RESEARCH ARTICLE
Arsenophonus insect symbionts are commonly infected with APSE, a bacteriophage involved in protective symbiosis Olivier Duron Institut des Sciences de l’Evolution, CNRS-UM2 (UMR5554), Montpellier Cedex 05, France
Correspondence: Olivier Duron, 911 Avenue Agropolis, Montpellier 34394, France. Tel.: 0033 4 6741 6158; fax: 0033 4 6741 6299; e-mail: [email protected] Present address: Olivier Duron, Laboratoire MIVEGEC (Maladies Infectieuses et Vecteurs: ^le), Ecologie, G en etique, Evolution et Contro CNRS-UM1-UM2 (UMR5290) – IRD (UR 224), Centre IRD, Montpellier, France Received 6 June 2014; revised 8 July 2014; accepted 9 July 2014. Final version published online 11 August 2014. DOI: 10.1111/1574-6941.12381
MICROBIOLOGY ECOLOGY
Editor: Max H€ aggblom Keywords heritable symbiosis; defensive symbionts; Arsenophonus; Hamiltonella; APSE bacteriophage.
Abstract Insects commonly have intimate associations with maternally inherited bacterial symbionts. While many inherited symbionts are not essential for host survival, they often act as conditional mutualists, conferring protection against certain environmental stresses. The defensive symbiont Hamiltonella defensa which protects aphids against attacks by parasitoid wasps is one of these conditional mutualists. The protection afforded by Hamiltonella depends on the presence of a lysogenic bacteriophage, called APSE, encoding homologs of toxins that are suspected to target wasp cells. In this study, an important diversity of APSE variants is reported from another heritable symbiont, Arsenophonus, which is exceptionally widespread in insects. APSE was found in association with two-thirds of the Arsenophonus strains examined and from a variety of insect groups such as aphids, white flies, parasitoid wasps, triatomine bugs, louse flies, and bat flies. No APSE was, however, found from Arsenophonus relatives such as the recently described Aschnera chinzeii and ALO-3 endosymbionts. Phylogenetic investigations revealed that APSE has a long evolutionary history in heritable symbionts, being secondarily acquired by Hamiltonella through lateral transfer from Arsenophonus. Overall, this highlights the role of lateral transfer as a major evolutionary process shaping the emergence of defensive symbiosis in heritable bacteria.
Introduction Insects commonly have associations with heritable bacterial endosymbionts that inhabit insect cells and depend on maternal transmission, through egg cytoplasm, to ensure their transmission (Moran et al., 2008; Werren et al., 2008; Oliver et al., 2010). While some heritable bacteria have evolved toward obligate mutualism and are required to support normal insect development, many others are facultative symbionts exerting more subtle effects such as protection against environmental stresses (Moran, 2007; Oliver et al., 2010; Hamilton & Perlman, 2013) or manipulation of host reproduction (Werren et al., 2008; Engelstadter & Hurst, 2009). These effects promote the spread of heritable bacteria through insect populations, but may also drive insect evolutionary ecology, contributing substantially to the acquisition of ecologically important traits to infected species (Moran, 2007; Oliver et al., 2010, 2014; Ferrari & Vavre, 2011; Jaenike, 2012). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
In recent years, a great deal of attention has been devoted to defensive symbionts that protect insects against attack by natural enemies (Oliver et al., 2003; Scarborough et al., 2005; Hedges et al., 2008; Jaenike et al., 2010). The pea aphid (Acyrthosiphon pisum), an organism intensively studied with respect to symbiotic interactions, was one the first insects revealed to have defensive symbionts (Oliver et al., 2003; Ferrari et al., 2004). Indeed, infection of the pea aphid by the heritable bacterium Hamiltonella defensa (c-Proteobacteria: Enterobacteriaceae) provides resistance to parasitoid wasps by killing developing wasp larvae or embryos in the aphid hemocoel (Oliver et al., 2003, 2005; Moran et al., 2005a; Bensadia et al., 2006). Through reducing the mortality risk after wasp oviposition, the pea aphid thus relies heavily on its Hamitonella for protection. Further studies suggest that the defensive function of Hamiltonella may be extended to other Hamiltonella-infected species, as it also provides another insect, the black bean aphid (Aphis fabae), with strong protection against parasitoid wasps FEMS Microbiol Ecol 90 (2014) 184–194
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(Vorburger et al., 2009, 2013; Cayetano & Vorburger, 2013). Moreover, parasitic wasps discriminate between Hamiltonella-infected and uninfected aphids of diverse species, thus showing that Hamiltonella reduces the risk of parasitism by making hosts less attractive to searching parasitoids (Oliver et al., 2012; Lukasik et al., 2013). Although Hamiltonella is rare in insects, it is commonly found in the sap-sucking hemipteran suborder Sternorrhyncha, including aphids, whiteflies, mealybugs, and psyllids (Zchori-Fein & Brown, 2002; Russell et al., 2003; Moran et al., 2005b). Remarkably, Hamiltonella can be occasionally exchanged between the Sternorrhyncha species through a process of horizontal transfer that may possibly result in the instantaneous acquisition of resistance to parasitoids (Oliver et al., 2010). The mechanistic basis of Hamlitonella’s protective effect has been actively studied in the pea aphid. The degree of protection was shown to vary among Hamiltonella strains, ranging from small reductions in successful wasp parasitism to nearly complete protection (Oliver et al., 2005, 2009). While this variation might depend on Hamiltonella chromosomal gene content, the degree of protection is also greatly influenced by a Hamiltonella lysogenic lambdoid bacteriophage (Moran et al., 2005a; Degnan et al., 2009), called APSE (‘A. pisum secondary endosymbiont phage’; van der Wilk et al., 1999): While Hamiltonella strains lacking APSE confer little protection, those harboring APSE increase aphid survival following wasp attack (Degnan & Moran, 2008b; Oliver et al., 2009). An additional variation in the degree of protection is associated with the nature of APSE gene products and especially of APSE-borne toxins: A variety of phage variants, each harboring a specific virulence cassette region, are known in Hamiltonella (Moran et al., 2005a; Degnan & Moran, 2008a). Remarkably, APSE was found to be common in Hamiltonella strains of aphids, whiteflies, and psyllids (Moran et al., 2005a; Degnan & Moran, 2008a, b), suggesting that APSE may have a significant influence in the evolution of symbiont-mediated protection in the Sternorrhyncha. Until recently, APSE was thought to be exclusively associated with Hamiltonella. However, APSE has been also occasionally detected in Arsenophonus, another facultative heritable bacterium but not closely related to Hamiltonella (Hansen et al., 2007; Taylor et al., 2011). Further analyses of the draft genome sequence of Arsenophonus type species, Arsenophonus nasoniae, also revealed similarity of its phage elements with the APSE sequences found in Hamiltonella (Wilkes et al., 2010). APSE may thus readily move between quite unrelated symbionts and be an immediate and powerful mechanism of rapid adaptation for symbionts and their hosts. Remarkably, while Hamiltonella is largely restricted to the Sternorrhyncha FEMS Microbiol Ecol 90 (2014) 184–194
suborder, Arsenophonus is more largely widespread and has been found in many insect orders including hemipterans, hymenopterans, and dipterans (Duron et al., 2008, 2010, 2014; Novakova et al., 2009; Taylor et al., 2011; Mouton et al., 2012; Russell et al., 2012; Jousselin et al., 2013), suggesting that APSE may possibly protect against parasitoid attacks in a variety of insect groups. A key question with regard to mutualistic symbiosis is the extent to which a source of adaptation, such as the defensive APSE phage, moves between symbiont taxa. Here, this topic was approached by undertaking an extensive screening for the presence, the diversity, and the evolutionary history of APSE infections in a collection of bacterial endosymbionts from 33 host species, including hemipterans, hymenopterans, and dipterans. This collection includes Arsenophonus and two other heritable bacteria phylogenetically allied to Arsenophonus (Arsenophonus and like organisms, ALOs hereafter) that have recently been characterized: Aschnera chinzeii (Hosokawa et al., 2012) and ALO-3 (Duron et al., 2014). While Arsenophonus is a widespread facultative endosymbiont, Aschnera chinzeii and ALO-3 are rather thought to be obligate endosymbionts which are restricted to few species of bat flies and louse flies, respectively (Hosokawa et al., 2012; Duron et al., 2014). Sequencing and phylogenetic analyses of the P3 (encoding for a putative P-loop ATPase) and P45 (DNA polymerase) phage genes were further used to characterize the evolutionary processes that shape APSE’s diversity in communities of heritable bacteria.
