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1 June 2014 Estimating Bacterial Diversity in Scirtothrips dorsalis (Thysanoptera: Thripidae) via Next Generation Sequencing
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Abstract

The last 2 decades have produced a better understanding of insect-microbial associations and yielded some important opportunities for insect control. However, most of our knowledge comes from model systems. Thrips (Thysanoptera: Thripidae) have been understudied despite their global importance as invasive species, plant pests and disease vectors. Using a culture and primer independent next-generation sequencing and metagenomics pipeline, we surveyed the bacteria of the globally important pest, Scirtothrips dorsalis Hood. The most abundant bacterial phyla identified were Actinobacteria and Proteobacteria and the most abundant genera were Propionibacterium, Stenotrophomonas, and Pseudomonas. A total of 189 genera of bacteria were identified. The absence of any vertically transferred symbiont taxa commonly found in insects is consistent with other studies suggesting that thrips primarilly acquire resident microbes from their environment. This does not preclude a possible beneficial/intimate association between S. dorsalis and the dominant taxa identified and future work should determine the nature of these associations.

Thrips, order Thysanoptera, are emerging as a globally important group of plant pests, damaging crops through direct feeding and transmission of tospoviruses and non-viral diseases (Morse & Hoddle 2006). Only a few hundred of the 5,500 identified thrips species are pests (Brunner et al. 2002), and of these, only 14 are documented virus vectors (Riley et al. 2011). Chilli thrips Scirtothrips dorsalis is both a virus vector (Chu et al. 2001; Meena et al. 2005; Gopal et al. 2010) and a pest of many crops around the world including tea (Saha & Mukhopadhyay 2013), mango (Aliakbarpour & Md. Rawi 2012; Choi et al. 2013), roses (Hegde et al. 2011; Mannion et al. 2013), and citrus (Gao et al. 2012; Hyun et al. 2012). It is a highly polyphagous species, feeding on >100 plant species in 40 different families, many containing important U.S. crops (Hodges et al. 2007; Kumar et al. 2013). S. dorsalis is globally invasive and has been established in Florida and Texas since 2005 (Mannion et al. 2013).

Bacterial associations with insects are ubiquitous and are often beneficial to the insect (Duron & Hurst 2013). Bacteria have been implicated in the manipulation of reproduction (Duron et al. 2008), body color (Tsuchida et al. 2010), disease transmission (Weiss & Aksoy 2009), development (Chouaia et al. 2012), and protection from parasites (Brownlie & Johnson 2009). Thrips on the whole have received little attention regarding this important aspect of arthropod ecology. Previous work has largely focused on the most frequently encountered bacterium (identified as a near-Erwinia species) within a single thrips species, the Western flower thrips Frankliniella occidentalis (de Vries et al. 2001a; de Vries et al. 2001b; de Vries et al. 2004; de Vries et al. 2006; Chanbusarakum & Ullman 2008, 2009; de Vries et al. 2012) with a few studies on other species (Wells et al. 2002; Gitaitis et al. 2003; de Vries et al. 2008).

In this study, next generation semiconductor sequencing was conducted on invasive chilli thrips in Florida from which we present the first metagenomic survey of any thrips. This expands our understanding of the bacterial symbioses of thrips in general and is a first step toward a bacterial transfection biocontrol strategy for this important pest.

Methods

Next Generation Sequencing

A single DNA extraction from 97 adult and nymph S. dorsalis was made using a DNeasy™ Blood and Tissue kit (Qiagen™ , Valencia, California). Eluted DNA was concentrated to a volume of ∼10 mL in a SpeedVac™ DNA 110 Concentrator (Savant, Farmingdale, New York), subjected to electrophoresis on a 1.5% agarose gel, and high molecular weight genomic DNA (> 10Kb) was cut out and purified with a Nucleospin Clean-up kit (Macherey-Nagel, Pennsylvania, USA). One hundred ng of purified DNA was used for library preparation using an Ion Xpress™ Plus Fragment Library Kit. End repair, adapter ligation, size selection, nick repair and amplification (16 cycles) were performed as described in the Ion Torrent protocol associated with the kit. 300 bp fragments were isolated using a SizeSelect 2% Gel in an E-Gel electrophoresis system (all Life Technologies, California, USA).

