Toxicity of Cry- and Vip3Aa-Class Proteins and Their Interactions against Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is one of the most important insect pests affecting corn crops worldwide. Although planting transgenic corn expressing Bacillus thuringiensis (Bt) toxins has been approved as being effective against FAW, its populations' resistance to Bt crops has emerged in different locations around the world. Therefore, it is important to understand the interaction between different Bt proteins, thereby delaying the development of resistance. In this study, we performed diet-overlay bioassays to evaluate the toxicity of Cry1Ab, Cry1Ac, Cry1B, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, Vip3Aa11, Vip3Aa19, and Vip3Aa20, as well as the interaction between Cry1Ab-, Cry1F-, Cry2Ab-, and Vip3Aa-class proteins against FAW. According to our results, the LC50 values of Bt proteins varied from 12.62 ng/cm2 to >9000 ng/cm2 (protein/diet), among which the Vip3Aa class had the best insecticidal effect. The combination of Cry1Ab and Vip3Aa11 exhibited additive effects at a 5:1 ratio. Cry1F and Vip3Aa11 combinations exhibited additive effects at 1:1, 1:2, and 5:1 ratios. The combination of Cry1F and Vip3Aa19 showed an antagonistic effect when the ratio was 1:1 and an additive effect when the ratio was 1:2, 2:1, 1:5, and 5:1. Additionally, the combinations of Cry1F and Vip3Aa20 showed antagonistic effects at 1:2 and 5:1 ratios and additive effects at 1:1 and 2:1 ratios. In addition to the above combinations, which had additive or antagonistic effects, other combinations exhibited synergistic effects, with variations in synergistic factors (SFs). These results can be applied to the establishment of new pyramided transgenic crops with suitable candidates, providing a basis for FAW control and resistance management strategies.

Secondary symbionts affect aphid fitness and the titer of primary symbiont.

Bacterial symbionts associated with aphids are important for their ecological fitness. The corn leaf aphid, Rhopalosiphum maidis (Fitch), is one of the most damaging aphid pests on maize and has been reported to harbor Hamiltonella defensa and Regiella insecticola while the effects of the secondary symbionts (S-symbionts) on host ecology and primary symbiont Buchnera aphidicola remain unclear. Here, four aphid strains were established, two of which were collected from Langfang - Hebei Province, China, with similar symbiont pattern except for the presence of H. defensa. Two other aphid strains were collected from Nanning - Guangxi Province, China, with the same symbiont infection except for the presence of R. insecticola. Phylogenetic analysis and aphid genotyping indicated that the S-symbiont-infected and free aphid strains from the same location had identical genetic backgrounds. Aphid fitness measurement showed that aphid strain infected with H. defensa performed shortened developmental duration for 1st instar and total nymph stages, reduced aphid survival rate, offspring, and longevity. While the developmental duration of H-infected strains was accelerated, and the adult weight was significantly higher compared to the H-free strain. Infection with R. insecticola did not affect the aphid's entire nymph stage duration and survival rate. As the H-strain does, aphids infected with R. insecticola also underwent a drop in offspring, along with marginally lower longevity. Unlike the H-infected strain, the R-infected strain performed delayed developmental duration and lower adult weight. The B. aphidicola titers of the H-infected strains showed a steep drop during the aphid 1st to 3rd instar stages, while the augmentation of B. aphidicola titers was found in the R-infected strain during the aphid 1st to 3rd instar. Our study investigated for the first time the effect of the S-symbionts on the ecology fitness and primary symbiont in R. maidis, indicating that infection with secondary symbionts leads to the modulation of aphid primary symbiont abundance, together inducing significant fitness costs on aphids with further impact on environmental adaptation and trophic interactions.

Seed Myco-priming improves crop yield and herbivory induced defenses in maize by coordinating antioxidants and Jasmonic acid pathway.