Materials and methods Insect collection
A collection of 114 DNA templates from 33 ALOinfected insect species was used (Table 1). For each DNA template, ALO infection had been formally characterized in previous studies through single- or multilocus DNA sequencing (Duron et al., 2010, 2014; Jousselin et al., 2013). Of the 33 insect species examined in this study, infection by Arsenophonus was found in 22, including five species of aphid (Aphididae), one of whitefly (Aleurodidae), one of psyllid (Psyllidae), two of triatomine bug (Reduvidae), three of bat fly (Nycteribiidae and Streblidae), nine of louse fly (Hippoboscidae), and one of parasitoid wasp (Pteromalidae) as detailed in Table 1. Infections by Aschnera chinzeii and by ALO-3 were found in six bat fly species and five louse fly species, respectively (Table 1). Each insect species was found infected by a genetically distinct ALO strain with the exception of two louse fly species pairs, Hippobosca equina/H. longipennis and Lipoptena cervi/Lipoptena sp., which were infected by genetically identical Arsenophonus ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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strains (Duron et al., 2010, 2014; Jousselin et al., 2013). Within each examined host species, there were 1–5 ALO-infected individuals separately investigated for the present analysis, with the exception of aphids and whiteflies for which 1–3 ALO-infected pools of five specimens were examined (Table 1).
Screening and sequencing for APSE
ALO DNA quality was systematically tested by specific PCR amplification of a region of the bacterial GroEL gene following Duron et al. (2014), as described in Supporting Information, Table S1. The APSE infections were next
Table 1. Distribution of APSE in ALO heritable bacteria and their host species APSE Host species Hemiptera Aleurodidae (whiteflies) Bemisia tabaci (Gennadius, 1889) Aphididae (aphids) Aphis craccivora (Koch, 1854) Aphis idaei (van der Goot, 1912) Aphis gossypii (Glover, 1877) €rner, 1932) Aphis ruborum (Bo Melanaphis donacis (Passerini, 1862) Psyllidae (psyllids) Cacopsylla alaterni (Foerster, 1848) Reduviidae (triatomine bugs) Triatoma guasayana (Wygodzinsky & Abalos, 1941) Triatoma infestans (Klug, 1834) Hymenoptera Pteromalidae (parasitoid wasps) Nasonia vitripennis (Walker, 1836) Diptera Hippoboscidae (louse flies) ndani, 1879) Crataerina melbae (Ro Crataerina pallida (Latreille, 1812) Hippobosca equina (Linnaeus, 1758) Hippobosca longipennis (Fabricius, 1805) Lipoptena capreoli (Rondani, 1878) Lipoptena cervi (Linnaeus, 1758) Lipoptena fortisetosa (Maa, 1965) Lipoptena sp. Pseudolynchia canariensis (Macquart, 1839) Ornithomya avicularia (Linnaeus, 1758) Ornithomya biloba (Dufour, 1827) Ornithomya chloropus (Bergroth, 1901) Ornithomya fringillina (Curtis, 1836) Stenepteryx hirundinis (Linnaeus, 1758) Nycteribiidae (bat flies) Basilia nov. sp. Eucampsipoda africana (Theodor, 1955) Nycteribia schmidlii scotti (Falcoz, 1923) Nycteribia stylidiopsis (Speicer, 1908) Nycteribia kolenatii (Theodor & Moscona, 1954) Penicillidia conspicua (Speiser, 1901) Penicillidia fulvida (Bigot, 1885) Penicillidia leptothrinax (Speiser, 1908) Streblidae (bat flies) Brachytarsina allaudi (Falcoz, 1923)
ALO heritable bacteria
n
P3
P45
Reference for ALO characterization of DNA samples
Arsenophonus
1*
Present
Present
Duron et al. (2010)
Arsenophonus Arsenophonus Arsenophonus Arsenophonus† Arsenophonus†
3* 3* 1* 3* 1*
_ _ _ Present Present
Present Present Present Present Present
Jousselin Jousselin Jousselin Jousselin Jousselin
Arsenophonus
3‡
Present
Present
Duron et al. (2010)
Arsenophonus Arsenophonus
1‡ 3‡
_ _
Present Present
Duron et al. (2010) Duron et al. (2010)
Arsenophonus
3‡
Present
Present
Duron et al. (2010)
Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus ALO-3 ALO-3 ALO-3 ALO-3 ALO-3
5‡ 5‡ 5‡ 5‡ 2‡ 5‡ 2‡ 2‡ 5‡ 5‡ 5‡ 5‡ 5‡ 5‡
Present Present _ _ _ _ _ _ Present _ _ _ _ _
Present Present Present Present _ _ _ _ Present _ _ _ _ _
Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron
et et et et et et et et et et et et et et
al. al. al. al. al. al. al. al. al. al. al. al. al. al.
(2014) (2014) (2010) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014)
Arsenophonus Arsenophonus Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii
2‡ 5‡ 5‡ 5‡ 1‡ 1‡ 4‡ 5‡
_ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _
Duron Duron Duron Duron Duron Duron Duron Duron
et et et et et et et et
al. al. al. al. al. al. al. al.
(2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014)
Arsenophonus
3‡
Present
Present
Duron et al. (2014)
et et et et et
al. al. al. al. al.
(2013) (2013) (2013) (2013) (2013)
*Number of pools of specimens examined. † In coinfection with Hamiltonella. ‡ Number of individuals separately investigated for analysis.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Ecol 90 (2014) 184–194
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screened and genotyped using independent PCR assays based on the P3 and P45 genes with a set of specific primers. Additional PCR assays were also conducted to check for coinfections with other bacterial endosymbionts. First, on all DNA samples, PCR assays on the bacterial 16S rRNA gene using Hamiltonella-specific primers were conducted. Second, all APSE-positive samples were further tested using specific primers for three other bacterial endosymbionts commonly found in insects: Wolbachia, Rickettsia, and Sodalis. Gene features and primers are listed in Table S1. All the PCR amplifications were performed under the following conditions: initial denaturation at 93 °C for 3 min, 30 cycles of denaturation (93 °C, 30 s), annealing (52–54 °C, depending on primers, cf. Table S1, 30 s), extension (72 °C, 1 min), and a final extension at 72 °C for 5 min. The PCR products were electrophoresed in a 1.5% agarose gel. Amplification products were sequenced in both directions at Eurofins. Genomics (Ebersberg, Germany). Chromatograms were checked and edited using CHROMAS LITE (http://www. technelysium.com.au), and sequence alignments were performed using CLUSTALW (Thompson et al., 2002), both implemented in MEGA (Kumar et al., 2004). New sequence data have been submitted to GenBank (Accession Numbers KJ774110–KJ774136; see Table S2 for details). Molecular phylogenetic analyses
Sequence alignments were carried out using CLUSTALW (Thompson et al., 2002) and corrected using MEGA (Tamura et al., 2007). The GBLOCKS program (Castresana, 2000) with default parameters was used to remove poorly aligned positions and to obtain unambiguous sequence alignments. All sequence alignments were also checked for putative recombinant regions using the GENECONV (Sawyer, 1989) and RDP (Martin & Rybicki, 2000) methods available in the RDP3 computer analysis package (Martin et al., 2010). Phylogenetic relationships were evaluated between APSE variants using P3 (771 unambiguously aligned nucleotide sites) and P45 (506 bp) sequences and between Hamiltonella defensa strains using partial 16S rRNA (428 bp) sequences. In addition to sequences produced in this study, additional P3 and P45 sequences of Hamiltonella and Arsenophonus, as well as a subset of more divergent phage sequences from other bacterial species available from GenBank, were also used (Table S2). The evolutionary model most closely fitting the sequence data was determined using Akaike information criterion with the MEGA program (Tamura et al., 2007). The best-fit approximation was K2+G for the APSE P3 and P45 data set and HKY+I for the Hamiltonella 16S rRNA data set. FEMS Microbiol Ecol 90 (2014) 184–194
Phylogenetic analyses were based on maximum likelihood (ML) analyses. A ML heuristic search, using a starting tree obtained by neighbor joining, was conducted, and clade robustness was further assessed by bootstrap analysis using 1000 replicates in MEGA (Tamura et al., 2007). Because the recombination between APSE variants may hamper the use of phylogenetic tree-based analyses, a network analysis was carried out, employing SplitsTree (Huson & Bryant, 2006) in conjunction with the agglomerating NeighborNet algorithm (Bryant & Moulton, 2004). The resulting phylogenetic networks generalize the trees by allowing cross-connections between branches which might visualize conflicting signals due to lateral gene transfers.
