualism (Zug and through gamete duplication (Fournier et al. 2016), a mode of Hammerstein 2015). ploidy restoration rare among animals and generally induced One of the most studied endosymbionts are the reproductive by Wolbachia (Suomalainen, Saura and Lokki 1987; Weeks and parasites, which have evolved various strategies to manipulate Breeuwer 2001; Ma and Schwander 2017). Second, these neotenic host reproduction in order to enhance their own transmission to queens display an empty digestive tract except from an enlarged the next generation: (i) by generating cytoplasmic incompatibil- pouch in the anterior part of the mesenteron (visible as a black ity (CI) between sperm and egg depending on the infection status dot; Fig. 2E). If a more or less distinct hump was described in of both partners, (ii) by killing males, (iii) by feminizing genetic the anterior end of the midgut of workers from higher termites males into functional females and (iv)byinducingthelytok- (Noirot 2001), an hypertrophy of this structure (Fig. 2CandD) ous parthenogenesis leading to the production of females only has never been reported so far, and could be regarded as a bac- (Weeks, Reynolds and Hoffman 2002; Werren, Baldo and Clark teriome playing a role in the nutrition by housing mutualistic 2008; Hurst and Frost 2015; Zug and Hammerstein 2015). As they symbionts. are maternally transmitted, such manipulations increase the We tested in the termite C. tuberosus the hypothesis of an proportion of infected females in the population of hosts, and infectious induction of parthenogenesis and/or of a mutualistic thus of the reproductive parasites in them. Although they are relationship with endosymbionts. First, over a range of 3500 km, usually vertically transmitted, these reproductive parasites can we screened C. tuberosus colonies for the presence of Wolbachia, also move horizontally within a host species or between differ- as well as Cardinium and Rickettsia, the two other bacteria known ent species, thereby explaining the success of their pandemics to induce parthenogenesis in insects (Zchori-Fein et al. 2004; (Zug, Koehncke and Hammerstein 2012). Among them, the bac- Perlman, Hunter and Zchori-Fein 2006; Engelstadter¨ and Hurst terium Wolbachia (Rickettsiales: Rickettsiaceae) is the most com- 2009). Wolbachia was found in all tested nests of C. tuberosus, mon and infects a considerable fraction of arthropod species but Cardinium and Rickettsia were absent. We also estimated the (Zug, Koehncke and Hammerstein 2012;Weinertet al. 2015). prevalence of infection in the populations, and sequenced and At the extreme end of this continuum of interactions, para- identified the strain(s) infecting C. tuberosus. Secondly, we deter- sitism may evolve towards less harmful relationships, and even mined the phenotype(s) of Wolbachia in C. tuberosus.Inorderto mutualisms (Zug and Hammerstein 2015). For instance, the par- link Wolbachia to parthenogenesis induction, we estimated the asitoid wasp Asobara tabida is infected by three Wolbachia strains: prevalence of infection according to the sex and the reproductive two manipulate host reproduction (CI), while the third is essen- origin of the individuals (i.e. sexually or parthenogenetically pro- tial for host oogenesis (Dedeine, Bouletreau´ and Vavre 2005). duced). To determine if the expansion connected to the mesen- Beside reproductive phenotypes, other types of obligate mutu- teron of C. tuberosus could be considered as a bacteriome, we alism can be selected in the case of long-term stable relation- investigated spatial aggregation of Wolbachia in different tissues ships, such as nutritional symbioses (Hosokawa et al. 2010; Zug by performing comparative qPCR to quantify expression levels and Hammerstein 2015). In the pea aphid Acyrthosiphon pisum, of a diagnostic fragment of the Wolbachia 16S rRNA gene, and we the bacterium Buchnera provides specific amino acids that are identified the bacterial communities present in this bacteriome- lacking in the host’s diet (Shigenobu et al. 2000). Additionally, the like expansion and in the gut by performing a targeted Illumina bacterium only lives in specialized cells, the bacteriocytes, local- high-throughput sequencing of the 16S rRNA gene. To highlight ized in the vicinity of the gut (Simonet et al. 2018). In the most a potential nutritional symbiosis, and as Wolbachia may fulfil a integrated associations, bacteria are concentrated in dedicated role of dietary provisioner of biotin (vitamin B7) (Nikoh et al. 2014; permanent organs, the bacteriomes. In the tsetse flies, the bac- Gerth and Bleidorn 2016), we amplified one of the five genes of terium Wigglesworthia lives in a midgut-associated bacteriome. the biotin operon. It provides vitamins lacking in the fly’s diet and are essential to its survival (Akman et al. 2002;Paiset al. 2008). Bacteriomes may take various shapes and locations, and are sometimes associ- ated with the digestive tract or to the gonads (Heddi et al. 2005; MATERIAL AND METHODS Kuechler et al. 2012; Douglas 2015). Samples collection Numerous studies have reported the evolution of complex symbiotic relationships in ants, social bees, wasps or termites Whole nests of the Neotropical termite Cavitermes tuberosus (Ter- (Ohkuma and Brune 2011; Treanor, Pamminger and Hughes mitidae: Termitinae) were collected in 2012, 2014, 2015, 2016, 2018). From the perspective of endosymbionts, eusocial insects 2017 and 2018 in three regions of French Guiana (the Petit Saut ◦ ◦ ◦ ◦ are primary targets as colonial organization and close inter- dam area: N 05.07 , W 52.87 ; Kaw Mountain: N 04.53 , W 52.15 ; ◦ ◦ actions between nestmates help to maintain the presence of and Saul:¨ N 03.62 , W 53.20 ), the island of Trinidad (Lopinot Hellemans et al. 3 Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019
Figure 1. Simplified developmental pathways and reproductive modes in C. tuberosus. After an undifferentiated larval instar, a bifurcation between the altruistic and reproductive castes occurs. Individuals in the altruistic caste (workers and soldiers) are sexually produced (purple). In the reproductive caste, a difference stems from the mode of reproduction: sexually produced individuals develop predominantly through five nymphal instars to the alate stage (84%) while somerentiate diffe into secondary reproductives (neotenics; 16%); parthenogenetically produced females (blue) develop predominantly into neotenic queens (93%) while some reach the alate imago (7%) (Fournier et al. 2016). We call aspirants the instars that differentiate into neotenics. Alates initiate new colonies after a nuptial flight and become primary reproductives while neotenics stay in the nest to replace primaries upon their death. Adapted from Hellemans et al. (2017b). To enhance its own transmission, Wolbachia could induce thelytokous parthenogenesis in C. tuberosus.
Figure 2. Worker digestive tract of C. tuberosus in dorsal (A) and left (B) view; with focus on the foregut-midgut junction in left view of worker (C) and neotenic queen (D), highlighting a pouch-shaped expansion at the antero-dorsal part of the mesenteron. Primary king with numerous neotenic queens (E): the pouch-shaped expansion is filled with particles and visible as a black dot through the cuticle of all neotenic queens. Foregut: Cr, crop; G, gizzard. Midgut:ppled). M,mesenteron(sti Hindgut: P1, ileum; P2, enteric valve; P3, paunch; P4, colon; P5, rectum. Scale bar for drawings = 0.5 mm. Nomenclature to describe the digestive tract follows Noirot (1995) and Noirot (2001).
Road: N 10.73◦, W 61.32◦) and in Brazil (campus of the Federal sequenced with BigDye Terminator Cycle Sequencing kit v3.1 University of Para´ıba, Joao˜ Pessoa: S 7.14◦, W 34.84◦) (see details (Applied Biosystems) according to the manufacturer’s recom- in Supplementary Table S1). Our sampling covers over 3500 km mendations (see SM-1). Sequencing products were purified with of the distribution of this species. an ethanol/EDTA/sodium acetate method. Sequence data were Termite species identity was ascertained using the mito- obtained with an ABI 3730 Genetic Analyzer (Applied Biosys- chondrial barcode gene cytochrome oxidase II (COII). DNA was tems), and were visualized and edited using the software Codon- extracted using a NucleoSpin Tissue kit (Macherey–Nagel). A Code Aligner (CodonCode Corporation, Dedham, MA.). fragment of approximately 780 bp of the COII was amplified using the modified forward primer A-tLeu (Miura, Roisin and Matsumoto 2000) and the reverse primer B-tLys (Simon et al. 1994). Amplification and cycling conditions are given in Sup- Screening of endosymbionts plementary materials SM-1. A fraction of the amplification We selected 20 nests of C. tuberosus representative for the pres- products was screened by electrophoresis on a 1% agarose gel ence of each caste and sex to screen for infection by Cardinium, and another fraction was then purified with the Nucleofast Rickettsia and Wolbachia. DNA extraction was performed on ter- PCR purification kit (Macherey–Nagel). Purified amplicons were mite heads of two individuals per nest (Supplementary Table S1) 4 FEMS Microbiology Ecology, 2019, Vol. 92, No. 2
using a NucleoSpin Tissue kit (Macherey–Nagel). To avoid con- sequences of coxA, fbpA, ftsZ, gatB and hcpA from representa- tamination and PCR inhibition from microorganisms present in tives of supergroups A, B, C, D, F, and H from the Wolbachia MLST the gut, and because our preliminary studies on C. tuberosus (https://pubmlst.org/wolbachia/) and GenBank databases (Sup- showed that infection can be detected using heads as well as plementary Table S3). We used Ehrlichia ruminantium as an out- gonads, we selected heads for the rest of this study. group for phylogeny reconstruction (Gerth et al. 2014). Each gene
We used species-specific primers targeting the 16S rRNA was aligned individually using the MUSCLE algorithm (Edgar Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 gene for each bacterium: the primer pair CLOf/CLOr1 specific 2004) implemented in CodonCode Aligner. The best nucleotide- to Cardinium (Weeks, Velten and Stouthamer 2003), the pair Rb- substitution models were determined using jModelTest 2 v2.1.10 F/Rb-R specific to Rickettsia (Gottlieb et al. 2006)andthepair (Darriba et al. 2012) according to the best Bayesian Information Wspecf/Wspecr (Werren and Windsor 2000), which proved to Criterion (BIC) for each gene (coxA:HKY+I; fbpA:HKY+I+G; ftsZ: be the most specific to Wolbachia (16S-2; Simoes˜ et al. 2011). TrN+G; gatB: GTR+I+G; hcpA:TIM+G). Positive controls (Cardinium-infected false spider mite Brevipal- Phylogenetic analyses with Bayesian inference were per- pus phoenicis (Acari), Rickettsia-infected whiteflies Bemisia tabaci formed using the software BEAST 2 (Bouckaert et al. 2014)with (Hemiptera) and Wolbachia-infected mosquito Culex pipiens pipi- the method ∗BEAST (Heled and Drummond 2010) for inference ens (Diptera)) and negative controls (PCR-grade water) were used from multilocus data. Because Wolbachia strains may recombine for each amplification. Cycling conditions followed specifica- (Jiggins et al. 2001) or there may be horizontal transfers between tions of each primer pair in the original publications. Amplifica- bacterial species (Duplouy et al. 2013; Duron 2013), molecular tion products were screened by electrophoresis on a 1% agarose clocks and gene trees were maintained unlinked between mark- gel. In order to discard false negatives, DNA extracts that did ers to allow for recombination. The ploidy level was set to ‘Y or not amplify were tested at the mitochondrial gene cytochrome mitochondrial’, strict molecular clocks were used and rates were oxidase I (COI) using the universal forward LCO (LCO1490) and assigned with gamma-distribution priors (shape = 2, scale = 2), reverse HCO (HCO2198) primers (Folmer et al. 1994). Cycling con- the species tree was modelled under a Yule model and birthrate ditions are given in Supplementary materials SM-2. Low-quality set to 1/X, and the species tree population size was set to ‘linear’ samples that did not amplify were discarded from the analyses with population mean estimated during the run. All other priors (8 individuals, 1.8% of the data). and operators were adjusted after a first run. We ran five independent runs of 10 million generations and trees were sampled every 10 000 generations. We followed traces Infection prevalence in the nests and among castes and with Tracer v1.7 (Rambaut et al. 2018) to ensure that all effective > sexes sample sizes were 200. We combined the five different runs and removed 20% of sampled trees for each run as burn-in (leav- Prevalence of Wolbachia was tested for a subset of 10 nests (420 ing a total of 5000 trees) using LogCombiner v1.8.3 (Bouckaert individuals; Supplementary Table S1), all collected in October et al. 2014). Bayesian posterior probabilities for the support of 2014. DNA was extracted from termite heads by a Chelex-based nodes were computed as maximum clade credibility retaining method (Walsh, Metzger and Higuchi 1991) and was screened median branch lengths using TreeAnnotator v2.4.2 (Bouckaert with amplification, cycling conditions and gel screening as et al. 2014). The resulting tree was visualized with the package above. GGTREE v3.5 in R (Yu et al. 2017). The developmental scheme of C. tuberosus outlined an altru- istic caste composed of workers and soldiers, and a reproduc- Wolbachia infection and impacts on phenotype tive caste composed of male and female dispersers (alates), pri- mary king and queen, aspirants (i.e. nymph-like pre-neotenics) In order to highlight a link between Wolbachia and the induction and female neotenics (Fig. 1). We analysed the infection rate for of parthenogenesis, we analysed the infection status of males all nests, and for each caste and sex under a generalized linear (n = 91) and females (n = 186) from the reproductive caste, distin- model framework with a binomial error structure and a logis- guishing females according to their reproductive origin, sexual tic link function to model the dichotomous infection status (0, (n = 100) or parthenogenetic (n = 86), using Fisher’s exact tests. non-infected; 1, infected). Analyses were performed with the R Reproductive origin was determined from genetic data retrieved software v3.0.1 (R Development Core Team 2013). from previous datasets (Fournier et al. 2016; Hellemans et al. In press), in which individuals were genotyped at 17 microsatel- lite markers (Fournier, Hanus and Roisin 2015). Additional geno- Strain identification of Wolbachia and supergroup typing was performed in this study using the same molecular assignment procedures. All genotypes used in this study are available in Appendix S1 (see Electronic Supporting Information). We per- Wolbachia strain was identified in all 20 nests by sequencing formed a factorial correspondence analysis (FCA) using GENETIX genes of the multilocus sequence typing (MLST), i.e. the con- v4.05.2 (Belkhir et al. 1998) in order to visualize the distribution served bacterial housekeeping genes coxA, ftsZ, gatB, hcpA and of alleles according to the reproductive origin and the infection fbpA (Baldo et al. 2006), and the 16S rRNA gene. Wolbachia strain status of females. determination was carried on DNA from heads of one individ- Because the sex ratio in eggs (primary sex ratio, PSR) is an ual per nest extracted using a NucleoSpin Tissue kit (Macherey– index of reproductive manipulation by endosymbionts (Werren, Nagel). Amplifications and cycling conditions of MLST genes Baldo and Clark 2008), we studied PSR in 11 nests of C. tubero- are given in Supplementary materials (SM-3). Sequences were sus (Supplementary Table S1). Sex determination was carried obtained with an ABI 3730 Genetic Analyzer and visualized with out using the diagnostic microsatellite locus Ctub-94, at which CodonCode Aligner. All sequences were deposited in GenBank females are systematically homozygous for one allele, whereas (see Supplementary Table S2). males are heterozygous (Fournier et al. 2016). The presence of From the 16 described supergroups of Wolbachia (A–F and Wolbachia in eggs was confirmed from the two tested colonies H–Q; Lindsey et al. 2016a;Lindseyet al. 2016b), we retrieved (Supplementary Table S1). Hellemans et al. 5
