Phenology model development for Neodryinus typhlocybae: Evaluation of phenological synchrony with its host, Metcalfa pruinosa.

The invasive species Metcalfa pruinosa has inflicted significant economic losses in various European and Asian regions. To combat this pest, the parasitoid wasp Neodryinus typhlocybae has been effectively introduced in Europe. Despite its success, research on the field occurrence patterns of N. typhlocybae, particularly its phenology, remains scarce. This study aims to develop a degree-day model for predicting the adult emergence of N. typhlocybae from overwintering cocoons and to assess the phenological synchrony between N. typhlocybae adults and the nymphal stages of M. pruinosa in Korea. In this study, we estimated the thermal parameters of N. typhlocybae under field temperatures and six constant temperatures (13.92, 17.71, 18.53, 20.53, 22.78, and 24.03 °C) conditions. The lower developmental temperature was estimated using the values of the coefficient of variation for the cumulative degree days of emerged individual adults. The estimated lower developmental threshold temperature was 12.3 °C. With this developmental threshold, a degree-day model was developed, and this model well-predicted emergence in field conditions. By simulating this developed model with the actual occurrence of the nymphal stages of its host, M. pruinosa, adult wasp emergence was estimated to be 1.5 weeks later than the first instar nymph of the host but faster than other nymphal stages of M. pruinosa. Thus, the findings in this study would be helpful in determining the possibility of establishing N. typhlocybae and improving the management efficiency of M. pruinosa.

Temperature-dependent development of Helicoverpa armigera (Lepidoptera: Noctuidae) at constant temperatures: instar pathways and stage transition models with semifield validation.

Temperature-dependent development of Helicoverpa armigera (Hüber) fed with an artificial diet was studied at different temperatures. The instar pathway (IPW) defined as the number of instars prior to pupation significantly affected larval development time, with higher IPW leading to longer larval development time. The IPW was determined at the fifth instar to proceed to 6-7 IPW, when the development time of fifth instar was largely shortened. Accordingly, the development time after the fourth instar was combined (i.e., the fifth-seventh instar) as a single stage to simplify the various IPW and applied to develop phenology models. In linear models, the lower threshold temperature (LT) and thermal constant (degree-days, DD) for each stage were estimated. DD based on the common LT of 10.7 °C were 43, 287, and 191 DD for eggs, larvae, and pupae, respectively. DD model (253.6 DD with LT 10.3 °C for larvae and 181.5 DD with 11.6 °C for pupae) showed good performance in predicting the 50% occurrences of pupae and adults. In nonlinear models, stage transition (ST) models were constructed using the development rate and distribution models to simulate the proportion of individuals shifted from one stage to the next stage. The ST model showed good performance, indicating an average discrepancy of 1.74 days at 25%, 50%, 75%, and 90% adult emergence. Our models developed here will be useful to predict the phenology of H. armigera in the field and to construct a deterministic population model in the future.

Rapid Assessment of Insect Pest Outbreak Using Drones: A Case Study with Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) in Soybean Fields.

Rapid assessment of crop damage is essential for successful management of insect pest outbreaks. In this study, we investigated the use of an unmanned aircraft system (UAS) and image analyses to assess an outbreak of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), that occurred in soybean fields in South Korea. A rotary-wing UAS was deployed to obtain a series of aerial images over 31 soybean blocks. The images were stitched together to generate composite imagery, followed by image analyses to quantify soybean defoliation. An economic analysis was conducted to compare the cost of the aerial survey with that of a conventional ground survey. The results showed that the aerial survey precisely estimated the defoliation compared to the ground survey, with an estimated defoliation of 78.3% and a range of 22.4-99.8% in the 31 blocks. Moreover, the aerial survey followed by image analyses was found to be more economical than the conventional ground survey when the number of target soybean blocks subject to the survey was more than 15 blocks. Our study clearly demonstrated the effectiveness of using an autonomous UAS and image analysis to conduct a low-cost aerial survey of soybean damage caused by S. exigua outbreaks, which can inform decision-making for S. exigua management.

Identification of a fall armyworm (Spodoptera frugiperda)-specific gene and development of a rapid and sensitive loop-mediated isothermal amplification assay.