Results Distribution of APSE
A total of 114 DNA templates with quality being confirmed by positive ALO-GroEL PCR amplification were used (Table 1). To check for ALO-Hamiltonella coinfection, the presence of Hamiltonella was first assayed through a specific 16S rRNA gene-based PCR assay. Of the 114 DNA templates, Hamiltonella was found in four templates from two Arsenophonus-infected aphid species, Aphis ruborum (three templates) and Melanaphis donacis (one), showing then that coinfection with Hamiltonella and Arsenophonus occurs in these species (Table 1). That these were double infections was ascertained through the sequencing of the Hamiltonella 16S rRNA PCR products and the observation of clean Hamiltonella sequences. Phylogenetically, the bacterial strains from A. ruborum and M. donacis clearly fall within the genus Hamiltonella and cluster with the strains found in other aphid species (Supporting Information, Fig. S1). The 114 DNA templates were next screened for the presence of APSE using P3 and P45-based PCR assays (Table 1). The P3 and P45 PCR screening produced similar results but with some differences: The P45 gene was successfully amplified from 50 of the 114 DNA templates while the P3 gene was only amplified from 29 templates (all P3-positive DNA templates were also P45 positive; Table 1). The failure of P3 PCR amplification in these cases may be due either to gene deletion or, possibly, to a consequence of mutations in the P3 priming regions. Overall, APSE was detected in 50 DNA templates, including the four of A. ruborum and M. donacis with Arsenophonus–Hamiltonella coinfection. In these last four cases, the detection of APSE suggests that APSE may be associated with Arsenophonus, Hamiltonella, or both; the association between APSE and Arsenophonus is thus here uncertain. However, in the 46 remaining cases, no ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Hamiltonella infection was detected, showing that APSE is associated with Arsenophonus infections. APSE was found in association with 14 of the 20 Arsenophonus strains (excluding the strains from A. ruborum and M. donacis in which coinfection with Hamitonella occurs), while none of the six Aschnera chinzeii strains and none of the five ALO-3 strains was detected to be APSE positive. The 14 APSE-positive Arsenophonus strains are from very diverse insect orders, including Hemiptera (three species of aphid, one of whitefly, one of psyllid and two of triatomine bugs), Hymenoptera (one species of parasitoid wasp), and Diptera (five species of louse fly and one of bat fly) (Table 1). For each positive host species, APSE was observed in all tested DNA templates (n = 1–5, depending on species; Table 1). Additional PCR screening did reveal other endosymbionts than Arsenophonus and Hamiltonella in nine of the 50 APSE-positive samples: Wolbachia was found in three templates from the wasp Nasonia vitripennis and in five templates from the louse fly Pseudolynchia canariensis; Rickettsia in one template from the white fly Bemisia tabaci; and Sodalis in the five templates from P. canariensis. Although the coinfection pattern suggests that APSE may be associated with other bacteria than with Arsenophonus in these three insect species, this hypothesis can be rejected in at least two cases. In N. vitripennis, the sequencing of the Arsenophonus genome has previously shown that APSE is integrated in the Arsenophonus chromosome (Wilkes et al., 2010). In B. tabaci, the Rickettsia genome has been also sequenced (Rao et al., 2012), but Basic Local Alignment Search Tool (BLAST) searches against this genome (GenBank Accession Number AJWD01000000) did not detect APSE, suggesting that APSE is rather associated with Arsenophonus. In P. canariensis, APSE may be associated either with Sodalis or with Arsenophonus, and the association between APSE and Arsenophonus should thus be treated with caution. However, this is unlikely that APSE is associated with Wolbachia in this insect as no APSE was found in all Wolbachia genomes sequenced to date from many insect species. Overall, the present data thus indicate that APSE is clearly associated with Arsenophonus infections in many cases. Diversity of APSE infections
The diversity of APSE was examined using P3 and P45 DNA sequences from the 50 positive DNA templates, including the A. ruborum and M. donacis DNA templates with Hamiltonella–Arsenophonus coinfection. The sequences were easily readable without double peaks, indicating that there were no multiple APSE variants in any DNA templates. This was also true for the A. ruborum ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
O. Duron
and M. donacis DNA templates, suggesting that only one APSE variant is present in Hamiltonella–Arsenophonus coinfected aphids. On the basis of nucleotide sequences, nine P3 distinct alleles (87.9–97.3% pairwise identity) and 14 P45 distinct alleles (85.0–98.6% pairwise identity) were characterized from the 50 DNA templates. Overall, 14 genetically different APSE variants were identified on the basis of both P3 and P45 sequences. No P3 and P45 sequence variation was observed between DNA templates from any given insect species. Each of the APSE-positive insect species harbored a different APSE variant with the exception of two species pairs, Hippobosca equina/H. longipennis and Triatoma infestans/T. guasayana, which were infected by genetically identical APSE variants on the basis of P45 sequences (no P3 PCR products were obtained from these templates; Table 1). The P45 sequence of the Arsenophonus strain from the whitefly Bemisia tabacci contained an internal stop codon, suggesting that this APSE gene may not be functional. In all other cases, P3 and P45 APSE sequences do not contain internal stop codons and are thus likely to code for putatively functional phage protein. The comparison between APSE sequences from this study and other APSE sequences available in GenBank (Table S2) revealed a contrasting pattern of nucleotide diversity between Hamiltonella APSE and Arsenophonus APSE (The APSE sequences from Hamiltonella–Arsenophonus coinfections were excluded from this comparison.) While the level of Hamiltonella APSE nucleotide diversity is moderate (96.8–99.6% and 98.4–99.6% pairwise identity for P3 and P45 alleles, respectively), the Arsenophonus ASPE variants are far more diverse (88.5–97.1% and 85.6–99.8% pairwise identity for P3 and P45 alleles, respectively). Recombination and phylogenetic analyses
The relationships between APSE variants were studied using the P3 and P45 sequences obtained in this study as well as additional sequences available in GenBank (Table S2). Although intergenic recombination was not found in the P3 and P45 data set, intragenic recombination was detected for the two APSE genes by both RDP and GENCONV methods (all P < 10 3), which are readily apparent through the examination of Arsenophonus and Hamiltonella APSE sequence alignments (Fig. S2). Intragenic recombination affects both P3 and P45, with clear evidence of horizontal DNA transfers between APSE variants from Arsenophonus but also between APSE variants from Arsenophonus and those from Hamiltonella. The network (Figs 1 and 2) and ML (Figs S3 and S4) phylogenetic analyses obtained for each of the two APSE genes were generally in agreement. The APSE variants FEMS Microbiol Ecol 90 (2014) 184–194
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(a) Arsenophonus - Hamiltonella defensa
Providencia alcalifaciens Providencia sneebia
Xenorhabdus nematophila Yersinia pseudotuberculosis
Yersinia pestis Enterobacter cloacae Escherichia coli
0.02
Sodalis glossinidius
Euplemus vesicularis
(b)
Nasonia vitripennis
Aphis cracivora
Nasonia vitripennis Spalangia cameroni
Pseudolynchia canariensis
Brachytarsina allaudi Nasonia vitripennis
Aphis idaei
Triatoma infestans Crataerina melbae Triatoma guasayana
Hippobosca longipennis Hippobosca equina
Cacopsylla alaterni Bemisia tabaci Crataerina pallida Trialeurodes vaporarium Aphis gossypii
Acyrtosiphon pisum
Glycapsis brimblecombei
Melanaphis donacis
Acyrtosiphon pisum Chaitophorus sp. Aphis ruborum
0.02
Fig. 1. (a) Phylogenetic network based on APSE P45 gene sequences from Hamiltonella defensa (black symbols), Arsenophonus (white symbols), Hamiltonella–Arsenophonus coinfections (gray symbols), as well as on more divergent phage sequences from other bacterial species. (b) Inset of Hamiltonella- and Arsenophonus-APSE P45 network. Circles: endosymbionts from Sternorrhyncha; diamonds: endosymbionts from triatomine bugs; squares: endosymbionts from parasitoid wasps; triangles: endosymbionts from louse flies; reverse triangles: endosymbionts from bat flies. New sequences from this study are in bold and underlined. The scale bar is in units of substitution/site.
FEMS Microbiol Ecol 90 (2014) 184–194
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(a) Arsenophonus - Hamiltonella defensa
Sodalis glossinidius
Providencia alcalifaciens
0.04
(b)
Crataerina melbae
Crataerina pallida
Nasonia vitripennis Pseudolynchia canariensis
Brachytarsina allaudi
Cacopsylla alaterni
Bemisia tabaci
Melanaphis donacis
Aphis ruborum
Bemisia tabaci Uroleucon rudbeckiae
Acyrtosiphon pisum
Chaitophorus sp.