BACKGROUND: Seed Myco-priming based on consortium of entomopathogenic fungi is very effective seed treatment against Ostrinia furnacalis herbivory. Maize regulates defense responses against herbivory by the production of defense-related enzymatic and non-enzymatic antioxidants, phytohormones, and their corresponding genes. Jasmonic acid (JA) plays a key role in plant-entomopathogenic fungi-herbivore interaction. RESULTS: To understand how a consortium of the entomopathogenic fungi Beauveria bassiana and Trichoderma asperellum induce changes in the response of maize to herbivory and increase the crop yield, 2-year field experiment, antioxidant enzymes, leaf transcriptome, and phytohormone were performed. Fungal inoculation enhanced the production of antioxidant enzymes and JA signaling pathway more than the normal herbivory. The comparison between single inoculated, consortium inoculated, and non-inoculated plants resulted in distinct transcriptome profiles representing a considerable difference in expression of antioxidant- and JA- responsive genes identified through Weighted gene co-expression network analysis (WGCNA) and expression analysis, respectively. Seed priming with a consortium of B. bassiana and T. asperellum significantly enhanced the expression of genes involved in antioxidants production and JA biosynthesis cascade, with the highest expression recorded at 24-h post O. furnacalis larval infestation. They reduced the larval nutritional indices and survival up to 87% and enhancing crop yield and gross return up to 82-96% over the year 2018 and 2019. CONCLUSION: From our results we suggest that a consortium of B. bassiana and T. asperellum can be used synergistically against O. furnacalis in maize under field condition and can mediate antioxidants- and JA- associated maize defense response by boosting up the expression of their responsive genes, thereby enhancing crop yield.

Revealing the difference of α-amylase and CYP6AE76 gene between polyphagous Conogethes punctiferalis and oligophagous C. pinicolalis by multiple-omics and molecular biological technique.

BACKGROUND: Conogethes pinicolalis has been thought as a Pinaceae-feeding variant of the yellow peach moth, Conogethes punctiferalis. The divergence of C. pinicolalis from the fruit-feeding moth C. punctiferalis has been reported in terms of morphology, ecology, and genetics, however there is a lack of detailed molecular data. Therefore, in this study, we investigated the divergence of C. pinicolalis from C. punctiferalis from the aspects of transcriptomics, proteomics, metabolomics and bioinformatics. RESULTS: The expression of 74,611 mRNA in transcriptome, 142 proteins in proteome and 218 metabolites in metabolome presented significantly differences between the two species, while the KEGG results showed the data were mainly closely related to metabolism and redox. Moreover, based on integrating system-omics data, we found that the α-amylase and CYP6AE76 genes were mutated between the two species. Mutations in the α-amylase and CYP6AE76 genes may influence the efficiency of enzyme preference for a certain substrate, resulting in differences in metabolic or detoxifying ability in both species. The qPCR and enzyme activity test also confirmed the relevant gene expression. CONCLUSIONS: These findings of two related species and integrated networks provide beneficial information for further exploring the divergence in specific genes, metabolism, and redox mechanism. Most importantly, it will give novel insight on species adaptation to various diets, such as from monophagous to polyphagous.

Toxicity of chemical pesticides commonly used in maize to Trichogramma ostriniae (Hymenoptera: Trichogrammatidae), an egg parasitoid of Asian corn borer.

Chemical pesticides are largely applied worldwide for the control of agricultural pests, and often cause lethal and sublethal side effects on natural enemies. To determine the compatibility of chemical pesticides, commonly used by maize growers, with Trichogramma ostriniae, the toxicities of four herbicides (pendimethalin, topramezone, glyphosate, and atrazine), three insecticides (imidacloprid, emamectin benzoate, and ethiofencarb), and three fungicides (propiconazole, benzoyl cyclazole, and difenoconazole) were tested under laboratory conditions, measuring residual toxicity to T. ostriniae adults, their lethal effects on the parasitoid's eggs inside the host egg, and on the reproduction of the parasitoid's female offspring. Three tested pesticides including ethiofencarb, glyphosate, and benzoyl cyclazole were slightly harmful to T. ostriniae adults, whereas the other seven pesticides were moderately harmful. The pesticide residues of ethiofencarb, and emamectin benzoate, showed slightly persistence of toxicity to T. ostriniae adults, in contrast to the remaining pesticides. Glyphosate, emamectin benzoate, imidacloprid, ethiofencarb, and propiconazole were classified as harmless, causing < 30% reduction in emergence rate of wasps from host eggs, while atrazine, pendimethalin, topramezone, benzoyl cyclazole, and difenoconazole were classified as slightly harmful, causing 31-52% reduction in parasitoid emergence rates. Furthermore, the number of eggs subsequently laid by T. ostriniae females that had emerged from parasitized host eggs treated with atrazine was significantly reduced compared to which of untreated parasitized host eggs. No negative effects were observed with atrazine treatment on the emergence rates of F1 wasps, their wing deformity rates and sex ratios. The data presented here indicate that the safety interval between pesticide applications and releases of T. ostriniae wasps is 2-3 weeks, which provide valuable information in designing a sound maize integrated pest management program that combines chemical pesticide applications with augmentative releases of T. ostriniae wasps.

