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

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.

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.

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.