Wolbachia is a nutritional mutualist that may supply B vita- individuals, bacteriome-like structure located at the anterior mins to its host (Nikoh et al. 2014). We investigated whether Wol- part of the mesenteron (Fig. 2) were separated, pooled and bachia could be associated in a nutritional mutualism with C. preserved. All samples were collected in triplicates. PowerVi- tuberosus by testing the presence of the bioA gene involved in ral Environmental RNA/DNA Isolation Kit (MO-BIO) was used the biotin (vitamin B7) biosynthetic pathway (Nikoh et al. 2014; for DNA extraction following the manufacturer’s protocol with
Gerth and Bleidorn 2016). Amplification and cycling conditions introduction of cell lysis step by bead-beating with 0.1 mm Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 are given in Supplementary materials (SM-4). Sequences were glass beads at 20 Hz for 2 min. The extracted DNA was treated obtained with an ABI 3730 Genetic Analyzer and visualized with with 1 μlof10μg/mL RNase A (Sigma) at room tempera- CodonCode Aligner. ture for 30 min and quantified. The microbial 16S rRNA gene libraries were prepared using an Illumina platform-optimized protocol (Marynowska et al. 2017). More specifically, universal Quantification of Wolbachia densities across tissues using modified primers S-D-Bact-0909-a-S-18 and∗ S- -Univ-∗-1392-a- quantitative PCR A-15 (Klindworth et al. 2013), targeting a 484 bp fragment of the V6–V8 region of the bacterial 16S rRNA gene, were used Wolbachia densities were assessed in five tissues—head, hind- in the first round PCR where 1 ng of template DNA was sub- leg, ovaries, bacteriome-like structure and the rest of the gut jected to 22 cycles of amplification. Subsequently, 1 μlofthe (fromP3toP5;seeFig.2)—of five neotenic queens from nest purified and quantified reaction was used as template in asec- M 82 (Supplementary Table S1) using quantitative PCRs (qPCRs). ond step 8 cycle-PCR, which allowed for the incorporation of qPCRs were performed on a Rotor-Gene Q cycler (Qiagen). Total index barcodes (Nextera XT Index Kit V2, Illumina) and Illu- DNA was extracted using a NucleoSpin Tissue kit (Macherey- mina adaptors. Purified (Agencourt AMPure XP) and quanti- Nagel). We used Wolbachia-specific primers (INTF2 and INTR2; fied (KAPA SYBR FAST Universal qPCR Kit, KapaBiosystems) (Sakamoto, Feinstein and Rasgon 2006) amplifying a small frag- libraries were pooled in equimolar ratios, spiked with PhiX ment of the 16S rRNA gene. Amplification and cycling condi- control (Illumina) and sequenced using MiSeq Reagent Kit V3– tions are given in Supplementary materials (SM-5). Each sam- 600 on the Illumina Platform. After demultiplexing, quality ple was run in triplicate, and we used mean threshold cycle trimming and chimera and singletons removal, resulting (C ) values of triplicates for each sample. Melt curves were t sequencing reads were assigned to Operational Taxonomic Units run after the last cycle for each reaction and always pro- (OTUs) at 97% similarity with Usearch pipeline v7.0.1090 win64 duced single products, ensuring primer specificity. We used con- (Edgar 2010) and further taxonomically annotated with SILVA trols (elution buffer from the DNA extraction kit) to ensure database v.128 (Pruesse et al. 2007) using the naive Bayesian that amplification did not occur in samples without template classifier (Wang et al. 2007) as implemented by the mothur DNA. For each tissue, primer efficiency (E) was determined software v.1.38.0 (Schloss et al. 2009). The resulting dataset is using standard curves with template dilutions of 1X, 2X, 4X available within GenBank repository under accession numbers and 8X. Optimal reaction efficiency occurred at 0.150 ng/μlof MG958889 - MG962339 (OTU sequences) and in Sequence Read stock template DNA for hindlegs and at 0.400 ng/μl for the Archive (SRA) under accession number SRP148861 (raw sequenc- other tissues. DNA was quantified in each sample using Qubit ing reads). dsDNA HS assay kit (Invitrogen). Each sample was diluted at the above stated optimal reaction efficiency for the final qPCR assay. All analyses were performed using the Rotor-Gene Q software v2.3.1.49. Results are reported as expression ratios slightly modified Termite genera phylogenetically close to Cavitermes and from Pfaffl (2001) (see SM-5). We used hindlegs as a control reported cases of facultative parthenogenesis in higher sample to which we compared all other tissues. We anal- termites ysed expression ratios as log10 transformed data in order We tested for the presence of the three endosymbionts in other to ensure normality, sphericity and homoscedasticity. Trans- termite species from the Cavitermes lineage sensu Hellemans formed ratios were compared between tissues (head, ovaries, et al. (2017a): Palmitermes impostor (Termitidae: Termitinae) that bacteriome-like structure and the rest of the gut) using one- facultatively reproduces asexually through gamete duplication way within subject (i.e. neotenic identity) ANOVA with tis- (heads and gonads of 25 neotenic queens distributed in five sue as the between-subject factor. Post-hoc comparisons were nests), and Spinitermes trispinosus (Termitidae: Termitinae), a performed using multiple paired t-tests and p values were cor- species known for the occurrence of neotenic queens (Carrijo rected for multiple comparisons using the False Discovery Rate 2009), and whose female aspirants arise from gamete dupli- (fdr) method (Benjamini and Hochberg 1995). Analyses were per- cation (Hellemans et al., unpublished data) (head and gonads formed with the R software v3.0.1. of one primary queen, and heads of two soldiers). Addition- ally, we tested Inquilinitermes inquilinus of the Termes lineage, Illumina 16S rRNA gene sequencing-based microbial the sister lineage to Cavitermes (Hellemans et al. 2017a), in community profiling which female aspirants and neotenic queens are also produced through gamete duplication (Hellemans et al., unpublished data) The microbial community composition of digestive tubes of (heads of six neotenic queens). workers and neotenic queens from nest A 147 (Supplemen- We also screened for the presence of the three endosym- tary Table S1) was investigated by means of Illumina sequenc- bionts in all other reported cases of facultative parthenogenesis ing of 16S rRNA gene. Individuals were cold-immobilized and in the Neotropics: Embiratermes neotenicus and Silvestritermes min- surface-sterilized using 80% ethanol and 1 X PBS. Using ster- utus (Termitidae: Syntermitinae), both restoring ploidy through ile forceps, whole guts were dissected and pooled from 20– central fusion (Fougeyrollas et al. 2015; Fougeyrollas et al. 2017) 30 workers and directly preserved in liquid nitrogen. In the (heads and gonads of 25 neotenic queens distributed in five case of the neotenics, after dissection of whole guts from 20 nests). 6 FEMS Microbiology Ecology, 2019, Vol. 92, No. 2
RESULTS Fig. 3), nor between sexually and parthenogenetically produced females (92.08% ± 21.01 and 95% ± 15.81, respectively; Wilcoxon Species identity rank sum test, W = 55, p = 0.58; Fig. 3). FCA revealed segregation All 20 nests used for the screening of endosymbionts and strain of allelic frequencies between (i) all nests and nest K 100 and (ii) identification belonged to C. tuberosus: sequences obtained from in nest K 100, between infected and uninfected individuals (Sup- 17 nests collected from the Petit Saut dam area, Kaw and Saul¨ plementary Fig. S1). All tested eggs were infected by Wolbachia. Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 have a 100% identity with the COII from the full mitochon- The primary sex ratio was balanced in all nests (Supplementary ± = ± drial genome of C. tuberosus (accession KP026294; E-value = 0); Table S1; mean SD 0.527 0.077, min–max: 0.431–0.700; one = = = sequences from individuals of the nest K 100 from the Petit Saut sample t-test: t 1.176, df 10, p 0.267). dam area differed by two nucleotide substitutions; sequences of To determine if Wolbachia could be involved in a nutritional the individuals collected in Trinidad (nest TT18–59) and Brazil mutualism by supplying B vitamins to its host, we tested for (nest BR18–06) showed, respectively, 98.8% and 97.4% identity thepresenceofthebioA gene involved in the synthesis of the with the COII of the full mitochondrial genome (see Supplemen- biotin. The bioA gene amplified in all 20 nests of C. tuberosus. tary Table S2 for GenBank accessions). Its sequence was 99.5% identical to the bioA gene from the endosymbiotic Wolbachia of the bedbug C. lectularius (E-value = 0; accession numbers MK053590 and AP013028, respectively; Sup- Infection prevalence in the nests and among castes plementary Table S2).
Wolbachia was found in all tested nests of C. tuberosus,butCar- dinium and Rickettsia were not detected. Infection rates were Quantification of Wolbachia densities across tissues analysed on 10 nests under a GLM framework in which the best using quantitative PCR model (according to the minimum theoretical Aikake Informa- tion Criterion; AIC; Supplementary Table S4) was the one con- The density of infection by Wolbachia significantly differed = < sidering nests and reproductive origin (i.e. sexually or partheno- among tissues (one-way ANOVA, F3,12 18.63, p 0.001). The genetically produced). GLM analysis determined a significant highest densities of Wolbachia were detected in gonads, the low- effect of nests on the infection rate (Likelihood-ratio Chi- est in the head. Pairwise comparisons showed that bacteriome- Squared test, χ 2 = 24.29, df = 9, p = 0.004), with nest K 100 like structure and digestive tract have similar densities of Wol- = strongly differing from all others (post-hoc pairwise compari- bachia (post-hoc pairwise t-test with fdr correction, p 0.180; son using the fdr method, all p < 0.004). In nest K 100, Wolbachia Fig. 5). infected 54.76% of individuals (Supplementary Table S1, Fig. 3), whereas for all other nests, virtually all individuals (372 out of Microbial community profiling of whole guts and 378, 98.41%) were positive for Wolbachia (mean per nest ± SD = bacteriome of C. tuberosus 98.20% ± 3.24; Supplementary Table S1, Fig. 3), whether they were from the altruistic or reproductive caste (97.22% ± 4.54 and Fifty-one OTUs were identified from the bacteriome-like struc- 98.70% ± 2.86 respectively; Wilcoxon rank sum test, W = 35, p = ture of the neotenics. Strikingly, one specific OTU (OTU 29) con- 0.576). stituted over 97.3% ± 0.7 reads in the three tested replicates. It was assigned with 100% identity to Wolbachia sp. of Proteobac- terium phylum (Fig. 6, Supplementary Table S5). Additionally, Strain determination and supergroup assignment OTU 29 was the only one assigned to this species. This points All six sequenced genes (16S rRNA, coxA, ftsZ, gatB, hcpA and towards the fact that the pouch-shaped expansion at the antero- fbpA) were identical in all 20 tested nests (except for one sub- dorsal part of the mesenteron in neotenics of C. tuberosus is stitution at base #364 and #352 for coxA and fbpA respectively a bacteriome that hosts endosymbiotic Wolbachia. The remain- for the sample TT18–59 collected in Trinidad) showing that one ing OTUs identified were characterized by very low abundance single strain of Wolbachia (hereafter referred to as wCtub) infects (Fig. 6, Supplementary Table S5), and might result from a possi- C. tuberosus. BLAST and phylogenetic results showed that all ble cross-contamination from the surrounding gut tissue. sequenced genes were highly similar to those of the Wolbachia Analyses of the whole gut of workers of C. tuberosus indi- endosymbionts of the bedbug Cimex lectularius (99% of identity, cated a more diverse and evenly distributed microbial commu- E-value = 0 for all genes), and belongs to supergroup F (Fig. 4; nity (Fig. 6, Supplementary Table S5). In total, 3457 OTUs were Supplementary Tables S2 and S3). identified and assigned to multiple prokaryotic taxa. The dom- inant phyla identified were: Firmicutes, Spirochaetae and Bac- teroidetes, with 38.8% ± 6.4, 24.2% ± 5.3 and 7.6% ± 0.6 relative Wolbachia infection and impacts on phenotype abundance, respectively. The Wolbachia-related OTU constituted a minor percentage of the total diversity (0.17% ± 0.04). In order to detect a possible connection between Wolbachia and the induction of parthenogenesis in C. tuberosus,weanal- ysed the infection status of males (n = 91 individuals) and Termite genera phylogenetically close to Cavitermes and = females (n 186) from the reproductive caste. We distinguished reported cases of facultative parthenogenesis in higher = females according to their reproductive origin, sexual (n 100) termites or parthenogenetic (n = 86). There was no association between sex and the infection status (Fisher’s exact tests, two-tailed; Endosymbionts (i.e. Wolbachia, Cardinium and Rickettsia)were p = 1), nor between the infection status of females and their totally absent in two species from the Cavitermes lineage (i.e. reproductive origin (Fisher’s exact tests, two-tailed; p = 0.580). in P. impostor and S. trispinosus), and in the two species where Accordingly, infection rates differed neither between males and facultative parthenogenesis has been reported, i.e. E. neotenicus females from reproductive castes (93.09% ± 14.41 and 93.47% ± and S. minutus (Syntermitinae). The quality of DNA extractions 18.57, respectively; Wilcoxon rank sum test, W = 45, p = 0.65; was verified by amplifying COI, which excluded false negatives. Hellemans et al. 7 Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019
Figure 3. Infection rate of Wolbachia per nest, between the altruistic and the reproductive castes, between males and females from the reproductive caste and between parthenogenetically and sexually produced females from the reproductive caste, in 10 nests of C. tuberosus. The total number of individuals tested is indicated above each bar.
