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Surveys for maternally-inherited endosymbionts reveal novel and variable infections within solitary bee species
Journal of Invertebrate Pathology 132 (2015) 111–114
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Surveys for maternally-inherited endosymbionts reveal novel and variable infections within solitary bee species q Abiya Saeed, Jennifer A. White ⇑ Department of Entomology, University of Kentucky, S-225 Agricultural Sciences Center North, Lexington, KY 40546, USA
a r t i c l e
i n f o
Article history: Received 15 July 2015 Revised 22 September 2015 Accepted 23 September 2015 Available online 26 September 2015 Keywords: Arsenophonus Bacterial endosymbionts Hymenoptera Sodalis Vertical transmission Wolbachia
a b s t r a c t Maternally-inherited bacteria can affect the fitness and population dynamics of their host insects; for solitary bees, such effects have the potential to influence bee efficacy as pollinators. We screened bee species for bacterial associates using 454-pyrosequencing (4 species) and diagnostic PCR (183 specimens across 29 species). The endosymbiont Wolbachia was abundant, infecting 18 species, including all specimens from the family Halictidae. Among commercially-supplied orchard bees (family Megachilidae), only 2/7 species were Wolbachia-infected, but one species showed variable infection among specimens. Two other maternally-inherited bacteria, Arsenophonus and Sodalis, were also detected, neither of which was fixed in infection frequency. Differential endosymbiont infection could potentially compromise fitness and reproductive compatibility among commercially redistributed pollinator populations. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Solitary bees are important pollinators of crops and native flora. Due to the ongoing decline of managed Apis mellifera populations, solitary bees act as a buffer to protect worldwide crop pollination operations (Winfree et al., 2007), and several species, particularly within the family Megachilidae, are commercially available for purchase and use in crop and orchard settings (Bosch and Kemp, 2002; Gruber et al., 2011). Given the rising economic and ecological importance of these bees, increasing attention is also being given to their biology, including their microbial associations (e.g., Gerth et al., 2011, 2013, 2015; McFrederick et al., 2012, 2013, 2014). Solitary bees are particularly prone to infection by maternallyinherited endosymbiotic bacteria in the genus Wolbachia (Gerth et al., 2011, 2013, 2015), bacteria that frequently manipulate host reproduction to increase bacterial transmission (Werren et al., 2008). The preponderance of Wolbachia among solitary bees suggests that these bees may be susceptible to infection by other
q The nucleotide sequence data reported in this manuscript have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers KT123206-42 and KT153630-43, with a public release date of 11 Dec, 2015, and the sequence read archive under project accession number PRJNA283198 with a public release date of 31 Aug, 2015. ⇑ Corresponding author. E-mail addresses: [email protected] (A. Saeed), [email protected] (J.A. White).
http://dx.doi.org/10.1016/j.jip.2015.09.011 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
maternally-inherited endosymbionts as well, yet the frequency of infection by other endosymbionts remains largely unexplored. Gerth et al. (2015) recently screened numerous bee species using taxon specific primers for three other maternally-inherited bacteria (Cardinium, Arsenophonus, and Rickettsia) and found the latter two within some bee clades. It is useful, however, to complement such diagnostic approaches with more exploratory techniques that can discover unexpected members of the microbial community. Here, we surveyed the microbial associates of several species of commercially available and wild-caught bees through 454-pyrosequencing, followed by diagnostic screening to determine frequency of infection by particular bacterial taxa. Commercial movement of solitary bees as pollinators means concurrent movement of their associated microbiota. Insect populations may be differentially infected with endosymbionts, which can affect host fitness and reproductive compatibility (e.g., Turelli and Hoffmann, 1991). For solitary bee species, such effects within commercially translocated species would have the potential to affect population dynamics and pollinator efficacy.
