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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. [ABSTRACT FROM AUTHOR]

BACKGROUND The whitefly Bemisia tabaci complex harbours over 40 cryptic species that have been placed in 11 phylogenetically distinct clades based on the molecular characterization of partial mitochondrial DNA COI (mtCOI) gene region. Four cryptic species are currently within the invasive clade, i.e. MED, MEAM1, MEAM2 and IO. Correct identification of these species is a critical step towards implementing reliable measures for plant biosecurity and border protection; however, no standardized B. tabaci -specific primers are currently available which has caused inconsistencies in the species identification processes. RESULTS We report three sets of polymerase chain reaction (PCR) primers developed to amplify the mtCOI region which can be used for genotyping MED, MEAM1 and IO species, and tested these primers on 91 MED, 35 MEAM1 and five IO individuals. PCR and sequencing of amplicons identified a total of 21, six and one haplotypes in MED, MEAM1 and IO respectively, of which six haplotypes were new to the B. tabaci database. CONCLUSION These primer pairs enabled standardization and robust molecular species identification via mtCOI screening of the targeted invasive cryptic species and will improve quarantine decisions. Use of this diagnostic tool could be extended to other species within the complex. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of themitochondrialDNAcytochromeoxidase I (mtDNACOI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problemis identified in the B. tabaci Africa/Middle East/AsiaMinor cladewhich comprises the globally invasive Mediterranean (MED) andMiddle East AsiaMinor I (MEAM1) species,Middle East AsiaMinor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion,MEAM2 has since been reported from Japan, Peru, Turkey and Iraq.We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (<3%) of MEAM1 genomes, whereas presence of internal stop codons, loss of expected stop codons and poor primer annealing sites, all suggested MEAM2 as a pseudogene artifact and so not a real species. [ABSTRACT FROM AUTHOR]