On the other hand, Wolbachia was detected in all six neotenic C. tuberosus (K 100), differs from all others: nearly one out of queens of I. inquilinus from the Termes lineage, the sister lineage two individuals is uninfected while the mean infection rate for of Cavitermes.TheWolbachia strain infecting I. inquilinus (wIinq) all other nests reaches 98.20%. While it shares the same strain shows 99.6%, 100% and 99.1% similarity with wCtub at loci coxA, wCtub with all other nests, our data showed that nest K 100 is gatB and hcpA, respectively (fbpA and ftsZ could not be amplified) in striking contrast to them regarding allele frequencies at 17 (see Supplementary Table S2 for GenBank accessions). nuclear microsatellite and mitochondrial profiles. These differ- ences could be in line with K 100 belonging to a distinct popula- tion in the course of losing its symbiosis with Wolbachia (Sicard DISCUSSION et al. 2014; Zug and Hammerstein 2015; Correa and Ballard 2016). Infection by Wolbachia was found in all nests of C. tuberosus, both Termite-infecting Wolbachia strains usually belong to super- altruistic and reproductive castes, males and females, sexually groups H and F, with basal termites infected by supergroup H and parthenogenetically produced individuals. One single strain and higher termites by supergroup F (Lo and Evans 2007). Wol- (wCtub), belonging to the supergroup F, infects all nests on a bachia from supergroups A and B sporadically infect Cubitermes wide geographic range (at least 3500 km). Wolbachia was found spp. (Roy and Harry 2007), supergroup B was reported in Odon- at different densities in reproductive and somatic tissues of C. totermes horni and Coptotermes heimi (Salunke et al. 2010)anda tuberosus and represents over 97% of bacterial reads in the bac- divergent clade within supergroup A was reported for Neoter- teriome associated with the mesenteron of neotenics, a struc- mes luykxi, N. jouteli and Serritermes serrifer (Lo and Evans 2007). ture here described in termites for the first time. One nest of While other termite-infecting Wolbachia are frequently reported 8 FEMS Microbiology Ecology, 2019, Vol. 92, No. 2 Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019
Figure 4. Rooted Wolbachia species tree estimated under Bayesian Inference using MLST genes (coxA, fbpA, ftsZ, gatB and hcpA) and highlighting the position of the new strain described from the termite C. tuberosus (wCtub) in supergroup F. Wolbachia strains are represented by the host species’ name followed by the Wolbachia strain in parentheses. Letters (A–D, F, H) stand for Wolbachia supergroups. Bayesian posterior probability support values are shown for each clade. from the supergroup F, wCtub is most closely related to the their digestion depends on a whole and complex community strain infecting the bedbug C. lectularius (wCle) (Fig. 4). The lack of symbiotic microorganisms (Ohkuma and Brune 2011). Inter- of phylogenetic congruence between Wolbachia and its hosts estingly, our results show that wCtub harbours the bioA gene evidences common horizontal transmission of Wolbachia;the involved in the biotin (vitamin B7) synthesis pathway. This hints mechanisms driving these transmissions have mostly remained that wCtub and wCle may share a similar genetic background of unclear (Oliver et al. 2010; Himler et al. 2011;Balv´ın et al. 2018). mutualism with their host (Gerth and Bleidorn 2016). Dissection of the digestive tract revealed that neotenic queens of C. tuberosus display a bacteriome-like structure in Wolbachia, a mutualistic partner of C. tuberosus the anterior part of the mesenteron (Fig. 2). Quantitative PCRs showed that Wolbachia densities are higher in this structure and Our data show that wCtub is 99% similar to the Wolbachia in the surrounding gut compared to other somatic tissues (Fig. 5). strain infecting the bedbug C. lectularius (wCle). In this species, Furthermore, high-throughput sequencing of the 16S rRNA gene Hosokawa et al. (2010) demonstrated that Wolbachia (i) resides in showed that Wolbachia represents over 97% of bacterial reads in bacteriomes located adjacent to the gonads and (ii)isanobli- the bacteriome associated with the mesenteron of C. tuberosus, gate nutritional mutualist: clearing of Wolbachia by using antibi- while it represents less than 1% in the gut (Fig. 6). In Cimex lec- otics led to sterility and reduced growth, which was rescued by B tularius, Wolbachia specifically inhabits the bacteriome and the vitamin supplementation. Nikoh et al. (2014) later demonstrated gonads of its host (Hosokawa et al. 2010), but in the leaf-cutting that wCle genome possesses operons involved in the biosynthe- ant Acromyrmex octospinosus, Wolbachia occurs abundantly in the sis of B vitamins. Unfortunately, such direct proofs using antibi- gut lumen (Andersen et al. 2012). In both cases, Wolbachia plays otics are practically impossible to obtain in termites because Hellemans et al. 9 Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019
Figure 5. Boxplots showing the density of infection of Wolbachia across four tissues (head; ovaries; bacteriome-like structure: digestive tube from P3 to P5; see Fig. 2) of five neotenic queens from nest M 82 of C. tuberosus using qPCR. Density of infection was estimated as the log10 of the expression ratio using hindlegs as a control sample to which we compared all other tissues (see SM-5). The black line within the box represents the median, the bottom and top of the box are the lower (Q1) and upper (Q3) quartiles respectively (the range gives the interquartile distance, IQR), the lower and upper whiskers of the box represent 1.5∗IQR below Q1 and above Q3 respectively, and outliers are found outside the range of whiskers. Letters denote significant differences between tissues (post-hoc multipleired pa t-tests with fdr correction of p values).
Figure 6. Taxonomic distribution into major phyla of prokaryotic OTUs from the guts of neotenic queens (bacteriome) and workers (whole guts) from nest A 147 of C. tuberosus. Relative abundances are derived from the percentage of reads assigned to specific OTUs (Supplementary Table S4). Microbial community profilessed areba on the 16S rRNA gene amplicon high-throughput sequencing and annotation is done according to SILVA database v.128. 10 FEMS Microbiology Ecology, 2019, Vol. 92, No. 2
a mutualistic nutritional role, by providing essential B vitamins feminization—can be definitively discarded as a same propor- for the former, or by interacting in the ant-fungus cultivation tion of males and females occur in eggs, and infected males for the latter. As interactions between two genomes may lead remain phenotypic males. to an extended phenotype shaping the morphology of the host Contrary to C. tuberosus, P. impostor and S. trispinosus,two (Hughes 2014), comparison with these two species may suggest species also restoring ploidy through gamete duplication and
an ongoing evolution of a specialized structure for which the belonging to the Cavitermes lineage, are not infected with Wol- Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 intracellular bacterium Wolbachia has a peculiar affinity. This bachia. At first glance, these observations could discard Wol- structure could house and maintain the endosymbiotic bacte- bachia as the inducer of parthenogenesis in species of the Caviter- ria before that it passes on to the host’s digestive tract. mes lineage. However, species with secondary loss of Wolbachia Diouf et al. (2018) reported that the relative abundance of can remain capable of thelytoky. For example, in the asexual Wolbachia in the gut of the wood-feeding termite Nasutitermes lineage of the thrips Aptinothrips rufus, Wolbachia is present in arborum is caste- and age-dependent and negatively correlated only 69% of females (van der Kooi and Schwander 2014), which withthediversityofthemicrobiota:Wolbachia was less abun- can probably be explained by the transfer of genes involved dant in the gut (fully colonized by true gut-resident bacteria) of in parthenogenesis from the Wolbachia genome into the host’s (old) castes directly feeding on their substrate, while it was dom- genome (Dunning Hotopp et al. 2007; Feldhaar and Gross 2009). inant in the gut of young individuals which are fed with salivary regurgitates of workers. In C. tuberosus, neotenic queens display Wolbachia and the inquiline lifestyle an empty digestive tube and receive nutriments through mouth- to-mouth exchanges (i.e. trophallaxis) with workers (Hellemans Taken together, the high similarity between Wolbachia strains in et al. 2017b). Composition of the gut community of workers C. tuberosus and Cimex lectularius, the high prevalence of a single shows a potential capacity allowing to feed on diverse ligno- strain of Wolbachia over a wide geographical area, the fact that cellulose rich biomass (Fig. 6), but the nutritional quality of the Wolbachia shows a high affinity for the bacteriome-like structure fluids transferred to neotenics is unknown and may be defi- located at the anterior part of the mesenteron of C. tuberosus, cient in some nutrients. Furthermore, C. tuberosus lives in arbo- combined to the behaviour and ecology of the species, suggest real nests constructed by other species (Mathews 1977)andis that the two partners are likely engaged in a nutritional mutual- not known to forage outside. Cavitermes tuberosus,asmostof ism. Whole genome sequencing of these partners may be useful inquiline species, feeds on their hosts’ nest material (Martius to elucidate the exact role of Wolbachia in the nutrition processes 1997; Florencio et al. 2013), which may be, once again, deficient of C. tuberosus, and to reveal horizontal gene transfers. Further in some nutrients that Wolbachia would synthesise. Actually, analyses on species with similar inquiline lifestyles, such as the in every known case, insects carrying bacteriome-associated Termitidae I. inquilinus (this study) and the Serritermitidae S. ser- endosymbionts have peculiar diet depleted in essential nutri- rifer (Lo and Evans 2007), respectively infected by Wolbachia from ents (Moran, McCutcheon and Nakabachi 2008). Buchnera pro- supergroups F and A, will be conducted to investigate whether vides essential amino acids to aphids, as Carsonella does to these intimate relationships evolved multiple times indepen- psyllids (Hemiptera) (Shigenobu et al. 2000; Nakabachi et al. dently from different Wolbachia supergroups and in unrelated 2006); Portiera is a source of carotenoids in whiteflies (Hemiptera) termite lineages. (Sloan and Moran 2012); Wiggleworthia synthesizes essential vitamins for the tsetse flies (Akman et al. 2002); and Blattabac- SUPPLEMENTARY DATA terium has allowed cockroaches, the closest relatives of termites, to subsist successfully on nitrogen-poor diets (Sabree, Kamb- Supplementary data are available at FEMSEC online. hampati and Moran 2009). ACKNOWLEDGMENTS
We are grateful for logistic support during fieldwork to the Wolbachia is not associated with parthenogenesis in C. late Philippe Cerdan and to Regis´ Vigouroux and the staff of tuberosus and related species the Laboratoire Environnement HYDRECO of Petit Saut (EDF- CNEH) in French Guiana; to Christopher K. Starr in the Repub- While we described here for the first time a potential nutri- lic of Trinidad and Tobago (University of the West Indies); and tional mutualism between Wolbachia and a termite, it is also to Alexandre Vasconcellos and Alane Ayana Vieira de Oliveira the first example of the occurrence of this bacterium in adiplo- Couto in Brazil (Federal University of Para´ıba). We thank Xavier diploid organism restoring ploidy through gamete duplication Goux for help in the field; Johannes A. J. Breeuwer and Martha during parthenogenesis. In haplo-diploids, Wolbachia was shown S. Hunter for the supply of positive controls for endosymbionts to restore ploidy through gamete duplication in Hymenoptera screening; Robert Hanus and Virginie Roy for the supply of (Rabeling and Kronauer 2013; Ma and Schwander 2017), and via termite samples from the Syntermitinae subfamily; and Sarah functional apomixis in several species of phytophagous mites of Cherasse´ for help in quantitative PCR laboratory work. We are the genus Bryobia (Weeks and Breeuwer 2001). For diplo-diploids, grateful to Johannes A. J. Breeuwer and Roman Zug for com- induction of parthenogenesis by Wolbachia is extremely difficult ments on an early version of this manuscript, and to the two to formally demonstrate, but has been suggested for the female- anonymous referees for their constructive comments. only springtail Folsomia candida in which ploidy is restored by terminal fusion (Pike and Kingcombe 2009; Ma and Schwander Data archiving 2017). However, the induction of parthenogenesis by Wolbachia is not obvious in C. tuberosus as Wolbachia infects all individ- Sequences produced for this study have been deposited in uals, whether they are male or female, sexually or partheno- GenBank repository under accessions MF953226 to MF953242, genetically produced. At the same time, two other reproductive MH522792, MH522793, MK053589 to MK053591 and MK064568 manipulations possibly induced by Wolbachia—male-killing and to MK064570 (Sanger sequencing; see Supplementary Table S2 Hellemans et al. 11
for details), MG958889 to MG962339 (Illumina sequencing; OTU (Hymenoptera, Braconidae) and evidence for intraspecific sequences); and in Sequence Read Archive (SRA) under acces- variation in A. tabida. Heredity 2005;95:394–400. sion number SRP148861 (Illumina sequencing; raw sequencing Diouf M, Miambi E, Mora P et al. Variations in the relative abun- reads). dance of Wolbachia in the gut of Nasutitermes arborum across life stages and castes. FEMS Microbiol Let 2018;365:fny046.