2. Materials and methods We obtained specimens of seven bee species from a variety of commercial suppliers and an additional 22 species through active and passive collection methods in central Kentucky, USA, between 2011 and 2013 (Table S1). Specimens were stored in 95% ethanol at 20 °C until identification and DNA extraction. Bees were
112
A. Saeed, J.A. White / Journal of Invertebrate Pathology 132 (2015) 111–114
identified morphologically using the Discoverlife IDnature guides for Apoidea (http://www.discoverlife.org/20/q?search=Apoidea) and/or molecularly using COI and/or EF-1 alpha sequences. Specimens were surface sterilized in 5% bleach (60 s); followed by rinses in 95% ethanol (3), and DI water. DNA was extracted from bee abdomens using DNeasy kits (Qiagen) following manufacturer’s instructions. COI and/or EF-1 alpha gene fragments from each specimen were amplified using PCR (primers and cycling conditions in Table S2) in 10 ll reactions as described in Wulff et al. (2013). Unsuccessful extractions (in which COI did not amplify; 3/186 extractions = 1.6%) were discarded from the dataset. Amplified product for a minimum of one specimen per morphospecies per population was sequenced at the Advanced Genetic Technologies Center (University of Kentucky, Lexington, KY, USA) and/or Beckman Coulter (Danvers, MA, USA). Sequences were manually trimmed and edited in Geneious v.6.0.6 (Biomatters Ltd., Auckland, NZ), and submitted to NCBI (Table S1). We identified specimens based on closest available sequences matched in the BOLD ID database (http://barcodinglife.org/index.php/IDS_OpenIdEngine) for COI sequences and NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for EF-1 alpha sequences. We used 454-pyrosequencing to evaluate the bacterial microbiomes of two commercial (Osmia aglaia and Osmia lignaria), and two locally captured (Halictus ligatus and Lasioglossum pilosum) bee species. For each species, DNA from 8 to 10 individuals was pooled at a DNA concentration of 20 ng/lL. Samples were multiplexed for bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) using 28F (50 -GAGTTTGATCNTGGCTCAG-30 ) and 519R (50 -GWNTTACNGCGGCKGCTG-30 ) primers for a segment of bacterial 16S rRNA using a 454 FLX instrument with Titanium chemistry (Research and Testing Laboratory, Lubbock, TX; Dowd et al., 2008). Resulting sequences underwent quality control and manual curation as described in Brady and White (2013). Final read depth ranged from 1700 to 6800 sequences per bee species. Based on pyrosequencing results, bee specimens were then individually screened for 3 bacterial endosymbiont taxa (Wolbachia, Sodalis and Arsenophonus) using previously published diagnostic primers (Table S2). Products were visualized on a 1% agarose gel stained with GelRed (Biotium, Hayward, CA) in comparison with both positive and negative controls. Putatively positive diagnoses were validated by Sanger sequencing whereas negative diagnoses were re-screened for confirmation; symbiont infections were confirmed
when sequences matched endosymbiont taxa within the NCBI nr database at >97% using the blastn algorithm. 