The fall armyworm [FAW, Spodoptera frugiperda (J E Smith)], a moth native to America, has spread throughout the world since it was first discovered in Africa in 2016. The FAW is a polyphagous migratory pest that can travel over long distances using seasonal winds or typhoons because of its excellent flying ability, causing serious damage to many crops. For effective FAW control, accurate species identification is essential at the beginning of the invasion. In this study, the FAW-specific gene Sf00067 was discovered by performing bioinformatics to develop a fast and accurate tool for the species-specific diagnosis of this pest. An Sf00067 loop-mediated isothermal amplification (LAMP) assay was developed, and optimal conditions were established. The Sf00067 6 primer LAMP (Sf6p-LAMP) assay established in this study was able to diagnose various genotype-based strains of FAW captured in Korea and FAWs collected from Benin, Africa. Our FAW diagnostic protocol can be completed within 30 min, from the process of extracting genomic DNA from an egg or a 1st instar larva to species determination.

A New Mitochondrial Genome of Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) and Mitogenome-Wide Investigation on Polymorphisms.

White-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the major sap-sucking rice pests in East Asia. We have determined a new complete mitochondrial genome of WBPH collected in the Korean peninsula using NGS technology. Its length and GC percentages are 16,613 bp and 23.8%, respectively. We observed one polymorphic site, a non-synonymous change, in the COX3 gene with confirmation heteroplasmy phenomenon within individuals of WBPH by PCR amplification and Sanger sequencing, the first report in this species. In addition, this heteroplasmy was not observed in wild WBPH populations, suggesting that it may be uncommon in fields. We analyzed single nucleotide polymorphisms, insertion, and deletions, and simple sequence repeats among the three WBPH mitogenomes from Korea and China and found diverse intraspecific variations, which could be potential candidates for developing markers to distinguish geographical populations. Phylogenetic analysis of 32 mitogenomes of Delphacidae including the three WBPH mitogenomes suggested that Delphacinae seems to be monophyletic and Sogatella species including WBPH are clearly formed as one clade. In the future, it is expected that complete mitogenomes of individuals of geographically dispersed WBPH populations will be used for further population genetic studies to understand the migration pathway of WBPH.

RNA-Seq and Electrical Penetration Graph Revealed the Role of Grh1-Mediated Activation of Defense Mechanisms towards Green Rice Leafhopper (Nephotettix cincticeps Uhler) Resistance in Rice (Oryza sativa L.).

The green rice leafhopper (GRH, Nephotettix cincticeps Uhler) is one of the most important insect pests causing serious damage to rice production and yield loss in East Asia. Prior to performing RNA-Seq analysis, we conducted an electrical penetration graph (EPG) test to investigate the feeding behavior of GRH on Ilpum (recurrent parent, GRH-susceptible cultivar), a near-isogenic line (NIL carrying Grh1) compared to the Grh1 donor parent (Shingwang). Then, we conducted a transcriptome-wide analysis of GRH-responsive genes in Ilpum and NIL, which was followed by the validation of RNA-Seq data by qPCR. On the one hand, EPG results showed differential feeding behaviors of GRH between Ilpum and NIL. The phloem-like feeding pattern was detected in Ilpum, whereas the EPG test indicated a xylem-like feeding habit of GRH on NIL. In addition, we observed a high death rate of GRH on NIL (92%) compared to Ilpum (28%) 72 h post infestation, attributed to GRH failure to suck the phloem sap of NIL. On the other hand, RNA-Seq data revealed that Ilpum and NIL GRH-treated plants generated 1,766,347 and 3,676,765 counts per million mapped (CPM) reads, respectively. The alignment of reads indicated that more than 75% of reads were mapped to the reference genome, and 8859 genes and 15,815,400 transcripts were obtained. Of this number, 3424 differentially expressed genes (DEGs, 1605 upregulated in Ilpum and downregulated in NIL; 1819 genes upregulated in NIL and downregulated in Ilpum) were identified. According to the quantile normalization of the fragments per kilobase of transcript per million mapped reads (FPKM) values, followed by the Student's t-test (p < 0.05), we identified 3283 DEGs in Ilpum (1935 upregulated and 1348 downregulated) and 2599 DEGs in NIL (1621 upregulated and 978 downregulated) with at least a log2 (logarithm base 2) twofold change (Log2FC ≥2) in the expression level upon GRH infestation. Upregulated genes in NIL exceeded by 13.3% those recorded in Ilpum. The majority of genes associated with the metabolism of carbohydrates, amino acids, lipids, nucleotides, the activity of coenzymes, the action of phytohormones, protein modification, homeostasis, the transport of solutes, and the uptake of nutrients, among others, were abundantly upregulated in NIL (carrying Grh1). However, a high number of upregulated genes involved in photosynthesis, cellular respiration, secondary metabolism, redox homeostasis, protein biosynthesis, protein translocation, and external stimuli response related genes were found in Ilpum. Therefore, all data suggest that Grh1-mediated resistance against GRH in rice would involve a transcriptome-wide reprogramming, resulting in the activation of bZIP, MYB, NAC, bHLH, WRKY, and GRAS transcription factors, coupled with the induction of the pathogen-pattern triggered immunity (PTI), systemic acquired resistance (SAR), symbiotic signaling pathway, and the activation of genes associated with the response mechanisms against viruses. This comprehensive transcriptome profile of GRH-responsive genes gives new insights into the molecular response mechanisms underlying GRH (insect pest)-rice (plant) interaction.

