Genetics‐based methods for agricultural insect pest management

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CRISPR/Cas9-based functional analysis of yellow gene in the diamondback moth,Plutella xylostella

The diamondback moth, Plutella xylostella (L.), is an economically important pest of cruciferous crops worldwide. This pest is notorious for rapid evolution of the resistance to different classes of insecticides, making it increasingly difficult to control. Genetics-based control approaches, through manipulation of target genes, have been reported as promising supplements or alternatives to traditional methods of pest management. Here we identified a gene of pigmentation (yellow) in P. xylostella, Pxyellow, which encodes 1674 bp complementary DNA sequence with four exons and three introns. Using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR-associated protein 9 system, we knocked out Pxyellow, targeting two sites in Exon III, to generate 272 chimeric mutants (57% of the CRISPR-treated individuals) with color-changed phenotypes of the 1st to 3rd instar larvae, pupae, and adults, indicating that Pxyellow plays an essential role in the body pigmentation of P. xylostella. Fitness analysis revealed no significant difference in the oviposition of adults, the hatchability of eggs, and the weight of pupae between homozygous mutants and wildtypes, suggesting that Pxyellow is not directly involved in regulation of growth, development, or reproduction. This work advances our understanding of the genetic and insect science molecular basis for body pigmentation of P. xylostella, and opens a wide avenue for development of the genetically based pest control techniques using Pxyellow as a screening marker.     

Microsatellites isolated from diamondback moth,Plutella xylostella (L.), for studies of dispersal in Australian populations

Diamondback moth, Plutella xylostella (L.), is a worldwide agricultural pest that has developed resistance to many insecticides used for its control. Population structure and gene flow are yet to be determined for P. xylostella in Australia, but are important factors for the design of effective control strategies. We have isolated six polymorphic microsatellite markers: three from a partial genomic library, two from an Expressed Sequence Tagged library and one from an aminopeptidase intron of P. xylostella. These microsatellites will be used to determine population structure and gene flow in Australian populations of P. xylostella to improve insecticide resistance management.     

Engineering of benzylglucosinolate in tobacco provides proof-of-concept for dead-end trap crops genetically modified to attract Plutella xylostella (diamondback moth)

Glucosinolates are biologically active natural products characteristic of crucifers, including oilseed rape, cabbage vegetables and the model plant Arabidopsis thaliana. Crucifer‐specialist insect herbivores, like the economically important pest Plutella xylostella (diamondback moth), frequently use glucosinolates as oviposition stimuli. This suggests that the transfer of a glucosinolate biosynthetic pathway to a non‐crucifer would stimulate oviposition on an otherwise non‐attractive plant. Here, we demonstrate that stable genetic transfer of the six‐step benzylglucosinolate pathway from A. thaliana to Nicotiana tabacum (tobacco) results in the production of benzylglucosinolate without causing morphological alterations. Benzylglucosinolate‐producing tobacco plants were more attractive for oviposition by female P. xylostella moths than wild‐type tobacco plants. As newly hatched P. xylostella larvae were unable to survive on tobacco, these results represent a proof‐of‐concept strategy for rendering non‐host plants attractive for oviposition by specialist herbivores with the long‐term goal of generating efficient dead‐end trap crops for agriculturally important pests.     

A stochastic model for predicting the stage emergence of Plutella xylostella under field conditions

The diamondback moth, Plutella xylostella, is a worldwide pest of brassicas, and its biology and ecology have been extensively studied over recent years. Despite the importance of mathematical models to the management of insect pests, no stochastic model has been developed to date for P. xylostella. In this context, the study aimed to develop a stochastic model capable of describing the stage emergence of P. xylostella under field conditions. The stochastic model was developed using simple nonlinear functions based on the laboratory data on development times under constant temperatures. Comparison between estimated and observed cumulative proportions of egg hatch, pupation and adult emergence recorded in the field in Southern Brazil shows that the model accurately describes the stage emergence of P. xylostella. The developed model shows potential to estimate the stage emergence of P. xylostella under field conditions, and can add significant advances to the management of this pest.     