3. Results and discussion Three of the four pyrosequenced bee samples were each dominated by a single bacterial taxon. In O. aglaia, 96% of bacterial reads came from Sodalis (Table 1), a genus of maternally-transmitted gram-negative bacteria whose members are facultative or obligate endosymbionts within a variety of insect hosts (Aksoy et al., 1997; Fukatsu et al., 2007). A 386bp 16S segment from this O. aglaia symbiont was identical to that of Sodalis symbionts from multiple stinkbugs and weevils (Toju et al., 2013; Hosokawa et al., 2015), and had greater than 99% similarity to many other insectassociated strains of Sodalis. We subsequently sequenced small fragments of genes encoding a molecular chaperonin, GroEL, and a ribosomal protein, rplB1, from O. aglaia (KT153640, KT153641), both of which also aligned with Sodalis symbionts at >99% identity. This is the first record of this genus of symbiotic bacteria within the order Hymenoptera. In L. pilosum, 88% of reads came from Arsenophonus, a genus that contains maternally-inherited arthropod-associated bacteria with a variety of functions, including reproductive manipulation (male killing; Ferree et al., 2008), insect vectored plant pathogens (Bressan et al., 2008), and obligate nutritional endosymbionts of some blood-feeding insects (Nováková et al., 2009). Our sequenced fragment (397bp of 16S) showed greater than 99% similarity to many insect-associated Arsenophonus strains, but intriguingly was identical to a strain isolated from a honeybee intestine (DQ837612; Babendreier et al., 2007). In H. ligatus, 94% of reads came from Lactobacillus, which is frequently associated with bees (e.g., Martinson et al., 2011; McFrederick et al., 2012), although it is likely an environmentally acquired rather than maternally inherited bacteria (McFrederick et al., 2012, 2013, 2014). Both the L. pilosum and H. ligatus samples also contained Wolbachia at relatively low prevalence of reads (2% of reads per sample; Table 1). In the final pyrosequenced species, O. lignaria, Acidovorax/Diaphorobacter reads were most prevalent, at 70% of reads. Very low prevalence reads from all three maternally-inherited bacteria (Sodalis, Arsenophonus, and Wolbachia) were detected in this sample (Table 1), but none could be validated through subsequent diagnostic PCR of individual specimens, suggesting that these sequences may have been
Table 1 High prevalence 454-pyrosequencing reads (and percentages) of bacterial taxa constituting >1% of total reads for at least one host species. Bacterial taxa
a b c d
Prevalence H. ligatus (N = 10)a
L. pilosum (N = 8)
O. lignaria (N = 10)
O. aglaia (N = 8)
Acidovorax/Diaphorobacterb Acinetobacter Arsenophonusc Enterobacteriaceae (unknown genus) Enterococcus Hafnia Lactobacillus Riemerella Sodalis Staphylococcus Streptococcus Wolbachia Xenorhabdus Otherd
8 (<1%) 0 1 (<1%) 0 0 98 (2%) 3773 (94%) 0 0 0 0 96 (2%) 0 58 (1%)
0 0 5980 (88%) 123 (2%) 0 0 584 (9%) 0 0 0 0 108 (2%) 0 25 (<1%)
1233 (70%) 26 (2%) 63 (4%) 0 81 (5%) 0 9 (1%) 64 (4%) 2 (<1%) 112 (6%) 22 (1%) 7 (<1%) 0 143 (8%)
10 (<1%) 0 5 (<1%) 90 (2%) 0 0 5 (<1%) 1 (<1%) 4035 (96%) 0 0 0 72 (2%) 17 (<1%)
Total bacterial reads
4033
6820
1762
4219
N = number of host specimens from which DNA was pooled. Genera indistinguishable from amplified sequence – both 100% matches. Bacterial genera in bold represent known maternally-inherited symbionts that were targeted in subsequent diagnostic screening. The taxonomic breakdown of this category is available in Table S3.
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A. Saeed, J.A. White / Journal of Invertebrate Pathology 132 (2015) 111–114 Table 2 Frequency of infection by maternally inherited endosymbionts in solitary bee species.
a b c d e
N
Arsenophonus
Sodalis
Wolbachia
Wolbachia strain (and Genbank accession #b)
barbara cressoni forbesii imitatrix nasonii sp. 1 sp. 2 sp. 3 sp. 4
2 1 1 5 4 1 3 2 4
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 4a 0 3 0 2
1 (KT153630)
10 (KT153638)
Apidae
Ceratina calcarata
1
0
1c
1
1 (KT153630)
Colletidae
Colletes sp.
