A high-throughput amplicon sequencing approach for population-wide species diversity and composition survey

Abstract

Management of agricultural pests requires an understanding of pest species diversity, their interactions with beneficial insects and spatial-temporal patterns of pest abundance. Invasive and agriculturally important insect pests can build up very high populations, especially in cropping landscapes. Traditionally, sampling effort for species identification involves small sample sizes and is labour intensive. Here, we describe a high throughput sequencing (HTS) PCR amplicon method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the Bemisia pest cryptic species complex as examples. The whiteflies Bemisia including the ′tabaci′ species are agriculturally important capable of vectoring diverse plant viruses that cause diseases and crop losses. We use our HTS amplicon platform to simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species and beneficial hymenopteran parasitoid species. Field-testing our HTS amplicon method across the Tanzania, Uganda and Malawi cassava cultivation landscapes, we identified the sub-Saharan Africa 1 Bemisia putative species as the dominant pest species, with other cryptic Bemisia species being detected at various abundances. We also provide evidence that Bemisia species compositions can be affected by sampling techniques that target either nymphs or adults. Our method is particularly suitable to molecular diagnostic surveys of cryptic insect species with high population densities. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving trans-boundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

Landscape factors and how they influence whitefly pests in cassava fields across East Africa

“…This large geographic gradient allowed us to examine Bemisia whitefly populations across a diversity of different cassava production contexts. Given that the species in the B. tabaci pest complex cannot be identified in the field using morphology alone, we developed a high-throughput sequencing method to identify the common species present in our focal fields (Tay et al 2020). Using this data set we asked:…”

Within-Season Changes in Land-Use Impact Pest Abundance in Smallholder African Cassava Production Systems

“…We also examined the net effect of parasitism rate, although we did not differentiate parasitoids. Nevertheless, there are potentially at least four species of parasitoids (i.e., Eretmocerus mundus , Encarsia Sophia , E. mineoi , and Encarsia sp-blackhead) that attack Bemisia species on cassava in Uganda [ 14 ], while our molecular diagnostics indicated likely high Eretmocerus and Encarisa species diversity in African cassava landscapes (also see Tay et al [ 22 ]). There remains a need to better understand if different Bemisia species present in the landscape are attacked by different parasitoid species.…”

“…Extracted gDNA was eluted twice in 100 µL (final volume 200 µL) of Elution Buffer (EB) (Qiagen). For downstream high throughput sequencing (HTS) of amplicon, we followed the protocol of Tay et al [ 22 ].…”

“…The concentration of individual gDNA samples was quantified and standardized to 5 ng/µL using EB buffer and kept at –18 °C until it was needed for PCR amplicon DNA library preparation for Illumina MiSeq NGS. The desired 3′ partial mitochondrial DNA cytochrome oxidase subunit I (mtCOI) gene widely used in molecular diagnostics of Bemisia species was amplified using specific HTS primer pairs [ 22 ]. Amplicon HTS runs were performed on Illumina MiSeq System, and sequence data processed using Trimmomatic [ 23 ] and analyzed as outlined in Tay et al [ 22 ] using the Geneious software version 10.0.8.…”

“…The desired 3′ partial mitochondrial DNA cytochrome oxidase subunit I (mtCOI) gene widely used in molecular diagnostics of Bemisia species was amplified using specific HTS primer pairs [ 22 ]. Amplicon HTS runs were performed on Illumina MiSeq System, and sequence data processed using Trimmomatic [ 23 ] and analyzed as outlined in Tay et al [ 22 ] using the Geneious software version 10.0.8. All mtCOI sequences were trimmed to 657 bp for molecular diagnostics of Bemisia species using reference sequences for Sub-Saharan Africa (SSA)1, SSA2, MED, IO and for pseudogene detection [ 24 ].…”