0.02
Fig. 2. (a) Phylogenetic network based on APSE P3 gene sequences from Hamiltonella defensa (black symbols), Arsenophonus (white symbols), Hamiltonella–Arsenophonus coinfections (gray symbols), as well as on more divergent phage sequences from other bacterial species. (b) Inset of Hamiltonella- and Arsenophonus-APSE P3 network. Circles: endosymbionts from Sternorrhyncha; diamonds: endosymbionts from triatomine bugs; squares: endosymbionts from parasitoid wasps; triangles: endosymbionts from louse flies; reverse triangles: endosymbionts from bat flies. New sequences from this study are in bold and underlined. The scale bar is in units of substitution/site.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Ecol 90 (2014) 184–194
APSE in Arsenophonus
found in Arsenophonus proved to be phylogenetically closely related to the APSE variants found in Hamiltonella. The phylogenetic analyses further showed the existence of one inner clade in which all APSE sequences from Hamiltonella cluster. This inner clade also contains APSE sequences found in the two Hamiltonella–Arsenophonus coinfected aphid species, suggesting that these sequences may be associated with Hamiltonella rather than with Arsenophonus. However, the clade of Hamiltonella APSE encompasses only a subpart of the APSE’s full diversity: Hamiltonella APSE belong to a vast phage group mainly delineated by Arsenophonus-APSE variants. Remarkably, some observations could suggest codivergence of APSE with Arsenophonus through cocladogenesis, resulting in sister bacterial strains hosting sister APSE variants. For instance, the sister Arsenophonus strains found in parasitoid wasps (Duron et al., 2010; Taylor et al., 2011) shared very closely related APSE variants (Figs 1 and S3). However, sister APSE variants can be also found in phylogenetically distant Arsenophonus strains, a pattern rather suggestive of APSE lateral transfer between Arsenophonus strains. This pattern is well shown by the sister APSE variants found in the bat fly Brachytarsina allaudi and parasitoid wasps (Figs 1, 2, S3 and S4) while their respective Arsenophonus strains are not sister strains (Duron et al., 2014). Similarly, sister Arsenophonus strains, such as the Arsenophonus strain of the aphid Aphis idaei and those found in parasitoid wasps (Jousselin et al., 2013), may also host divergent APSE variants (Figs 1 and S3), suggesting a recent acquisition through lateral transfer.
Discussion When originally described, APSE had only been found in Hamiltonella defensa, but this present work and a few early studies (Hansen et al., 2007; Wilkes et al., 2010; Taylor et al., 2011) show that a wide range of APSE variants exists in another facultative heritable symbiont of insects, Arsenophonus. The incidence of APSE is exceptionally high for Arsenophonus, with two-thirds of surveyed strains harboring APSE, compared with none among the surveyed Arsenophonus-related groups, Aschnera chinzeii and ALO-3. Many APSE variants were found in Arsenophonus strains hosted by insects distantly related to the typical hosts of Hamiltonella (i.e. the Sternorrhyncha), as shown with triatomine bugs, parasitoid wasps, louse flies, and bat flies. Through Arsenophonus infections, APSE has thus a broad distribution across insects and possibly contributes to the ecology of many host species. APSE has a long evolutionary history in heritable endosymbionts and, although primarily described in Hamiltonella, may more likely originate from the ArsenFEMS Microbiol Ecol 90 (2014) 184–194
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ophonus genus. Specifically, the broad distribution of APSE across the Arsenophonus strains, the extensive sequence divergence in APSE genes (far greater in Arsenophonus than in Hamiltonella), and the finding that the Hamiltonella APSE variants cluster in an inner subclade embedded in a vast clade delineated by ArsenophonusAPSE variants suggest that the origin of APSE extends deep into the evolutionary past of Arsenophonus. Worthy of note is that an undiscovered source of APSE diversity may also exist in another bacterium, which could be the primary origin of this phage. Moreover, such a diversity pattern of APSE in Hamiltonella and Arsenophonus, which are distantly related within the Enterobacteriaceae (Husnik et al., 2011), combined with its apparent absence in other bacteria, supports an origin from lateral transfer with a secondary and recent acquisition of APSE by Hamiltonella from an Arsenophonus ancestor. Repeated lateral transfer among Arsenophonus strains and among Hamiltonella strains also led to the APSE’s present distribution. In Arsenophonus, phylogenetic evidence also suggests that APSE had often transferred horizontally between strains. This scenario is also likely for Hamiltonella, as shown by the incongruence between APSE and Hamiltonella phylogenies (Degnan & Moran, 2008b). However, although lateral transfers of APSE are common in this system, not all strains of Arsenophonus (this study) or Hamiltonella (Degnan & Moran, 2008b) are infected by APSE. This is suggestive of a complex APSE infection dynamic, and its present distribution may indicate a widespread equilibrium between processes of gain from lateral transfers and losses, resulting from balance of benefits and costs associated with phage infection, as recently observed in Hamiltonella-infecting laboratory colonies of the pea aphid (Oliver et al., 2009). Forces other than horizontal transfers of entire phage genomes drive APSE’s evolutionary dynamics: APSE has a dynamic and mosaic genome with repeated recombination events that have resulted in a diversity of variants, as shown in Hamiltonella (Degnan & Moran, 2008a, b). Recombination in bacteriophages hosted by heritable symbionts is, however, not surprising and was reported in other systems, as in the WO bacteriophage of Wolbachia (Bordenstein & Wernegreen, 2004). Remarkably, WO is a widespread source of genomic instability in Wolbachia (Duron et al., 2006; Atyame et al., 2011) and putatively involved in host-adaptation processes (Kent & Bordenstein, 2010). Such a pattern may also occur in Hamiltonella: Recombination was also shown to affect the evolution of APSE virulence cassettes and then to play a key role in the evolution of defensive symbiosis: by reassorting toxin genes and by acquiring novel toxin genes from distantly related bacteria, recombination is thought to contribute substantially to variation among Hamiltonella ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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strains’ protective abilities (Degnan & Moran, 2008a). As a result, recombination between APSE variants contributes to its adaptive dynamics, hence possibly facilitating the emergence of new phenotypes. Ecological connections between Arsenophonus and Hamiltonella are probably one of the keys to elucidate APSE distribution. Different heritable symbionts, as Arsenophonus and Hamiltonella, commonly reside within the same individual insect as coinfections (Russell et al., 2003, 2013; Weinert et al., 2007; Duron et al., 2008; Gueguen et al., 2010; Ferrari et al., 2012) providing opportunities for genetic exchanges as shown with the phage WO between Wolbachia strains (Bordenstein & Wernegreen, 2004; Kent et al., 2011) and with transposable elements between a variety of heritable endosymbionts (Cordaux et al., 2008; Duron, 2013). It is thus likely that Sternorrhyncha cells, or Sternorrhyncha hemocoel to some extent, have served as primary ecological area for exchange of APSE (and possibly for other genetic elements) within and between Arsenophonus and Hamiltonella strains. Coinfections may thus be major catalysts for APSE dispersal, dictating global incidence across endosymbiont communities. While some case studies have demonstrated the importance of APSE in Hamiltonella defensive symbioses (Degnan & Moran, 2008b; Oliver et al., 2009), its role in Arsenophonus symbioses remains to be characterized. Worthy of note is that APSE was not found in the Arsenophonus relatives that are thought to be obligate endosymbionts, Aschnera chinzeii and ALO-3, suggesting that APSE may play a significant role only in facultative symbionts as Arsenophonus. The widespread distribution of APSE in Arsenophonus may indicate the existence of many defensive Arsenophonus types in insects, as suggested for the red lerp psyllid, Glycaspis brimblecombei (Hansen et al., 2007). In this insect, variation of infection frequency by an Arsenophonus strain associated with APSE was found to be significantly related to parasitism pressure: Arsenophonus reached higher frequencies in psyllid populations where parasitoids are more common, suggesting that this Arsenophonus strain may here act as a defensive symbiont protecting against parasitoid attacks, like Hamiltonella. In contrast, Arsenophonus did not protect the soybean aphid, Aphis glycines, from parasitoids, although it is not known whether it is infected with APSE (Wulff et al., 2013). Other phenotypes than defensive symbiosis are also possible: Homologs of toxins that are suspected to target eukaryotic cells and thus to induce wasp mortality (Moran et al., 2005a; Degnan & Moran, 2008b) are present in the APSE variant of the Arsenophonus type species, the male killer Arsenophonus nasoniae (Wilkes et al., 2010). This Arsenophonus species manipulates the reproduction of various parasitoid wasps by ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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distorting the progeny sex ratio toward the production of females through male killing (Skinner, 1985; Werren et al., 1986; Duron et al., 2010). Although the biochemical mechanism by which A. nasoniae kills wasp male offspring remains unknown, the similarity between A. nasoniae APSE’s and Hamiltonella APSE’s virulence factors may suggest a role of APSE in male killing. The effects of APSE remain, however, to be characterized indepth in this system, prompting further investigations on Arsenophonus biology. To conclude, APSE evolutionary dynamics illustrate the complex webs of genetic information exchange existing within heritable symbiosis. Notably, acquisition of heritable symbionts by a new host species is now viewed as an immediate mechanism of adaptation (Moran, 2007; Oliver et al., 2010; Jaenike, 2012; Henry et al., 2013). The case of APSE further shows that other genetic connections are possible. Given that phage presence determines the defensive capacity of Hamiltonella, phage transfers may move important traits between symbiont taxa, resulting in insect host species acquiring new adaptive traits without the need for a new heritable symbiont.
Acknowledgements I am are grateful to U. Schneppat, B. Droz, C. Paupy, P. Tortosa, E. Jousselin, A. Coeur d’Acier, F. VanlerbergheMasutti, J. Barabas, J.C. Beaucournu, S. Marcoux, J.F. Noblet, M. Raymond, and B. Godelle for providing specimens. This work was supported by recurrent funding from the CNRS.