1
0
0
1
11 (fbpA = KT153639)
Halictidae
Agapostemon virescens Augochlorella aurata Halictus confusus Halictus ligatus Lasioglossum hitchensi Lasioglossum imitatum Lasioglossum paradmirandum Lasioglossum pilosum Lasioglossum pruinosum Lasioglossum tegulare Lasioglossum trigeminum
5 2 1 37 3 8 1 9 3 1 1
0 0 0 0 0 0 0 1 0 0 0
0 2c 0 0 0 0 0 0 0 0 0
5 2 1 37 3 8 1 9 3 1 1
1 (KT153630) 4 (KT153633) Ud 3 (KT153632) 6 (KT153635) 9 (KT153637) Me 2 (KT153631) 1 (KT153630) 5 (KT153634) M
Megachilidae
Megachile pugnata Megachile rotunda Osmia aglaia Osmia caerulescens Osmia californica Osmia lignaria Osmia taurus
8 1 38 1 5 30 4
0 0 0 0 0 0 0
0 0 10 0 0 0 0
0 0 0 1 0 0 1
7 (fbpA = KP265901)
Family
Species
Andrenidae
Andrena Andrena Andrena Andrena Andrena Andrena Andrena Andrena Andrena
8 (KT153636)
U
Bold text highlights the positive detection of endosymbionts. Unless otherwise specified, accessions reference Wolbachia surface protein (wsp) sequence. Denotes the detection of a Sodalis-like endosymbiont, with 90% best match to various Sodalis or Sodalis-like bacterial strains. U = unable to determine Wolbachia strain. M = multiple co-infecting Wolbachia strains.
erroneously allocated to O. lignaria due to sequencing barcode errors or sample contamination (White et al., 2015). Subsequent diagnostic screening across all bee specimens for Sodalis and Arsenophonus corroborated the presence of these bacteria, although neither was widespread. We found 10/38 O. aglaia individuals (from 2/3 populations) were infected with Sodalis (Table 2). Two additional species had Sodalis-like symbionts, based on >90% sequence similarity of either groEl (Ceratina calcarata, KT153642), or rplB1 (Augochlorella aurata, KT153643). For Arsenophonus, diagnostic screening showed this endosymbiont to be present in only 1 out of 9 screened individuals of L. pilosum (Table 2), despite the disproportionately high prevalence of Arsenophonus reads in the pyrosequenced L. pilosum sample, indicating that percentage composition within a pyrosequenced sample may not be reflective of unamplified bacterial titer across pooled specimens. Arsenophonus was not detected in any of the other screened solitary bee species (Table 2); however, the low frequency of infected individuals in L. pilosum suggests that screening efforts may need to include a large number of specimens to detect this endosymbiont. In contrast, Wolbachia was found in all individual specimens of L. pilosum and H. ligatus, despite the relatively low prevalence of reads in the pyrosequenced samples (Tables 1 and 2). Wolbachia was additionally found in 16 other species, including all screened species within the family Halictidae, consistent with previous studies (Table 2; Gerth et al., 2013). Based on sequencing of genes encoding the Wolbachia surface protein, wsp, and/or fructose-bisphosphate aldolase, fbpA, at least 11 strains of Wolbachia were represented among the bee specimens. The phylogenetic relationships among most of these Wolbachia strains, based on a multilocus sequence typing (MLST) protocol, are discussed elsewhere (Gerth et al., 2015). Most infected species showed a
100% infection frequency (Table 2), except Andrena sp. 4, in which two out of four specimens were infected, and Osmia taurus, in which one out of four specimens was infected. The latter species was commercially-obtained, demonstrating the potential for differentially-infected individuals to come into contact with one another as a consequence of commercial translocation. Further study of the function of Wolbachia in solitary bees, as well as Arsenophonus and Sodalis, will determine whether such differential infections could be detrimental to the reproductive compatibility, fitness, or efficacy of these important pollinators. Acknowledgments We thank A. Dehnel, A. Maldonado, and A. Styer for technical support, and K. Clark, E. Dobbs, D. Hunter, R. Lee, D. Shreeve, K. Strickland, and E. Sugden for assistance in acquiring specimens. In addition, we thank L. Ayres, T. Boyd, K. Evans, C. Peek, and D. Reed for the use of their orchards in bee collections. The information reported in this paper (No. 15-08-113) is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. This research was funded by the University of Kentucky Department of Entomology, and USDA National Institute of Food and Agriculture Hatch project KY008052. AS was supported by Kentucky’s National Science Foundation Experimental Program to Stimulate Competitive Research, grant 0814194. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip.2015.09.011.