The Agilent 2100 Bioanalyzer and the associated High Sensitivity DNA kit (Agilent Technologies, Englewood, Colorado) were used to determine quality and concentration of the library. The amount of library required for template preparation was calculated using the Template Dilution Factor calculation described in the protocol. Next-generation sequencing was conducted using the Ion Torrent Personal Genome Machine using an Ion PGM 200 Sequencing kit following sequencing template preparation with the OneTouch™ 2 System and Ion PGM™ Template OT2 200 Kit according to manufacturer's instructions (all products: Life Technologies). The library was sequenced on a single Ion 318 semiconductor chip (Life Technologies) with a barcode.

Contig Assembly and Metagenomics

Contigs were assembled from the raw reads using the following parameters in Geneious v6; word length, 12; index word length, 11; maximum gap size, 5; maximum gaps per read, 10%; maximum mismatches, 5%; and maximum ambiguity, 16. Assembled contigs were compared against the NCBI nt database (Retrieved 9-XII-2012) and to a custom combined LSU and SSU Silva database (Release SSURef_lll and LSURef_lll) (Quast et al. 2013) with BLASTN 2.2.27+ (Camacho et al. 2009) with an E-value of 1e-10 with BLAST data outputted in XML format. XML outputs from BLAST coupled with the corresponding assembly input data were provided as input to MEGAN v4.70.4 (Huson et al. 2011) and analyzed with default lowest common ancestor parameters. The minimum support filter was set to one ensuring rare taxa were represented. Contigs compared to the Silva database were mapped to taxa using the silva_2_ncbi roadmap. The taxonomic tree generated by MEGAN was manually parsed to present the data at the level of microbial Genus. To accomplish this, the tree was uncollapsed at the level of Genus, followed by the collapse of all non-microbial taxa at the level of Kingdom. Using the summarize feature of MEGAN the number of both direct and summed (assigned to a more specific taxon) contigs were exported in a tabular format, including non-microbial Kingdoms, microbial Genera, as well as nodes representing low complexity sequences, sequences which had no BLAST hit, and those not assigned to a given taxa. Contigs assigned to nodes below the Genus threshold and lost/not processed contigs were calculated using total reads in raw files, the number of reads placed in the MEGAN root taxon, and the total assigned to selected nodes. Summary data was exported to Microsoft Excel and graphed. A listing of the various implicated bacterial species is provided in supplementary material for this article in Suppl. Table 1 in Florida Entomologist 97(2) (2014) online at  http://purl.fcla.edu/fcla/entomologist/browse.

Results and Discussion

The sequencing yield was 6,204,869 million raw reads averaging 164 bases. From these reads, 615,073 contigs were assembled averaging 483 bases. More than 99.99% of contigs were processed successfully through the metagenomics pipeline. 28,416 contigs were identified as having microbial origin and 189 bacterial genera were identified as the best match for a minimum of 4 different contigs (Table 1, Suppl. Table 1). The relative abundance of sequences from identified genera was highest for the phyla Actinobacteria, and Proteobacteria (Fig. 1). This is consistent with the dominant bacterial phyla found for other animals (Jones et al. 2013).

Table 1.

Abundance of the 15 most commonly identified bacterial genera in Scirtothrips dorsalis. The remaining 174 species are listed in suppl. Table 1.

t01_362.gif

Fig. 1.

Relative abundance of dominant bacterial taxa in Scirtothrips dorsalis. Abundance based on 23,068 contigs classified to 189 genera (supplimental table). Genera were included if identified as the best match for >3 contigs.

f01_362.jpg

Eight genera identified by this study have previously been associated with other thrips species. Erwinia, Pantoea, Enterobacter, Serratia, Escherichia and Pseudomonas have been identified internally from thrips (de Vries et al. 2001a; Gitaitis et al. 2003; Chanbusarakum & Ullman 2008; de Vries et al. 2008; de Vries et al. 2012). Erwinia herbicola and Enterobacter agglomerans have been synonymized with Pantoea agglomerans so these 3 genera identified in chilli thrips could represent a single taxon found in association with several insects (Medina et al. 2011). Agrobacterium, Methylobacterium, Pseudomonas and Escherichia have been isolated from the exoskeleton of Sericothrips staphylinus (Yamoah et al. 2008).

Many of the genera identified by this study have members with documented insect associations (Table 1, Suppl. Table 1), though a thorough literature search on all 189 genera was not conducted. Notably absent from the list are heritable genera reported as globally common in insects by Duron & Hurst (2013), Arsenophonus, Wolbachia, Rickettsia, Spiroplasma, and Cardinium. In the 3 thrips species with literature records of endosymbiotic bacterial associations, it has generally been argued that the bacteria are facultative, and acquired from the environment through feeding (de Vries et al. 2001b; Gitaitis et al. 2003; de Vries et al. 2008). Our results suggest this may also be the case for S. dorsalis. In Frankliniella occidentalis, a geographically ubiquitous association with a near Erwinia species provisionally identified as Pantoea agglomerans, is promoted behaviorally since thrips individuals prefer thrips-damaged leaves (de Vries et ah 2006). Future work should investigate whether any of the dominant bacteria identified in S. dorsalis could be maintained in a similar fashion as this could provide a mechanism for the spread of transfected bacteria through the thrips population for the purpose of biological control.

