Metatranscriptomics and Pyrosequencing Facilitate Discovery of Potential Viral Natural Enemies of the Invasive Caribbean Crazy Ant, Nylanderia pubens

BACKGROUND: Nylanderia pubens (Forel) is an invasive ant species that in recent years has developed into a serious nuisance problem in the Caribbean and United States. A rapidly expanding range, explosive localized population growth, and control difficulties have elevated this ant to pest status. Professional entomologists and the pest control industry in the United States are urgently trying to understand its biology and develop effective control methods. Currently, no known biological-based control agents are available for use in controlling N. pubens. METHODOLOGY AND PRINCIPAL FINDINGS: Metagenomics and pyrosequencing techniques were employed to examine the transcriptome of field-collected N. pubens colonies in an effort to identify virus infections with potential to serve as control agents against this pest ant. Pyrosequencing (454-platform) of a non-normalized N. pubens expression library generated 1,306,177 raw sequence reads comprising 450 Mbp. Assembly resulted in generation of 59,017 non-redundant sequences, including 27,348 contigs and 31,669 singlets. BLAST analysis of these non-redundant sequences identified 51 of potential viral origin. Additional analyses winnowed this list of potential viruses to three that appear to replicate in N. pubens. CONCLUSIONS: Pyrosequencing the transcriptome of field-collected samples of N. pubens has identified at least three sequences that are likely of viral origin and, in which, N. pubens serves as host. In addition, the N. pubens transcriptome provides a genetic resource for the scientific community which is especially important at this early stage of developing a knowledgebase for this new pest

Impacts of Antibiotic and Bacteriophage Treatments on the Gut-Symbiont-Associated Blissus insularis (Hemiptera: Blissidae)

The Southern chinch bug, Blissus insularis, possesses specialized midgut crypts that harbor dense populations of the exocellular symbiont Burkholderia. Oral administration of antibiotics suppressed the gut symbionts in B. insularis and negatively impacted insect host fitness, as reflected by retarded development, smaller body size, and higher susceptibility to an insecticide, bifenthrin. Considering that the antibiotics probably had non-lethal but toxic effects on host fitness, attempts were conducted to reduce gut symbionts using bacteriophage treatment. Soil-lytic phages active against the cultures of specific Burkholderia ribotypes were successfully isolated using a soil enrichment protocol. Characterization of the BiBurk16MC_R phage determined its specificity to the Bi16MC_R_vitro ribotype and placed it within the family Podoviridae. Oral administration of phages to fifth-instar B. insularis, inoculated with Bi16MC_R_vitro as neonates had no deleterious effects on host fitness. However, the ingested phages failed to impact the crypt-associated Burkholderia. The observed inactivity of the phage was likely due to the blockage of the connection between the anterior and posterior midgut regions. These findings suggest that the initial colonization by Burkholderia programs the ontogeny of the midgut, providing a sheltered residence protected from microbial antagonists

Environmental Transmission of the Gut Symbiont <i>Burkholderia</i> to Phloem-Feeding <i>Blissus insularis</i>

<div><p>The plant-phloem-feeding <i>Blissus insularis</i> possesses specialized midgut crypts, which harbor a dense population of the exocellular bacterial symbiont <i>Burkholderia</i>. Most individual <i>B</i>. <i>insularis</i> harbor a single <i>Burkholderia</i> ribotype in their midgut crypts; however, a diverse <i>Burkholderia</i> community exists within a host population. To understand the mechanism underlying the consistent occurrence of various <i>Burkholderia</i> in <i>B</i>. <i>insularis</i> and their specific association, we investigated potential gut symbiont transmission routes. PCR amplification detected a low titer of <i>Burkholderia</i> in adult reproductive tracts; however, fluorescence <i>in situ</i> hybridization assays failed to produce detectable signals in these tracts. Furthermore, no <i>Burkholderia</i>-specific PCR signals were detected in eggs and neonates, suggesting that it is unlikely that <i>B</i>. <i>insularis</i> prenatally transmits gut symbionts via ovarioles. In rearing experiments, most nymphs reared on St. Augustinegrass treated with cultured <i>Burkholderia</i> harbored the cultured <i>Burkholderia</i> strains. <i>Burkholderia</i> was detected in the untreated host grass of <i>B</i>. <i>insularis</i>, and most nymphs reared on untreated grass harbored a <i>Burkholderia</i> ribotype that was closely related to a plant-associated <i>Burkholderia</i> strain. These findings revealed that <i>B</i>. <i>insularis</i> neonates acquired <i>Burkholderia</i> primarily from the environment (<i>i</i>.<i>e</i>., plants and soils), even though the possibility of acquisition via egg surface cannot be excluded. In addition, our study explains how the diverse <i>Burkholderia</i> symbiont community in <i>B</i>. <i>insularis</i> populations can be maintained.</p></div

The survivorship and symbiont transmission rate of <i>Blissus insularis</i> reared on the live plant inoculated with <i>Burkholderia</i> isolates.

<p>The survivorship and symbiont transmission rate of <i>Blissus insularis</i> reared on the live plant inoculated with <i>Burkholderia</i> isolates.</p

Phylogenetic relationship of <i>Burkholderia</i> obtained from <i>Blissus insularis</i> nymph midgut crypts.

<p>16S rRNA gene sequences (1.3 kb) obtained in the current study are shown in bold. Numbers at the tree nodes represent the maximum-likelihood bootstrap values obtained after 100 repetitions; only values over 50 are shown. In brackets are shown nucleotide sequence accession numbers in the GenBank. Clear and gray circles denote the <i>Burkholderia</i> isolates detected previously in the <i>B</i>. <i>insularis</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref016" target="_blank">16</a>] and in <i>C</i>. <i>saccharivorus</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref012" target="_blank">12</a>], respectively; squares denote the <i>Burkholderia</i> isolates detected in other heteropteran hosts. The sequences of nymph midgut crypts (denoted as BiNym01 to BiNym32) derived from the same treatment are highlighted by same colors. The clades SBE, PBE, and BCC correspond to those described in references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref012" target="_blank">12</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref023" target="_blank">23</a>], and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref024" target="_blank">24</a>], respectively.</p

Visualization of <i>Burkholderia</i> in the digestive tract of <i>Blissus insularis</i>.

<p>(A) An adult <i>B</i>. <i>insularis</i>. (B) Dissected digestive tract. (C) Epifluorescence micrograph of FISH targeting <i>Burkholderia</i> 16S rRNA gene (red) in M4. (D) Phase contrast micrograph of the same areas. (E-F) Focus stacked laser scanning confocal micrographs of FISH localization of <i>Burkholderia</i> (red) in M4. Cyan signals indicate host nuclei stained with DAPI. Abbreviations: M3, midgut third section; M4, midgut fourth section with crypts; M4B, M4 bulb; H, hindgut. The labeling corresponds to that used for <i>Cavelerius saccharivorus</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161699#pone.0161699.ref012" target="_blank">12</a>].</p

Stepwise decision tree employed as a guide to assess the likelihood that a given sequence was of RNA (A) or DNA (B) viral origin.

<p>Stepwise decision tree employed as a guide to assess the likelihood that a given sequence was of RNA (A) or DNA (B) viral origin.</p