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Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Surveys for maternally-inherited endosymbionts reveal novel and variable infections within solitary bee species q Abiya Saeed, Jennifer A. White ⇑ Department of Entomology, University of Kentucky, S-225 Agricultural Sciences Center North, Lexington, KY 40546, USA
a r t i c l e
i n f o
Article history: Received 15 July 2015 Revised 22 September 2015 Accepted 23 September 2015 Available online 26 September 2015 Keywords: Arsenophonus Bacterial endosymbionts Hymenoptera Sodalis Vertical transmission Wolbachia
a b s t r a c t Maternally-inherited bacteria can affect the fitness and population dynamics of their host insects; for solitary bees, such effects have the potential to influence bee efficacy as pollinators. We screened bee species for bacterial associates using 454-pyrosequencing (4 species) and diagnostic PCR (183 specimens across 29 species). The endosymbiont Wolbachia was abundant, infecting 18 species, including all specimens from the family Halictidae. Among commercially-supplied orchard bees (family Megachilidae), only 2/7 species were Wolbachia-infected, but one species showed variable infection among specimens. Two other maternally-inherited bacteria, Arsenophonus and Sodalis, were also detected, neither of which was fixed in infection frequency. Differential endosymbiont infection could potentially compromise fitness and reproductive compatibility among commercially redistributed pollinator populations. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Solitary bees are important pollinators of crops and native flora. Due to the ongoing decline of managed Apis mellifera populations, solitary bees act as a buffer to protect worldwide crop pollination operations (Winfree et al., 2007), and several species, particularly within the family Megachilidae, are commercially available for purchase and use in crop and orchard settings (Bosch and Kemp, 2002; Gruber et al., 2011). Given the rising economic and ecological importance of these bees, increasing attention is also being given to their biology, including their microbial associations (e.g., Gerth et al., 2011, 2013, 2015; McFrederick et al., 2012, 2013, 2014). Solitary bees are particularly prone to infection by maternallyinherited endosymbiotic bacteria in the genus Wolbachia (Gerth et al., 2011, 2013, 2015), bacteria that frequently manipulate host reproduction to increase bacterial transmission (Werren et al., 2008). The preponderance of Wolbachia among solitary bees suggests that these bees may be susceptible to infection by other
q The nucleotide sequence data reported in this manuscript have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers KT123206-42 and KT153630-43, with a public release date of 11 Dec, 2015, and the sequence read archive under project accession number PRJNA283198 with a public release date of 31 Aug, 2015. ⇑ Corresponding author. E-mail addresses: [email protected] (A. Saeed), [email protected] (J.A. White).
http://dx.doi.org/10.1016/j.jip.2015.09.011 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
maternally-inherited endosymbionts as well, yet the frequency of infection by other endosymbionts remains largely unexplored. Gerth et al. (2015) recently screened numerous bee species using taxon specific primers for three other maternally-inherited bacteria (Cardinium, Arsenophonus, and Rickettsia) and found the latter two within some bee clades. It is useful, however, to complement such diagnostic approaches with more exploratory techniques that can discover unexpected members of the microbial community. Here, we surveyed the microbial associates of several species of commercially available and wild-caught bees through 454-pyrosequencing, followed by diagnostic screening to determine frequency of infection by particular bacterial taxa. Commercial movement of solitary bees as pollinators means concurrent movement of their associated microbiota. Insect populations may be differentially infected with endosymbionts, which can affect host fitness and reproductive compatibility (e.g., Turelli and Hoffmann, 1991). For solitary bee species, such effects within commercially translocated species would have the potential to affect population dynamics and pollinator efficacy.