Douglas AE. Multiorganismal insects: diversity and function of Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 Authors’ contribution resident microorganisms. Annu Rev Entomol 2015;60:17–34. SH and DF designed the study. SH, NK, MM, YR and DF collected Dunning Hotopp JC, Clark ME, Oliveira DCSG et al. Widespread the material. SH and NK performed the microsatellite genotyp- lateral gene transfer from intracellular bacteria to multicel- ing, single gene sequencing and qPCR. MM and MC performed lular eukaryotes. Science 2007;317:1753–6. Illumina sequencing and subsequent analyses. SH, NK and DF Duplouy A, Iturbe-Ormaetxe I, Beatson SA et al. Draft genome carried out the statistical analyses. All authors contributed sig- sequence of the male-killing Wolbachia strain wBol1 reveals nificantly to the manuscript and approved the final version. recent horizontal gene transfers from diverse sources. BMC Genomics 2013;14:20. Duron O. Lateral transfers of insertion sequences between Wol- FUNDING bachia, Cardinium and Rickettsia bacterial endosymbionts. This work was supported by a PhD fellowship and a travel Heredity 2013;111:330–7. grant from the KMDA Royal Zoological Society of Antwerp (S.H.), Edgar RC. MUSCLE: multiple sequence alignment with grants from the Belgian National Fund for Scientific Research high accuracy and high throughput. Nucleic Acids Res FRS-FNRS (D.F. and Y.R.: FRFC grant nos. 2.4594.12; D.F.: J.0110.17), 2004;32:1792–7. from the Fonds Defay (D.F.) and by an FNR 2014 CORE project Edgar RC. Search and clustering orders of magnitude faster than OPTILYS (Exploring the higher termite lignocellulolytic system BLAST. Bioinformatics 2010;26:2460–1. to optimize the conversion of biomass into energy and useful Embley TM, Martin W. Eukaryotic evolution, changes and chal- platform molecules/C14/SR/8286517). lenges. Nature 2006;440:623–30. Engelstadter¨ J, Hurst GDD. The ecology and evolution of Conflicts of interest. None declared. microbes that manipulate host reproduction. Annu Rev Ecol Evol Syst 2009;40:127–49. REFERENCES Feldhaar H, Gross R. Insects as hosts for mutualistic bacteria. Int J Med Microbiol 2009;299:1–8. Akman L, Yamashita A, Watanabe H et al. Genome sequence of Florencio DF, Marins A, Rosa CS et al. Diet segregation between the endocellular obligate symbiont of tsetse flies, Wiggleswor- cohabiting builder and inquiline termite species. PLoS One thia Glossinidia Nat Genet 2002;32:402–7. 2013;8:e66535. Andersen SB, Boye M, Nash DR et al. Dynamic Wolbachia preva- Folmer O, Black M, Hoeh W et al. DNA primers for amplifica- lence in Acromyrmex leaf-cutting ants: potential for a nutri- tion of mitochondrial cytochrome c oxidase subunit I from tional symbiosis. J Evol Biol 2012;25:1340–50. diverse metazoan invertebrates. Mol Marine Biol Biotechnol Baldo L, Dunning Hotopp JC, Jolley KA et al. Multilocus sequence 1994;3:294–9. typing system for the endosymbiont Wolbachia pipientis. Appl Fougeyrollas R, Dolejsovˇ a´ K, Sillam-Dusses` D et al. Asexual Environ Microbiol 2006;72:7098–110. queen succession in the higher termite Embiratermes neoteni- Balv´ın O, Roth S, Talbot B et al. Co-speciationinbedbugWolbachia cus. Proc Biol Sci 2015;282:20150260. parallel the pattern in nematode hosts. Sci Rep 2018;8:8797. FougeyrollasR,Krivˇ anek´ J, Roy V et al. Asexual Queen Succes- Belkhir K, Borsa P, Goudet J et al. Genetix, logiciel sous Win- sion mediates an accelerated colony life cycle in the termite dowsTM pour la gen´ etique´ des populations, Windows 3.1 Silvestritermes minutus. Mol Ecol 2017;26:3295–308. ou ulterieur:´ 3.0 Edition. Montpellier (France): Laboratoire Fournier D, Hanus R, Roisin Y. Development and characteriza- Genome´ et Populations, CNRS UPR 9060, UniversitedeMont-´ tion of microsatellite markers from the humivorous termite pellier II, 1998. Cavitermes tuberosus (Isoptera: Termitinae) using pyrose- Benjamini Y, Hochberg Y. Controlling the false discovery rate: a quencing technology. Conserv Genet Resour 2015;7:521–4. practical and powerful approach to multiple testing. J R Stat Fournier D, Hellemans S, Hanus R et al. Facultative asex- Soc Series B Stat Methodol 1995;57:289–300. ual reproduction and genetic diversity of populations in BoltonB,AlpertG,WardPSet al. Bolton’s Catalogue of Ants of the the humivorous termite Cavitermes tuberosus. Proc Biol Sci World: 1758–2005. Harvard: Harvard University Press, 2007. 2016;283:20160196. Bouckaert R, Heled J, Kuhnert¨ D et al. BEAST 2: a software plat- Gerth M, Bleidorn C. Comparative genomics provides a time- form for Bayesian evolutionary analysis. PLoS Comput Biol frame for Wolbachia evolution and exposes a recent biotin 2014;10:e1003537. synthesis operon transfer. Nature Microbiology 2016;2:16241. Carrijo TF. Revisao˜ taxonomicaˆ do generoˆ Spinitermes Wasmann, Gerth M, Gansauge M-T, Weigert A et al. Phylogenomic analyses 1897 (Isoptera, Termitidae, Termitinae): University of Sao˜ uncover origin and spread of the Wolbachia pandemic. Nat Paulo, 2009. Commun 2014;5:5117. Correa CC, Ballard JWO. Wolbachia associations with insects: Gottlieb Y, Ghanim M, Chiel E et al. Identification and localization winning or losing against a master manipulator. Front Ecol of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae). Evol 2016;3:153. Appl Environ Microbiol 2006;72:3646–52. Darriba D, Taboada GL, Doallo R et al. jModelTest 2: more mod- Heddi A, Vallier A, Anselme C et al. Molecular and cellular els, new heuristics and parallel computing. Nat Methods profiles of insect bacteriocytes: mutualism and harmat 2012;9:772. the initial evolutionary step of symbiogenesis. Cell Microbiol Dedeine F, Bouletreau´ M, Vavre F. Wolbachia requirement 2005;7:293–305. for oogenesis: occurrence within the genus Asobara 12 FEMS Microbiology Ecology, 2019, Vol. 92, No. 2
Heled J, Drummond AJ. Bayesian inference of species trees from Marynowska M, Goux X, Sillam-Dusses` D et al. Optimization multilocus data. Mol Biol Evol 2010;27:570–80. of a metatranscriptomic approach to study the lignocellu- Hellemans S, Bourguignon T, KyjakovaP´ et al. Mitochondrial and lolytic potential of the higher termite gut microbiome. BMC chemical profiles reveal a new genus and species of Neotrop- Genomics 2017;18:681. ical termite with snapping soldiers, Palmitermes impostor (Ter- Mathews AGA. Studies on Termites from the Mato Grosso State,
mitidae: Termitinae). Invertebr Syst 2017a;31:394–405. Brazil. Rio de Janeiro, Brazil: Academia Brasileira de Ciencias,ˆ Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 Hellemans S, Fournier D, Hanus R et al. Secondary queens in 1977, 267. the parthenogenetic termite Cavitermes tuberosus develop Maynard Smith J, Szathmary´ E. The Major Transitions in Evolution. through a transitional helper stage. Evol Dev 2017b;19:253– New York: W.H. Freeman and Company, 1995. 62. Miura T, Roisin Y, Matsumoto T. Molecular phylogeny and Hellemans S, Fournier D, Hanus R et al. Sex ratio varia- biogeography of the nasute termite genus Nasutitermes tions among years and breeding systems in a faculta- (Isoptera: Termitidae) in the Pacific Tropics. Mol Phylogenet tively parthenogenetic termite. Insectes soc In press, DOI Evol 2000;17:1–10. 10.1007/s00040-018-0667-y. Moran NA, McCutcheon JP, Nakabachi A. Genomics and evo- Himler AG, Adachi-Hagimori T, Bergen JE et al. Rapid spread of a lution of heritable bacterial symbionts. Annu Rev Genet bacterial symbiont in an invasive whitefly is driven by fitness 2008;42:165–90. benefits and female bias. Science 2011;332:254–6. Nakabachi A, Yamashita A, Toh H et al. The 160-Kilobase Hosokawa T, Koga R, Kikuchi Y et al. Wolbachia as a bacteriocyte- genome of the bacterial endosymbiont Carsonella. Science associated nutritional mutualist. Proc Natl Acad Sci USA 2006;314:267. 2010;107:769–74. Nikoh N, Hosokawa T, Moriyama M et al. Evolutionary origin Hughes DP. On the origins of parasite-extended phenotypes. of insect-Wolbachia nutritional mutualism. Proc Natl Acad Sci Integr Comp Biol 2014;54:210–7 USA 2014;111:10257–62. Hurst GDD, Frost CL. Reproductive parasitism: maternally inher- Noirot C. The gut of termites (Isoptera). Comparative anatomy, ited symbionts in a biparental world. Cold Spring Harbor Per- systematics, phylogeny. I. Lower termites. Ann Soc Entomol Fr spectives in Biology 2015;7:a017699. 1995;31:197–226. Jiggins FM, von der Schulenburg JHG, Hurst GDD et al. Recombi- Noirot C. The gut of termites (Isoptera). Comparative anatomy, nation confounds interpretations of Wolbachia evolution. Proc systematics, phylogeny. II. Higher termites (Termitidae). Ann Biol Sci 2001;268:1423–7. Soc Entomol Fr 2001;37:431–71. Jones DT, Eggleton P. Global biogeography of termites: a compi- Ohkuma M, Brune A. Diversity, structure and evolution of the lation of sources. In: Bignell DE Roisin Y Lo N (eds.) Biology of termite gut microbial community. In: Bignell DE Roisin Y Termites: A Modern Synthesis. Dordrecht: Springer, 2011, 477– Lo N (eds.) Biology of termites: a modern synthesis. Dordrecht: 98. Springer; 2011, 413–38. Klindworth A, Pruesse E, Schweer T et al. Evaluation of general Oliver KM, Degnan PH, Burke GR et al. Facultative symbionts in 16S ribosomal RNA gene PCR primers for classical and next- aphids and the horizontal transfer of ecologically important generation sequencing-based diversity studies. Nucleic Acids traits. Annu Rev Entomol 2010;55:247–66. Res 2013;41:e1. Pais R, Lohs C, Wu Y et al. The obligate mutualist Wigglesworthia Krishna K, Grimaldi DA, Krishna V et al. Treatise on the Isoptera glossinidia influences reproduction, digestion, and immunity of the world. Bull Am Mus Nat Hist 2013;377:1–2704. processes of its host, the tsetse fly. Appl Environ Microbiol Kuechler SM, Renz P, Dettner K et al. Diversity of symbiotic 2008;74:5965–74. organs and bacterial endosymbionts of Lygaeoid bugs of the Perlman SJ, Hunter MS, Zchori-Fein E. The emerging diversity of families Blissidae and Lygaeidae (Hemiptera: Heteroptera: Rickettsia. Proc Biol Sci 2006;273:2097–106. Lygaeoidea). Appl Environ Microbiol 2012;78:2648–59. Pfaffl MW. A new mathematical model for relative quantification Lach L Parr C Abbott K (eds.) Ant Ecology. Oxford: Oxford Univer- in real-time RT–PCR. Nucleic Acids Res 2001;29:e45. sity Press, 2009, 432. Pike N, Kingcombe R. Antibiotic treatment leads to the elim- Lindsey ARI, Bordenstein SR, Newton ILG et al. Wolbachia pipien- ination of Wolbachia endosymbionts and sterility in the tis should not be split into multiple species: A response to diplodiploid collembolan Folsomia candida. BMC Biol 2009;7:54. Ram´ırez-Puebla et al., “Species in Wolbachia? Proposal for the Pruesse E, Quast C, Knittel K et al. SILVA: a comprehensive designation of ‘Candidatus Wolbachia bourtzisii’, ‘Candidatus online resource for quality checked and aligned ribosomal Wolbachia onchocercicola’, ‘Candidatus Wolbachia blaxteri’, RNA sequence data compatible with ARB. Nucleic Acids Res ‘Candidatus Wolbachia brugii’, ‘Candidatus Wolbachia taylori’, 2007;35:7188–96. ‘Candidatus Wolbachia collembolicola’ and ‘Candidatus Wol- R Development Core Team. R: A Language and Environment for Sta- bachia multihospitum’ for the different species within Wol- tistical Computing. Vienna, Austria: R Foundation for Statisti- bachia supergroups”. Syst Appl Microbiol 2016a;39:220–2. cal Computing; 2013. Lindsey ARI, Werren JH, Richards S et al. Comparative genomics Rabeling C, Kronauer DJC. Thelytokous parthenogenesis in euso- of a parthenogenesis-inducing Wolbachia symbiont. G3: cial Hymenoptera. Annu Rev Entomol 2013;58:273–92. Genes, Genomes, Genetics 2016b;6:2113–23. Rambaut A, Drummond AJ, Xie D et al. Posterior summarisa- Lo N, Evans TA. Phylogenetic diversity of the intracellular sym- tion in Bayesian phylogenetics using Tracer 1.7. Syst Biol biont Wolbachia in termites. Mol Phylogenet Evol 2007;44:461–6. 2018;67:901–4. Ma WJ, Schwander T. Patterns and mechanisms in instances Roy V, Harry M. Diversity of Wolbachia isolated from the of endosymbiont-induced parthenogenesis. J Evol Biol Cubitermes sp. affinis subarquatus complex of species (Termi- 2017;30:868–88. tidae), revealed by multigene phylogenies. FEMS Microbiol Let Martius C. The termites. In: Junk WJ (ed.) The Central Amazon 2007;274:102–11. Floodplain. Berlin: Springer-Verlag, 1997, 361–71. Rust J, Wappler T. Palaeontology: the point of no return in the fossil record of eusociality. Curr Biol 2016;26:R159–R61. Hellemans et al. 13
Sabree ZL, Kambhampati S, Moran NA. Nitrogen recycling and Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for nutritional provisioning by Blattabacterium,thecockroach simple extraction of DNA for PCR-based typing from forensic endosymbiont. Proc Natl Acad Sci USA 2009;106:19521–6. material. BioTechniques 1991;10:506–13. Sakamoto JM, Feinstein J, Rasgon JL. Wolbachia infections in the Wang Q, Garrity GM, Tiedje JM et al. Naive Bayesian classifier for Cimicidae: museum specimens as an untapped resource for rapid assignment of rRNA sequences into the new bacterial
endosymbiont surveys. Appl Environ Microbiol 2006;72:3161–7. taxonomy. Appl Environ Microbiol 2007;73:5261–7. Downloaded from https://academic.oup.com/femsec/article-abstract/95/2/fiy235/5247714 by University of British Columbia Library user on 04 January 2019 Salunke BK, Salunkhe RC, Dhotre DP et al. Diversity of Wol- Weeks AR, Breeuwer JAJ. Wolbachia-induced parthenogenesis bachia in Odontotermes spp. (Termitidae) and Coptotermes in a genus of phytophagous mites. Proc R Soc Lond, B, Biol heimi (Rhinotermitidae) using the multigene approach. FEMS 2001;268:2245–51. Microbiol Let 2010;307:55–64. Weeks AR, Reynolds KT, Hoffman AA. Wolbachia dynamics and Schloss PD, Westcott SL, Ryabin T et al. Introducing mothur: open- host effects: what has (and has not) been demonstrated? source, platform-independent, community-supported soft- Trends Ecol Evol 2002;17:257–62. ware for describing and comparing microbial communities. Weeks AR, Turelli M, Harcombe WR et al. From parasite to mutu- Appl Environ Microbiol 2009;75:7537–41. alist: rapid evolution of Wolbachia in natural populations of Shigenobu S, Watanabe H, Hattori M et al. Genome sequence of Drosophila. PLoS Biol 2007;5:e114. the endocellular bacterial symbiont of aphids Buchnera sp. Weeks AR, Velten R, Stouthamer R. Incidence of a new sex-ratio- APS. Nature 2000;407:81–6. distorting endosymbiotic bacterium among arthropods. Proc Sicard M, Dittmer J, Greve` P et al. A host as an ecosystem: Wol- R Soc Lond, B, Biol 2003;270:1857–65. bachia coping with environmental constraints. Environ Micro- Weinert LA, Araujo-Jnr EV, Ahmed MZ et al. The incidence of bac- biol 2014;16:3583–607. terial endosymbionts in terrestrial arthropods. Proc Biol Sci Simoes˜ PM, Mialdea G, Reiss D et al. Wolbachia detection: an 2015;282:20150–249. assessment of standard PCR Protocols. Mol Ecol Resources Werren JH, Baldo L, Clark ME. Wolbachia: master manipulators of 2011;11:567–72. invertebrate biology. Nat Rev Microbiol 2008;6:741–51. Simon C, Frati F, Beckenbach A et al. Evolution, weighting, Werren JH, Windsor DM. Wolbachia infection frequencies in and phylogenetic utility of mitochondrial gene sequences insects: evidence of a global equilibrium. Proc R Soc Lond, B, and a compilation of conserved polymerase chain reaction Biol 2000;267:1277–85. primers. Ann Entomol Soc Am 1994;87:651–701. Yu G, Smith DK, Zhu H et al. ggtree: anrpackage for visualization Simonet P, Gaget K, Balmand S et al. Bacteriocyte cell death in and annotation of phylogenetic trees with their covariates the pea aphid Buchnera symbiotic system. Proc Natl Acad Sci and other associated data. Methods Ecol Evol 2017;8:28–36. USA 2018;115:E1819–E28. Zchori-Fein E, Perlman SJ, Kelly SE et al. Characterization of Sloan DB, Moran NA. Endosymbiotic bacteria as a source of a‘Bacteroidetes’ symbiont in Encarsia wasps (Hymenoptera: carotenoids in whiteflies. Biol Lett 2012;8:986–9. Aphelinidae): proposal of ‘Candidatus Cardinium hertigii’. Int Suomalainen E, Saura A, Lokki J. Cytology and Evolution in J Syst Evol Microbiol 2004;54:961–8. Parthenogenesis. Boca Raton, FL: CRC Press; 1987. Zug R, Hammerstein P. Bad guys turned nice? A critical assess- Treanor D, Pamminger T, Hughes WOH. The evolution of caste- ment of Wolbachia mutualisms in arthropod hosts. Biol Rev biasing symbionts in the social hymenoptera. Insectes Soc 2015;90:89–111. 2018;65:513–9. Zug R, Koehncke A, Hammerstein P. Epidemiology in evolu- van der Kooi CJ, Schwander T. Evolution of asexuality via tionary time: the case of Wolbachia horizontal transmission different mechanisms in grass thrips (Thysanoptera: between arthropod host species. J Evol Biol 2012;25:2149–60. Aptinothrips). Evolution 2014;68:1883–93. Symbiosis between Wolbachia and a termite
Bacteriome-associated Wolbachia of the parthenogenetic termite Cavitermes tuberosus
Electronic Supplementary Materials
Running title: Symbiosis between Wolbachia and a termite
Simon Hellemans1,†,*, Nicolas Kaczmarek1,†,*, Martyna Marynowska2, Magdalena
Calusinska2, Yves Roisin1, Denis Fournier1,*
1Evolutionary Biology & Ecology, Université libre de Bruxelles, Avenue F.D. Roosevelt 50,
CP 160/12, B-1050 Brussels, Belgium.
2Environmental Research and Innovation Department, Luxembourg Institute of Science and
Technology, L-4422 Belvaux, Luxembourg.
*Corresponding authors: [email protected], [email protected],
†These authors contributed equally to this study.
1 Symbiosis between Wolbachia and a termite
Supplementary Materials and Methods
SM-1 Termite species identity – mitochondrial barcode gene COII
Amplification was carried in 25 µl reactions containing 0.1 µl (0.5 U) TopTaq DNA polymerase
(Qiagen), 2.5 µl 10x TopTaq PCR Buffer, 1 µl 25 mM MgCl2, 0.6 µl dNTP mix (10 mM of each), 0.25 µl of each forward and reverse primer (20 µM of each), 1.5 µl of template DNA and PCR-grade water (q.s.). Cycling conditions were as follow: an initial denaturing step at
94°C for 3 min; 35 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; with a final extension step at 72°C for 10 min.
Amplicons were sequenced with BigDye Terminator Cycle Sequencing kit v3.1. (Applied
Biosystems) in 11.2 µl reactions containing 1.0 µl BigDye, 2.1 µl 5x Sequencing Buffer, 0.1 µl of forward or reverse primer (20 µM of each), 3-8 µl of amplicon and PCR-grade water (q.s.).
Cycling conditions were as follow: an initial denaturing step at 96°C for 1 min, 25 cycles of denaturing at 96°C for 10 s, annealing at 50°C for 5 s, and extension at 60°C for 4 min.
