Symbiont-mediated insecticide resistance.

Development of insecticide resistance has been a serious concern worldwide, whose mechanisms have been attributed to evolutionary changes in pest insect genomes such as alteration of drug target sites, up-regulation of degrading enzymes, and enhancement of drug excretion. Here, we report a previously unknown mechanism of insecticide resistance: Infection with an insecticide-degrading bacterial symbiont immediately establishes insecticide resistance in pest insects. The bean bug Riptortus pedestris and allied stinkbugs harbor mutualistic gut symbiotic bacteria of the genus Burkholderia, which are acquired by nymphal insects from environmental soil every generation. In agricultural fields, fenitrothion-degrading Burkolderia strains are present at very low densities. We demonstrated that the fenitrothion-degrading Burkholderia strains establish a specific and beneficial symbiosis with the stinkbugs and confer a resistance of the host insects against fenitrothion. Experimental applications of fenitrothion to field soils drastically enriched fenitrothion-degrading bacteria from undetectable levels to >80% of total culturable bacterial counts in the field soils, and >90% of stinkbugs reared with the enriched soil established symbiosis with fenitrothion-degrading Burkholderia. In a Japanese island where fenitrothion has been constantly applied to sugarcane fields, we identified a stinkbug population wherein the insects live on sugarcane and ≈8% of them host fenitrothion-degrading Burkholderia. Our finding suggests the possibility that the symbiont-mediated insecticide resistance may develop even in the absence of pest insects, quickly establish within a single insect generation, and potentially move around horizontally between different pest insects and other organisms.

Evidence of environmental and vertical transmission of Burkholderia symbionts in the oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae).

The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ∼89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects.

Mating disruption for control of Melanotus okinawensis (Coleoptera: elateridae) with synthetic sex pheromone.

A mating disruption experiment to control Melanotus okinawensis Ohira (Coleoptera: Elateridae) was conducted at a sugarcane (Saccharum spp.) field and a wild Japanese pampas, Miscanthus sinensis Anderss, grassland on Minami-Daito Island (3,057 ha) from 2001 to 2007. The sugarcane field and the pampas grassland were treated with synthetic sex pheromone that evaporated from a polyethylene tube dispenser. The mean total catches obtained by monitoring traps in the sugarcane fields decreased by 96.1% in 2001 from the previous year on Minami-Daito Island. The mean total trap catches in the treated area further decreased by 74.0% from 2001 until 2007 as cumulative effects. Simultaneously, the number of adults captured by hand decreased from 4.7 per sugarcane field in 2001 to 0.5 in 2007 (89.3% reduction), whereas those captured in the untreated area (Miyagi Island) did not show such a decrease. The mating rates were significantly lower in the females captured in the treated area (14.3-71.4%) than those in the untreated area (96.9-100%). However, the amount of the decrease in the trap catches was relatively small at first (39.6% reduction) in the Japanese pampas grassland on the periphery of the Island. This was probably due to the loss of pheromone substance caused by the strong seasonal wind in the periphery. However, mean total trap catches at the periphery also decreased within several years; significant decreases were detected until 2003, 2006, and 2007. These results indicated that the mating disruption effectively reduced an isolated population of M. okinawensis.

Infection dynamics of insecticide-degrading symbionts from soil to insects in response to insecticide spraying.

Insecticide resistance is a serious concern in modern agriculture, and an understanding of the underlying evolutionary processes is pivotal to prevent the problem. The bean bug Riptortus pedestris, a notorious pest of leguminous crops, acquires a specific Burkholderia symbiont from the environment every generation, and harbors the symbiont in the midgut crypts. The symbiont's natural role is to promote insect development but the insect host can also obtain resistance against the insecticide fenitrothion (MEP) by acquiring MEP-degrading Burkholderia from the environment. To understand the developing process of the symbiont-mediated MEP resistance in response to the application of the insecticide, we investigated here in parallel the soil bacterial dynamics and the infected gut symbionts under different MEP-spraying conditions by culture-dependent and culture-independent analyses, in conjunction with stinkbug rearing experiments. We demonstrate that MEP application did not affect the total bacterial soil population but significantly decreased its diversity while it dramatically increased the proportion of MEP-degrading bacteria, mostly Burkholderia. Moreover, we found that the infection of stinkbug hosts with MEP-degrading Burkholderia is highly specific and efficient, and is established after only a few times of insecticide spraying at least in a field soil with spraying history, suggesting that insecticide resistance could evolve in a pest bug population more quickly than was thought before.