Inheritance and Fitness Costs of Vip3Aa19 Resistance in Mythimna separata.

The "high-dose/refuge" strategy is expected to work most effectively when resistance is inherited as a functionally recessive trait and the fitness costs associated with resistance are present. In the present study, a laboratory selected Mythimna separata strain that have evolved >634.5-fold resistance to Vip3Aa19 was used to determine the mode of inheritance. To determine if fitness costs were associated with the resistance, life history parameters (larva stage, pupa stage, pupal weight, adult longevity and fecundity) of resistant (RR), -susceptible (SS) and heterozygous (R♂S♀ and R♀S♂) strains on nontoxic diet were assayed. The LC50 values of R♀S♂ were significantly higher than that of R♂S♀ (254.58 µg/g vs. 14.75 µg/g), suggesting that maternal effects or sex linkage were present. The effective dominance h of F1 offspring decreased as concentration increased, suggesting the resistance was functionally dominant at low concentration and recessive at high concentration. The analysis of observed and expected mortality of the progeny from a backcross suggested that more than one locus is involved in conferring Vip3Aa19 resistance. The results showed that significant differences in many life history traits were observed among the four insect genotypes. In short, resistance to Vip3Aa19 in M. separata was inherited as maternal and multigene and the resistance in the strain was associated with significant fitness costs. The results described here provide useful information for understanding resistance evolution and for developing resistance management strategies.

Baseline Susceptibility and Resistance Allele Frequency in Ostrinia furnacalis in Relation to Cry1Ab Toxins in China.

Asian corn borer (ACB), Ostrinia furnacalis (Guenée) is a destructive pest of corn and major target of transgenic corn expressing Bt (Bacillus thuringiensis) toxins in China. It is necessary to establish the baseline susceptibility of geographically distinct ACB populations to Cry1Ab protein and estimate the resistance alleles frequency. The median lethal concentration (LC50) and LC95 values of Bt toxin Cry1Ab for 25 geographically distinct populations collected in 2018-2019 ranged from 0.86 to 71.33, 18.58 to 5752.34 ng/cm2, respectively. The median effective concentration (EC50) and EC95 values ranged from 0.03 to 10.40 ng/cm2 and 3.75 to 172.86 ng/cm2, respectively. We used the F2 screening method for estimating the expected frequency of resistance alleles of the 13 ACB populations, to Bt corn (Bt11 × GA21) expressing the Cry1Ab toxin. The neonates could not survive on the leaves of transgenic maize Bt11 × GA21 with cry1Ab gene, the Cry1Ab resistance allele frequency was still low in each geographic population in the field, ranging from 0.0032-0.0048, indicating that the sensitivity of ACB to Cry1Ab was still at a high level, and there were no viable resistant individuals in the field at present. The susceptibility of 25 populations of ACB collected in China showed regional differences, although the Cry1Ab resistance allele frequency in these ACB populations is still at a low level. This provides essential knowledge for making the decision to commercialize Bt maize, and monitoring resistance development and evaluating resistance management strategies in the future in China.

Myco-Synergism Boosts Herbivory-Induced Maize Defense by Triggering Antioxidants and Phytohormone Signaling.