SM-2 Screening of endosymbionts – mitochondrial gene COI
We used the same amplification conditions as in SM-1, with the following cycling conditions: an initial denaturation step at 94°C for 2 min; 40 cycles of denaturation at 94°C for 1 min, annealing at 47°C for 1 min, and extension at 72°C for 75 s; with a final extension step at 72°C for 7 min.
SM-3 Strain identification of Wolbachia – MLST genes
We used the same amplification conditions as in SM-1. Cycling conditions of MLST genes were performed in two sets: an initial denaturation step at 94°C for 2 min; 36 cycles of denaturation at 94°C for 30 s, annealing at 54°C (coxA, ftsZ, gatB and hcpA) or at 59°C (fbpA) for 45 s, and extension at 72°C for 90 s; with a final extension step at 72°C for 10 min (Simões et al. 2011). Amplicons were sequenced as in SM-1.
2 Symbiosis between Wolbachia and a termite
SM-4 Wolbachia infection and impacts on phenotype - Biotine BioA
We used the same amplification conditions as in SM-1, using the primers designed by (Gerth and Bleidorn 2016) to amplify the BioA gene. Cycling conditions were as follow: an initial denaturation step at 95°C for 2 min; 35 cycles of denaturation at 95°C for 1 min, annealing at
51°C for 1 min, and extension at 72°C for 1 min; with a final extension step at 72°C for 5 min.
Amplicons were sequenced as in SM-1.
SM-5 Quantification of Wolbachia densities across tissues using quantitative PCR
Amplifications were carried in 20 µl reactions containing 10 µl SYBR Premix Ex Taq
(TaKaRa), 0.8 µl of each forward and reverse primer (10 µM of each), 2 µl of template DNA and PCR-grade water (q.s.). Cycling conditions were as follow: an initial denaturing step at
95°C for 1 min 45s; 40 cycles of denaturing at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min.
Expression ratios were calculated using the mean threshold cycle (Ct) values of sample triplicate and primer efficiencies (E) per tissue using the equation (1) slightly modified from
Pfaffl (2001):
89:;<=>?@ (/0123456) Expression ratio = 89EFGH>? (/ABCD45)
We used hindlegs as a control in order to allow comparisons between head, ovaries, bacteriome-like structure (i.e., the anterior part of the mesenteron) and the digestive tube without its anterior part.
3 Symbiosis between Wolbachia and a termite
Supplementary Figure
1000 Infected and Sexually-produced Infected and Parthenogenetically-produced 800 Non infected and Parthenogenetically-produced Non infected and Sexually-produced K100 Infected 600 K100 Non infected
400
200
0 -500 0 500 1000 1500 2000 2500 19.08% -200
-400
-600
-800
-1000 70.48% Figure S1 Factorial correspondence analysis on allelic frequencies in females of C. tuberosus from the reproductive caste.
4 Symbiosis between Wolbachia and a termite
) n 90.63 (32) 95.65 (46) 97.50 (40) 54.76 (42) Prevalence per nest % ( 100 (41) 100 (72) 100 (40) 100 (31) 100 (42) 100 (34) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 8/8 8/8 8/8 5/10 6/6 2/2 2/2 Female neotenics Female 10/10 / / / / / / / / / 8/8 8/8 8/8 7/7 10/10 Female aspirants Female / / / / 1/1 1/1 1/1 1/1 1/1 1/1 Primary king / / / / / / 8/8 8/8 7/8
10/10 infection in the nest. the in infection Male alates / / / 8/8 8/8 7/7 7/8 5/10 10/10 Male nymphs (stage 5) / / / / / / / 0/1 1/1 1/1 1/1 1/1 Primary queen screened) / / / / n 8/8 8/8 2/2 2/2 2/2 2/2 10/10 12/12 10/10 Female alates Female infected/ n / / / / 8/8 8/8 7/8 2/2 2/10 10/10 Reproductive caste Reproductive 5) (stage nymphs Female / / / 0/2 8/8 8/8 8/8 8/8 8/8 10/10 10/10 Soldiers 0/8 0/8 0/8 0/8 0/8 0/8 8/9 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 7/8 7/8 7/8 5/5 6/6 6/8 10/10 10/10 Infection per developmental stage ( Altruistic caste Workers screened) n 5/5 5/5 infected/ n ( Infection eggs ) n PSR (
0.480 (50) 0.523 (44) 0.463 (54) 0.545 (55) 0.511 (47) 0.615 (52) 0.561 (57) 0.694 (49) 0.431 (51) 0.533 (45) 0.444 (45) Supplementary Tables Supplementary Strain, BioA Strain, BioA Strain, BioA Strain, BioA Strain, BioA Use Infection rate, Infection strain, BioA rate, Infection strain, BioA rate, Infection strain, BioA rate, Infection strain, BioA, qPCR rate, Infection strain, BioA rate, Infection strain, BioA rate, Infection strain, BioA rate, Infection strain, BioA, PSR rate, Infection strain, BioA, PSR rate, Infection strain, BioA, PSR PSR PSR PSR PSR PSR PSR PSR, statusInfection PSR, statusInfection Horizontal transfer Horizontal transfer Horizontal transfer Horizontal transfer Horizontal transfer (thrips) Illumina 16S rRNA sequencing status,Infection strain, BioA status,Infection strain, BioA status,Infection strain, BioA status,Infection strain, BioA status,Infection strain, BioA Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction strain Parthenogenesis induction, Horizontal transfer Horizontal transfer Horizontal transfer Horizontal transfer transfer, Horizontal strain Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Horizontal transfer Horizontal transfer Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction Parthenogenesis induction January 2012 January 2012 January 2016 June 2017 January 2017 January 2012 January 2017 January 2017 January 2017 January 2017 January 2016 June 2017 January 2018 July 2017 January 2017 January 2017 January 2017 January 2017 January 2016 June 2016 June 2016 June 2017 January 2017 January 2017 January 2016 June April April 2017 April 2015 September 2012 September 2012 September Collection date October 2014 October 2014 October 2014 October 2014 October 2014 October 2014 October 2014 October 2014 October 2014 October 2014 October November 2015 November 2015 November 2015 November 2015 November 2015 November May 2018 May 2018 May 2018 May 2018 March 2014 March 2014 March 2014 March 2014 March 2014 March 2014 2015 November 2015 November 2015 November 2015 November 52.98158 53.02177 53.05422 52.97978 52.97992 52.98003 52.97865 52.15177 53.05362 52.96523 52.98093 53.02652 52.97948 52.99858 53.05597 52.99853 52.99802 52.97060 52.97713 52.99847 53.02787 52.97902 53.05142 52.99890 52.96745 52.99898 52.99892 52.97960 52.98043 53.1997 53.20393 53.20417 52.97928 53.05327 53.05313 52.96453 53.0535 52.979 53.05528 52.97058 52.99875 52.99898 52.99892 52.97905 52.9791 52.97227 52.99778 52.99792 52.99890 52.96745 52.9707 53.0548 52.97967 52.97975 52.97198 52.97960 34.84388 61.31548 Longitude (W) Longitude 4.54331 -7.13833 5.06808 5.07612 5.06828 5.07387 5.07323 5.07308 5.07005 5.07007 5.11993 5.06843 5.07642 5.06987 5.06712 5.07257 5.06702 5.06533 5.04693 5.06060 5.06725 5.07157 5.07000 5.09885 5.06455 5.11048 5.06448 5.06393 5.07257 5.06563 5.06945 5.06892 5.06938 5.08885 5.0699 5.06942 5.07022 5.04727 5.0653 5.06448 5.06393 5.07062 5.0715 5.07683 5.06625 5.06633 5.06455 5.11048 5.04622 5.06942 5.06977 5.0698 5.04537 5.07368 3.61953 3.6369 3.63677 Latitude (N)Latitude 10.7297 S. heyeri S. heyeri S. minutus S. minutus S. minutus S. minutus S. minutus S. trispinosus Species I. inquilinus taracua N. taracua N. P. impostor P. impostor P. impostor P. impostor P. impostor E. neotenicus E. neotenicus E. neotenicus E. neotenicus E. neotenicus E. L. labralis L. labralis L. labralis
C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus C. tuberosus Collection Collection data on nests used in this study with GPS coordinates, use of the nest in 1 Nest K_100 M_82 M_83 M_90 I_56 K_81 N_96 F_67 H_108 R_150 B_17 BR18-06 M_027 J_70 TT18-59 A_95 A_43 A_138 A_147 S_156a S_156b G_88 G18-156 G18-175b G18-179 G14-125 G14-41 G14-54 G14-27 G14-64 G14-111 G17-038 G17-043 G17-062 G17-050 G17-05 G15-251 G15-253 G15-89 G16-125 C_119 C_136 C_143 C_118 C_153a C_154a C_158a C_154b C_158b C_029 C_030 C_153b O_98 O_113 O_99 Q_126 O_008 O_001 Table Table S Region UFPB, MataBiolério do Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Kaw-Roura National Nature Reserve Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area road Lopinot Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Petit Saut dam area Saül Saül Saül the the work, primary sex ratio (PSR), infection per developmental stage and prevalence of Country French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Tobago and Trinidad of Republic Brazil Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French Guiana French
5 Symbiosis between Wolbachia and a termite
Table S2 Information on sequences generated by Sanger sequencing and deposited in GenBank.
Genome w Strain Gene Country Nest Isolation source GenBank ID mitochondrial NA COII Brazil BR18-06 Cavitermes tuberosus MK053589 mitochondrial NA COII French Guiana K_100 Cavitermes tuberosus MF953241 mitochondrial NA COII French Guiana O_98 Cavitermes tuberosus MF953242 mitochondrial NA COII Trinidad and Tobago TT18-59 Cavitermes tuberosus MH522792 mitochondrial NA COI French Guiana A_138 ectoparasitic Thysanoptera sp. MF953240 Wolbachia w Ctub 16S rRNA French Guiana O_98 Cavitermes tuberosus MF953226 Wolbachia w Ctub bioA French Guiana O_98 Cavitermes tuberosus MK053590 Wolbachia w Ctub coxA French Guiana O_98 Cavitermes tuberosus MF953228 Wolbachia w Ctub fbpA French Guiana O_98 Cavitermes tuberosus MF953231 Wolbachia w Ctub ftsZ French Guiana O_98 Cavitermes tuberosus MF953230 Wolbachia w Ctub gatB French Guiana O_98 Cavitermes tuberosus MF953227 Wolbachia w Ctub hcpA French Guiana O_98 Cavitermes tuberosus MF953229 Wolbachia w Ctub coxA Trinidad and Tobago TT18-59 Cavitermes tuberosus MK053591 Wolbachia w Ctub fbpA Trinidad and Tobago TT18-59 Cavitermes tuberosus MH522793 Wolbachia w Pip 16S rRNA NA NA Culex pipiens pipiens MF953232 Wolbachia w Pip coxA NA NA Culex pipiens pipiens MF953234 Wolbachia w Pip fbpA NA NA Culex pipiens pipiens MF953237 Wolbachia w Pip ftsZ NA NA Culex pipiens pipiens MF953236 Wolbachia w Pip gatB NA NA Culex pipiens pipiens MF953233 Wolbachia w Pip hcpA NA NA Culex pipiens pipiens MF953235 Wolbachia w Iinq coxA French Guiana G14-111 Inquilinitermes inquilinus MK064568 Wolbachia w Iinq gatB French Guiana G14-111 Inquilinitermes inquilinus MK064569 Wolbachia w Iinq hcpA French Guiana G14-111 Inquilinitermes inquilinus MK064570 Wolbachia w Ncap 16S rRNA French Guiana C_158b Neocapritermes taracua MF953238 Wolbachia w Ncap coxA French Guiana C_158b Neocapritermes taracua MF953239
6 Symbiosis between Wolbachia and a termite
References (2006) al. et Baldo (2006) al. et Baldo study This (2006) al. et Baldo (2006) al. et Baldo (2006) al. et Baldo (2006) al. et Baldo (2006) al. et Baldo Lindsey et al. (2016) (2006) al. et Baldo (2009) al. et Russell (2010) al. et Stahlhut & Bordenstein Rosengaus (2005), et Bordenstein al. (2009), databasePubMLST (2006) al. et Baldo study This (2010) al. et Salunke (2010) al. et Salunke (2010) al. et Salunke (2010) al. et Salunke PubMLST database PubMLST database PubMLST database PubMLST database PubMLST database Darby et al. (2012) (2006) al. et Baldo (2014) al. et Gerth Genome ------LKEQ00000000 ------HE660029 - CR925678 fbpA DQ842378 DQ842359 MF953237 DQ842351 DQ842361 DQ842349 DQ842367 DQ842371 - DQ842376 EU127822 EU126428 69 DQ842366 MF953231 GQ422843 GQ422842 GQ422841 GQ422844 33 31 32 57 70 - DQ842347 - ftsZ DQ842340 DQ842323 MF953236 DQ842313 DQ842322 DQ842311 DQ842329 DQ842333 - DQ842338 EU127715 EU126324 AY764283 DQ842328 MF953230 GQ422852 GQ422851 GQ422850 GQ422853 48 27 28 60 48 - DQ842341 - hcpA DQ842415 DQ842396 MF953235 DQ842388 DQ842398 DQ842386 DQ842404 DQ842408 - DQ842413 EU127660 EU126267 FJ390174 DQ842403 MF953229 GQ422872 GQ422870 GQ422869 GQ422873 35 34 36 58 72 - DQ842384 - PubMLST ID coxA DQ842304 DQ842285 MF953234 DQ842277 DQ842287 DQ842275 DQ842293 DQ842297 - DQ842302 EU127606 EU126210 FJ390248 DQ842292 MF953228 GQ422835 GQ422833 GQ422832 GQ422836 31 30 30 56 55 - DQ842273 - GenBank accession or or accession GenBank gatB DQ842452 DQ842433 MF953233 DQ842425 DQ842435 DQ842423 DQ842441 DQ842445 - DQ842450 EU127714 EU126153 64 DQ842440 MF953227 GQ422862 GQ422861 GQ422860 GQ422863 30 29 31 29 30 - DQ842421 - Mel Ri Pip Pip No Cle Uni VitB Tpre Zoo Ctub Oo Bm Strain w w w w w w w w w TdeiB Opic Apal w Isny w Ohor_T21 Ohor_T1 Ohor_RA Osp_T3 Ogra Ocap Olat Olit Ocha w w Welgevonden Wolbachia Supergroup A A B B B F A B B B F F H A F F F F F F F F F F C D / Accession phylogeny. numbers of in Accession genes used the 3
Table S Order Diptera Diptera Diptera Diptera Diptera Hemiptera Hymenoptera Hymenoptera Hymenoptera Hymenoptera Hymenoptera Hymenoptera Isoptera, Archotermopsidae Isoptera, Kalotermitidae Isoptera, Termitidae Isoptera, Termitidae Isoptera, Termitidae Isoptera, Termitidae Isoptera, Termitidae Scorpiones Scorpiones Scorpiones Scorpiones Scorpiones Spirurida Spirurida Rickettsiale Phylum Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Arthropoda Nematoda Nematoda Proteobacteria (control) sp. sp.
Host Species Drosophila melanogaster Drosophila simulans Culex pipiens pipiens Culex pipiens pipiens Drosophila simulans Cimex lectularius Muscidifurax uniraptor vitripennis Nasonia pretiosum Trichogramma deion Trichogramma picardi Ocymyrmex Apoica angusticollis Zootermopsis Incisitermes snyderi Cavitermes tuberosus horni Odontotermes horni Odontotermes horni Odontotermes Odontotermes granifrons Opistophthalmus capensis Opistophthalmus latimanus Opistophthalmus litoralis Opistophthalmus chaperi Opistophthalmus ochengi Onchocerca malayi Brugia ruminantium Ehrlichia
7 Symbiosis between Wolbachia and a termite
Table S4 Results from model comparison of the infection status versus nest, caste, sex and reproductive origin of the individuals, with and without including interactions using maximum likelihood estimate of the model (log10L), Akaike’s information criterion (AIC), delta AIC (ΔAIC) and Akaike weights.
