New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a.

BACKGROUND: Khapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. RESULTS: We designed and selected the first khapra beetle-specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA = 100 nM/500 nM). With only a 37 °C-heat-source and a blue light torch, RPA and CRISPR/CAS12a-based visualization assays can be completed within 40 min to differentiate between khapra beetle and nine similar Dermestidae species. After DNA extraction using a kit (4-5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min RPA reaction at 37 °C, followed by a 15 min color reaction under 37 °C in dark conditions using a CRISPR/CAS12a system and a fluorescent probe (5'-FAM/3'-BHQ1 labeled). This method is ingenious to low levels of DNA (10-1 ng µL-1 ) and meets the sensitivity requirements for detecting a single khapra beetle's egg (≈0.7 mm). CONCLUSION: Our specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37 °C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management, and provides value as a reference for monitoring and identification of other pests. © 2023 Society of Chemical Industry.

Revalidation of morphological characteristics and multiplex PCR for the identification of three congener invasive Liriomyza species (Diptera: Agromyzidae) in China.

Due to varietal differences, diminutive size, and similar morphological characters, it is difficult to classify and identify Liriomyza spp., a genus comprised of economically-important, highly-polyphagous insect pests. In this study, we reconfirmed the morphological characteristics of three closely-related invasive leafminers, L. trifolii, L. sativae, and L. huidobrensis. Morphological results showed that characteristics imparted by the male genitalia were the most reliable morphological features for identification. The colors exhibited by vertical setae were variable among species, and the ratio of the length of the ultimate section of vein CuA1 divided by penultimate section also varied within species. Although the patterns of abdominal tergites were diverse among Liriomyza spp., L. trifolii exhibited a unique pattern with a yellow patch at the 5th black visible tergite; this pattern can be profiled as a prominent characteristic for morphological identification. In order to identify the three Liriomyza spp. quickly and accurately, we developed an improved molecular identification method using multiplex PCR based on the gene encoding mitochondrial cytochrome oxidase I (COI); this method enabled direct identification based on the size of amplified products. The results of this study provide a valuable reference for the identification of Liriomyza spp., which will ultimately improve our ability to control individual species.

Population genetics of Liriomyza trifolii (Diptera: Agromyzidae) and comparison with four Liriomyza species in China based on COI, EF-1a and microsatellites loci.

Liriomyza is a large genus that includes polyphagous and invasive species (L. trifolii, L. sativae, and L. huidobrensis), and oligophagous species such as L. Chinensis in China. Effective control of these invasive and oligophagous species is not easy due to the fast invasion rate, interspecific competition, and pesticide resistance. In this study, we investigated population genetics of five Liriomyza species L. trifolii, L. sativae, L. huidobrensis, L. bryoniae, and L. chinensis based on COI and EF-1a genes, and microsatellite DNA. These five Liriomyza species revealed highly conservative characteristics in the COI gene among populations collected from different geographical regions and host plants. By contrast, the mutation rate of the EF-1a gene was higher than COI, and phylogenetic tree based on EF-1a showed that haplotypes of L. trifolii and L. sativae were not distinguished well. Genetic differentiation in microsatellite loci was obvious among the five species. Our results also indicated that geographic isolation had a greater impact on genetic differentiation in L. trifolii than the host plant. Populations of L. trifolii in China showed a high to moderate level of genetic differentiation and they had divided into two groups representing the coastal areas of southern China and northern regions. The genetic diversity of the southern group was higher than the northern group. We speculated that the invasion of L. trifolii likely occurred in southern regions of China and then spread northward. Bottleneck analyses revealed that the L. trifolii population in China was in a steady growth period.

Cryphalus eriobotryae sp. nov. (Coleoptera: Curculionidae: Scolytinae), a New Insect Pest of Loquat Eriobotrya japonica in China.

A previously unknown bark beetle species, Cryphalus eriobotryae sp. nov. Johnson, 2019 has emerged as a lethal pest of loquat (Eriobotrya japonica) in China. The description of new species has been provided. The new species is distinguished from the other Cryphalus by the weakly aciculate frons, by the antennae, with unevenly spaced procurved sutures, by the short pronotal disc, with hair-like setae, and by the widely spaced mesocoxae. The survey of plantation records from around Suzhou suggests that this beetle was introduced from another area not long before 2017. In the surveyed loquat plantation in 2018, 20-90% of trees showed signs of infestation, and 5% were killed in 2018, resulting in the death of over 1000 trees. Outbreaks of the apparently loquat-specific Cryphalus eriobotryae can be diagnosed by hundreds of cankers on the trunk, and wilted foliage. This pest is of concern as a loquat plantation pest and as a pest of fruit production and ornamental trees within Suzhou, and globally.

Comparative analysis of the Liriomyza chinensis mitochondrial genome with other Agromyzids reveals conserved genome features.

Liriomyza chinensis is a serious pest of onions in many countries, especially in East Asia. We sequenced the complete mitochondrial genome of this species and compared it with five other Agromyzidae species. The L. chinensis mitogenome is a double-stranded 16,175 bp circular molecule with an A + T content of 78.3%. It contains 37 genes and a control region as do the sequenced Liriomyza species. The mitogenomes of L. chinensis and other Agromyzidae species showed a clear bias in nucleotide composition with a positive AT-skew. Most PCGs used standard ATN as start codons, and TAN as termination codons. The tRNAs exhibited the typical clover-leaf structure, except for tRNASer(AGN) and the two rRNA genes are conserved with those of other Agromyzids. The L. chinensis mitogenome control region included several conserved regions, including a poly-T, two (TA)n and one poly-A stretch, which are considered important replication and transcription. The 13 PCGs were used to study the phylogeny of L. chinensis and five related Agromyzids. Analysis by maximum likelihood, Bayesian inference and genetic distance suggest congruent phylogenetic relationships in Liriomyza spp. and provide a useful supplement to taxonomic classification by morphology.

DNA barcoding identification of Pseudococcidae (Hemiptera: Coccoidea) using the mitochondrial COI gene.

DNA barcoding is a recently developed technique for species-level identification that involves the use of short, standard DNA sequences as species labels. It is an effective complement to traditional taxonomic classification based on morphology. At present, research and applications involving the DNA barcoding of the Pseudococcidae are focused primarily on the cytochrome coxidase subunit I (COI) gene, but there is not yet a consensus on the preferred gene region for barcoding. The purpose of this study was to explore the effectiveness of identification of Pseudococcidae beetles using DNA barcoding technology. The COI gene sequences of 97 samples from 21 species of Asemini were analysed, followed by evaluation of the ability to identify species using a tree-building method and distance evaluation. The COI sequences (500 bp) exhibited distinct distributions of intra-specific and inter-specific variation and a significant barcoding gap. The success rate of identification was 97.84%. These results demonstrate the feasibility of using this segment of COI to identify most species of Pseudococcidae.