BACKGROUND: Biocontrol strategies are the best possible and eco-friendly solution to develop resistance against O furnacalis and improve the maize yield. However, the knowledge about underlying molecular mechanisms, metabolic shifts, and hormonal signaling is limited. METHODS: Here, we used an axenic and a consortium of entomopathogenic Beauveria bassiana OFDH1-5 and a pathogen-antagonistic Trichoderma asperellum GDFS1009 in maize and observed that consortium applications resulted in higher chlorophyll contents and antioxidants activities [superoxide dismutase (SOD), peroxidase (POD), proline, protease, and polyphenol oxidase (PPO)] with a decrease in O. furnacalis survival. We performed a comprehensive transcriptome and an untargeted metabolome profiling for the first time at a vegetative stage in fungal inoculated maize leaves at 0-, 12-, 24-, 48-, and 72-h post insect infestation. RESULTS: The consortium of B. bassiana and T. asperellum leads to 80-95% of O. furnacalis mortality. A total of 13,156 differentially expressed genes were used for weighted gene coexpression network analysis. We identified the six significant modules containing thirteen candidate genes [protein kinase (GRMZM2G025459), acyl-CoA dehydrogenase (GRMZM5G864319), thioredoxin gene (GRMZM2G091481), glutathione S-transferase (GRMZM2G116273), patatin-like phospholipase gene (GRMZM2G154523), cytochrome P450 (GRMZM2G139874), protease inhibitor (GRMZM2G004466), (AC233926.1_FG002), chitinase (GRMZM2G453805), defensin (GRMZM2G392863), peroxidase (GRMZM2G144153), GDSL- like lipase (AC212068.4_FG005), and Beta-glucosidase (GRMZM2G031660)], which are not previously reported that are highly correlated with Jasmonic acid - Ethylene (JA-ET) signaling pathway and antioxidants. We detected a total of 130 negative and 491 positive metabolomic features using a ultrahigh-performance liquid chromatography ion trap time-of-flight mass spectrometry (UHPLC-QTOF-MS). Intramodular significance and real time-quantitative polymerase chain reaction (RT-qPCR) expressions showed that these genes are the true candidate genes. Consortium treated maize had higher jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) levels. CONCLUSION: Our results provide insights into the genetics, biochemicals, and metabolic diversity and are useful for future biocontrol strategies against ACB attacks.

Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis.

Ostrinia furnacalis is an important borer on maize. Long-term and large-scale planting of transgenic corn has led O. furnacalis evolving resistance and reducing the control effect. Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily G gene ABCG4 in O. furnacalis. In order to further determine the relationship between ABCG4 gene and the resistance to Cry1 toxins in O. furnacalis, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct ABCG4-KO knockout homozygous strain. Bioassay results indicated that an ABCG4-KO strain had a higher resistance to Cry1 proteins compared with a susceptible strain (ACB-BtS). The result indicates that the ABCG4 gene may act as a receptor of the Bt Cry1 toxin in O. furnacalis. Furthermore, the development time was significantly changed in the early stage ABCG4-KO larvae, and the population parameters were also significantly changed. In summary, our CRISPR/Cas9-mediated genome editing study presents evidence that ABCG4 gene is a functional receptor for Bt Cry1 toxins, laying the foundation for further clarification of the Bt resistance mechanism.

Selection of the Most Effective Trichogramma Strains (Hymenoptera: Trichogrammatidae) From Myanmar to Control Asian Corn Borer, Ostrinia furnacalis (Lepidoptera: Crambidae).

Selecting the candidate agents or species is a fundamental step in developing effective biological control programs. However, no attempts have been made to evaluate the efficacy of Trichogramma Westwood (Hymenoptera: Trichogrammatidae) strains against the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) in Myanmar. In this study, Trichogramma strains were investigated through a series of glass tube bioassays and under a semifield condition. Ostrinia furnacalis or Corcyra cephalonica egg preferences were compared among six strains of Trichogramma ostriniae, three of Trichogramma chilonis, and four of Trichogramma dendrolimi using a choice-test assay design. Significant differences were observed at the inter- and intraspecific levels. Four strains of T. ostriniae and two of T. dendrolimi showed a strong preference for O. furnacalis eggs, while two strains of T. chilonis and one T. dendrolimi strain preferred Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) eggs. The remaining strains showed no preference. Eleven strains were examined on O. furnacalis eggs only, wherein parasitism, sex ratio, and total progeny per female were highest for two strains of T. ostriniae (respectively: 67.6 ± 3.0%, 82.7 ± 2.3%, 49.6 ± 2.8, and 67.6 ± 3.6%, 90.0 ± 2.4%, 42.7 ± 2.6), and one strain of T. dendrolimi (65.2 ± 3.2%, 84.7 ± 2.6%, 46.3 ± 2.8). Parasitism and searching capacity were evaluated for the three, best performing strains in cages containing maize plants. One T. ostriniae from Southern Shan State, Myanmar parasitized ~81% of egg masses, and should be considered a candidate biological control agent against O. furnacalis in Myanmar.

Genome-Wide Analysis of WRKY Gene Family and the Dynamic Responses of Key WRKY Genes Involved in Ostrinia furnacalis Attack in Zea mays.