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Arsenophonus insect symbionts are commonly infected with APSE, a bacteriophage involved in protective symbiosis Olivier Duron Institut des Sciences de l’Evolution, CNRS-UM2 (UMR5554), Montpellier Cedex 05, France
Correspondence: Olivier Duron, 911 Avenue Agropolis, Montpellier 34394, France. Tel.: 0033 4 6741 6158; fax: 0033 4 6741 6299; e-mail: [email protected] Present address: Olivier Duron, Laboratoire MIVEGEC (Maladies Infectieuses et Vecteurs: ^le), Ecologie, G en etique, Evolution et Contro CNRS-UM1-UM2 (UMR5290) – IRD (UR 224), Centre IRD, Montpellier, France Received 6 June 2014; revised 8 July 2014; accepted 9 July 2014. Final version published online 11 August 2014. DOI: 10.1111/1574-6941.12381
MICROBIOLOGY ECOLOGY
Editor: Max H€ aggblom Keywords heritable symbiosis; defensive symbionts; Arsenophonus; Hamiltonella; APSE bacteriophage.
Abstract Insects commonly have intimate associations with maternally inherited bacterial symbionts. While many inherited symbionts are not essential for host survival, they often act as conditional mutualists, conferring protection against certain environmental stresses. The defensive symbiont Hamiltonella defensa which protects aphids against attacks by parasitoid wasps is one of these conditional mutualists. The protection afforded by Hamiltonella depends on the presence of a lysogenic bacteriophage, called APSE, encoding homologs of toxins that are suspected to target wasp cells. In this study, an important diversity of APSE variants is reported from another heritable symbiont, Arsenophonus, which is exceptionally widespread in insects. APSE was found in association with two-thirds of the Arsenophonus strains examined and from a variety of insect groups such as aphids, white flies, parasitoid wasps, triatomine bugs, louse flies, and bat flies. No APSE was, however, found from Arsenophonus relatives such as the recently described Aschnera chinzeii and ALO-3 endosymbionts. Phylogenetic investigations revealed that APSE has a long evolutionary history in heritable symbionts, being secondarily acquired by Hamiltonella through lateral transfer from Arsenophonus. Overall, this highlights the role of lateral transfer as a major evolutionary process shaping the emergence of defensive symbiosis in heritable bacteria.
Introduction Insects commonly have associations with heritable bacterial endosymbionts that inhabit insect cells and depend on maternal transmission, through egg cytoplasm, to ensure their transmission (Moran et al., 2008; Werren et al., 2008; Oliver et al., 2010). While some heritable bacteria have evolved toward obligate mutualism and are required to support normal insect development, many others are facultative symbionts exerting more subtle effects such as protection against environmental stresses (Moran, 2007; Oliver et al., 2010; Hamilton & Perlman, 2013) or manipulation of host reproduction (Werren et al., 2008; Engelstadter & Hurst, 2009). These effects promote the spread of heritable bacteria through insect populations, but may also drive insect evolutionary ecology, contributing substantially to the acquisition of ecologically important traits to infected species (Moran, 2007; Oliver et al., 2010, 2014; Ferrari & Vavre, 2011; Jaenike, 2012). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
In recent years, a great deal of attention has been devoted to defensive symbionts that protect insects against attack by natural enemies (Oliver et al., 2003; Scarborough et al., 2005; Hedges et al., 2008; Jaenike et al., 2010). The pea aphid (Acyrthosiphon pisum), an organism intensively studied with respect to symbiotic interactions, was one the first insects revealed to have defensive symbionts (Oliver et al., 2003; Ferrari et al., 2004). Indeed, infection of the pea aphid by the heritable bacterium Hamiltonella defensa (c-Proteobacteria: Enterobacteriaceae) provides resistance to parasitoid wasps by killing developing wasp larvae or embryos in the aphid hemocoel (Oliver et al., 2003, 2005; Moran et al., 2005a; Bensadia et al., 2006). Through reducing the mortality risk after wasp oviposition, the pea aphid thus relies heavily on its Hamitonella for protection. Further studies suggest that the defensive function of Hamiltonella may be extended to other Hamiltonella-infected species, as it also provides another insect, the black bean aphid (Aphis fabae), with strong protection against parasitoid wasps FEMS Microbiol Ecol 90 (2014) 184–194
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(Vorburger et al., 2009, 2013; Cayetano & Vorburger, 2013). Moreover, parasitic wasps discriminate between Hamiltonella-infected and uninfected aphids of diverse species, thus showing that Hamiltonella reduces the risk of parasitism by making hosts less attractive to searching parasitoids (Oliver et al., 2012; Lukasik et al., 2013). Although Hamiltonella is rare in insects, it is commonly found in the sap-sucking hemipteran suborder Sternorrhyncha, including aphids, whiteflies, mealybugs, and psyllids (Zchori-Fein & Brown, 2002; Russell et al., 2003; Moran et al., 2005b). Remarkably, Hamiltonella can be occasionally exchanged between the Sternorrhyncha species through a process of horizontal transfer that may possibly result in the instantaneous acquisition of resistance to parasitoids (Oliver et al., 2010). The mechanistic basis of Hamlitonella’s protective effect has been actively studied in the pea aphid. The degree of protection was shown to vary among Hamiltonella strains, ranging from small reductions in successful wasp parasitism to nearly complete protection (Oliver et al., 2005, 2009). While this variation might depend on Hamiltonella chromosomal gene content, the degree of protection is also greatly influenced by a Hamiltonella lysogenic lambdoid bacteriophage (Moran et al., 2005a; Degnan et al., 2009), called APSE (‘A. pisum secondary endosymbiont phage’; van der Wilk et al., 1999): While Hamiltonella strains lacking APSE confer little protection, those harboring APSE increase aphid survival following wasp attack (Degnan & Moran, 2008b; Oliver et al., 2009). An additional variation in the degree of protection is associated with the nature of APSE gene products and especially of APSE-borne toxins: A variety of phage variants, each harboring a specific virulence cassette region, are known in Hamiltonella (Moran et al., 2005a; Degnan & Moran, 2008a). Remarkably, APSE was found to be common in Hamiltonella strains of aphids, whiteflies, and psyllids (Moran et al., 2005a; Degnan & Moran, 2008a, b), suggesting that APSE may have a significant influence in the evolution of symbiont-mediated protection in the Sternorrhyncha. Until recently, APSE was thought to be exclusively associated with Hamiltonella. However, APSE has been also occasionally detected in Arsenophonus, another facultative heritable bacterium but not closely related to Hamiltonella (Hansen et al., 2007; Taylor et al., 2011). Further analyses of the draft genome sequence of Arsenophonus type species, Arsenophonus nasoniae, also revealed similarity of its phage elements with the APSE sequences found in Hamiltonella (Wilkes et al., 2010). APSE may thus readily move between quite unrelated symbionts and be an immediate and powerful mechanism of rapid adaptation for symbionts and their hosts. Remarkably, while Hamiltonella is largely restricted to the Sternorrhyncha FEMS Microbiol Ecol 90 (2014) 184–194
suborder, Arsenophonus is more largely widespread and has been found in many insect orders including hemipterans, hymenopterans, and dipterans (Duron et al., 2008, 2010, 2014; Novakova et al., 2009; Taylor et al., 2011; Mouton et al., 2012; Russell et al., 2012; Jousselin et al., 2013), suggesting that APSE may possibly protect against parasitoid attacks in a variety of insect groups. A key question with regard to mutualistic symbiosis is the extent to which a source of adaptation, such as the defensive APSE phage, moves between symbiont taxa. Here, this topic was approached by undertaking an extensive screening for the presence, the diversity, and the evolutionary history of APSE infections in a collection of bacterial endosymbionts from 33 host species, including hemipterans, hymenopterans, and dipterans. This collection includes Arsenophonus and two other heritable bacteria phylogenetically allied to Arsenophonus (Arsenophonus and like organisms, ALOs hereafter) that have recently been characterized: Aschnera chinzeii (Hosokawa et al., 2012) and ALO-3 (Duron et al., 2014). While Arsenophonus is a widespread facultative endosymbiont, Aschnera chinzeii and ALO-3 are rather thought to be obligate endosymbionts which are restricted to few species of bat flies and louse flies, respectively (Hosokawa et al., 2012; Duron et al., 2014). Sequencing and phylogenetic analyses of the P3 (encoding for a putative P-loop ATPase) and P45 (DNA polymerase) phage genes were further used to characterize the evolutionary processes that shape APSE’s diversity in communities of heritable bacteria.