Model log10L AIC ΔAIC Relative likelihood Akaike weights Nest -51.78 123.60 65.64 5.58E-15 5.57E-15 Caste -92.60 189.20 131.24 3.17E-29 3.17E-29 Sex -74.35 152.70 94.74 2.68E-21 2.67E-21 Origin -49.33 102.70 44.74 1.93E-10 1.93E-10 Nest + Caste -51.12 124.20 66.24 4.13E-15 4.13E-15 Nest + Sex -34.01 90.10 32.14 1.05E-07 1.05E-07 Nest + Origin -17.98 57.96 0 1 1 Nest + Caste + Nest *Caste -47.64 135.30 77.34 1.61E-17 1.61E-17 Nest + Sex + Nest *Sex -32.36 104.70 46.74 7.09E-11 7.08E-11 Nest + Origin + Nest *Origin -17.58 73.17 15.21 4.98E-04 4.98E-04
8 Symbiosis between Wolbachia and a termite
Table S5 Microbial community profiles of whole guts of workers (gut) and bacteriome-like structures of neotenics (bls) of C. tuberosus (nest A_147) based on the 16S rRNA gene amplicon high-throughput sequencing – Taxonomic distribution of prokaryotic OTUs into major phyla (annotated according to SILVA database v.128) and their relative abundance including standard deviation for triplicates.
Host Average relative abundance (%) Standard deviation* Phylum relative abundance bls gut bls gut Unclassified 0.00 16.45 0.00 0.76 Other 0.03 2.96 0.04 0.11 Tenericutes 0.00 1.08 0.00 0.12 Spirochaetae 0.07 24.15 0.07 5.33 Wolbachia (Proteobacteria) 97.29 0.17 0.66 0.04 Other Proteobacteria 0.50 3.30 0.07 0.40 Planctomycetes 0.00 2.05 0.00 0.07 Firmicutes 0.04 38.74 0.04 6.41 Fibrobacteres 0.02 2.59 0.02 1.77 Bacteroidetes 1.96 7.59 0.58 0.60 Actinobacteria 0.08 0.92 0.02 0.04
*Derived from triplicates
9 Symbiosis between Wolbachia and a termite
Supplementary File
Appendix S1 Genotypes at 17 microsatellite markers of C. tuberosus used to determine the reproductive origin of females (s: sexually produced; p: parthenogenetically produced) with their infection status by Wolbachia (1: infected; 0: uninfected).
10 Reproductive Pop Nest Sample name Developmental stage Caste Genotype source Infection status origin Ctub21 Ctub42 Ctub43 Ctub45 Ctub60 Ctub70 Ctub72 Ctub74 Ctub77 Ctub80 Ctub84 Ctub85 Ctub86 Ctub90 Ctub91 Ctub94 Ctub95 A A_95 A_95_n3f_1 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 175 175 245 245 121 121 144 144 129 129 149 149 293 293 236 236 163 163 112 112 101 101 175 175 103 103 0 0 A A_95 A_95_n3f_2 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 175 175 245 245 131 131 146 146 129 129 168 168 280 280 250 250 163 163 103 103 101 101 175 175 103 103 0 0 A A_95 A_95_n3f_3 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 175 175 239 239 131 131 144 144 129 129 149 149 293 293 236 236 163 163 103 103 101 101 172 172 103 103 0 0 A A_95 A_95_n3f_4 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 175 175 239 239 131 131 144 144 129 129 149 149 280 280 250 250 163 163 112 112 119 119 175 175 103 103 0 0 A A_95 A_95_n3f_5 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 169 169 245 245 131 131 144 144 129 129 149 149 293 293 236 236 163 163 112 112 119 119 172 172 103 103 0 0 A A_95 A_95_n3f_6 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 169 169 245 245 131 131 146 146 129 129 168 168 280 280 250 250 163 163 103 103 119 119 172 172 103 103 0 0 A A_95 A_95_n3f_8 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 148 148 169 169 239 239 131 131 146 146 129 129 168 168 293 293 236 236 163 163 103 103 119 119 172 172 103 103 0 0 A A_95 A_95_if-9 Female alate Female disperser This study 1 s 165 191 247 247 134 148 156 175 239 242 131 131 144 148 129 129 149 149 280 282 250 269 163 163 110 112 101 119 175 206 103 103 0 0 A A_95 A_95_if-10 Female alate Female disperser This study 1 s 165 195 247 247 134 148 156 175 239 245 131 134 144 148 129 129 149 149 280 282 250 269 163 163 112 112 101 119 169 172 103 103 0 0 A A_95 A_95_if-11 Female alate Female disperser This study 1 s 165 191 247 247 134 148 156 169 242 245 131 131 146 146 129 129 145 168 293 293 236 267 163 163 110 112 101 119 169 172 103 103 0 0 A A_95 A_95_if-12 Female alate Female disperser This study 1 s 165 195 247 247 134 148 0 0 0 0 0 0 146 148 129 129 149 168 0 0 250 267 163 163 103 112 101 101 0 0 103 103 0 0 A A_95 A_95_if-13 Female alate Female disperser This study 1 s 165 195 247 247 134 148 169 172 239 245 121 134 146 148 129 129 149 168 282 293 236 269 163 163 112 112 101 119 169 172 103 103 0 0 A A_95 A_95_if-14 Female alate Female disperser This study 1 s 165 195 247 247 134 148 172 175 239 242 121 134 146 146 129 129 145 149 280 282 250 269 163 163 110 112 101 101 169 175 103 103 0 0 A A_95 A_95_if-15 Female alate Female disperser This study 1 s 165 195 0 0 0 0 0 0 0 0 0 0 0 0 129 129 149 149 0 0 0 0 163 163 103 112 101 101 0 0 103 103 0 0 A A_95 A_95_if-16 Female alate Female disperser This study 1 s 165 195 247 247 0 0 0 0 0 0 0 0 0 0 129 129 149 168 0 0 236 267 163 163 112 112 119 119 0 0 103 103 0 0 A A_95 A_95_if-17 Female alate Female disperser This study 1 s 165 191 247 247 134 148 172 175 239 242 121 131 144 148 129 129 149 149 280 293 250 267 163 163 103 112 101 119 169 172 103 103 0 0 A A_95 A_95_if-18 Female alate Female disperser This study 1 s 165 191 247 247 134 148 156 175 0 0 121 131 144 146 0 0 0 0 0 0 0 0 0 0 103 112 0 0 0 0 103 103 0 0 A A_95 A_95_Nf_1 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 247 247 148 148 175 175 245 245 121 121 144 144 129 129 149 149 293 293 236 236 163 163 112 112 101 101 172 172 103 103 0 0 A A_95 A_95_Nf_2 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 247 247 148 148 169 169 245 245 131 131 144 144 129 129 149 149 280 280 250 250 163 163 112 112 119 119 172 172 103 103 0 0 G G_88 if_88_n3f_1 Female aspirant Female aspirant This study 1 p 165 165 247 247 148 148 0 0 245 245 134 134 146 146 129 129 168 168 0 0 271 271 163 163 103 103 123 123 0 0 103 103 0 0 G G_88 if_88_n3f_2 Female aspirant Female aspirant This study 1 p 191 191 247 247 148 148 166 166 245 245 134 134 146 146 129 129 168 168 293 293 271 271 163 163 103 103 123 123 209 209 103 103 0 0 G G_88 if_88_n3f_3 Female aspirant Female aspirant This study 1 p 165 165 247 247 134 134 166 166 245 245 121 121 144 144 129 129 168 168 293 293 267 267 163 163 103 103 101 101 172 172 103 103 0 0 G G_88 if_88_n3f_4 Female aspirant Female aspirant This study 1 p 165 165 254 254 148 148 0 0 245 245 121 121 144 144 129 129 168 168 293 293 267 267 163 163 103 103 123 123 172 172 103 103 0 0 G G_88 if_88_n3f_5 Female aspirant Female aspirant This study 1 p 191 191 254 254 148 148 0 0 245 245 121 121 144 144 129 129 168 168 293 293 271 271 163 163 103 103 101 101 209 209 103 103 0 0 G G_88 if_88_n3f_6 Female aspirant Female aspirant This study 1 p 165 165 254 254 0 0 0 0 0 0 0 0 144 144 129 129 168 168 0 0 0 0 163 163 0 0 101 101 0 0 103 103 0 0 G G_88 if_88_n3f_7 Female aspirant Female aspirant This study 1 p 191 191 247 247 148 148 166 166 245 245 134 134 144 144 129 129 168 168 293 293 271 271 163 163 103 103 123 123 209 209 103 103 0 0 G G_88 if_88_n3f_8 Female aspirant Female aspirant This study 1 p 191 191 247 247 148 148 166 166 245 245 134 134 146 146 129 129 168 168 293 293 271 271 163 163 103 103 123 123 172 172 103 103 0 0 G G_88 if_88_n3f_9 Female aspirant Female aspirant This study 1 p 191 191 254 254 148 148 0 0 245 245 121 121 146 146 129 129 168 168 293 293 271 271 163 163 103 103 101 101 209 209 103 103 0 0 G G_88 if_88_n3f_10 Female aspirant Female aspirant This study 1 p 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 103 103 0 0 0 0 103 103 0 0 G G_88 if_88_if_11 Female alate Female disperser This study 1 s 165 191 247 254 148 148 156 166 245 245 121 121 144 146 114 129 149 168 293 293 250 267 163 163 103 112 101 119 169 209 103 103 0 0 G G_88 if_88_if_12 Female alate Female disperser This study 1 s 191 195 247 247 148 148 166 172 245 245 121 134 144 144 129 129 149 168 276 293 265 271 163 163 103 112 101 123 169 172 103 103 0 0 G G_88 if_88_if_1 Female alate Female disperser This study 1 s 165 195 247 247 134 148 166 172 245 245 115 115 144 144 129 129 149 168 293 293 250 267 163 163 103 112 119 123 209 221 103 103 0 0 G G_88 if_88_if_2 Female alate Female disperser This study 1 s 165 195 247 254 148 148 166 172 245 245 115 127 144 146 114 129 149 168 293 293 250 267 163 163 103 112 101 119 169 209 103 103 0 0 G G_88 if_88_if_3 Female alate Female disperser This study 1 s 165 191 247 247 134 148 156 166 245 245 115 115 144 146 114 129 149 168 276 293 265 271 163 163 103 112 101 123 169 172 103 103 0 0 G G_88 if_88_if_4 Female alate Female disperser This study 1 s 191 191 247 247 148 148 172 172 245 245 115 115 144 144 129 129 149 168 276 293 265 271 163 163 103 112 101 123 169 172 103 103 0 0 G G_88 if_88_if_5 Female alate Female disperser This study 1 s 191 195 247 247 134 134 156 166 245 245 115 115 144 146 114 129 149 168 0 0 265 267 163 163 103 112 101 123 0 0 103 103 0 0 G G_88 if_88_if_6 Female alate Female disperser This study 1 s 191 195 247 247 134 148 156 166 245 245 115 127 144 146 129 129 168 168 276 293 265 267 163 163 103 112 119 123 169 172 103 103 0 0 G G_88 if_88_if_7 Female alate Female disperser This study 1 s 165 195 247 247 134 134 156 166 245 245 115 127 144 144 114 129 149 168 0 0 265 271 163 163 103 112 119 123 172 172 103 103 0 0 G G_88 if_88_if_8 Female alate Female disperser This study 1 s 191 191 247 247 148 148 156 166 245 245 115 127 144 144 114 129 149 168 276 293 265 267 163 163 103 112 119 123 169 172 103 103 0 0 G G_88 if_88_if_9 Female alate Female disperser This study 1 s 165 191 247 247 134 148 156 156 245 245 121 134 144 144 114 129 168 168 293 293 250 267 163 163 103 112 119 123 172 221 103 103 0 0 G G_88 if_88_if_10 Female alate Female disperser This study 1 s 191 195 247 247 134 148 156 166 245 245 121 121 144 144 129 129 168 168 276 293 265 271 163 163 103 112 101 119 172 221 103 103 0 0 G G_88 if_88_n5f_1 Female nymph 5th instar Female disperser This study 1 s 165 195 247 247 148 148 156 166 245 245 121 134 144 146 114 129 149 168 293 293 250 267 163 163 103 112 101 119 169 172 103 103 0 0 G G_88 if_88_n5f_2 Female nymph 5th instar Female disperser This study 1 s 165 191 247 247 134 148 156 166 245 245 121 121 144 146 114 129 168 168 293 293 250 271 163 163 103 112 119 123 209 221 103 103 0 0 G G_88 if_88_n5f_3 Female nymph 5th instar Female disperser This study 1 s 191 191 247 247 134 148 166 172 245 245 121 134 144 146 114 129 168 168 276 293 265 267 163 163 103 112 119 123 209 221 103 103 0 0 G G_88 if_88_n5f_4 Female nymph 5th instar Female disperser This study 1 s 191 191 247 254 134 134 156 156 245 245 121 121 144 146 129 129 149 168 293 293 250 271 163 163 103 112 119 123 169 172 103 103 0 0 G G_88 if_88_n5f_5 Female nymph 5th instar Female disperser This study 1 s 191 191 247 247 148 148 166 172 245 245 121 121 144 144 129 129 149 168 293 293 250 267 163 163 103 112 101 123 169 209 103 103 0 0 G G_88 if_88_n5f_6 Female nymph 5th instar Female disperser This study 1 s 191 195 247 247 134 148 166 172 245 245 121 121 144 144 129 129 0 0 0 0 0 0 163 163 103 112 101 101 169 209 103 103 0 0 G G_88 if_88_n5f_7 Female nymph 5th instar Female disperser This study 1 s 165 191 247 247 148 148 166 172 245 245 121 134 144 146 0 0 0 0 0 0 0 0 0 0 103 112 101 123 172 172 103 103 0 0 G G_88 if_88_n5f_8 Female nymph 5th instar Female disperser This study 1 s 191 191 247 254 134 148 166 172 245 245 121 121 144 146 114 129 149 168 276 293 265 271 163 163 103 112 101 119 172 221 103 103 0 0 G G_88 if_88_n5f_9 Female nymph 5th instar Female disperser This study 1 s 165 191 247 247 134 148 156 166 245 245 121 121 144 146 114 129 149 168 0 0 265 267 163 163 103 112 119 123 0 0 103 103 0 0 G G_88 if_88_n5f_10 Female nymph 5th instar Female disperser This study 1 s 165 191 247 254 134 148 172 172 245 245 121 134 144 144 129 129 149 168 293 293 250 267 163 163 103 112 101 119 172 221 103 103 0 0 I I_56 I_56_Nf_9 Female neotenic Female neotenic This study 1 p 195 195 254 254 140 140 0 0 242 242 121 121 148 148 114 114 168 168 282 282 267 267 163 163 112 112 101 101 157 157 103 103 0 0 I I_56 I_56_Nf_10 Female neotenic Female neotenic This study 1 p 195 195 254 254 142 142 0 0 242 242 121 121 148 148 114 114 168 168 282 282 267 267 163 163 112 112 101 101 157 157 103 103 0 0 I I_56 I_56_Nf_11 Female neotenic Female neotenic This study 1 p 195 195 254 254 140 140 0 0 245 245 121 121 144 144 114 114 168 168 282 282 267 267 163 163 112 112 119 119 157 157 103 103 0 0 I I_56 I_56_Nf_12 Female neotenic Female neotenic This study 1 p 0 0 0 0 0 0 0 0 0 0 121 121 0 0 0 0 0 0 0 0 0 0 163 163 0 0 0 0 0 0 103 103 0 0 I I_56 I_56_Nf_13 Female neotenic Female neotenic This study 1 p 195 195 254 254 140 140 0 0 242 242 121 121 148 148 114 114 168 168 282 282 267 267 163 163 112 112 119 119 157 157 103 103 0 0 I I_56 I_56_Nf_14 Female neotenic Female neotenic This study 1 p 195 195 254 254 142 142 0 0 245 245 121 121 144 144 114 114 168 168 282 282 267 267 163 163 112 112 119 119 157 157 103 103 0 0 I I_56 I_56_Nf_15 Female neotenic Female neotenic This study 1 p 195 195 254 254 142 142 0 0 245 245 121 121 144 144 114 114 168 168 0 0 267 267 163 163 112 112 119 119 0 0 103 103 0 0 I I_56 I_56_Nf_16 Female neotenic Female neotenic This study 1 p 0 0 0 0 0 0 0 0 0 0 121 121 144 144 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 103 103 0 0 I I_56 I_56_Nf_17 Female neotenic Female neotenic This study 1 p 0 0 0 0 0 0 0 0 0 0 0 0 144 144 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 103 103 0 0 I I_56 I_56_Nf_18 Female neotenic Female neotenic This study 1 p 0 0 0 0 142 142 0 0 245 245 121 121 148 148 0 0 0 0 0 0 0 0 0 0 112 112 0 0 