Alteration of unfolded protein responses and autophagy signaling represented the molecular basis underlying saccharin toxicity to Drosophila (Diptera: Drosophilidae).

The purpose of this study was to develop a new control method for Drosophila using saccharin sodium dihydrate (saccharin), an artificial sweetener that is safe for humans and the environment, and to elucidate its mode of action. In this study, we confirmed that saccharin can dose-dependently inhibit the development of or kill vinegar flies (VFs) and spotted wing Drosophila (SWDs). In addition, we found that low concentrations of saccharin induced a similar effect as starvation in Drosophila, whereas high concentrations of saccharin induced changes in the unfolded protein response (UPR) and autophagy signaling that were unlike starvation and inhibited development or killed the VF and the SWD by performing real-time quantitative polymerase chain reaction analyses. Spinosad is a widely used plant protection agent for SWD control. When saccharin was cotreated with 0.25-1.0 ppm spinosad, an additive insecticidal activity was observed only at high concentrations of saccharin. However, when saccharin was cotreated with 2.0 ppm spinosad, an additive insecticidal activity was observed at low concentrations of saccharin. Taken together, alteration of UPR and autophagy signaling represented the molecular basis underlying saccharin toxicity to Drosophila and concurrent spraying of an insecticide with saccharin could enhance the insecticidal activities.

Detection of Monema flavescens (Lepidoptera: Limacodidae) Cocoons Using Small Unmanned Aircraft System.

Current unmanned aircraft system (a.k.a. drone) technology is an effective tool for aerial survey of pests including weeds, plant diseases, and insects. This study was conducted to develop an aerial survey method that can locate cocoons of the oriental moth, Monema flavescens Walker, for precise and accurate detection of the cocoons in winter to prevent defoliation in the subsequent summer. We used a rotary-wing drone for an aerial survey of M. flavescens cocoons on 15 trees at 3-5 m above the tree canopy. We also conducted a conventional ground survey of M. flavescens cocoons on the same trees for two different conditions of cocoons: open (i.e., adult moths already emerged from cocoons) and closed (i.e., adult moths were not emerged yet). A validation census with destructive sampling was conducted to determine the precision and accuracy of the aerial and ground survey methods. The results of this study showed that from the aerial survey with the drone, images of open cocoons differed from those of closed cocoons. We found higher precision and accuracy and lower type I and II error rates for closed cocoons by the aerial survey with the drone than those by the ground survey. No significant relationships of the number of cocoons with tree height and diameter at breast height were found. This is the first study to demonstrate direct detection of insects with an aerial survey by using a drone.

Thermal Effects on the Population Parameters and Growth of Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae).

The pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) is a cosmopolitan and polyphagous species. An evaluation of A. pisum's demographic parameters and growth was carried out after rearing aphids on faba bean plants (Vicia faba) under five different temperature conditions (10 °C, 15 °C, 20 °C, 25 °C and 30 °C). We analyzed the raw life history data, including developmental time, survival, longevity and reproduction, using an age-stage, two-sex life table to consider variable developmental rates among individuals. The population fluctuation of A. pisum determined the stage-specific population structure and potential population growth under different temperature conditions. A. pisum individuals developed successfully from nymphs to adults at all temperatures in this study. The developmental rate of A. pisum increased as the temperature increased. Our results indicated that A. pisum showed a higher pre-adult mortality, lower total fecundity and a negative intrinsic rate of increase at 30 °C. The highest intrinsic rate of increase (0.30) and finite rate of increase (1.35) were observed at 25 °C. Comparisons of population parameters and their analytical methods between different A. pisum populations from other geographic areas are also discussed.