2. Materials and methods We obtained specimens of seven bee species from a variety of commercial suppliers and an additional 22 species through active and passive collection methods in central Kentucky, USA, between 2011 and 2013 (Table S1). Specimens were stored in 95% ethanol at 20 °C until identification and DNA extraction. Bees were
112
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identified morphologically using the Discoverlife IDnature guides for Apoidea (http://www.discoverlife.org/20/q?search=Apoidea) and/or molecularly using COI and/or EF-1 alpha sequences. Specimens were surface sterilized in 5% bleach (60 s); followed by rinses in 95% ethanol (3), and DI water. DNA was extracted from bee abdomens using DNeasy kits (Qiagen) following manufacturer’s instructions. COI and/or EF-1 alpha gene fragments from each specimen were amplified using PCR (primers and cycling conditions in Table S2) in 10 ll reactions as described in Wulff et al. (2013). Unsuccessful extractions (in which COI did not amplify; 3/186 extractions = 1.6%) were discarded from the dataset. Amplified product for a minimum of one specimen per morphospecies per population was sequenced at the Advanced Genetic Technologies Center (University of Kentucky, Lexington, KY, USA) and/or Beckman Coulter (Danvers, MA, USA). Sequences were manually trimmed and edited in Geneious v.6.0.6 (Biomatters Ltd., Auckland, NZ), and submitted to NCBI (Table S1). We identified specimens based on closest available sequences matched in the BOLD ID database (http://barcodinglife.org/index.php/IDS_OpenIdEngine) for COI sequences and NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for EF-1 alpha sequences. We used 454-pyrosequencing to evaluate the bacterial microbiomes of two commercial (Osmia aglaia and Osmia lignaria), and two locally captured (Halictus ligatus and Lasioglossum pilosum) bee species. For each species, DNA from 8 to 10 individuals was pooled at a DNA concentration of 20 ng/lL. Samples were multiplexed for bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) using 28F (50 -GAGTTTGATCNTGGCTCAG-30 ) and 519R (50 -GWNTTACNGCGGCKGCTG-30 ) primers for a segment of bacterial 16S rRNA using a 454 FLX instrument with Titanium chemistry (Research and Testing Laboratory, Lubbock, TX; Dowd et al., 2008). Resulting sequences underwent quality control and manual curation as described in Brady and White (2013). Final read depth ranged from 1700 to 6800 sequences per bee species. Based on pyrosequencing results, bee specimens were then individually screened for 3 bacterial endosymbiont taxa (Wolbachia, Sodalis and Arsenophonus) using previously published diagnostic primers (Table S2). Products were visualized on a 1% agarose gel stained with GelRed (Biotium, Hayward, CA) in comparison with both positive and negative controls. Putatively positive diagnoses were validated by Sanger sequencing whereas negative diagnoses were re-screened for confirmation; symbiont infections were confirmed
when sequences matched endosymbiont taxa within the NCBI nr database at >97% using the blastn algorithm. 