0 0 103 103 0 0 K K_100 K_100_if_9 Female alate Female disperser This study 1 s 165 193 247 254 142 142 156 172 242 245 121 134 144 144 129 132 149 168 293 293 267 271 157 163 112 112 119 123 157 172 103 103 0 0 K K_100 K_100_if_10 Female alate Female disperser This study 1 s 191 201 247 254 142 142 156 156 245 245 131 131 144 144 129 132 149 168 282 293 250 271 157 157 103 112 101 123 157 218 103 103 0 0 K K_100 K_100_n5f_9 Female nymph 5th instar Female disperser This study 0 s 191 193 247 247 148 148 156 172 242 245 121 134 144 144 129 132 149 168 0 0 250 267 157 163 103 110 0 0 0 0 103 103 0 0 Reproductive Pop Nest Sample name Developmental stage Caste Genotype source Infection status origin Ctub21 Ctub42 Ctub43 Ctub45 Ctub60 Ctub70 Ctub72 Ctub74 Ctub77 Ctub80 Ctub84 Ctub85 Ctub86 Ctub90 Ctub91 Ctub94 Ctub95 K K_100 K_100_n5f_10 Female nymph 5th instar Female disperser This study 0 s 0 0 247 247 142 142 156 156 0 0 121 134 144 144 0 0 0 0 0 0 0 0 0 0 110 112 0 0 0 0 103 103 0 0 K K_100 K_100_n5f_11 Female nymph 5th instar Female disperser This study 1 s 165 201 247 254 142 148 156 156 245 245 121 134 144 144 129 132 149 168 282 293 250 271 157 157 112 112 101 123 157 172 103 103 0 0 K K_100 K_100_n5f_12 Female nymph 5th instar Female disperser This study 1 s 191 201 247 254 142 142 156 172 242 245 131 131 144 144 129 132 149 168 282 293 250 271 157 163 103 112 119 123 157 218 103 103 0 0 K K_100 K_100_n5f_13 Female nymph 5th instar Female disperser This study 0 s 191 193 247 254 142 142 156 156 245 245 131 131 144 144 129 129 149 149 282 293 250 267 163 163 112 112 101 123 157 218 103 103 0 0 K K_100 K_100_n5f_14 Female nymph 5th instar Female disperser This study 0 s 191 201 247 247 142 148 156 156 245 245 121 134 144 144 129 132 147 149 0 0 0 0 159 163 110 112 0 0 0 0 103 103 0 0 K K_100 K_100_n5f_15 Female nymph 5th instar Female disperser This study 0 s 191 193 247 247 142 142 156 156 242 242 121 134 144 144 129 132 147 149 293 293 267 267 159 163 110 112 101 101 157 157 103 103 0 0 K K_100 K_100_n5f_16 Female nymph 5th instar Female disperser This study 0 s 191 201 247 247 142 142 156 156 245 245 121 134 144 144 129 129 147 149 282 293 250 271 157 159 103 110 101 101 157 160 103 103 0 0 K K_100 K_100_n5f_17 Female nymph 5th instar Female disperser This study 0 s 191 201 247 247 142 148 172 172 242 242 121 134 144 144 129 132 149 168 293 293 267 267 157 163 103 110 101 119 157 157 103 103 0 0 K K_100 K_100_n5f_18 Female nymph 5th instar Female disperser This study 0 s 191 193 247 247 142 142 156 156 242 245 121 131 144 144 129 132 147 149 282 293 250 271 157 159 110 112 101 101 157 160 103 103 0 0 K K_100 K_100_Nf_9 Female neotenic Female neotenic This study 0 p 191 191 254 254 142 142 156 156 245 245 131 131 144 144 129 129 149 149 282 282 250 250 163 163 112 112 123 123 218 218 103 103 0 0 K K_100 K_100_Nf_10 Female neotenic Female neotenic This study 0 p 0 0 247 247 148 148 172 172 242 242 121 121 144 144 0 0 0 0 0 0 0 0 0 0 110 110 0 0 157 157 103 103 0 0 K K_100 K_100_Nf_11 Female neotenic Female neotenic This study 0 p 191 191 247 247 142 142 156 156 242 242 121 121 144 144 129 129 147 147 293 293 267 267 159 159 110 110 101 101 157 157 103 103 0 0 K K_100 K_100_Nf_12 Female neotenic Female neotenic This study 1 p 165 165 254 254 142 142 156 156 245 245 121 121 144 144 129 129 168 168 293 293 271 271 163 163 112 112 123 123 172 172 103 103 0 0 K K_100 K_100_Nf_13 Female neotenic Female neotenic This study 0 p 191 191 247 247 142 142 156 156 245 245 121 121 144 144 129 129 147 147 293 293 271 271 159 159 110 110 101 101 160 160 103 103 0 0 K K_100 K_100_Nf_14 Female neotenic Female neotenic This study 1 p 191 191 254 254 142 142 156 156 245 245 131 131 144 144 129 129 149 149 0 0 250 250 163 163 112 112 123 123 0 0 103 103 0 0 K K_100 K_100_Nf_15 Female neotenic Female neotenic This study 1 p 165 165 254 254 142 142 156 156 245 245 131 131 144 144 129 129 168 168 282 282 250 250 157 157 112 112 123 123 218 218 103 103 0 0 K K_100 K_100_Nf_16 Female neotenic Female neotenic This study 1 p 165 165 254 254 142 142 156 156 245 245 121 121 144 144 129 129 168 168 293 293 271 271 157 157 112 112 123 123 172 172 103 103 0 0 K K_100 K_100_Nf_17 Female neotenic Female neotenic This study 0 p 191 191 247 247 142 142 156 156 242 242 121 121 144 144 129 129 147 147 293 293 267 267 159 159 110 110 101 101 157 157 103 103 0 0 K K_100 K_100_Nf_18 Female neotenic Female neotenic This study 1 p 191 191 247 247 142 142 156 156 245 245 121 121 144 144 129 129 147 147 293 293 271 271 159 159 110 110 101 101 160 160 103 103 0 0 K K_81 K_81_n3f_1 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 140 140 159 159 245 245 134 134 146 146 129 129 149 149 293 293 267 267 157 157 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_2 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 148 148 169 169 245 245 121 121 144 144 129 129 149 149 293 293 267 267 163 163 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_3 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 148 148 169 169 242 242 134 134 144 144 129 129 149 149 293 293 267 267 163 163 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_4 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 140 140 169 169 245 245 134 134 144 144 114 114 149 149 293 293 267 267 163 163 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_5 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 148 148 159 159 242 242 121 121 146 146 129 129 149 149 293 293 267 267 157 157 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_6 Female aspirant Female aspirant Fournier et al . (2016) 1 p 193 193 247 247 148 148 169 169 245 245 121 121 146 146 129 129 149 149 293 293 267 267 157 157 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_7 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 140 140 159 159 242 242 134 134 144 144 129 129 149 149 293 293 267 267 163 163 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n3f_8 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 148 148 159 159 245 245 134 134 146 146 129 129 149 149 293 293 267 267 157 157 103 103 119 119 157 157 103 103 0 0 K K_81 K_81_n5f_9 Female nymph 5th instar Female disperser This study 1 s 165 191 247 247 142 148 169 172 242 245 121 134 146 146 129 129 149 149 293 293 267 271 157 163 103 103 119 119 157 218 103 103 0 0 K K_81 K_81_n5f_10 Female nymph 5th instar Female disperser This study 1 s 165 191 247 247 148 148 169 172 242 245 131 134 146 146 114 129 149 149 293 293 267 271 157 163 103 103 119 119 157 178 103 103 0 0 K K_81 K_81_n5f_3 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 193 247 247 148 148 169 172 242 245 121 131 144 146 129 129 149 149 282 293 267 267 163 163 103 103 119 119 157 178 103 103 0 0 K K_81 K_81_n5f_4 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 193 247 247 142 148 159 169 242 245 121 131 146 146 129 129 149 149 293 293 267 271 157 163 103 103 101 119 157 178 103 103 0 0 K K_81 K_81_n5f_5 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 0 0 247 247 140 142 159 169 242 245 131 134 144 144 114 129 149 149 293 293 267 271 163 163 103 103 0 0 157 178 103 103 0 0 K K_81 K_81_n5f_6 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 193 247 247 148 148 159 169 245 245 131 134 144 146 114 129 149 149 293 293 267 271 163 163 103 103 101 119 157 178 103 103 0 0 K K_81 K_81_n5f_7 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 193 247 247 140 148 159 172 242 245 121 134 146 146 129 129 149 149 293 293 267 271 157 163 103 103 119 119 157 218 103 103 0 0 K K_81 K_81_n5f_8 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 191 247 247 140 142 169 172 245 245 121 134 144 146 129 129 149 149 293 293 267 271 157 163 103 103 119 119 157 178 103 103 0 0 K K_81 K_81_Q_1 Primary queen Primary queen Fournier et al . (2016) 1 s 191 193 247 247 140 148 159 169 242 245 121 134 144 146 114 129 149 149 293 293 267 267 157 163 103 103 119 119 157 157 103 103 0 0 M M_82 M_82_n5f_9 Female nymph 5th instar Female disperser This study 1 s 187 191 247 247 134 134 172 175 242 248 134 134 144 146 114 132 149 168 293 293 250 271 163 163 112 112 101 119 157 212 103 103 0 0 M M_82 M_82_n5f_10 Female nymph 5th instar Female disperser This study 1 s 187 187 247 254 134 134 166 175 245 248 121 134 144 146 114 129 149 168 280 293 250 271 163 163 112 112 101 119 172 215 103 103 0 0 M M_82 M_82_n5f_3 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 187 187 254 254 134 140 172 175 242 242 121 134 144 146 114 129 149 168 280 293 250 271 163 163 112 112 101 119 172 215 103 103 0 0 M M_82 M_82_n5f_4 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 187 191 247 254 134 134 166 175 242 248 121 134 144 146 114 129 149 168 280 293 250 271 163 163 103 112 101 119 157 212 103 103 0 0 M M_82 M_82_n5f_5 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 187 187 247 254 134 140 166 175 242 242 121 134 146 146 114 129 149 149 280 293 250 271 163 163 103 112 101 119 212 215 103 103 0 0 M M_82 M_82_n5f_6 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 187 191 247 254 134 140 169 172 242 248 121 134 144 146 114 132 149 168 280 293 250 271 163 163 112 112 101 119 172 215 103 103 0 0 M M_82 M_82_n5f_7 Female nymph 5th instar Female disperser Fournier et al . (2016) 0 s 187 191 247 247 134 140 166 169 242 242 134 134 144 148 114 132 149 168 280 293 250 271 157 163 112 112 101 119 157 172 103 103 0 0 M M_82 M_82_n5f_8 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 187 191 254 254 134 134 169 172 245 248 121 134 144 148 114 132 149 168 293 293 250 271 163 163 112 112 101 119 157 172 103 103 0 0 M M_82 M_82_Nf_1 Female neotenic Female neotenic Fournier et al . (2016) 1 p 187 187 254 254 134 134 175 175 248 248 134 134 146 146 114 114 149 149 280 280 271 271 163 163 112 112 119 119 157 157 103 103 0 0 M M_82 M_82_Nf_2 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 247 247 134 134 169 169 248 248 134 134 146 146 114 114 149 149 280 280 271 271 163 163 112 112 119 119 215 215 103 103 0 0 M M_82 M_82_Nf_3 Female neotenic Female neotenic Fournier et al . (2016) 1 p 187 187 254 254 134 134 175 175 248 248 134 134 144 144 114 114 168 168 280 280 271 271 163 163 112 112 119 119 215 215 103 103 0 0 M M_82 M_82_Nf_4 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 254 254 134 134 175 175 248 248 134 134 144 144 114 114 168 168 293 293 271 271 163 163 112 112 119 119 215 215 103 103 0 0 M M_82 M_82_Nf_5 Female neotenic Female neotenic Fournier et al . (2016) 1 p 187 187 254 254 134 134 175 175 242 242 134 134 146 146 114 114 149 149 280 280 271 271 163 163 112 112 119 119 215 215 103 103 0 0 M M_82 M_82_Nf_6 Female neotenic Female neotenic Fournier et al . (2016) 1 p 187 187 254 254 134 134 175 175 242 242 134 134 144 144 114 114 168 168 280 280 271 271 163 163 112 112 119 119 215 215 103 103 0 0 M M_82 M_82_Nf_7 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 247 247 134 134 169 169 248 248 134 134 146 146 114 114 149 149 280 280 271 271 163 163 112 112 119 119 215 215 103 103 0 0 M M_82 M_82_Nf_8 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 254 254 134 134 175 175 248 248 134 134 146 146 114 114 149 149 293 293 271 271 163 163 112 112 119 119 157 157 103 103 0 0 M M_83 M_83_n3f_1 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 134 134 172 172 245 245 134 134 144 144 114 114 168 168 293 293 250 250 163 163 112 112 119 119 160 160 103 103 0 0 M M_83 M_83_n3f_2 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 134 134 172 172 245 245 134 134 144 144 129 129 168 168 293 293 250 250 163 163 112 112 101 101 160 160 103 103 0 0 M M_83 M_83_n3f_3 Female aspirant Female aspirant Fournier et al . (2016) 1 p 195 195 247 247 134 134 172 172 245 245 134 134 144 144 114 114 149 149 293 293 250 250 163 163 112 112 119 119 200 200 103 103 0 0 M M_83 M_83_n3f_4 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 134 134 172 172 245 245 134 134 144 144 114 114 149 149 293 293 250 250 163 163 112 112 119 119 200 200 103 103 0 0 M M_83 M_83_n3f_5 Female aspirant Female aspirant Fournier et al . (2016) 1 p 195 195 247 247 134 134 172 172 245 245 134 134 144 144 114 114 149 149 293 293 250 250 163 163 103 103 119 119 200 200 103 103 0 0 M M_83 M_83_n3f_6 Female aspirant Female aspirant Fournier et al . (2016) 1 p 191 191 247 247 134 134 172 172 245 245 121 121 144 144 114 114 168 168 293 293 250 250 163 163 112 112 119 119 200 200 103 103 0 0 M M_83 M_83_n3f_7 Female aspirant Female aspirant Fournier et al . (2016) 1 p 195 195 247 247 134 134 172 172 245 245 134 134 144 144 114 114 149 149 293 293 250 250 163 163 112 112 119 119 160 160 103 103 0 0 M M_83 M_83_n3f_8 Female aspirant Female aspirant Fournier et al . (2016) 1 p 195 195 247 247 134 134 172 172 245 245 121 121 144 144 129 129 149 149 293 293 250 250 163 163 103 103 101 101 200 200 103 103 0 0 M M_83 M_83_if_1 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 247 254 134 140 0 0 239 245 121 121 144 144 114 129 168 168 293 293 250 250 163 163 103 112 101 119 0 0 0 0 144 144 M M_83 M_83_if_2 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 254 134 134 0 0 245 245 121 134 144 144 114 129 149 168 293 293 250 250 163 163 112 112 101 101 0 0 0 0 144 144 M M_83 M_83_if_3 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 254 134 134 0 0 239 245 121 134 144 144 129 129 149 168 282 293 236 250 163 163 112 112 101 101 0 0 0 0 144 144 M M_83 M_83_if_4 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 247 254 134 140 0 0 239 245 121 134 144 144 129 129 149 168 