WRKY transcription factors comprise one of the largest gene families and serve as key regulators of plant defenses against herbivore attack. However, studies related to the roles of WRKY genes in response to herbivory are limited in maize. In this study, a total of 128 putative maize WRKY genes (ZmWRKYs) were identified from the new maize genome (v4). These genes were divided into seven subgroups (groups I, IIa-e, and III) based on phylogenomic analysis, with distinct motif compositions in each subgroup. Syntenic analysis revealed that 72 (56.3%) of the genes were derived from either segmental or tandem duplication events (69 and 3, respectively), suggesting a pivotal role of segmental duplication in the expansion of the ZmWRKY family. Importantly, transcriptional regulation prediction showed that six key WRKY genes contribute to four major defense-related pathways: L-phenylalanine biosynthesis II and flavonoid, benzoxazinoid, and jasmonic acid (JA) biosynthesis. These key WRKY genes were strongly induced in commercial maize (Jingke968) infested with the Asian corn borer, Ostrinia furnacalis, for 0, 2, 4, 12 and 24 h in the field, and their expression levels were highly correlated with predicted target genes, suggesting that these genes have important functions in the response to O. furnacalis. Our results provide a comprehensive understanding of the WRKY gene family based on the new assembly of the maize genome and lay the foundation for further studies into functional characteristics of ZmWRKY genes in commercial maize defenses against O. furnacalis in the field.

Genetic knockout and general odorant-binding/chemosensory protein interactions: Revealing the function and importance of GOBP2 in the yellow peach moth's olfactory system.

The GOBP2 protein has a unique function in the yellow peach moth (Conogethes punctiferalis (Guenée)). Several general odorant-binding proteins (GOBPs) have been identified in various lepidopteran species, but the functional difference between GOBP1 and GOBP2 in recognition of host plant odorants is still unknown. The functions of GOBP1 and GOBP2 in the yellow peach moth were evaluated in this study by using the CRISPR-Cas9 system. The results revealed the importance of GOBP2 in the olfaction mechanism in the yellow peach moth. The perception of the GOBP1-knockout larvae toward feeding decreased but did not reach a significant level while knocking out the GOBP2 and GOBP1/2 genes resulted in huge differences. On the other hand, electroantennograms (EAGs) and wind tunnel tests showed that the sensitivity of GOBP2 knockout adults to odorants decreased more than that of GOBP1 knockout individuals. The results of STRING database text mining grabbed our attention in protein-protein interaction studies. In this research, we first proved the existence of physical interactions between GOBPs and chemosensory proteins (CSPs) through the surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) methods. Interestingly, GOBP1 and GOBP2 could not interact with each other, but they could interact with CSPs. The interaction results indicated that GOBP2 could physically interact with CSP15, CSP5, and OBP17, whereas GOBP1 could bind only with CSP5 and CSP10, and its association constant (ka) was also more substantial than that of GOBP1. These results strongly suggest the importance of the function of GOBP2 in the perception of host plant odorants by the yellow peach moth.

Effects of brief exposure to high temperatures on the survival and fecundity of Athetis lepigone (Lepidoptera: Noctuidae).

In Huang-Huai-Hai Summer Corn Region of China, brief periods of high summer temperatures have been reported with increasing frequency in recent years. Athetis lepigone is a cosmopolitan insect which causes severe damage on summer corn seedlings. To understand how high summer temperatures may affect the population dynamics of A. lepigone, we exposed different developmental stages (1, 2 and 4-day old eggs; 1, 6, 12 and 18-day old larvae; 1, 3 and 6-day old pupae; and 1 and 2-day old female and male adults) to 41 °C for periods of various length (0.5, 1, 2, 4 and 6 h): The rearing temperature (constant 26 °C) was used as control. After heat treatment, all individuals were transferred to a 26 °C climate chamber for further development. The effects on immediate survival, maturation success to adulthood, and female fecundity were studied. Eggs, young larvae, late pupae and newly emerged adults had relatively higher immediate survival rates than the other experimental groups. Heat treatment at the egg and larval stages had no impact on development to adulthood and on female fecundity, while it significantly reduced the survival rate of larvae but not of eggs. Brief exposure to high temperature during the early pupal stage and as adults depressed female fecundity whereas exposure during the late pupal stage had no effect.

