Gut Bacterial Communities of Two Insect Species Feeding on Toxic Plants Are Dominated by Enterococcus Sp
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fmicb-07-01005 June 24, 2016 Time: 16:20 # 1 ORIGINAL RESEARCH published: 28 June 2016 doi: 10.3389/fmicb.2016.01005 The Generalist Inside the Specialist: Gut Bacterial Communities of Two Insect Species Feeding on Toxic Plants Are Dominated by Enterococcus sp. Cristina Vilanova1,2, Joaquín Baixeras1, Amparo Latorre1,2,3* and Manuel Porcar1,2* 1 Cavanilles Institute of Biodiversity and Evolutionary Biology, Universitat de València, Valencia, Spain, 2 Institute for Integrative Systems Biology (I2SysBio), University of Valencia-CSIC, Valencia, Spain, 3 Unidad Mixta de Investigación en Genómica y Salud, Centro Superior de Investigación en Salud Pública, Valencia, Spain Some specialist insects feed on plants rich in secondary compounds, which pose a major selective pressure on both the phytophagous and the gut microbiota. However, microbial communities of toxic plant feeders are still poorly characterized. Here, we Edited by: Mark Alexander Lever, show the bacterial communities of the gut of two specialized Lepidoptera, Hyles ETH Zürich, Switzerland euphorbiae and Brithys crini, which exclusively feed on latex-rich Euphorbia sp. and Reviewed by: alkaloid-rich Pancratium maritimum, respectively. A metagenomic analysis based on Virginia Helena Albarracín, CONICET, Argentina high-throughput sequencing of the 16S rRNA gene revealed that the gut microbiota Jeremy Dodsworth, of both insects is dominated by the phylum Firmicutes, and especially by the common California State University, gut inhabitant Enterococcus sp. Staphylococcus sp. are also found in H. euphorbiae San Bernardino, USA though to a lesser extent. By scanning electron microscopy, we found a dense ring- *Correspondence: Manuel Porcar shaped bacterial biofilm in the hindgut of H. euphorbiae, and identified the most [email protected]; prominent bacterium in the biofilm as Enterococcus casseliflavus through molecular Amparo Latorre [email protected] techniques. Interestingly, this species has previously been reported to contribute to the immobilization of latex-like molecules in the larvae of Spodoptera litura, a highly Specialty section: polyphagous lepidopteran. The E. casseliflavus strain was isolated from the gut and its This article was submitted to Extreme Microbiology, ability to tolerate natural latex was tested under laboratory conditions. This fact, along a section of the journal with the identification of less frequent bacterial species able to degrade alkaloids and/or Frontiers in Microbiology latex, suggest a putative role of bacterial communities in the tolerance of specialized Received: 01 April 2016 Accepted: 13 June 2016 insects to their toxic diet. Published: 28 June 2016 Keywords: : lepidoptera, gut communities, metagenomics, Enterococcus sp., secondary metabolites Citation: Vilanova C, Baixeras J, Latorre A and Porcar M (2016) The Generalist INTRODUCTION Inside the Specialist: Gut Bacterial Communities of Two Insect Species Plants have biochemical and molecular mechanisms to defend themselves from insects attack. Feeding on Toxic Plants Are Dominated by Enterococcus sp. Among those, plants produce a vast range of secondary metabolites with anti-herbivore effects, Front. Microbiol. 7:1005. which are produced either constitutively or in response to tissue damage (War et al., 2012). Some doi: 10.3389/fmicb.2016.01005 plant biochemicals are toxic, repellent, or antinutritive for herbivores. Among these compounds, Frontiers in Microbiology | www.frontiersin.org 1 June 2016 | Volume 7 | Article 1005 fmicb-07-01005 June 24, 2016 Time: 16:20 # 2 Vilanova et al. The Generalist Inside the Specialist alkaloids, terpenoids, and complex mixtures of macromolecules littoralis (Tang et al., 2012), or Ostrinia nubilalis (Belda et al., such as latex are among the most frequent plant biochemical 2011). defense barriers. Latex- and alkaloid-rich plants constitute a particularly strong Plant alkaloids, are toxic to a wide range of insects (Nuringtyas selection pressure not only for phytophagous insects (Kirk et al., et al., 2014). However, a few species of insects are unaffected 2012; Ramos et al., 2015), but also for their gut microbiota, which by even high concentrations of alkaloids. The Amaryllidaceae, is subjected to a constant flow of toxic compounds. The gut of with more than 300 alkaloids isolated to date (Bastida et al., insects feeding on toxic plants is thus a unique and extreme 2011), are among the most deterrent plants. Alkaloids present habitat. We present here a complete characterization of the in Pancratium maritimum have demonstrated both cytotoxic and bacterial gut symbionts of two monophagous Lepidoptera feeding antimicrobial activity (Hetta and Shafei, 2013). The aposematic