3. Results and discussion Three of the four pyrosequenced bee samples were each dominated by a single bacterial taxon. In O. aglaia, 96% of bacterial reads came from Sodalis (Table 1), a genus of maternally-transmitted gram-negative bacteria whose members are facultative or obligate endosymbionts within a variety of insect hosts (Aksoy et al., 1997; Fukatsu et al., 2007). A 386bp 16S segment from this O. aglaia symbiont was identical to that of Sodalis symbionts from multiple stinkbugs and weevils (Toju et al., 2013; Hosokawa et al., 2015), and had greater than 99% similarity to many other insectassociated strains of Sodalis. We subsequently sequenced small fragments of genes encoding a molecular chaperonin, GroEL, and a ribosomal protein, rplB1, from O. aglaia (KT153640, KT153641), both of which also aligned with Sodalis symbionts at >99% identity. This is the first record of this genus of symbiotic bacteria within the order Hymenoptera. In L. pilosum, 88% of reads came from Arsenophonus, a genus that contains maternally-inherited arthropod-associated bacteria with a variety of functions, including reproductive manipulation (male killing; Ferree et al., 2008), insect vectored plant pathogens (Bressan et al., 2008), and obligate nutritional endosymbionts of some blood-feeding insects (Nováková et al., 2009). Our sequenced fragment (397bp of 16S) showed greater than 99% similarity to many insect-associated Arsenophonus strains, but intriguingly was identical to a strain isolated from a honeybee intestine (DQ837612; Babendreier et al., 2007). In H. ligatus, 94% of reads came from Lactobacillus, which is frequently associated with bees (e.g., Martinson et al., 2011; McFrederick et al., 2012), although it is likely an environmentally acquired rather than maternally inherited bacteria (McFrederick et al., 2012, 2013, 2014). Both the L. pilosum and H. ligatus samples also contained Wolbachia at relatively low prevalence of reads (2% of reads per sample; Table 1). In the final pyrosequenced species, O. lignaria, Acidovorax/Diaphorobacter reads were most prevalent, at 70% of reads. Very low prevalence reads from all three maternally-inherited bacteria (Sodalis, Arsenophonus, and Wolbachia) were detected in this sample (Table 1), but none could be validated through subsequent diagnostic PCR of individual specimens, suggesting that these sequences may have been
Table 1 High prevalence 454-pyrosequencing reads (and percentages) of bacterial taxa constituting >1% of total reads for at least one host species. Bacterial taxa
a b c d
Prevalence H. ligatus (N = 10)a
L. pilosum (N = 8)
O. lignaria (N = 10)
O. aglaia (N = 8)
Acidovorax/Diaphorobacterb Acinetobacter Arsenophonusc Enterobacteriaceae (unknown genus) Enterococcus Hafnia Lactobacillus Riemerella Sodalis Staphylococcus Streptococcus Wolbachia Xenorhabdus Otherd
8 (<1%) 0 1 (<1%) 0 0 98 (2%) 3773 (94%) 0 0 0 0 96 (2%) 0 58 (1%)
0 0 5980 (88%) 123 (2%) 0 0 584 (9%) 0 0 0 0 108 (2%) 0 25 (<1%)
1233 (70%) 26 (2%) 63 (4%) 0 81 (5%) 0 9 (1%) 64 (4%) 2 (<1%) 112 (6%) 22 (1%) 7 (<1%) 0 143 (8%)
10 (<1%) 0 5 (<1%) 90 (2%) 0 0 5 (<1%) 1 (<1%) 4035 (96%) 0 0 0 72 (2%) 17 (<1%)
Total bacterial reads
4033
6820
1762
4219
N = number of host specimens from which DNA was pooled. Genera indistinguishable from amplified sequence – both 100% matches. Bacterial genera in bold represent known maternally-inherited symbionts that were targeted in subsequent diagnostic screening. The taxonomic breakdown of this category is available in Table S3.
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A. Saeed, J.A. White / Journal of Invertebrate Pathology 132 (2015) 111–114 Table 2 Frequency of infection by maternally inherited endosymbionts in solitary bee species.
a b c d e
N
Arsenophonus
Sodalis
Wolbachia
Wolbachia strain (and Genbank accession #b)
barbara cressoni forbesii imitatrix nasonii sp. 1 sp. 2 sp. 3 sp. 4
2 1 1 5 4 1 3 2 4
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 4a 0 3 0 2
1 (KT153630)
10 (KT153638)
Apidae
Ceratina calcarata
1
0
1c
1
1 (KT153630)
Colletidae
Colletes sp.