Environmental factors shaping the community structure of ammonia-oxidizing bacteria and archaea in sugarcane field soil.

The effects of environmental factors such as pH and nutrient content on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in soil has been extensively studied using experimental fields. However, how these environmental factors intricately influence the community structure of AOB and AOA in soil from farmers' fields is unclear. In the present study, the abundance and diversity of AOB and AOA in soils collected from farmers' sugarcane fields were investigated using quantitative PCR and barcoded pyrosequencing targeting the ammonia monooxygenase alpha subunit (amoA) gene. The abundances of AOB and AOA amoA genes were estimated to be in the range of 1.8 × 10(5)-9.2 × 10(6) and 1.7 × 10(6)-5.3 × 10(7) gene copies g dry soil(-1), respectively. The abundance of both AOB and AOA positively correlated with the potential nitrification rate. The dominant sequence reads of AOB and AOA were placed in Nitrosospira-related and Nitrososphaera-related clusters in all soils, respectively, which varied at the level of their sub-clusters in each soil. The relationship between these ammonia-oxidizing community structures and soil pH was shown to be significant by the Mantel test. The relative abundances of the OTU1 of Nitrosospira cluster 3 and Nitrososphaera subcluster 7.1 negatively correlated with soil pH. These results indicated that soil pH was the most important factor shaping the AOB and AOA community structures, and that certain subclusters of AOB and AOA adapted to and dominated the acidic soil of agricultural sugarcane fields.

Insecticide-degrading Burkholderia symbionts of the stinkbug naturally occupy various environments of sugarcane fields in a Southeast island of Japan.

The stinkbug Cavelerius saccharivorus, which harbors Burkholderia species capable of degrading the organophosphorus insecticide, fenitrothion, has been identified on a Japanese island in farmers' sugarcane fields that have been exposed to fenitrothion. A clearer understanding of the ecology of the symbiotic fenitrothion degraders of Burkholderia species in a free-living environment is vital for advancing our knowledge on the establishment of degrader-stinkbug symbiosis. In the present study, we analyzed the composition and abundance of degraders in sugarcane fields on the island. Degraders were recovered from field samples without an enrichment culture procedure. Degrader densities in the furrow soil in fields varied due to differences in insecticide treatment histories. Over 99% of the 659 isolated degraders belonged to the genus Burkholderia. The strains related to the stinkbug symbiotic group predominated among the degraders, indicating a selection for this group in response to fenitrothion. Degraders were also isolated from sugarcane stems, leaves, and rhizosphere in fields that were continuously exposed to fenitrothion. Their density was lower in the plant sections than in the rhizosphere. A phylogenetic analysis of 16S rRNA gene sequences demonstrated that most of the degraders from the plants and rhizosphere clustered with the stinkbug symbiotic group, and some were identical to the midgut symbionts of C. saccharivorus collected from the same field. Our results confirmed that plants and the rhizosphere constituted environmental reservoirs for stinkbug symbiotic degraders. To the best of our knowledge, this is the first study to investigate the composition and abundance of the symbiotic fenitrothion degraders of Burkholderia species in farmers' fields.

A fine-scale phylogenetic analysis of free-living Burkholderia species in sugarcane field soil.

The diversity and abundance of Burkholderia species in sugarcane field soils were investigated by a 16S rRNA gene-based approach using genus-specific primers. A total of 365,721 sequences generated by the Illumina MiSeq platform were assigned to the genus Burkholderia. Nearly 58% of these sequences were placed in a previously defined cluster, including stinkbug symbionts. Quantitative PCR analysis revealed a consistent number of 16S rRNA gene copies for Burkholderia species (10(7) g(-1) soil) across the sampled fields. C/N, pH, and nitrate concentrations were important factors shaping the Burkholderia community structure; however, their impacts were not significant considering the overall genus size.