There are several factors limiting the inferences that can be drawn from this dataset. First, we are unable to distinguish internal symbionts from those associated with the surface of the insect or possible contaminants (see Hail et al. 2012). Second, we cannot gauge the bacterial communities of individuals or life-stages. Both of these limitations can be overcome within the context of this sequencing and bioinformatics framework at an increased cost. That being said, the abundance of genera identified in the order: Enterobacteriales and those with documented insect associations (Table 1, Suppl. Table 1) argue that many of the identified taxa are thrips associated.

Massively parallel, high throughput, primer independent sequencing has some benefits over bacterial culturing, traditional PCR with cloning, and target enriched next-generation sequencing for estimating bacterial diversity. It not only does away with the assumption of culturability, but also with the assumption of equivalent primer-template compatibility for all bacteria. As such, it has the ability to recover taxa that may not have been detected with primer dependent methods (Mao et al. 2012). The primer independent method presented here identified a large number of bacterial taxa, free of these assumptions, for an insect with no prior information about its microbiome.

Acknowledgments

We thank John Prokop and Florian Grant for technical support.

References Cited

1.

H. Aliakbarpour , and C. S. Md. Rawi 2012. Seasonal abundance of Thrips hawaiiensis (Morgan) and Scirtothrips dorsalis (Hood) (Thysanoptera: Thripidae) in mango orchards in Malaysia. Petranika J. Trop. Agrie. Sci. 35(3): 637–645. Google Scholar

2.

J. C. Brownlie , and K. N. Johnson 2009. Symbiontmediated protection in insect hosts. Trends Microbiol. 17: 348–354. Google Scholar

3.

P. C. Brunner , C. Fleming , and J. E. Frey 2002. A molecular identification key for economically important thrips species (Thysanoptera: Thripidae using direct sequencing and a per-rflp-based approach. Agrie. For. Entomol. 4: 127–136. Google Scholar

4.

C. Camacho , G. Colulouris , V. Avagyan , N. Ma , J. Papadopoulos , K. Bealer , and T. Madden 2009. Blast+: Architecture and applications. BMC Bioinforma 10: 421. Google Scholar

5.

L. Chanbusarakum , and D. Ullman 2008. Characterization of bacterial symbionts in Frankliniella occidentalis (Pergande), western flower thrips. J. Invertebr. Pathol. 99(3): 318–325. Google Scholar

6.

L. J. Chanbusarakum , and D. E. Ullman 2009. Distribution and ecology of Frankliniella occidentalis (Thysanoptera: Thripidae) bacterial symbionts. Environ. Entomol. 38(4): 1069–1077. Google Scholar

7.

K. S. Choi , J. Y. Yang , Y. M. Park , S. Kim , H. Choi , D. Lyu , and D. S. Kim 2013. Pest lists and their damages on mango, dragon fruit and atemoya in Jeju, Korea. Korean J. Appl. Entomol. 52(1): 45–51. Google Scholar

8.

B. Chouaia, P. Rossi, S. Epis, M. Mosca, I. Ricci, C. Damiani, U. Ulissi, E. Crotti, D. Daffonchio, I. C. Band, and G. Favia 2012. Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts. BMC Microbiol. 12(Suppl 1): S2. Google Scholar

9.

F. H. Chu , C. H. Chao , Y. C. Peng , S. S. Lin , C. C. Chen , and S. D. Yeh 2001. Serological and molecular characterization of Peanut chlorotic fan-spot virus, a new species of the genus Tospovirus. Phytopathology 91(9): 856–863. Google Scholar

10.

E. J. De Vries , J. A. J. Breeuwer , G. Jacobs , and C. Mollema 2001a. The association of western flower thrips, Frankliniella occidentalis, with a near Erwinia species gut bacteria: Transient or permanent? J. Invertebr. Pathol. 77(2): 120–128. Google Scholar

11.

E. J. De Vries , G. Jacobs , and J. A. J. Breeuwer 2001b. Growth and transmission of gut bacteria in the western flower thrips, Frankliniella occidentalis. J, Invertebr. Pathol. 77(2): 129–137. Google Scholar

12.