Development, Reproduction, and Life Table Parameters of the Foxglove Aphid, Aulacorthum solani Kaltenbach (Hemiptera: Aphididae), on Soybean at Constant Temperatures.

We investigated several characteristics of the development and reproduction of the aphid Aulacorthum solani raised on soybean (Glycine max) at 10 constant temperatures between 2.5 and 30 °C, and described the relationship between temperature and several critical biological characteristics using mathematical models. We found that A. solani could survive and reproduce on soybean at temperatures ranging from 5 to 27.5 °C. High fecundity was observed at temperatures from 12.5 to 20 °C. The lower developmental threshold and thermal constant for this species' nymphal stages were estimated to be 5.02 °C and 131.2 degree-days, respectively, using a linear model. The upper developmental threshold was estimated to be 33.9 °C using the Lactin-2 model. The optimum temperature for nymphal development was estimated to be 26.9 °C. The maximum total fecundity was estimated as ca. 76.9 nymphs per adult at 18.1 °C. The daily fecundity sharply increased at earlier adult ages, and slowly decreased thereafter until final parthenogenesis occurred, over a range of temperatures from 12.5 to 25 °C. The maximum daily fecundity was estimated to be ca. 6.1 nymphs per adult per day for a 5.2 day old of adult at 21.3 °C using an age- and temperature-dependent model of adult fecundity. In terms of life table statistics, the intrinsic rates of increase and the finite rate of increase were both highest at 25 °C, while the net reproductive rate was highest at 20 °C.

The complete mitochondrial genome of Laodelphax striatellus (Fallén, 1826) (Hemiptera: Delphacidae) collected in a mid-Western part of Korean peninsula.

Laodelphax striatellus (Fallén, 1826) is one of key rice pests in Northeast Asia. We have determined the mitochondrial genome of L. striatellus collected in a mid-western part of Korean peninsula. The circular mitogenome of L. striatellus is 16,359 bp long including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and a single large non-coding region of 1,972 bp. The base composition was AT-biased (77.3%). In comparison of the two Chinese L. striatellus mitogenomes with Korean mitogenome, total 140 and 40 single nucleotide polymorphisms and 166 and 118 insertions and deletions were identified, presenting intra-species variations based on geographical distribution.

The complete mitochondrial genome of Laodelphax striatellus (Fallén, 1826) (Hemiptera: Delphacidae) collected in a southern part of Korean peninsula.

A 16,359 bp mitochondrial genome of Laodelphax striatellus collected in a southern part of Korean peninsula was completed and their intraspecies variations were compared with Korean and Chinese L. striatellus mitogenomes. The circular mitogenome of L. striatellus contains 13 protein-coding genes, two rRNA genes, 22 tRNAs, and a single large non-coding region of 1,972 bp. The base composition was AT-biased (77.3%). It has only one single nucleotide polymorphism (SNP) in AT-rich region compared to other Korean mitogenome, but total 41 and 141 SNPs and 118 and 166 insertions and deletions, respectively, compared to two Chinese mitogenomes, suggesting the possibility of tracing migration based on geographic genetic diversity.

The complete mitochondrial genome of Exorista japonica (Townsend, 1909) (Diptera:Tachinidae).

Exorista japonica (Townsend, 1909), a dipteran tachinid fly, is an endoparasitoid of lepidopteran larvae as a potential biological agent. We have determined a 17,663 bp mitogenome of E. japonica which includes 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNAs, and a single large non-coding region of 2,773 bp. The base composition was AT-biased (81.4%). Phylogenetic trees present monophyly of Tachinidae and Exoristinae, where two Exorista species are clustered in single clade, Exoristini. The E. japonica mitogenome will be a good resource for understanding phylogenetic relationship among species of Tachinidae presenting morphological and ecological complexities.

The complete mitochondrial genome of the fall armyworm, Spodoptera frugiperda Smith, 1797 (Lepidoptera; Noctuidae), firstly collected in Korea.

The fall armyworm, Spodoptera frugiperda, is a serious pest in large numbers on more than 350 plant species in the world. We have determined a 15,388 bp mitogenome of S. frugiperda which includes 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNAs. The base composition was AT-biased (81.3%). Phylogenetic trees present that Korean S. frugiperda placed in basal position of S. frugiperda clade. S. frugiperda mitochondrial genome can be used for understanding recent active migration of S. frugiperda.