Materials and methods Insect collection
A collection of 114 DNA templates from 33 ALOinfected insect species was used (Table 1). For each DNA template, ALO infection had been formally characterized in previous studies through single- or multilocus DNA sequencing (Duron et al., 2010, 2014; Jousselin et al., 2013). Of the 33 insect species examined in this study, infection by Arsenophonus was found in 22, including five species of aphid (Aphididae), one of whitefly (Aleurodidae), one of psyllid (Psyllidae), two of triatomine bug (Reduvidae), three of bat fly (Nycteribiidae and Streblidae), nine of louse fly (Hippoboscidae), and one of parasitoid wasp (Pteromalidae) as detailed in Table 1. Infections by Aschnera chinzeii and by ALO-3 were found in six bat fly species and five louse fly species, respectively (Table 1). Each insect species was found infected by a genetically distinct ALO strain with the exception of two louse fly species pairs, Hippobosca equina/H. longipennis and Lipoptena cervi/Lipoptena sp., which were infected by genetically identical Arsenophonus ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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strains (Duron et al., 2010, 2014; Jousselin et al., 2013). Within each examined host species, there were 1–5 ALO-infected individuals separately investigated for the present analysis, with the exception of aphids and whiteflies for which 1–3 ALO-infected pools of five specimens were examined (Table 1).
Screening and sequencing for APSE
ALO DNA quality was systematically tested by specific PCR amplification of a region of the bacterial GroEL gene following Duron et al. (2014), as described in Supporting Information, Table S1. The APSE infections were next
Table 1. Distribution of APSE in ALO heritable bacteria and their host species APSE Host species Hemiptera Aleurodidae (whiteflies) Bemisia tabaci (Gennadius, 1889) Aphididae (aphids) Aphis craccivora (Koch, 1854) Aphis idaei (van der Goot, 1912) Aphis gossypii (Glover, 1877) €rner, 1932) Aphis ruborum (Bo Melanaphis donacis (Passerini, 1862) Psyllidae (psyllids) Cacopsylla alaterni (Foerster, 1848) Reduviidae (triatomine bugs) Triatoma guasayana (Wygodzinsky & Abalos, 1941) Triatoma infestans (Klug, 1834) Hymenoptera Pteromalidae (parasitoid wasps) Nasonia vitripennis (Walker, 1836) Diptera Hippoboscidae (louse flies) ndani, 1879) Crataerina melbae (Ro Crataerina pallida (Latreille, 1812) Hippobosca equina (Linnaeus, 1758) Hippobosca longipennis (Fabricius, 1805) Lipoptena capreoli (Rondani, 1878) Lipoptena cervi (Linnaeus, 1758) Lipoptena fortisetosa (Maa, 1965) Lipoptena sp. Pseudolynchia canariensis (Macquart, 1839) Ornithomya avicularia (Linnaeus, 1758) Ornithomya biloba (Dufour, 1827) Ornithomya chloropus (Bergroth, 1901) Ornithomya fringillina (Curtis, 1836) Stenepteryx hirundinis (Linnaeus, 1758) Nycteribiidae (bat flies) Basilia nov. sp. Eucampsipoda africana (Theodor, 1955) Nycteribia schmidlii scotti (Falcoz, 1923) Nycteribia stylidiopsis (Speicer, 1908) Nycteribia kolenatii (Theodor & Moscona, 1954) Penicillidia conspicua (Speiser, 1901) Penicillidia fulvida (Bigot, 1885) Penicillidia leptothrinax (Speiser, 1908) Streblidae (bat flies) Brachytarsina allaudi (Falcoz, 1923)
ALO heritable bacteria
n
P3
P45
Reference for ALO characterization of DNA samples
Arsenophonus
1*
Present
Present
Duron et al. (2010)
Arsenophonus Arsenophonus Arsenophonus Arsenophonus† Arsenophonus†
3* 3* 1* 3* 1*
_ _ _ Present Present
Present Present Present Present Present
Jousselin Jousselin Jousselin Jousselin Jousselin
Arsenophonus
3‡
Present
Present
Duron et al. (2010)
Arsenophonus Arsenophonus
1‡ 3‡
_ _
Present Present
Duron et al. (2010) Duron et al. (2010)
Arsenophonus
3‡
Present
Present
Duron et al. (2010)
Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus Arsenophonus ALO-3 ALO-3 ALO-3 ALO-3 ALO-3
5‡ 5‡ 5‡ 5‡ 2‡ 5‡ 2‡ 2‡ 5‡ 5‡ 5‡ 5‡ 5‡ 5‡
Present Present _ _ _ _ _ _ Present _ _ _ _ _
Present Present Present Present _ _ _ _ Present _ _ _ _ _
Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron Duron
et et et et et et et et et et et et et et
al. al. al. al. al. al. al. al. al. al. al. al. al. al.
(2014) (2014) (2010) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014)
Arsenophonus Arsenophonus Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii Aschnera chinzeii
2‡ 5‡ 5‡ 5‡ 1‡ 1‡ 4‡ 5‡
_ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _
Duron Duron Duron Duron Duron Duron Duron Duron
et et et et et et et et
al. al. al. al. al. al. al. al.
(2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014)
Arsenophonus
3‡
Present
Present
Duron et al. (2014)
et et et et et
al. al. al. al. al.
(2013) (2013) (2013) (2013) (2013)
*Number of pools of specimens examined. † In coinfection with Hamiltonella. ‡ Number of individuals separately investigated for analysis.
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FEMS Microbiol Ecol 90 (2014) 184–194
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APSE in Arsenophonus
screened and genotyped using independent PCR assays based on the P3 and P45 genes with a set of specific primers. Additional PCR assays were also conducted to check for coinfections with other bacterial endosymbionts. First, on all DNA samples, PCR assays on the bacterial 16S rRNA gene using Hamiltonella-specific primers were conducted. Second, all APSE-positive samples were further tested using specific primers for three other bacterial endosymbionts commonly found in insects: Wolbachia, Rickettsia, and Sodalis. Gene features and primers are listed in Table S1. All the PCR amplifications were performed under the following conditions: initial denaturation at 93 °C for 3 min, 30 cycles of denaturation (93 °C, 30 s), annealing (52–54 °C, depending on primers, cf. Table S1, 30 s), extension (72 °C, 1 min), and a final extension at 72 °C for 5 min. The PCR products were electrophoresed in a 1.5% agarose gel. Amplification products were sequenced in both directions at Eurofins. Genomics (Ebersberg, Germany). Chromatograms were checked and edited using CHROMAS LITE (http://www. technelysium.com.au), and sequence alignments were performed using CLUSTALW (Thompson et al., 2002), both implemented in MEGA (Kumar et al., 2004). New sequence data have been submitted to GenBank (Accession Numbers KJ774110–KJ774136; see Table S2 for details). Molecular phylogenetic analyses
Sequence alignments were carried out using CLUSTALW (Thompson et al., 2002) and corrected using MEGA (Tamura et al., 2007). The GBLOCKS program (Castresana, 2000) with default parameters was used to remove poorly aligned positions and to obtain unambiguous sequence alignments. All sequence alignments were also checked for putative recombinant regions using the GENECONV (Sawyer, 1989) and RDP (Martin & Rybicki, 2000) methods available in the RDP3 computer analysis package (Martin et al., 2010). Phylogenetic relationships were evaluated between APSE variants using P3 (771 unambiguously aligned nucleotide sites) and P45 (506 bp) sequences and between Hamiltonella defensa strains using partial 16S rRNA (428 bp) sequences. In addition to sequences produced in this study, additional P3 and P45 sequences of Hamiltonella and Arsenophonus, as well as a subset of more divergent phage sequences from other bacterial species available from GenBank, were also used (Table S2). The evolutionary model most closely fitting the sequence data was determined using Akaike information criterion with the MEGA program (Tamura et al., 2007). The best-fit approximation was K2+G for the APSE P3 and P45 data set and HKY+I for the Hamiltonella 16S rRNA data set. FEMS Microbiol Ecol 90 (2014) 184–194
Phylogenetic analyses were based on maximum likelihood (ML) analyses. A ML heuristic search, using a starting tree obtained by neighbor joining, was conducted, and clade robustness was further assessed by bootstrap analysis using 1000 replicates in MEGA (Tamura et al., 2007). Because the recombination between APSE variants may hamper the use of phylogenetic tree-based analyses, a network analysis was carried out, employing SplitsTree (Huson & Bryant, 2006) in conjunction with the agglomerating NeighborNet algorithm (Bryant & Moulton, 2004). The resulting phylogenetic networks generalize the trees by allowing cross-connections between branches which might visualize conflicting signals due to lateral gene transfers.