293 293 250 250 163 163 112 112 101 119 0 0 0 0 144 144 Reproductive Pop Nest Sample name Developmental stage Caste Genotype source Infection status origin Ctub21 Ctub42 Ctub43 Ctub45 Ctub60 Ctub70 Ctub72 Ctub74 Ctub77 Ctub80 Ctub84 Ctub85 Ctub86 Ctub90 Ctub91 Ctub94 Ctub95 M M_83 M_83_if_5 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 254 134 140 0 0 239 245 121 121 144 144 129 129 149 149 293 293 250 250 163 163 103 112 101 101 0 0 0 0 144 144 M M_83 M_83_if_6 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 247 254 134 134 0 0 245 245 121 121 144 144 129 129 168 168 282 293 236 250 163 163 112 112 101 119 0 0 0 0 144 144 M M_83 M_83_if_7 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 247 254 134 134 0 0 245 245 121 134 144 144 129 129 149 168 293 293 250 250 163 163 103 112 101 119 0 0 0 0 144 144 M M_83 M_83_if_8 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 254 134 134 0 0 245 245 121 121 144 144 129 129 149 168 293 293 250 250 163 163 103 112 101 101 0 0 0 0 144 144 M M_83 M_83_if_9 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 247 254 134 134 0 0 239 245 121 134 144 144 114 129 0 0 282 293 236 250 163 163 103 112 101 119 0 0 0 0 144 144 M M_83 M_83_if_10 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 254 134 140 0 0 239 245 121 134 144 144 129 129 0 0 282 293 236 250 163 163 112 112 101 119 0 0 0 0 144 144 M M_83 M_83_n5f_9 Female nymph 5th instar Female disperser This study 1 s 165 195 247 254 134 140 172 175 239 245 121 134 144 144 129 129 149 168 293 293 250 250 163 163 112 112 101 119 157 160 103 103 0 0 M M_83 M_83_n5f_10 Female nymph 5th instar Female disperser This study 1 s 165 195 247 254 134 140 163 172 239 245 121 134 144 144 114 129 168 168 293 293 250 250 163 163 103 112 101 101 160 172 103 103 0 0 M M_83 M_83_Q_1 Primary queen Primary queen Fournier et al . (2016) 1 s 191 195 247 247 134 134 172 172 245 245 121 134 144 144 114 129 149 168 293 293 250 250 163 163 103 112 101 119 160 200 0 0 144 144 M M_90 M_90_n5f_9 Female nymph 5th instar Female disperser This study 1 s 165 191 247 254 134 134 172 172 245 245 134 134 144 148 114 129 149 168 293 293 250 267 163 163 103 112 119 119 160 200 103 103 0 0 M M_90 M_90_n5f_10 Female nymph 5th instar Female disperser This study 1 s 165 195 247 254 134 140 172 175 245 245 121 121 144 144 126 129 149 168 293 293 250 267 157 167 103 112 119 119 200 200 103 103 0 0 M M_90 M_90_n5f_3 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 191 195 247 247 134 134 0 0 245 245 134 134 144 148 126 129 0 0 293 293 250 267 157 165 103 112 101 119 0 0 0 0 144 144 M M_90 M_90_n5f_4 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 191 247 247 134 134 0 0 245 245 121 121 144 144 114 126 0 0 293 293 250 267 163 163 103 103 119 119 0 0 0 0 144 144 M M_90 M_90_n5f_5 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 191 195 247 254 134 134 0 0 245 245 134 134 144 148 126 129 0 0 293 293 250 267 163 163 103 112 119 119 0 0 0 0 144 144 M M_90 M_90_n5f_6 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 165 191 247 254 134 134 0 0 245 245 134 134 148 148 126 129 0 0 293 293 267 267 157 163 103 112 119 119 0 0 0 0 144 144 M M_90 M_90_n5f_7 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 191 195 247 254 134 134 0 0 245 245 121 134 148 148 114 129 0 0 293 293 250 250 157 165 103 112 119 119 0 0 0 0 144 144 M M_90 M_90_n5f_8 Female nymph 5th instar Female disperser Fournier et al . (2016) 1 s 191 191 247 254 134 134 0 0 245 245 121 134 144 148 126 129 0 0 293 293 250 267 163 165 103 103 119 119 0 0 0 0 144 144 M M_90 M_90_Nf_1 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 254 254 134 134 0 0 245 245 134 134 148 148 129 129 0 0 293 293 267 267 163 163 112 112 119 119 0 0 0 0 144 144 M M_90 M_90_Nf_2 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 254 254 134 134 0 0 245 245 121 121 144 144 129 129 0 0 293 293 250 250 163 163 112 112 119 119 0 0 0 0 144 144 M M_90 M_90_Nf_3 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 254 254 134 134 0 0 245 245 134 134 148 148 126 126 0 0 293 293 267 267 165 165 112 112 101 101 0 0 0 0 144 144 M M_90 M_90_Nf_4 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 247 247 134 134 0 0 245 245 134 134 144 144 129 129 0 0 293 293 250 250 165 165 112 112 101 101 0 0 0 0 144 144 M M_90 M_90_Nf_5 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 247 247 134 134 0 0 245 245 121 121 144 144 126 126 0 0 293 293 250 250 163 163 103 103 119 119 0 0 0 0 144 144 M M_90 M_90_Nf_6 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 254 254 134 134 0 0 245 245 121 121 144 144 129 129 0 0 293 293 250 250 163 163 112 112 119 119 0 0 0 0 144 144 M M_90 M_90_Nf_7 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 247 247 134 134 0 0 245 245 134 134 144 144 126 126 0 0 293 293 267 267 163 163 103 103 101 101 0 0 0 0 144 144 M M_90 M_90_Nf_8 Female neotenic Female neotenic Fournier et al . (2016) 1 p 191 191 247 247 134 134 0 0 245 245 134 134 144 144 129 129 0 0 293 293 250 250 163 163 112 112 101 101 0 0 0 0 144 144 N N_96 N_96_n3f_1 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 114 114 149 149 293 293 250 250 163 163 103 103 119 119 157 157 103 103 0 0 N N_96 N_96_n3f_2 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 129 129 149 149 282 282 250 250 163 163 103 103 119 119 157 157 103 103 0 0 N N_96 N_96_n3f_3 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 129 129 149 149 293 293 250 250 163 163 103 103 119 119 157 157 103 103 0 0 N N_96 N_96_n3f_4 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 114 114 149 149 293 293 250 250 163 163 103 103 119 119 160 160 103 103 0 0 N N_96 N_96_n3f_5 Female aspirant Female aspirant Fournier et al . (2016) 1 s 165 191 247 247 134 140 166 169 242 242 121 134 146 148 129 129 149 168 282 293 250 250 163 163 103 112 101 119 157 206 103 103 0 0 N N_96 N_96_n3f_6 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 114 114 149 149 293 293 250 250 163 163 103 103 119 119 160 160 103 103 0 0 N N_96 N_96_n3f_7 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 114 114 149 149 282 282 250 250 163 163 103 103 119 119 160 160 103 103 0 0 N N_96 N_96_n3f_8 Female aspirant Female aspirant Fournier et al . (2016) 1 p 165 165 247 247 140 140 166 166 242 242 121 121 146 146 129 129 149 149 293 293 250 250 163 163 103 103 119 119 157 157 103 103 0 0 N N_96 N_96_if_9 Female alate Female disperser This study 1 s 165 191 247 247 140 148 166 169 242 242 121 134 144 146 114 129 149 149 282 282 250 250 157 163 103 112 101 119 157 206 103 103 0 0 N N_96 N_96_if_10 Female alate Female disperser This study 1 s 165 165 247 247 134 140 166 169 242 242 121 134 144 146 129 129 149 149 282 293 250 250 157 163 103 112 119 119 163 172 103 103 0 0 N N_96 N_96_if_3 Female alate Female disperser Fournier et al . (2016) 1 s 165 165 247 247 134 140 166 169 242 242 121 121 146 148 114 129 149 168 282 282 250 250 163 163 103 112 119 119 157 206 103 103 0 0 N N_96 N_96_if_4 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 247 140 148 166 169 242 242 121 121 144 146 114 129 149 149 282 293 248 250 157 163 103 112 119 119 157 206 103 103 0 0 N N_96 N_96_if_5 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 247 140 148 166 169 242 245 121 121 144 146 114 129 149 149 282 282 250 250 157 163 103 112 101 119 157 206 103 103 0 0 N N_96 N_96_if_6 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 247 134 140 166 169 242 245 121 121 146 148 129 129 149 168 282 293 250 250 163 163 103 112 101 119 157 206 103 103 0 0 N N_96 N_96_if_7 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 247 140 148 166 169 242 242 121 134 144 146 114 114 149 149 293 293 248 250 157 163 103 112 119 119 160 172 103 103 0 0 N N_96 N_96_if_8 Female alate Female disperser Fournier et al . (2016) 1 s 165 191 247 247 134 140 166 169 242 242 121 134 144 146 129 129 149 149 282 293 248 250 157 163 103 112 119 119 160 206 103 103 0 0 M N_96 N_96_Q_1 Primary queen Primary queen Fournier et al . (2016) 1 s 165 165 247 247 140 140 166 166 242 242 121 121 146 146 114 129 149 149 282 293 250 250 163 163 103 103 119 119 157 160 103 103 0 0 O O_98 O_98_if_11 Female alate Female disperser This study 1 s 165 195 247 254 134 134 166 172 245 245 121 121 144 148 114 114 149 168 293 293 248 271 163 163 110 112 101 119 154 160 103 103 0 0 O O_98 O_98_if_12 Female alate Female disperser This study 1 s 165 193 247 254 134 134 0 0 242 245 0 0 0 0 114 114 149 149 0 0 250 271 163 163 110 112 101 119 154 157 103 103 0 0 O O_98 O_98_if_3 Female alate Female disperser Fournier et al . (2016) 1 s 165 193 247 254 134 134 156 172 242 245 121 121 144 144 114 114 149 149 293 293 248 271 163 163 103 112 101 119 160 215 103 103 144 144 O O_98 O_98_if_4 Female alate Female disperser Fournier et al . (2016) 1 s 165 165 247 254 134 134 156 175 245 245 121 121 144 144 114 114 168 168 276 293 250 271 163 163 103 112 101 119 154 160 103 103 144 144 O O_98 O_98_if_5 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 247 254 134 134 166 172 245 245 121 121 144 144 114 114 149 168 293 293 248 271 163 163 103 112 101 119 157 215 103 103 144 144 O O_98 O_98_if_6 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 254 254 134 134 156 172 242 245 121 121 144 148 114 114 168 168 276 293 250 250 163 163 110 112 119 119 160 215 103 103 144 144 O O_98 O_98_if_7 Female alate Female disperser Fournier et al . (2016) 1 s 193 195 247 254 134 134 166 175 242 245 121 121 144 144 114 114 149 149 276 293 250 271 163 163 110 112 101 119 160 215 103 103 144 144 O O_98 O_98_if_8 Female alate Female disperser Fournier et al . (2016) 1 s 165 195 254 254 134 134 156 175 242 245 121 121 144 144 114 114 149 149 276 293 250 271 163 163 103 112 119 119 160 215 103 103 144 144 O O_98 O_98_Nf_1 Female neotenic Female neotenic Fournier et al . (2016) 1 p 193 193 247 247 134 134 166 166 245 245 121 121 144 144 114 114 168 168 293 293 250 250 163 163 110 110 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_2 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 254 254 134 134 166 166 242 242 121 121 144 144 114 114 168 168 276 276 250 250 163 163 103 103 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_3 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 247 247 134 134 166 166 245 245 121 121 144 144 114 114 168 168 293 293 248 248 163 163 103 103 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_4 Female neotenic Female neotenic Fournier et al . (2016) 1 p 193 193 254 254 134 134 156 156 242 242 121 121 144 144 114 114 168 168 293 293 248 248 163 163 110 110 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_5 Female neotenic Female neotenic Fournier et al . (2016) 1 p 193 193 254 254 134 134 156 156 245 245 121 121 144 144 114 114 168 168 293 293 248 248 163 163 110 110 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_6 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 254 254 134 134 156 156 242 242 121 121 144 144 114 114 149 149 293 293 248 248 163 163 110 110 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_7 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 254 254 134 134 156 156 245 245 121 121 144 144 114 114 149 149 293 293 248 248 163 163 103 103 119 119 215 215 103 103 144 144 O O_98 O_98_Nf_8 Female neotenic Female neotenic Fournier et al . (2016) 1 p 165 165 254 254 134 134 156 156 242 242 121 121 144 144 114 114 168 168 276 276 250 250 163 163 110 110 119 119 215 215 103 103 144 144 Symbiosis between Wolbachia and a termite
Supplementary References
Baldo L, Dunning Hotopp JC, Jolley KA et al. Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl Environ Microbiol 2006;72: 7098-110 Bordenstein S, Rosengaus RB. Discovery of a novel Wolbachia supergroup in Isoptera. Curr Microbiol 2005;51: 393-8 Bordenstein SR, Paraskevopoulos C, Dunning Hotopp JC et al. Parasitism and mutualism in Wolbachia: what the phylogenomic trees can and cannot say. Mol Biol Evol 2008;26: 231-41 Darby AC, Armstrong SD, Bah GS et al. Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis. Genome Res 2012;22: 2467-77 Gerth M, Bleidorn C. Comparative genomics provides a timeframe for Wolbachia evolution and exposes a recent biotin synthesis operon transfer. Nature Microbiology 2016;2: 16241. Gerth M, Gansauge M-T, Weigert A et al. Phylogenomic analyses uncover origin and spread of the Wolbachia pandemic. Nat Commun 2014;5: 5117. Lindsey ARI, Werren JH, Richards S et al. Comparative genomics of a parthenogenesis- inducing Wolbachia symbiont. G3: Genes, Genomes, Genetics 2016;6: 2113-23 Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 2001;29: e45. Russell JA, Goldman-Huertas B, Moreau CS et al. Specialization and geographic isolation among Wolbachia symbionts from ants and lycaenid butterflies. Evolution 2009;63: 624-40 Salunke BK, Salunkhe RC, Dhotre DP et al. Diversity of Wolbachia in Odontotermes spp. (Termitidae) and Coptotermes heimi (Rhinotermitidae) using the multigene approach. FEMS Microbiol Let 2010;307: 55-64 Simões PM, Mialdea G, Reiss D et al. Wolbachia detection: an assessment of standard PCR Protocols. Mol Ecol Resources 2011;11: 567-72 Stahlhut JK, Desjardins CA, Clark ME et al. The mushroom habitat as an ecological arena for global exchange of Wolbachia. Mol Ecol 2010;19: 1940-52
(Baldo et al. 2006; Bordenstein and Rosengaus 2005; Bordenstein et al. 2008; Darby et al. 2012; Gerth et al. 2014; Lindsey et al. 2016; Russell et al. 2009; Salunke et al. 2010; Simões et al. 2011; Stahlhut et al. 2010)
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