E. J. De Vries , G. Jacobs , M. W. Sabelis , S. B. J. Menken , and J. A. J. Breeuwer 2004. Diet-dependent effects of gut bacteria on their insect host: The symbiosis of Erwinia sp and western flower thrips. Proc. R. Soc. B-Biol. Sci. 271(1553): 2171–2178. Google Scholar

13.

E. J. De Vries , F. Van De Wetering , M. M. Van Der Hoek , G. Jacobs , and J. A. J. Breeuwer 2012. Symbiotic bacteria (Erwinia sp.) in the gut of Frankliniella occidentalis (Thysanoptera: Thripidae) do not affect its ability to transmit tospovirus. European J. Entomol. 109(2): 261–266. Google Scholar

14.

E. J. De Vries , A. W. G. Van Der Wurfe , G. Jacobs , and J. A. J. Breeuwer 2008. Onion thrips, Thrips tabaci, have gut bacteria that are closely related to the symbionts of the western flower thrips, Frankliniella occidentalis. J. Insect Sci. 8(23): 1–11. Google Scholar

15.

E. J. De Vries , R. A. Vos , G. Jacobs , and H. A. J. Breeuwer 2006. Western flower thrips (Thysanoptera:Thripidae) preference for thripsdamaged leaves over fresh leaves enables uptake of symbiotic gut bacteria. European J. Entomol. 103(4): 779–786. Google Scholar

16.

O. Duron , D. Bouchon , S. Boutin , L. Bellamy , L. Zhou , J. Engelstadter , and G. D. Hurst 2008. The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone. BMC Biol 6: 27. Google Scholar

17.

O. Duron , and G. D. D. Hurst 2013. Arthropods and inherited bacteria: From counting the symbionts to understanding how symbionts count. BMC Biol 11: 45. Google Scholar

18.

J. Y. Gao , J. Guo , Z. R. Wang , D. G. Zhou , M. X. Peng , and J. Q. Yue 2012. Study on insect pest species and occurrence rule of main species in Dehong lemon orchard of Yunnan province. Acta Agrie. Jiangxi 24(6): 70–73. Google Scholar

19.

R. D. Gitaitis , R. R. Walcott , M. L. Wells , J. C. D. Perez , and F. H. Sanders 2003. Transmission of Pantoea ananatis, causal agent of center rot of onion, by tobacco thrips, Frankliniella fusca. Plant Dis. 87(6): 675–678. Google Scholar

20.

K. Gopal , M. K. Reddy , D. V. R. Reddy , and V. Muniyappa 2010. Transmission of peanut yellow spot vims (PYSV) by thrips, Scirtothrips dorsalis Hood in groundnut. Arch. Phytopathol. Plant Prot. 43: 421–429. Google Scholar

21.

J. N. Hegde , A. K. Chakravarthy , M. K. Nagamani , and M. S. Prabhakar 2011. Management of thrips, Scirtothrips dorsalis Hood, on rose under open-field and protected conditions. J. Hort. Sci. 62(2): 118–122. Google Scholar

22.

A. Hodges, S. Ludwig, L. Osborne, M. Ciomperlik, and G. Hodges 2007. National pest alert: Scirtothrips dorsalis Hood. USDA-CSREES. Google Scholar

23.

D. H. Huson , S. Mitra , N. Weber , H. Ruscheweyh , and S. C. Schuster 2011. Integrative analysis of environmental sequences using MEGAN4. Genome Res. 21: 1552–1560. Google Scholar

24.

J. W. Hyun , R. Y. Hwang , K. S. Lee , J. H. Song , P. H. Yi , H. M. Kwon , D. H. Hyun , and K. S. Kim 2012. Seasonal occurrence of yellow tea thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) in citrus orchards and its damage on citrus fruits. Korean J. Appl. Entomol. 51(1): 1–7. Google Scholar

25.

R. T. Jones , L. G. Sanchez , and N. Fierer 2013. A cross-taxon analysis of insect-associated bacterial diversity. PLoS ONE 8(4): e61218. Google Scholar

26.

V. Kumar, G. Kakkar, C. L. Mckenzie, D. R. Seal, and L. S. Osborne 2013. An overview of chilli thrips, Scirtothrips dorsalis (Thysanoptera: Thripidae) biology, distribution and management In S. Soloneski and M. Larramendy [eds.], Weed and pest control - conventional and new challenges. InTech, DOI: 10.5772/55045. Available from:  http://www.intechopen.com/books/weed-and-pest-control-conventional-and-new-challenges/an-overview-of-chillithrips-scirtothrips-dorsalis-thysanoptera-thripidaebiology-distribution-and-m Google Scholar

27.