Results Distribution of APSE
A total of 114 DNA templates with quality being confirmed by positive ALO-GroEL PCR amplification were used (Table 1). To check for ALO-Hamiltonella coinfection, the presence of Hamiltonella was first assayed through a specific 16S rRNA gene-based PCR assay. Of the 114 DNA templates, Hamiltonella was found in four templates from two Arsenophonus-infected aphid species, Aphis ruborum (three templates) and Melanaphis donacis (one), showing then that coinfection with Hamiltonella and Arsenophonus occurs in these species (Table 1). That these were double infections was ascertained through the sequencing of the Hamiltonella 16S rRNA PCR products and the observation of clean Hamiltonella sequences. Phylogenetically, the bacterial strains from A. ruborum and M. donacis clearly fall within the genus Hamiltonella and cluster with the strains found in other aphid species (Supporting Information, Fig. S1). The 114 DNA templates were next screened for the presence of APSE using P3 and P45-based PCR assays (Table 1). The P3 and P45 PCR screening produced similar results but with some differences: The P45 gene was successfully amplified from 50 of the 114 DNA templates while the P3 gene was only amplified from 29 templates (all P3-positive DNA templates were also P45 positive; Table 1). The failure of P3 PCR amplification in these cases may be due either to gene deletion or, possibly, to a consequence of mutations in the P3 priming regions. Overall, APSE was detected in 50 DNA templates, including the four of A. ruborum and M. donacis with Arsenophonus–Hamiltonella coinfection. In these last four cases, the detection of APSE suggests that APSE may be associated with Arsenophonus, Hamiltonella, or both; the association between APSE and Arsenophonus is thus here uncertain. However, in the 46 remaining cases, no ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Hamiltonella infection was detected, showing that APSE is associated with Arsenophonus infections. APSE was found in association with 14 of the 20 Arsenophonus strains (excluding the strains from A. ruborum and M. donacis in which coinfection with Hamitonella occurs), while none of the six Aschnera chinzeii strains and none of the five ALO-3 strains was detected to be APSE positive. The 14 APSE-positive Arsenophonus strains are from very diverse insect orders, including Hemiptera (three species of aphid, one of whitefly, one of psyllid and two of triatomine bugs), Hymenoptera (one species of parasitoid wasp), and Diptera (five species of louse fly and one of bat fly) (Table 1). For each positive host species, APSE was observed in all tested DNA templates (n = 1–5, depending on species; Table 1). Additional PCR screening did reveal other endosymbionts than Arsenophonus and Hamiltonella in nine of the 50 APSE-positive samples: Wolbachia was found in three templates from the wasp Nasonia vitripennis and in five templates from the louse fly Pseudolynchia canariensis; Rickettsia in one template from the white fly Bemisia tabaci; and Sodalis in the five templates from P. canariensis. Although the coinfection pattern suggests that APSE may be associated with other bacteria than with Arsenophonus in these three insect species, this hypothesis can be rejected in at least two cases. In N. vitripennis, the sequencing of the Arsenophonus genome has previously shown that APSE is integrated in the Arsenophonus chromosome (Wilkes et al., 2010). In B. tabaci, the Rickettsia genome has been also sequenced (Rao et al., 2012), but Basic Local Alignment Search Tool (BLAST) searches against this genome (GenBank Accession Number AJWD01000000) did not detect APSE, suggesting that APSE is rather associated with Arsenophonus. In P. canariensis, APSE may be associated either with Sodalis or with Arsenophonus, and the association between APSE and Arsenophonus should thus be treated with caution. However, this is unlikely that APSE is associated with Wolbachia in this insect as no APSE was found in all Wolbachia genomes sequenced to date from many insect species. Overall, the present data thus indicate that APSE is clearly associated with Arsenophonus infections in many cases. Diversity of APSE infections
The diversity of APSE was examined using P3 and P45 DNA sequences from the 50 positive DNA templates, including the A. ruborum and M. donacis DNA templates with Hamiltonella–Arsenophonus coinfection. The sequences were easily readable without double peaks, indicating that there were no multiple APSE variants in any DNA templates. This was also true for the A. ruborum ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
O. Duron
and M. donacis DNA templates, suggesting that only one APSE variant is present in Hamiltonella–Arsenophonus coinfected aphids. On the basis of nucleotide sequences, nine P3 distinct alleles (87.9–97.3% pairwise identity) and 14 P45 distinct alleles (85.0–98.6% pairwise identity) were characterized from the 50 DNA templates. Overall, 14 genetically different APSE variants were identified on the basis of both P3 and P45 sequences. No P3 and P45 sequence variation was observed between DNA templates from any given insect species. Each of the APSE-positive insect species harbored a different APSE variant with the exception of two species pairs, Hippobosca equina/H. longipennis and Triatoma infestans/T. guasayana, which were infected by genetically identical APSE variants on the basis of P45 sequences (no P3 PCR products were obtained from these templates; Table 1). The P45 sequence of the Arsenophonus strain from the whitefly Bemisia tabacci contained an internal stop codon, suggesting that this APSE gene may not be functional. In all other cases, P3 and P45 APSE sequences do not contain internal stop codons and are thus likely to code for putatively functional phage protein. The comparison between APSE sequences from this study and other APSE sequences available in GenBank (Table S2) revealed a contrasting pattern of nucleotide diversity between Hamiltonella APSE and Arsenophonus APSE (The APSE sequences from Hamiltonella–Arsenophonus coinfections were excluded from this comparison.) While the level of Hamiltonella APSE nucleotide diversity is moderate (96.8–99.6% and 98.4–99.6% pairwise identity for P3 and P45 alleles, respectively), the Arsenophonus ASPE variants are far more diverse (88.5–97.1% and 85.6–99.8% pairwise identity for P3 and P45 alleles, respectively). Recombination and phylogenetic analyses
The relationships between APSE variants were studied using the P3 and P45 sequences obtained in this study as well as additional sequences available in GenBank (Table S2). Although intergenic recombination was not found in the P3 and P45 data set, intragenic recombination was detected for the two APSE genes by both RDP and GENCONV methods (all P < 10 3), which are readily apparent through the examination of Arsenophonus and Hamiltonella APSE sequence alignments (Fig. S2). Intragenic recombination affects both P3 and P45, with clear evidence of horizontal DNA transfers between APSE variants from Arsenophonus but also between APSE variants from Arsenophonus and those from Hamiltonella. The network (Figs 1 and 2) and ML (Figs S3 and S4) phylogenetic analyses obtained for each of the two APSE genes were generally in agreement. The APSE variants FEMS Microbiol Ecol 90 (2014) 184–194
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APSE in Arsenophonus
(a) Arsenophonus - Hamiltonella defensa
Providencia alcalifaciens Providencia sneebia
Xenorhabdus nematophila Yersinia pseudotuberculosis
Yersinia pestis Enterobacter cloacae Escherichia coli
0.02
Sodalis glossinidius
Euplemus vesicularis
(b)
Nasonia vitripennis
Aphis cracivora
Nasonia vitripennis Spalangia cameroni
Pseudolynchia canariensis
Brachytarsina allaudi Nasonia vitripennis
Aphis idaei
Triatoma infestans Crataerina melbae Triatoma guasayana
Hippobosca longipennis Hippobosca equina
Cacopsylla alaterni Bemisia tabaci Crataerina pallida Trialeurodes vaporarium Aphis gossypii
Acyrtosiphon pisum
Glycapsis brimblecombei
Melanaphis donacis
Acyrtosiphon pisum Chaitophorus sp. Aphis ruborum
0.02
Fig. 1. (a) Phylogenetic network based on APSE P45 gene sequences from Hamiltonella defensa (black symbols), Arsenophonus (white symbols), Hamiltonella–Arsenophonus coinfections (gray symbols), as well as on more divergent phage sequences from other bacterial species. (b) Inset of Hamiltonella- and Arsenophonus-APSE P45 network. Circles: endosymbionts from Sternorrhyncha; diamonds: endosymbionts from triatomine bugs; squares: endosymbionts from parasitoid wasps; triangles: endosymbionts from louse flies; reverse triangles: endosymbionts from bat flies. New sequences from this study are in bold and underlined. The scale bar is in units of substitution/site.
FEMS Microbiol Ecol 90 (2014) 184–194
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O. Duron
(a) Arsenophonus - Hamiltonella defensa
Sodalis glossinidius
Providencia alcalifaciens
0.04
(b)
Crataerina melbae
Crataerina pallida
Nasonia vitripennis Pseudolynchia canariensis
Brachytarsina allaudi
Cacopsylla alaterni
Bemisia tabaci
Melanaphis donacis
Aphis ruborum
Bemisia tabaci Uroleucon rudbeckiae
Acyrtosiphon pisum
Chaitophorus sp.