Baseline Susceptibility and Laboratory Selection of Resistance to Bt Cry1Ab Protein of Chinese Populations of Yellow Peach Moth, Conogethes punctiferalis (Guenée).

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.

Voltine Ecotypes of the Asian Corn Borer and Their Response to Climate Warming.

In the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), diapause is governed by a multigenetic constitution that responds to daylength and temperature with seasonality. The ACB displays uni- or multivoltinism, depending on its geographic specificity. Hence, warmer temperatures may result in alternation of voltinism in the ACB, which will help in understanding the ecological consequences of climate warming on insects. In the present study, we investigated the voltinism in two natural populations from Harbin (H) and Gongzhuling (G) as well as a laboratory (L) population (established from the H population in 2017) of the ACB, at ambient and elevated atmospheric CO2 (aCO2 390 µL/L and eCO2 750 µL/L) and temperature (aT and Et = At + 2 °C). From the diapause response, both the uni- and multivoltine ecotypes were coexisting in the H population. The neonate occurrence date of 50% individuals that induced diapause was ca. 10 days later in the G population than in the H population, but it was about 10 days earlier than in the L population. Comparing to the dates of onset and the peak of diapause induction, the G and L populations were less variable than the H population in response to a short and/or shortening daylength in the field. The univoltine individuals could not be eliminated completely after 19 generations of selection. Diapause incidence decreased with a climate-warming scenario, which was temporally specific and could be overridden by significantly low daily average temperatures. The eCO2 did not directly impact the voltinism. On the basis of voltinism, the H population was sympatric for uni- and multivoltine ecotypes, with multivoltinism being dominant. The univoltinism trait was recessive. Climate warming could significantly override the effect of photoperiod, which was yearly dependent. Warmer temperatures and a decreased latitude (shortened daylength), and their interaction, would drive ACB evolution toward diapause homogeneity for multivoltinism.

Molecular and Morphological Identification of Trichogramma (Hymenoptera: Trichogrammatidae) Species From Asian Corn Borer (Lepidoptera: Crambidae) in Myanmar.

The Asian corn borer, Ostrinia furnacalis (Guenée), is the most devastating maize pest in Myanmar. The damage caused by this pest has become a significant obstacle to farmers' income and the national economy. Control of O. furnacalis is challenging due to its protected feeding sites and the crop's height during later phases of the pest's attack. Trichogramma (Hymenoptera: Trichogrammatidae) parasitoids have been used successfully against corn borers in other countries, but knowledge on the species composition in Myanmar is limited. As a first step for potential biological control of Asian corn borer in Myanmar, Trichogramma were collected from major maize growing regions. Identification was performed based on both morphological and molecular techniques. The majority of collected specimens were identified as Trichogramma ostriniae Pang and Chen, T. chilonis Ishii, and T. dendrolimi Matsumura. This is the first report on the composition of Trichogramma species, and an important step towards the establishment of biological control against O. furnacalis in Myanmar.

Toxicity of Cry1-Class, Cry2Aa, and Vip3Aa19 Bt proteins and their interactions against yellow peach Moth, Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae).

Transgenic plants expressing insecticidal proteins from the Bacillus thuringiensis (Bt) have provided an effective way to control target pests. However, the toxicity of Bt proteins against yellow peach moth (YPM), Conogethes punctiferalis (Guenée), one of the most serious maize pests in China, has not received much study. Therefore, we performed diet-overlay bioassays to evaluate the insecticidal activities of Cry1Ab, Cry1Ac, Cry1Fa, Cry1Ah, Cry1Ie, Cry2Aa, and Vip3Aa19, as well as the interaction between Cry1-Class, Cry2Aa, and Vip3Aa19 against YPM. Results showed that the LC50 values ranged from 1.08 to 178.12 ng/cm2 (protein/diet). Among these proteins, Cry1Ab and Cry1Ac had lower LC50 values and LC90 values. In YPM bioassays, the combinations of Cry2Aa with Cry1Ac, Cry1Ie, and Cry1Ab showed antagonism while a mixture of Cry2Aa with Cry1Fa and Cry1Ah exhibited synergism. When Vip3Aa19 was combined with Cry proteins, all combinations interacted positively, with variation in synergistic factors (SF). Three ratios 1:1, 1:2, and 2:1 of Cry1Ah and Vip3Aa19 protein combination showed SF values of 5.20, 5.63, and 8.98, respectively. These findings can be applied in the establishment of new pyramided transgenic crops with suitable candidates as well as in resistance management strategies.