Evaluation of the impact of natural enemies on Plutella xylostella L. (Lepidoptera: Yponomeutidae) populations on commercial Brassica farms

Abstract 1 Accurate assessment of the impact of natural enemies on pest populations is fundamental to the design of robust integrated pest management programmes. In most situations, diseases, predators and parasitoids act contemporaneously on insect pest populations and the impact of individual natural enemies, or specific groups of natural enemies, is difficult to interpret. These problems are exacerbated in agro‐ecosystems that are frequently disrupted by the application of insecticides. 2 A combination of life‐table and natural enemy exclusion techniques was utilized to develop a method for the assessment of the impact of endemic natural enemies on Plutella xylostella populations on commercial Brassica farms. 3 At two of the experimental sites, natural enemies had no impact on P. xylostella survival, at two other sites, natural enemy impact was low but, at a fifth site, natural enemies drastically reduced the P. xylostella population. 4 The calculation of marginal death rates and associated k‐values allowed the comparison of mortality factors between experimental sites, and indicated that larval disappearance was consistently the most important mortality factor, followed by egg disappearance, larval parasitism and pupal parasitism. The appropriateness of the methods and assumptions made to calculate the marginal death rates are discussed. 5 The technique represents a robust and easily repeatable method for the analysis of the activity of natural enemies of P. xylostella, which could be adapted for the study of other phytophagous pests.     

Formulation and application of the entomopathogenic fungus: Zoophthora radicans (Brefeld) Batko (Zygomycetes: Entomophthorales)

Aims: To isolate and formulate a native strain of Zoophthora radicans naturally infecting larvae of diamondback moth, Plutella xylostella, existing in South Australia and to provide evidence that formulation of the fungus is effective against P. xylostella larvae, and therefore, it could be used as a tool in pest management of this insect. Methods and Results: Dose–response bioassays using formulated and unformulated forms of the fungus strain were carried out against third instar larvae of P. xylostella. Results obtained have indicated a significant increase in the larval mortality when higher concentrations of a formulated form of the fungus strain were applied compared to the treatments with the unformulated form (85·0 vs 57·5% of larval mortality, respectively, at the top concentration of 10⁷ conidia/ml). The median lethal concentration (LC50) for a formulated form was 100 times less than that of the unformulated form when they were applied against the third instar larvae of P. xylostella. In addition, the formulation used in the present bioassays has preserved the viability of introduced fungus conidia for longer time in comparison with the unformulated conidia. Conclusions: The effective application of a formulated fungus strain against P. xylostella larvae constitutes the first step towards its use in pest management of this insect. Significance and Impact of the Study: The formulated fungus in inverted emulsion could be used as an alternative tool to insecticides in pest management of P. xylostella larvae because of the development of resistance to insecticides in the treated larvae.     

CRISPR/Cas9‐mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni

The Queensland fruit fly, Bactrocera tryoni (Froggatt), is a polyphagous horticultural pest in Australia that is capable of causing significant damage to more than 100 different host fruits and vegetables. Chemical applications and ecological control strategies, such as the sterile insect technique (SIT), are commonly used to suppress established populations and eradicate invasive outbreaks following migration. The recently published B. tryoni draft genome provides new opportunities to identify candidate genes for targeted genome modification in order to generate advanced genetic strains for management using sterile insect strategies. Here, we demonstrate CRISPR/Cas‐mediated mutagenesis in B. tryoni through generating a series of frame‐shift mutations in the ATP‐dependent binding cassette transporter, white, causing a classic white‐eye phenotype. This work establishes methods for CRISPR/Cas genome editing in tephritids and demonstrates its potential for developing genetic sexing strains which could be used for SIT‐based pest control.     

Natal insect experience with Arabidopsis thaliana plant genotypes influences plasticity in oviposition behavior

Previous studies have shown that insect experience with secondary chemicals present in different plant species can induce behavioral changes in female oviposition preferences. However, there is a lack of information on whether insect experience with intraspecific plant variation may influence oviposition behavior. The prediction that experience with plant genotypes would affect the oviposition behavior of two crucifer insect pests was tested using a wild ecotype of Arabidopsis thaliana (L.) Heynh. (Brassicaceae) (Col‐0) and two of its genetically modified lines (35S:ESP and tgg1 tgg2), which differ in their glucosinolate hydrolysis profiles. Choice oviposition assays were performed using both naïve and experienced females of the specialist Plutella xylostella (L.) (Lepidoptera: Plutellidae) and the generalist Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae). In addition to oviposition preferences, the effect of plant genetic lines was assessed on insect traits related to development, growth, survival, and fecundity. Experience with different natal treatments (i.e., artificial diet and plant genotypes) led to changes in oviposition behavior of the diamondback moth P. xylostella; however, this effect was dependent on the specific genetic lines included in dual‐choice oviposition assays. In addition, for both moth species, experience led to female oviposition choices that would have maximized fitness of their offspring. In summary, this article suggests that insect experience with plant genotypes varying in their secondary metabolites can influence subsequent oviposition behavior. This outcome may have implications for plant‐insect coevolution and integrated pest management.     

