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Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).

ABSTRACT: The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

SUBMITTER: Ma L

PROVIDER: S-EPMC8174695 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Invasions of leaf beetles can cause tremendous economic consequences because some of these insects become major pests in invaded territories. We present the first inventory of alien Chrysomelidae of European Russia that appeared in the region in the 20th and 21st centuries (9 species) with analysis of the history of their invasions and detailed maps of distribution. This case study revealed some general tendencies of invasions of leaf beetles: (1) Recently, a dramatic increase in the rate of Chrysomelidae invasions is observed, which reflects the increase in international trade of living plants; (2) Alien leaf beetles can spread quickly, occupying almost all of Europe within several decades; (3) When the range of some leaf beetle species is quickly expanding, or when the species has been recorded established somewhere outside the native range, this species should be regarded as a potential invader worldwide. and (4) Alien leaf beetles usually occur on alien or cultivated plants, but some become naturalized in native communities. The specific information was the following. Two species native to the Mediterranean region, Chrysolina americana (feeds on Rosmarinus and Lavandula) and Leptomona erythrocephala (feeds on Lotus corniculatus) were recorded in European Russia for the first time. A polyphagous pest of floriculture Luperomorpha xanthodera native to China and Korea and a pest of soybeans Medythia nigrobilineata native to east Asia have been in the region since 2016. A pest of tobacco Epitrix hirtipennis native to North America has occurred since 2011. A pest of corn Diabrotica virgifera was intercepted at the border of Russia in 2011 but has not established. Three alien species have been in the region since the 20th century: Zygogramma suturalis introduced from North America for control of Ambrosia, Phyllotreta reitteri native to Afghanistan and Tajikistan and feeding on Lepidium latifolium, and the Colorado potato beetle Leptinotarsa decemlineata.

Project description:BACKGROUND:Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates. METHODOLOGY/PRINCIPAL FINDINGS:Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284-289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469-481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation. CONCLUSIONS/SIGNIFICANCE:Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing.

Dataset Information

Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).

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