Synergistic Effect of Beauveria bassiana and Trichoderma asperellum to Induce Maize (Zea mays L.) Defense against the Asian Corn Borer, Ostrinia furnacalis (Lepidoptera, Crambidae) and Larval Immune Response.

Ostrinia furnacalis, is the major pest of maize causing significant yield losses. So far, many approaches have been used to increase the virulence of entomopathogenic fungal isolates. The current study is an attempt to estimate synergistic effect of Beauveria bassiana and Trichoderma asperellum in order to explore larval immune response through RNA sequencing and differentially expression analysis. In vivo synergism was examined in seven proportions (B. bassiana: T. asperellum = 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1) and in the in vitro case, two inoculation methods were applied: seed coating and soil drenching. Results revealed significant decrease in plant damage and high larval mortality in fungal treatments. Fungal isolates mediated the plant defense by increasing proline, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and protease activities. Seed coating method was proved to be the most effective in case of maize endophytic colonization. In total, 59 immune-related differentially expressed genes DEGs were identified including, cytochrome P450, heat shock protein, ABC transporter, cadherin, peptidoglycan recognition protein (PGRP), cuticlular protein, etc. Further, transcriptomic response was confirmed by qRT-PCR. Our results concluded that, coculture of B. bassiana and T. asperellum has the synergistic potential to suppress the immune response of O. furnacalis and can be used as sustainable approach to induce plant resistance through activation of defense-related enzymes.

Sexual-biased gene expression of olfactory-related genes in the antennae of Conogethes pinicolalis (Lepidoptera: Crambidae).

BACKGROUND: Conogethes pinicolalis (Lepidoptera: Crambidae), is similar to Conogethes punctiferalis (yellow peach moth) and its host plant is gymnosperms, especially for masson pine. So far, less literature was reported on this pest. In the present study, we sequenced and characterized the antennal transcriptomes of male and female C. pinicolalis for the first time. RESULTS: Totally, 26 odorant-binding protein (OBP) genes, 19 chemosensory protein (CSP) genes, 55 odorant receptor (OR) genes and 20 ionotropic receptor (IR) genes were identified from the C. pinicolalis antennae transcriptome and amino sequences were annotated against homologs of C. punctiferalis. The neighbor-joining tree indicated that the amino acid sequence of olfactory related genes is highly homologous with C. punctiferalis. Furthermore, the reference genes were selected, and we recommended the phosphate dehydrogenase gene (GAPDH) or ribosomal protein 49 gene (RP49) to verify the target gene expression during larval development stages and RP49 or ribosomal protein L13 gene (RPL13) for adult tissues. CONCLUSIONS: Our study provides a starting point on the molecular level characterization between C. pinicolalis and C. punctiferalis, which might be supportive for pest management studies in future.

Molecular characterization and volatile binding properties of pheromone binding proteins and general odorant binding proteins in Conogethes pinicolalis (Lepidoptera: Crambidae).

Conogethes pinicolalis (Lepidoptera: Crambidae), a major pine pest, possesses a sensitive olfactory system to locate its host. Pheromone binding proteins (PBPs) and general odorant binding proteins (GOBPs) are two types of proteins involved in the process. In this work, we used phylogenetic analysis, gene expression, fluorescence competitive binding assay, and molecular docking to characterize PBPs and GOBPs in C. pinicolalis. The phylogenetic unrooted tree revealed the C. pinicolalis GOBPs and PBPs amino acid sequences showed very close relation with Conogethes punctiferalis (yellow peach moth). Meanwhile, both the PBPs and GOBPs were specifically expressed in the antennae. Binding affinities of PBPs and GOBPs to 19 volatile compounds were tested. PBP2 shows the strongest binding to E10-16: Ald with a Ki value of 0.28/1.66 µM; GOBP1 to Z10-16:Ald with a Ki value of 3.11/4.24 µM. Furthermore, molecular docking reveals potential active sites in PBP2 and GOBP1 to interact with most of the tested volatiles. Finally, we demonstrate that PBP2 and GOBP1 are the dominant genes in their respective families using several different assays. In conclusion, PBP2 and GOBP1 genes may play similar roles in detecting and transporting sex pheromones and host plant volatiles in C. pinicolalis.