C. M. Mannion , A. I. Derksen , D. R. Seal , L. S. Osborne , and C. G. Martin 2013. Effects of rose cultivars and fertilization rates on populations of Scirtothrips dorsalis (Thysanoptera: Thripidae) in southern Florida. Florida Entomol. 96(2): 403–411. Google Scholar

28.

D. P. Mao , Q. Zhou , C. Y. Chen , and Z. X. Quan 2012. Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC Microbiol. 12(66). Google Scholar

29.

R. F. Medina , P. Nachappa , and C. Tamborindeguy 2011. Differences in bacterial diversity of host-associated populations of Phylloxera notabilis Pergande (Hemiptera: Phylloxeridae) in pecan and water hickory. J. Evol. Biol. 24(4): 761–771. Google Scholar

30.

R. L. Meena , T. Ramasubramanian , S. Venkatesan , and S. Mohankumar 2005. Molecular characterization of Tospovirus transmitting thrips populations from India. American J. Biochem. Biotechnol. 1(3): 167–172. Google Scholar

31.

G. Minard , P. Mavingui , and C. V. Moro 2013. Diversity and function of bacterial microbiota in the mosquito holobiont. Parasites and Vectors 6: 146. Google Scholar

32.

J. G. Morse , and M. S. Hoddle 2006. Invasion biology of thrips. Annu Rev Entomol 51: 67–89. Google Scholar

33.

C. Quast , E. Pruesse , P. Yilmaz , J. Gerken , T. Schweer , P. Yarza , J. Peplies , and F. O. Glockner 2013. The SILVA ribosomal rna gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41(D1): D590–D596. Google Scholar

34.

D. G. Riley , S. V. Joseph , R. Srinivasan , and S. Dieppe 2011. Thrips vectors of tospoviruses. J. Integ. Pest Mgt. 1(2). Google Scholar

35.

D. Saha , and A. Mukhopadhyay 2013. Insecticide resistance mechanisms in three sucking insect pests of tea with reference to north-east India: An appraisal. Inti. J. Trop. Insect Sci. 33(1): 46–70. Google Scholar

36.

M. Shelomi , W. S. Lo , L. S. Kimsey , and C. H. Kuo 2013. Analysis of the gut microbiota of walking sticks (Phasmatodea). BMC Res. Notes 6: 368. Google Scholar

37.

T. Tsuchida , R. Koga , M. Horikawa , T. Tsunoda , T. Maoka , S. Matsumoto , J. C. Simon , and T. Fukatsu 2010. Symbiotic bacterium modifies aphid body color. Science 330: 1102–1104. Google Scholar

38.

B. Weiss , and S. Aksoy 2009. Microbiome influences in insect host vector competence. Trends Parasitol. 27(11): 514–522. Google Scholar

39.

M. L. Wells , R. D. Gitaitis , and F. H. Sanders 2002. Association of tobacco thrips, Frankliniella fusca (Thysanoptera : Thripidae) with 2 species of bacteria of the genus Pantoea. Ann. Entomol. Soc. America 95(6): 719–723. Google Scholar

40.

E. Yamoah , E. E. Jones , R. J. Weld , D. M. Suckling , N. Waipara , G. W. Bourdot , A. K. W. Hee ,and A. Stewart 2008. Microbial population and diversity on the exoskeletons of four insect species associated with gorse (Ulex europaeus L.). Australian J. Entomol. 47: 370–379. Google Scholar

Notes

[1] Summarized from a presentation and discussions at the “Thrips: small players with big damage”, Symposium at the Annual Meeting of the Florida Entomological Society, 16 July 2013, Naples, Florida.

[2] Supplementary material for this article in Florida Entomologist 97(2) (2014) is online at  http://purl.fcla.edu/fcla/entomologist/browse

Aaron M. Dickey, Andrew J. Trease, Antonella Jara-Cavieres, Vivek Kumar, Matthew K. Christenson, LAkshmi-Prasad Potluri, J. Kent Morgan, Robert G. Shatters, Cindy L. Mckenzie, Paul H. Davis, and Lance S. Osborne "Estimating Bacterial Diversity in Scirtothrips dorsalis (Thysanoptera: Thripidae) via Next Generation Sequencing," Florida Entomologist 97(2), 362-366, (1 June 2014). https://doi.org/10.1653/024.097.0204
Published: 1 June 2014
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