Life stage and population density of Plutella xylostella affect the predation behavior of Euborellia annulipes

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the main pest of brassica crops worldwide. The ringlegged earwig, Euborellia annulipes (Lucas) (Dermaptera: Anisolabididae), has been reported as a potential predator of lepidopteran larvae, including this pest, and may therefore be used for biological control. Knowledge about predator–prey interactions is important to establish pest management strategies. Therefore, the objective of this work was to evaluate the influence of the developmental stage (larva and pupa) and density of P. xylostella on the preference and functional response of E. annulipes adult females. We used choice and no‐choice tests to evaluate the foraging behavior and preference of E. annulipes on DBM life stages and varied prey density to assess the type of functional response of the ringlegged earwig. Larvae were preferred over pupae, and the predator’s functional response was type II for both prey stages. Our results report the potential of E. annulipes as a biocontrol agent of P. xylostella. Understanding their interactions may help in decision‐making and optimization of integrated management strategies.     

Implication for DNA methylation involved in the host transfer of diamondback moth,Plutella xylostella (L.)

DNA methylation exerts extensive impacts on gene expression of various living organisms exposed to environmental variation. However, little is known whether DNA methylation is involved in the host transfer of diamondback moth, Plutella xylostella (L.), a worldwide destructive pest of crucifers. In this study, we found that P. xylostella genome exhibited a relatively low level of DNA methylation on the basis of the CpG O/E prediction and experimental validation. A significant positive linear correlation was observed between the stage‐specific expressions of PxDNMT1 and DNA methylation levels (5mC content). Particularly, high levels of DNA methylation and gene expression of PxDNMT1 were observed in eggs and mature females of P. xylostella. After host transfer of P. xylostella from Raphanus sativus to Arabidopsis thaliana, we identified some potential genomic loci that might have changed methylation levels. Using the method of fluorescence‐labeled methylation‐sensitive amplified polymorphism (F‐MSAP), we also found the corresponding genes primarily involved in neural system and signaling. The expressions of six candidate genes were verified by qRT‐PCR. One of the genes, Px009600, might be regulated by a DNA methylation‐mediated mechanism in response to host transfer. Our study provides evidence for a functional system of DNA methylation in P. xylostella and its possible role in adaptation during host transfer. Further studies should examine methylation as responsive factors to different host plants and environmental cues in insect pests.     

Isolation and characterization of Pseudomonas cedrina infecting Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth, Plutella xylostella, is one of the most destructive pests worldwide and its management relies exclusively on frequent application of chemical insecticides. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. Entomopathogenic bacteria and their related products play an important role in the management of this pest. In the present work, one bacterial strain was separated from infected pupae of P. xylostella collected from field and its pathogenicity was evaluated. On the basis of the 16S ribosomal RNA sequencing, BLASTN, and phylogenetic analysis, this bacterial isolate was identified as Pseudomonas cedrina. Oral administration of P. cedrina at levels above 10,000 CFU/ml gave significant mortality to P. xylostella larvae. The pathogenicity was also observed by reduced longevity and fecundity in adult females. However, when live bacterial cells were removed, the cultured broth lost any pathogenicity. In response to the bacterial infection, P. xylostella expressed antimicrobial and stress‐associated genes. A mixture treatment of P. cedrina and Bacillus thuringiensis showed an additive effect on larval mortality of P. xylostella. These results indicated that P. cedrina is an opportunistic entomopathogen without secretion of toxins. Furthermore, the additive effect of P. cedrina and B. thuringiensis provide a new insight to develop new strategy for controlling P. xylostella.     

Can imitation companion planting interfere with host selection by Brassica pest insects?

