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.

Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.

Evolutionary Shift of Insect Diapause Strategy in a Warming Climate: An Intra-Population Evidence from Asian Corn Borer.

Herbivorous insects having variable numbers of generations annually depending on climate and day length conditions are increasingly breeding additional generations driven by elevated temperature under the scenario of global warming, which will increase insect abundance and result in more frequent damage events. Theoretically, this relies on two premises, i.e., either an evolutionary shift to facultative diapause for an insect behaving an obligatory diapause or developmental plasticity to alter voltinism productively for an insect with facultative diapause before shortening photoperiods inducing diapause. Inter-population evidence supporting the premise (theory) comes primarily from a model system with voltinism linked to thermal gradients across latitude. We examined the intra-population evidence in the field (47°24' N, 123°68' E) with Ostrinia furnacalis, one of the most destructive pests, on corn in Asia and Pacific islands. The species was univoltine in high latitudinal areas (≤46° N). Divergence of the diapause feature (obligatory and facultative) was observed within the field populations from 2016 to 2021. Warmer climates would provoke more facultative diapause individuals to initiate a second generation, which will significantly drive the population to evolve toward facultative diapause (multi-voltinism). Both divergent diapause and temperature must be considered for accurate prediction of phenology and population dynamics in ACB.

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.

Intra-Population Alteration on Voltinism of Asian Corn Borer in Response to Climate Warming.

The Asian corn borer (ACB) Ostrinia furnacalis (Guenée) can occur in one to seven generations annually from cool (48°00' N) to warm (18°10' N) region of corn cultivation in China. Although ACB is commonly known as a facultative larval diapause insect, the co-existence of various voltinism suggests that intra-population variation may have evolved for the nature of diapause, i.e., voltinism plasticity. Here, we conducted recurrent selection efforts to establish three strains of, respectively, univoltine (with obligate diapause), multivoltine (with facultative diapause), and non-diapausing ACB under various temperature and photoperiod environments. The univoltine (Lu) strain has evolved a stable univoltinism under a diapause suppressing condition (16 h daylength at 28 °C), with the diapause incidence constantly over 80% after three generations of selection. The multivoltine strain (Lm) under the high temperature (28 °C) was shown to have a typical facultative diapause induced by a range of short-day lengths (11-13.5 h). Diapause incidence was constantly <2.6% under the long day length (16 h) when the temperature was from 18 to 28 °C, i.e., low temperature could not enhance the diapause response in the Lm strain. However, the development was prolonged from 14.2 ± 0.3 d to 46.0 ± 0.8 d when the temperature was reduced from 28 °C to 18 °C. The majority (94.4%) of the developed Ln strain still maintained the non-diapausing nature under a diapause enhancing condition, i.e., a short (13 h) daylength at a low temperature (22 °C). Lm and Ln were able to complete their second generation in Heihe (50°14' N) if the first-generation moth oviposits before 18 June. The study suggests that ACB has evolutionary intra-population variation in voltinism. Under the climate change scenario warmer spring and summer might affect the proportion of sympatric voltine biotype populations that evolve toward being multivoltine.

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.

RNA Interference of Phenoloxidases of the Fall Armyworm, Spodoptera frugiperda, Enhance Susceptibility to Bacillus thuringiensis Protein Vip3Aa19.

Phenoloxidase (PO) is an important enzyme in the cellular immune system and is involved in defense against a wide range of pathogens, including Bacillus thuringiensis. Vip3Aa19 is secreted and expressed by Bacillus thuringiensis (Bt) at the middle exponential growth phase and is a kind of protein with efficient insecticidal activity against Spodoptera frugiperda. However, immune responses of the target insects have been regarded as a hindrance to Bt pathogenicity. This paper reports two phenoloxidase (PO) genes (SfPAE and SfPO2) identified from the hemocyte transcriptome data of the fall armyworm, Spodoptera frugiperda. qRT-PCR validation results showed that the expression levels of two PO genes were significantly upregulated after Vip3Aa19 (LC50 = 4.98 µg/g) toxin treatment compared with those of S. frugiperda fed an insecticide-free artificial diet. Meanwhile, two PO genes were expressed from the egg to adult stages even without an immune challenge. We noticed that at all developmental stages investigated in the S. frugiperda, SfPAE was generally expressed at a higher level than SfPO2. However, after Vip3Aa19 treatment, the SfPO2 gene mRNA expression level was significantly elevated in response to the toxin challenge. An injection of a specific double-stranded RNA (dsRNA) against POs could suppress its expression. The third instar larvae of S. frugiperda treated with dsRNA were much more susceptible to Vip3Aa19 toxin than the control larvae were. Notably, the mortality rate was nearly 90% after a dsPO2 injection. These results proved that SfPO2 was more important for the survival of S. frugiperda. Finally, RNA interference and then PO activity detection revealed that PO genes mainly existed in the hemolymph and played an important role in immune defense against Bt toxin.

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.

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.

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.

Phenotypic responses and potential genetic mechanism of lepidopteran insects under exposure to graphene oxide.

Clarification of the interactions between engineered nanomaterials and multiple generations of insects is crucial to understanding the impact of nanotechnology on the environment and agriculture, particularly in toxicity management, pest management and genetic engineering. To date, there has been very limited information about nanoparticle-insect interactions at the genetic and proteomic levels. Here, we examined the phenotypic responses and potential mechanism of a lepidopteran insect Asian corn borer (ACB) to graphene oxide (GO). It was demonstrated that GO could significantly promote the growth of ACB. The transcriptomic and proteomic results consistently verified that GO might activate trypsin-like serine protease, glutathione S-transferase, heat shock protein and glycosyltransferase to further influence the development of ACB. RNA interference results indicated that the trypsin gene was one of the critical genes to accelerate the growth of ACB fed with GO diet. Moreover, physiological analysis showed potential alterations of the expression levels of genes and proteins, and more cholesterol (CE), triacylglycerides (TG) and lipids were accumulated in GO-exposed ACB. Our findings may help to reveal the phenotypic, physiological and genetic responses of insects under exposure to nanomaterials and to assess the environmental risks of other nanomaterials.

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.

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.