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Molecular Identification of Trissolcus japonicus, Parasitoid of the Brown Marmorated Stink Bug, by Species-Specific PCR.

ABSTRACT: The samurai wasp, Trissolcus japonicus (Ashmead), has been proposed as a biocontrol agent against brown marmorated stink bugs (BMSB), due to its ability to parasitize and kill BMSB eggs. However, the wasps' small size makes it challenging for those untrained in morphological identification to determine the wasps' species. To circumvent this problem, a molecular method was created to identify T. japonicus. The method uses species-specific primers, designed in this study, which target the variable region of the mitochondrial Cytochrome Oxidase 1 (CO1) locus. After confirming successful DNA extraction from samples, the PCR amplification using our primers produced 227-bp PCR products for all T. japonicus specimens and no amplification in other microhymenoptera candidates. Additionally, DNA from BMSB-parasitized eggs gave positive PCR amplification, while the control BMSB samples showed no amplification. This indicates that PCR with our primers specifically and sensitively differentiates T. japonicus specimens from other similar wasp species and discriminates between T. japonicus-parasitized and non-parasitized BMSB eggs. Finally, an in silico analysis of CO1 sequences demonstrated that our primers match the sequences of four different haplotypes of T. japonicus, indicating that our diagnostic method could potentially be applied to analyze T. japonicus populations throughout North America, Europe, and parts of Asia.

SUBMITTER: Chen MN

PROVIDER: S-EPMC8157830 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug, is an invasive agricultural and nuisance pest rapidly expanding its incidence in North America. This voracious pest poses a significant threat to rural and urban agriculture, especially to specialty crops such as apples, grapes and ornamentals, as well as staple crops including soybean and corn. The object of this study was to generate transcript sequence resources for H. halys. RNA-seq libraries derived from distinct developmental stages and sexes were sequenced and assembled into 248,569 putatively unique transcripts (PUTs). PUTs were segmented into three disjoint tiers of varying reliability, with 4,794 classified as gold tier (highest quality), 16,878 as silver, and 14,357 as bronze. The gold-tier PUTs associated with 2,580 distinct non-redundant protein sequences from the NCBI NR database--1,785 of these (69%) mapped to annotated UniProtKB database proteins, from which 1,273 unique Pfam families and 459 unique Molecular Function GO terms were encountered. Of the silver tier's 6,527 PUTs associated with unique proteins, 4,193 mapped to UniProtKB (64%), from which 1,941 and 640 unique Pfam and Molecular Function GO terms were extracted. H. halys PUTs related to important life processes like immunity, endocrinology, reproduction, development, behavior, neurotransmission, neurotoxicity, olfaction, and small RNA pathways were validated through quantitative Real-Time PCR (qRT-PCR) for differential expression during distinct life stages (eggs, 2nd instar nymphs, 4th instar nymphs, female adults, male adults). PUTs similar to hypothetical proteins identified in symbiont microbes, including Pantoea and Nosema species, were more abundantly expressed in adults versus nymphs. These comprehensive H. halys transcriptomic resources can be utilized to aid development of novel control methodologies to disrupt life processes; to conduct reverse genetic screens to determine host gene function; and to design environmentally unobtrusive means to control host populations or target specific H. halys life stages, such as molecular biopesticides.

Project description:Halyomorpha halys (Stål) (Insecta:Hemiptera;Pentatomidae), commonly known as the Brown Marmorated Stink Bug (BMSB), is an invasive pest of the mid-Atlantic region of the United States, causing economically important damage to a wide range of crops. Native to Asia, BMSB was first observed in Allentown, PA, USA, in 1996, and this pest is now well-established throughout the US mid-Atlantic region and beyond. In addition to the serious threat BMSB poses to agriculture, BMSB has become a nuisance to homeowners, invading home gardens and congregating in large numbers in human-made structures, including homes, to overwinter. Despite its significance as an agricultural pest with limited control options, only 100 bp of BMSB sequence data was available in public databases when this project began.Transcriptome sequencing was undertaken to provide a molecular resource to the research community to inform the development of pest control strategies and to provide molecular data for population genetics studies of BMSB. Using normalized, strand-specific libraries, we sequenced pools of all BMSB life stages on the Illumina HiSeq. Trinity was used to assemble 200,000 putative transcripts in >100,000 components. A novel bioinformatic method that analyzed the strand-specificity of the data reduced this to 53,071 putative transcripts from 18,573 components. By integrating multiple other data types, we narrowed this further to 13,211 representative transcripts.Bacterial endosymbiont genes were identified in this dataset, some of which have a copy number consistent with being lateral gene transfers between endosymbiont genomes and Hemiptera, including ankyrin-repeat related proteins, lysozyme, and mannanase. Such genes and endosymbionts may provide novel targets for BMSB-specific biocontrol. This study demonstrates the utility of strand-specific sequencing in generating shotgun transcriptomes and that rapid sequencing shotgun transcriptomes is possible without the need for extensive inbreeding to generate homozygous lines. Such sequencing can provide a rapid response to pest invasions similar to that already described for disease epidemiology.

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Molecular Identification of Trissolcus japonicus, Parasitoid of the Brown Marmorated Stink Bug, by Species-Specific PCR.

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