• 1 Companion planting with nonhosts may offer a non‐insecticidal means of controlling pests, although the results of studies can be variable and species‐dependent.
• 2 The effect of companion planting on two pests of Brassica crops, Plutella xylostella (L.) and Brevicoryne brassicae (L.), was examined using Brussels sprout as the host plant and imitation cereal plants made from green plastic as the nonhost. For P. xylostella, the effect of nonhost density was also investigated.
• 3 Oviposition (P. xylostella) and abundance (B. brassicae) were lower on Brussels sprout plants presented on a background of high‐density imitation cereal plants (reductions of 59% and 85%, respectively).
• 4 The results are discussed in the context of host location by pest insects and the selection of nonhost companion plants for pest management.
• 5 It is concluded that nonhost plants interfere with pest host selection through disruption to visual host location processes.

     

Feasibility,limitation and possible solutions of RNAi‐based technology for insect pest control

Abstract Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi‐mediated crop protection. However, there are many limitations using RNAi‐based technology for pest control, with the effectiveness target gene selection and reliable double‐strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome‐scale high‐throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro‐injection or by feeding as a dietary component. However, micro‐injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi‐mediated crop protection has been considered as a potential new‐generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi‐based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Tritrophic Choice Experiments with Bt Plants,the Diamondback Moth (Plutella xylostella) and the parasitoid Cotesia plutellae

Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes. Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests. A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implementing measures for the conservation of parasitoid populations. It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations. The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins. However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P. xylostella. Cotesia plutellae is an important endoparasitoid of P. xylostella larvae. Although unable to survive in Bt-susceptible P. xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C. plutellae is able to complete its larval development in Bt-resistant P. xylostella larvae. Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C. plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts. This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage. Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P. xylostella larvae on Bt plants as on wildtype plants. In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant. The implications for integrated pest management and the evolution of resistance to Bt in P. xylostella are discussed.     

Protease inhibitors:recent advancement in its usage as a potential biocontrol agent for insect pest management

Plant derived protease inhibitors(PIs)are a promising defensin for crop im-provement and insect pest management.Although agronomist made significant efforts in utilizing PIs for managing insect pests.the potentials of PIs are still obscured.Insect ability to compensate nutrient starvation induced by dietary PI feeding using different strategies,that is,overexpression of PI-sensitive protease,expression of PI-insensitive proteases,degradation of PI,has made this innumerable collection of PIs worthless.A practical challenge for agronomist is to identify potent PI candidates,to limit insect compensatory responses and to elucidate insect compensatory and resistance mechanisms activated upon herbivory.This knowledge could be further efficiently utilized to identify potential targets for RNAi-mediated pest control.These vital genes of insects could be functionally anno-tated using the advanced gene-editing technique,CRISPR/Cas9.Contemporary research is exploiting different in silico and modern molecular biology techniques to utilize PIs in controlling insect pests efficiently.This review is structured to update recent advancements in this field,along with is chronological background.     

Disentangling factors limiting diamondback moth,Plutella xylostella (L.), spatio‐temporal population abundance: A tool for pest forecasting

Data‐mining techniques play an important role in hyperparameter optimization of heterogeneous environmental factors and their relative contribution as determinants of incidences in insect pest ecological studies. A multidimensional field‐based surveillance was conducted in two seasons (24 months), July–June of each season (2015/2016 ‐ season 1 and 2016/2017 ‐ season 2) using sex‐pheromone‐baited traps and Thermocron i‐Buttons to identify key determinants of population abundance of diamondback moth, Plutella xylostella L., across spatial horticultural hotspots of Botswana. The moth is a notorious global brassica pest. Pearson's product moment correlation matrix showed month of the year (M), mean temperature (T_mean) and maximum temperature (T_max) as positively correlated (p < 0.001) to number of moths (N), while minimum temperature (T_min), minimum relative humidity (RH_min), mean relative humidity (RH_mean), maximum relative humidity (RH_max) and host plant (h) were negatively correlated (p < 0.001) to N. Using Waikato Environment for Knowledge Analysis (WEKA) data‐mining techniques, two models were developed: (a) M5P decision‐tree algorithm associated with nine linear models (LMs) and (b) principal component analysis (PCA) based on four principal components. Both approaches identified M as the major predictor of moth abundance, followed by h and farming region (R). However, R was a function of T_max (positive auto‐correlation) and RH_max (negative auto‐correlation). These results provide simplified relative contribution of heterogeneous factors in influencing P. xylostella spatio‐temporal abundance, essential for early warning systems in pest management. This is an important component of sustainable pest management aimed at managing insect pests and minimizing pesticides abuse in brassica production systems.