0.02
Fig. 2. (a) Phylogenetic network based on APSE P3 gene sequences from Hamiltonella defensa (black symbols), Arsenophonus (white symbols), Hamiltonella–Arsenophonus coinfections (gray symbols), as well as on more divergent phage sequences from other bacterial species. (b) Inset of Hamiltonella- and Arsenophonus-APSE P3 network. Circles: endosymbionts from Sternorrhyncha; diamonds: endosymbionts from triatomine bugs; squares: endosymbionts from parasitoid wasps; triangles: endosymbionts from louse flies; reverse triangles: endosymbionts from bat flies. New sequences from this study are in bold and underlined. The scale bar is in units of substitution/site.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Ecol 90 (2014) 184–194
APSE in Arsenophonus
found in Arsenophonus proved to be phylogenetically closely related to the APSE variants found in Hamiltonella. The phylogenetic analyses further showed the existence of one inner clade in which all APSE sequences from Hamiltonella cluster. This inner clade also contains APSE sequences found in the two Hamiltonella–Arsenophonus coinfected aphid species, suggesting that these sequences may be associated with Hamiltonella rather than with Arsenophonus. However, the clade of Hamiltonella APSE encompasses only a subpart of the APSE’s full diversity: Hamiltonella APSE belong to a vast phage group mainly delineated by Arsenophonus-APSE variants. Remarkably, some observations could suggest codivergence of APSE with Arsenophonus through cocladogenesis, resulting in sister bacterial strains hosting sister APSE variants. For instance, the sister Arsenophonus strains found in parasitoid wasps (Duron et al., 2010; Taylor et al., 2011) shared very closely related APSE variants (Figs 1 and S3). However, sister APSE variants can be also found in phylogenetically distant Arsenophonus strains, a pattern rather suggestive of APSE lateral transfer between Arsenophonus strains. This pattern is well shown by the sister APSE variants found in the bat fly Brachytarsina allaudi and parasitoid wasps (Figs 1, 2, S3 and S4) while their respective Arsenophonus strains are not sister strains (Duron et al., 2014). Similarly, sister Arsenophonus strains, such as the Arsenophonus strain of the aphid Aphis idaei and those found in parasitoid wasps (Jousselin et al., 2013), may also host divergent APSE variants (Figs 1 and S3), suggesting a recent acquisition through lateral transfer.
Discussion When originally described, APSE had only been found in Hamiltonella defensa, but this present work and a few early studies (Hansen et al., 2007; Wilkes et al., 2010; Taylor et al., 2011) show that a wide range of APSE variants exists in another facultative heritable symbiont of insects, Arsenophonus. The incidence of APSE is exceptionally high for Arsenophonus, with two-thirds of surveyed strains harboring APSE, compared with none among the surveyed Arsenophonus-related groups, Aschnera chinzeii and ALO-3. Many APSE variants were found in Arsenophonus strains hosted by insects distantly related to the typical hosts of Hamiltonella (i.e. the Sternorrhyncha), as shown with triatomine bugs, parasitoid wasps, louse flies, and bat flies. Through Arsenophonus infections, APSE has thus a broad distribution across insects and possibly contributes to the ecology of many host species. APSE has a long evolutionary history in heritable endosymbionts and, although primarily described in Hamiltonella, may more likely originate from the ArsenFEMS Microbiol Ecol 90 (2014) 184–194
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ophonus genus. Specifically, the broad distribution of APSE across the Arsenophonus strains, the extensive sequence divergence in APSE genes (far greater in Arsenophonus than in Hamiltonella), and the finding that the Hamiltonella APSE variants cluster in an inner subclade embedded in a vast clade delineated by ArsenophonusAPSE variants suggest that the origin of APSE extends deep into the evolutionary past of Arsenophonus. Worthy of note is that an undiscovered source of APSE diversity may also exist in another bacterium, which could be the primary origin of this phage. Moreover, such a diversity pattern of APSE in Hamiltonella and Arsenophonus, which are distantly related within the Enterobacteriaceae (Husnik et al., 2011), combined with its apparent absence in other bacteria, supports an origin from lateral transfer with a secondary and recent acquisition of APSE by Hamiltonella from an Arsenophonus ancestor. Repeated lateral transfer among Arsenophonus strains and among Hamiltonella strains also led to the APSE’s present distribution. In Arsenophonus, phylogenetic evidence also suggests that APSE had often transferred horizontally between strains. This scenario is also likely for Hamiltonella, as shown by the incongruence between APSE and Hamiltonella phylogenies (Degnan & Moran, 2008b). However, although lateral transfers of APSE are common in this system, not all strains of Arsenophonus (this study) or Hamiltonella (Degnan & Moran, 2008b) are infected by APSE. This is suggestive of a complex APSE infection dynamic, and its present distribution may indicate a widespread equilibrium between processes of gain from lateral transfers and losses, resulting from balance of benefits and costs associated with phage infection, as recently observed in Hamiltonella-infecting laboratory colonies of the pea aphid (Oliver et al., 2009). Forces other than horizontal transfers of entire phage genomes drive APSE’s evolutionary dynamics: APSE has a dynamic and mosaic genome with repeated recombination events that have resulted in a diversity of variants, as shown in Hamiltonella (Degnan & Moran, 2008a, b). Recombination in bacteriophages hosted by heritable symbionts is, however, not surprising and was reported in other systems, as in the WO bacteriophage of Wolbachia (Bordenstein & Wernegreen, 2004). Remarkably, WO is a widespread source of genomic instability in Wolbachia (Duron et al., 2006; Atyame et al., 2011) and putatively involved in host-adaptation processes (Kent & Bordenstein, 2010). Such a pattern may also occur in Hamiltonella: Recombination was also shown to affect the evolution of APSE virulence cassettes and then to play a key role in the evolution of defensive symbiosis: by reassorting toxin genes and by acquiring novel toxin genes from distantly related bacteria, recombination is thought to contribute substantially to variation among Hamiltonella ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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strains’ protective abilities (Degnan & Moran, 2008a). As a result, recombination between APSE variants contributes to its adaptive dynamics, hence possibly facilitating the emergence of new phenotypes. Ecological connections between Arsenophonus and Hamiltonella are probably one of the keys to elucidate APSE distribution. Different heritable symbionts, as Arsenophonus and Hamiltonella, commonly reside within the same individual insect as coinfections (Russell et al., 2003, 2013; Weinert et al., 2007; Duron et al., 2008; Gueguen et al., 2010; Ferrari et al., 2012) providing opportunities for genetic exchanges as shown with the phage WO between Wolbachia strains (Bordenstein & Wernegreen, 2004; Kent et al., 2011) and with transposable elements between a variety of heritable endosymbionts (Cordaux et al., 2008; Duron, 2013). It is thus likely that Sternorrhyncha cells, or Sternorrhyncha hemocoel to some extent, have served as primary ecological area for exchange of APSE (and possibly for other genetic elements) within and between Arsenophonus and Hamiltonella strains. Coinfections may thus be major catalysts for APSE dispersal, dictating global incidence across endosymbiont communities. While some case studies have demonstrated the importance of APSE in Hamiltonella defensive symbioses (Degnan & Moran, 2008b; Oliver et al., 2009), its role in Arsenophonus symbioses remains to be characterized. Worthy of note is that APSE was not found in the Arsenophonus relatives that are thought to be obligate endosymbionts, Aschnera chinzeii and ALO-3, suggesting that APSE may play a significant role only in facultative symbionts as Arsenophonus. The widespread distribution of APSE in Arsenophonus may indicate the existence of many defensive Arsenophonus types in insects, as suggested for the red lerp psyllid, Glycaspis brimblecombei (Hansen et al., 2007). In this insect, variation of infection frequency by an Arsenophonus strain associated with APSE was found to be significantly related to parasitism pressure: Arsenophonus reached higher frequencies in psyllid populations where parasitoids are more common, suggesting that this Arsenophonus strain may here act as a defensive symbiont protecting against parasitoid attacks, like Hamiltonella. In contrast, Arsenophonus did not protect the soybean aphid, Aphis glycines, from parasitoids, although it is not known whether it is infected with APSE (Wulff et al., 2013). Other phenotypes than defensive symbiosis are also possible: Homologs of toxins that are suspected to target eukaryotic cells and thus to induce wasp mortality (Moran et al., 2005a; Degnan & Moran, 2008b) are present in the APSE variant of the Arsenophonus type species, the male killer Arsenophonus nasoniae (Wilkes et al., 2010). This Arsenophonus species manipulates the reproduction of various parasitoid wasps by ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
O. Duron
distorting the progeny sex ratio toward the production of females through male killing (Skinner, 1985; Werren et al., 1986; Duron et al., 2010). Although the biochemical mechanism by which A. nasoniae kills wasp male offspring remains unknown, the similarity between A. nasoniae APSE’s and Hamiltonella APSE’s virulence factors may suggest a role of APSE in male killing. The effects of APSE remain, however, to be characterized indepth in this system, prompting further investigations on Arsenophonus biology. To conclude, APSE evolutionary dynamics illustrate the complex webs of genetic information exchange existing within heritable symbiosis. Notably, acquisition of heritable symbionts by a new host species is now viewed as an immediate mechanism of adaptation (Moran, 2007; Oliver et al., 2010; Jaenike, 2012; Henry et al., 2013). The case of APSE further shows that other genetic connections are possible. Given that phage presence determines the defensive capacity of Hamiltonella, phage transfers may move important traits between symbiont taxa, resulting in insect host species acquiring new adaptive traits without the need for a new heritable symbiont.
Acknowledgements I am are grateful to U. Schneppat, B. Droz, C. Paupy, P. Tortosa, E. Jousselin, A. Coeur d’Acier, F. VanlerbergheMasutti, J. Barabas, J.C. Beaucournu, S. Marcoux, J.F. Noblet, M. Raymond, and B. Godelle for providing specimens. This work was supported by recurrent funding from the CNRS.
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