1
0
0
1
11 (fbpA = KT153639)
Halictidae
Agapostemon virescens Augochlorella aurata Halictus confusus Halictus ligatus Lasioglossum hitchensi Lasioglossum imitatum Lasioglossum paradmirandum Lasioglossum pilosum Lasioglossum pruinosum Lasioglossum tegulare Lasioglossum trigeminum
5 2 1 37 3 8 1 9 3 1 1
0 0 0 0 0 0 0 1 0 0 0
0 2c 0 0 0 0 0 0 0 0 0
5 2 1 37 3 8 1 9 3 1 1
1 (KT153630) 4 (KT153633) Ud 3 (KT153632) 6 (KT153635) 9 (KT153637) Me 2 (KT153631) 1 (KT153630) 5 (KT153634) M
Megachilidae
Megachile pugnata Megachile rotunda Osmia aglaia Osmia caerulescens Osmia californica Osmia lignaria Osmia taurus
8 1 38 1 5 30 4
0 0 0 0 0 0 0
0 0 10 0 0 0 0
0 0 0 1 0 0 1
7 (fbpA = KP265901)
Family
Species
Andrenidae
Andrena Andrena Andrena Andrena Andrena Andrena Andrena Andrena Andrena
8 (KT153636)
U
Bold text highlights the positive detection of endosymbionts. Unless otherwise specified, accessions reference Wolbachia surface protein (wsp) sequence. Denotes the detection of a Sodalis-like endosymbiont, with 90% best match to various Sodalis or Sodalis-like bacterial strains. U = unable to determine Wolbachia strain. M = multiple co-infecting Wolbachia strains.
erroneously allocated to O. lignaria due to sequencing barcode errors or sample contamination (White et al., 2015). Subsequent diagnostic screening across all bee specimens for Sodalis and Arsenophonus corroborated the presence of these bacteria, although neither was widespread. We found 10/38 O. aglaia individuals (from 2/3 populations) were infected with Sodalis (Table 2). Two additional species had Sodalis-like symbionts, based on >90% sequence similarity of either groEl (Ceratina calcarata, KT153642), or rplB1 (Augochlorella aurata, KT153643). For Arsenophonus, diagnostic screening showed this endosymbiont to be present in only 1 out of 9 screened individuals of L. pilosum (Table 2), despite the disproportionately high prevalence of Arsenophonus reads in the pyrosequenced L. pilosum sample, indicating that percentage composition within a pyrosequenced sample may not be reflective of unamplified bacterial titer across pooled specimens. Arsenophonus was not detected in any of the other screened solitary bee species (Table 2); however, the low frequency of infected individuals in L. pilosum suggests that screening efforts may need to include a large number of specimens to detect this endosymbiont. In contrast, Wolbachia was found in all individual specimens of L. pilosum and H. ligatus, despite the relatively low prevalence of reads in the pyrosequenced samples (Tables 1 and 2). Wolbachia was additionally found in 16 other species, including all screened species within the family Halictidae, consistent with previous studies (Table 2; Gerth et al., 2013). Based on sequencing of genes encoding the Wolbachia surface protein, wsp, and/or fructose-bisphosphate aldolase, fbpA, at least 11 strains of Wolbachia were represented among the bee specimens. The phylogenetic relationships among most of these Wolbachia strains, based on a multilocus sequence typing (MLST) protocol, are discussed elsewhere (Gerth et al., 2015). Most infected species showed a
100% infection frequency (Table 2), except Andrena sp. 4, in which two out of four specimens were infected, and Osmia taurus, in which one out of four specimens was infected. The latter species was commercially-obtained, demonstrating the potential for differentially-infected individuals to come into contact with one another as a consequence of commercial translocation. Further study of the function of Wolbachia in solitary bees, as well as Arsenophonus and Sodalis, will determine whether such differential infections could be detrimental to the reproductive compatibility, fitness, or efficacy of these important pollinators. Acknowledgments We thank A. Dehnel, A. Maldonado, and A. Styer for technical support, and K. Clark, E. Dobbs, D. Hunter, R. Lee, D. Shreeve, K. Strickland, and E. Sugden for assistance in acquiring specimens. In addition, we thank L. Ayres, T. Boyd, K. Evans, C. Peek, and D. Reed for the use of their orchards in bee collections. The information reported in this paper (No. 15-08-113) is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. This research was funded by the University of Kentucky Department of Entomology, and USDA National Institute of Food and Agriculture Hatch project KY008052. AS was supported by Kentucky’s National Science Foundation Experimental Program to Stimulate Competitive Research, grant 0814194. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip.2015.09.011.
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