on plants rich in, among other toxic compounds, a cocktail of larvae of the noctuid moth Brithys crini –the “lily borer”– alkaloids or terpenes (Figure 1). This is the first report of the are well known to feed monophagously on the sand lily microbial larval gut communities associated to such toxic diets. Pancratium maritimum. Sequestration of alkaloids by this species has never been tested but the larvae of the closely related species complex Xanthopastis timais – the “Spanish moth”, another MATERIALS AND METHODS Amaryllidaceae specialist – was early included by Rothschild (1973) in her seminal work on insect chemical defense and is Sampling a typical example of sequestration of phenanthridine alkaloids Larvae of B. crini and H. euphorbiae (Figure 1) were obtained (Nishida, 2002). Terpenes are chemical compounds that are in the field (coastal dunes from Pinedo and El Saler, Valencia, present in large amounts in a large variety of plants: in conifers, Spain) by direct inspection of the food plants P. maritimum and for example, they are the main components of resin. Plant Euphorbia sp., respectively, in the adequate moment of the year: terpenes are involved in defense against herbivory, even at spring for most larvae of B. crini and autumn for H. euphorbiae. the belowground level (Vaughan et al., 2013). Terpenes, along with alkaloids, natural gum and many other compounds, are Gut Dissection and DNA Extraction also present in Euphorbiaceae and other plants exuding latex. Last instar larvae from B. crini and H. euphorbiae were kept Unsurprisingly, latex-producing plants are particularly resistant in starvation for one day to promote the elimination of plant to many insects and other pests (Hagel et al., 2008). The larvae of material from the gut. Larvae were immobilized by placing the sphingid moth Hyles euphorbiae – the “spurge hawk moth” – them on ice and both the midgut and hindgut were dissected feed on a broad variety of Euphorbia plants from which they under sterile conditions. Guts from three different individuals sequester the cytotoxic ingenane diterpene esters (Marsh and of each species were independently disaggregated and manually Rothschild, 1984). homogenized in PBS buffer (NaCl 8 g/L, KCl 0.2 g/L, Na HPO Beyond biochemicals, plant defenses against herbivory also 2 4 1.44 g/L, and KH PO 0.24 g/L, pH adjusted to 7.4) with an involve mutualistic microorganisms. Endophytic fungi, plant 2 4 Eppendorf-adapted pestle. Total DNA was obtained from the symbionts living asymptomatically within the host tissues, homogenate with a standard purification protocol consisting of produce alkaloid-based herbivore deterrents that contribute to alkaline lysis followed by precipitation with potassium acetate the defense of the plant (Koh and Hik, 2007). Reciprocally, and isopropanol (Latorre et al., 1986). An initial incubation step bacteria associated with insects can play a role in disturbing with 2 mg/mL lysozyme at 37◦C for 30 min was performed plant defensive barriers (Hansen and Moran, 2014; Hammer and to ensure the lysis of Gram-positive bacteria. The quality of Bowers, 2015). For example, Colorado potato beetle (Leptinotarsa the DNA was finally checked on a 0.8% (w/v) agarose gel decemlineata) larvae have been reported to bear bacteria in their and quantified with Nanodrop-1000 Spectophotometer (Thermo oral secretions that suppress antiherbivore defenses in tomato Scientific, Wilmington, DE, USA). (Solanum lycopersicum), the plant the beetle feeds on (Chung et al., 2013). In summary, plant–insect interactions are complex ecological processes mediated by secondary metabolites, alkaloids PCR Amplification and 16S rRNA and terpenes among them, but also by microorganisms, which Profiling play key roles as both defense and attack allies for the plants A 700 bp fragment of the V1–V3 hypervariable region of the and phytophagous insects, respectively. Surprisingly enough, 16S rRNA genes was polymerase chain reaction (PCR)-amplified though, there are few reports on that topic. The few studies from all the samples with universal primers 28F (50-GAG TTT available on the microbial communities associated to the gut of GAT CNT GGC TCA G-30) and 519R (50-GTN TTA CNG CGG insects, and particularly to Lepidoptera, are focused on species CKG CTG-30). A short (9–11 nucleotides) barcode sequence considered as agricultural or forest pests worldwide. This is followed by a four-nucleotide spacer (CGAT) was included at the case of the gypsy moth Lymantria dispar (Broderick et al., the 50 end of the oligonucleotides used as forward primers to 2004; Mason and Raffa, 2014), the diamondback moth Plutella enable assignment of sequences to samples after high-throughput xylostella (Lin et al., 2015), or the cotton bollworm Helicoverpa sequencing. All the amplifications were performed under the armigera (Xiang et al., 2006). Also,