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 multiprimer high throughput sequencing (HTS) metabarcoding method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the taxonomically challenging 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. Our multiprimer metabarcoding HTS amplicon approach simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species, while our improved whitefly primers for metabarcoding also detected beneficial hymenopteran parasitoid species from whitefly nymphs. Field-testing our redesigned Bemisia metabarcoding primer sets 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 host crops and sampling techniques that target either nymphs or adults. Our multiprimer HTS metabarcoding method incorporated two overlapping amplicons of 472 bp and 518 bp that spanned the entire 657 bp 3' barcoding region for Bemisia, and is particularly suitable to molecular diagnostic surveys of this highly cryptic insect pest species complex that also typically exhibited high population densities in heavy crop infestation episodes. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving transboundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

Keywords: Bemisia; African cassava whitefly; Aphelinidae; metabarcoding; multiprimer molecular diagnostics; parasitoids.

FIGURE 1

(a) Bemisia cryptic species sampling sites from Uganda, Tanzania and Malawi, with field site codes from Tanzania (i.e., T1–T6) provided in a, Uganda (i.e., U1–U9) in panel (b), and Malawi (i.e., M1–M11) in panel (c). GPS coordinates of each sampling site are provided in Table 3. The maps of Africa were produced using Mapchart <https://mapchart.net>

FIGURE 2

Workflow for analysis of MiSeq generated high‐throughput sequencing of mtCOI amplicon reads for estimating proportions of Bemisia species in the African cassava cultivation landscape, including assessment of hymenopteran parasitoid species. Optional steps are indicated in dashed box depending on whether amplicon libraries were gel‐purified (refer to Supporting Information for Online Publication I) to remove small fragments. Quality of amplicons can also be improved if desired, by using an Illumina sequence trimming program such as Trimmomatic (Bolger et al., 2014). The de novo assembly step enables novel Trialeurodes‐, parasitoid‐ and bacterial‐related sequences, as well as NUMTs, to be identified. NUMTs were identified through detection of INDELs, premature stop codons, and presence of unexpected amino acid residues at highly conserved COI regions following the methods of Kunz, Tay, Elfekih, et al. (2019)

FIGURE 3

Summary results of B. tabaci cryptic species from Uganda (a), Tanzania (b), and Malawi (c). Site codes are as provided in Table 3. Bemisia species are sub‐Saharan Africa 1 (SSA1), sub‐Saharan Africa 2 (SSA2), Mediterranean (MED), Uganda 1 (UG), Indian Ocean (IO), B. afer (Baf), and unknown (“Uknw”) species from Bemisia and Trialeurodes genera. Low detection rates of COI‐related pseudogene, parasitoid (Eretmocerus and Encarsia genera), and bacterial/fungal sequences are grouped in the “others” category. Whitefly species compositions are also compared between cassava and noncassava host plants (d and e, respectively), and between nymphal (M1, M3, M5, M7) and adult (M2, M4, M6, M8) life stages from four cassava sites in Malawi (f)

FIGURE 4

Summaries of efficacies of species delimitation powers based on the p‐dist method across the 657 bp 3' mtCOI barcoding gene region in the cryptic B. tabaci and non‐tabaci species complex. The metabarcoding primer pair “wfly‐PCR‐F1/R1” (472 bp) was divided into four overlapping regions of 315 bp across a 50 bp sliding window size, and the 518 bp amplicon generated by the “wfly‐PCR‐F2/R2” primer pair was divided into five overlapping regions of 315 bp. We also assessed full amplicon lengths from both primer pairs (i.e., F1/R1: 472 bp; F2/R2: 518 bp, blue coloured cells). Cryptic species that could not be defined were represented by grey coloured cells, and sequences that were successfully defined into their respective species clades were shown by green cells. Dark grey cells indicated failed species delimitation by both primer pairs and full species delimitation will require the complete 657 bp barcoding gene. The number of clean mtCOI sequences in the database (db) of Kunz, Tay, Elfekih, et al. (2019) for each species analysed are indicated (total: 224 sequences)