Forecasting Spring Emergence of the Asian Corn Borer, Ostrinia furnacalis (Lepidoptera: Crambidae), Based on Postdiapause Development Rate.

This study was conducted to develop temperature-dependent postdiapause development models of overwintering larvae of the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Crambidae), and to evaluate the models' forecasting accuracy using spring adult emergence data. Overwintering larvae were collected at three different times: 24 February (first), 23 March (second), and 25 April (third), 2005. The developmental periods of each collection colony were measured at eight constant temperatures, and those developmental rates were modeled with linear and nonlinear regression. One linear and three nonlinear models provided good fits of developmental rate to temperature across all colonies (r2 = 0.96-0.99). The distribution of development completion time was modeled with a Weibull equation that fit data from the second (r2 = 0.92) and third (r2 = 0.97) colonies better than the first (r2 = 0.87). A Lactin 2 model based on data from the first colony was statistically the best model to describe the relationship between temperature and the postdiapause development rate of O. furnacalis (r2adj = 0.99). However, validation results based on the field data showed that the Logan 6 model combined with the Weibull model (based on the second colony) was well describing spring adult emergence patterns up to 50% cumulative emergence date.

Time-Course RNA-Seq Analysis Reveals Transcriptional Changes in Rice Plants Triggered by Rice stripe virus Infection.

Rice stripe virus (RSV) has become a major pathogen of rice. To determine how the rice transcriptome is modified in response to RSV infection, we used RNA-Seq to perform a genome-wide gene expression analysis of a susceptible rice cultivar. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 3, 7, and 15 days post-infection (dpi). From 8 to 11% of the genes were differentially expressed (>2-fold difference in expression) in RSV-infected vs. noninfected rice. Among them, 532 genes were differentially expressed at all three time points. Surprisingly, 37.6% of the 532 genes are related to transposons. Gene ontology enrichment analysis revealed that many chloroplast genes were down-regulated in infected plants at 3 and 15 dpi. Expression of genes associated with cell differentiation and flowering was significantly down-regulated in infected plants at 15 dpi. In contrast, most of the up-regulated genes in infected plants concern the cell wall, plasma membrane, and vacuole and are known to function in various metabolic pathways and stress responses. In addition, transcripts of diverse transcription factors gradually accumulated in infected plants with increasing infection time. We also confirmed that the expression of gene subsets (including NBS-LRR domain-containing genes, receptor-like kinase genes, and genes involving RNA silencing) was changed by RSV infection. Taken together, we demonstrated that down-regulation of genes related to photosynthesis and flowering was strongly associated with disease symptoms caused by RSV and that up-regulation of genes involved in metabolic pathways, stress responses, and transcription was related to host defense mechanisms.

Mutational analysis of interaction between coat protein and helper component-proteinase of Soybean mosaic virus involved in aphid transmission.

Soybean mosaic virus (SMV), a member of the genus Potyvirus, is transmitted by aphids in a non-persistent manner. It has been well documented that the helper component-proteinase (HC-Pro) plays a role as a 'bridge' between virion particles and aphid stylets in the aphid transmission of potyviruses. Several motifs, including the KITC and PTK motifs on HC-Pro and the DAG motif on the coat protein (CP), have been found to be involved in aphid transmission. Previously, we have shown strong interaction between SMV CP and HC-Pro in a yeast two-hybrid system (YTHS). In this report, we further analysed this CP-HC-Pro interaction based on YTHS and an in vivo binding assay to identify crucial amino acid residues for this interaction. Through this genetic approach, we identified two additional amino acid residues (H256 on CP and R455 on HC-Pro), as well as G12 on the DAG motif, crucial for the CP-HC-Pro interaction. We introduced mutations into the identified residues using an SMV infectious clone and showed that these mutations affected the efficiency of aphid transmission of SMV. We also investigated the involvement of the PTK and DAG motifs in the CP-HC-Pro interaction and aphid transmission of SMV. Our results support the concept that physical interaction between CP and HC-Pro is important for potyviral aphid transmission. Based on the combination of our current results with previous findings, the possibility that aphid transmission may be regulated by more complex molecular interactions than the simple involvement of HC-Pro as a bridge is discussed.

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices.

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.