Transcriptome differences between Cry1Ab resistant and susceptible strains of Asian corn borer.

BACKGROUND: Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is the major insect pest of maize in China and countries of East and Southeast Asia, the Pacific and Australasia. ACB can develop strong resistance to the transgenic Bt maize expressing Cry1Ab, the most widely commercialized Bt maize worldwide. However, the molecular basis for the resistance mechanisms of ACB to Cry1Ab remained unclear. Two biological replicates of the transcriptome of Bt susceptible (ACB-BtS) and Cry1Ab resistant (ACB-AbR) strains of ACB were sequenced using Solexa/Illumina RNA-Seq technology to identify Cry1Ab resistance-relevant genes. RESULTS: The numbers of unigenes for two biological replications were 63,032 and 53,710 for ACB-BtS and 57,770 and 54,468 for ACB-AbR. There were 35,723 annotated unigenes from ACB reads found by BLAST searching NCBI non-redundant, NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology databases. Based on the NOISeq method, 3,793 unigenes were judged to be differentially expressed between ACB-BtS and ACB-AbR. Cry1Ab resistance appeared to be associated with change in the transcription level of enzymes involved in growth regulation, detoxification and metabolic/catabolic process. Among previously described Bt toxin receptors, the differentially expressed unigenes associated with aminopeptidase N and chymotrypsin/trypsin were up-regulated in ACB-AbR. Whereas, other putative Cry receptors, cadherin-like protein, alkaline phosphatase, glycolipid, actin, V-type proton ATPase vatalytic, heat shock protein, were under-transcripted. Finally, GPI-anchor biosynthesis was found to be involved in the significantly enriched pathway, and all genes mapped to the pathway were substantially down-regulated in ACB-AbR. CONCLUSION: To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in ACB Bt resistance. This study identified differentially expressed unigenes related to general Bt resistance in ACB. The assembled, annotated transcriptomes provides a valuable genomic resource for further understanding of the molecular basis of ACB Bt resistance mechanisms.

Effects of host plants on aphid feeding behavior, fitness, and Buchnera aphidicola titer.

Aphids are sap-feeding plant pests that depend on their symbiotic relationships with the primary endosymbiont Buchnera aphidicola to adapt to impoverished diets. However, how the host plant affects the aphid primary symbiont and aphid adaptation to host plant transfer are poorly known. In this study, aphid symbiont screening and genotype identification were used to establish 2 aphid strains (Rhopalosiphum maidis [Rm] and Rhopalosiphum padi [Rp] strains) containing only Buchnera without any secondary symbionts for both wheat aphid species (R. maidis and R. padi). Aphid fitness and Buchnera titers were unstable on some of these host plants after transferring to novel host plants (G1-G5), which were influenced by host plant species and generations; however, they stabilized after prolonged feeding on the same plants for 10 generations. The electropenetrography (EPG) records showed that the allocation of aphid feeding time was significantly distinct in the 6 host plants; aphids had more intracellular punctures and spent more nonprobing time on green bristlegrass which was not conducive to its growth compared with other plants. The content of soluble sugar, soluble protein, and amino acid in the leaves of the 6 host plants were also clearly separated. The correlation coefficient analysis showed that the nutrient contents of host plants had significant correlations with aphid feeding behaviors, fitness, and Buchnera titers. In the meantime, aphid fitness, and Buchnera titers were also affected by aphid feeding behaviors. Also, Buchnera titers of aphid natural populations on 6 host plants showed a visible difference. Our study deepened our understanding of the interaction among aphids, endosymbionts, and host plants, indicating that the host plant nutrient content is a predominant factor affecting aphid adaptation to their diet, initially affecting aphid feeding behaviors, and further affecting aphid fitness and Buchnera titers, which would further contribute to exploiting new available strategies for aphid control.

Comparison of larval performance and oviposition preference of Spodoptera frugiperda among three host plants: Potential risks to potato and tobacco crops.

Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a major polyphagous pest with the potential to seriously damage various crops. A better understanding of FAW's performance on different hosts may help to predict which plants will be attacked when the preferred host is absent, and facilitate the development of effective pest management practices. We compared the larval performance of FAW fed on maize with that of FAW fed on potato and tobacco, which are important crops in China, using an age-stage two-sex life table and adult female oviposition preference experiments. In cage experiments with potato, tobacco, or maize as the host, FAW reared on maize exhibited the strongest performance with shorter developmental time in the larval stage, longer longevity, and a higher reproductive rate in adults. Females oviposited on maize in preference to potato or tobacco. Compared with larvae fed on maize, those fed on potato and tobacco exhibited significantly lower survival, with only 31.61% and 8.13% developing to the adult stage, respectively. Several life table parameters, including the mean generation time (T), net reproductive rate (R0 ), finite rate of increase (λ), and intrinsic rate of natural increase (r) were negatively affected in FAW fed on potato and tobacco. Our results support the preference-performance hypothesis, that is, that herbivore females maximize fitness by choosing host plants associated with strong larval performance. Although larvae and adults performed poorly on potato and tobacco, their offspring will be capable of establishing populations on them, posing a potential threat to these crops in China.

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.

Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques.

The fall armyworm, Spodoptera frugiperda, is a species native to the Americas and has spread to many countries in Africa and Asia in recent years. Proactive actions for potential invasion of S. frugiperda to China coordinated by government agencies and agricultural extension systems resulted in timely detection in January 2019 in Yunnan province neighboring onto Myanmar. The extensive monitoring in southern provinces of China since February 2019 resulted in dynamic tracking of S. frugiperda spreading to 13 provincial regions in China within 4 months by May 10, 2019, which is crucial for timely management actions in the fields. The first detections of S. frugiperda (corn strain) in China were confirmed using cytochrome oxidase subunit 1 (CO1) and triosephosphate isomerase (Tpi) genes molecular marker method. In addition to S. frugiperda, larvae of three other noctuid species with similar morphological appearance (S. litura, S. exigua and Mythimna separata) can occur simultaneously and cause similar damage in cornfields in southern China. Thus, we can use both morphological and molecular marker methods to compare larval stages of four noctuid species. Further, we discuss the risk of potential spread of invasive S. frugiperda to other regions and impact on corn production in China.

Behavioral Evidence and Olfactory Reception of a Single Alarm Pheromone Component in Halyomorpha halys.

Halyomorpha halys is a major herbivore insect in the fruit orchards of China that has become a devastating invasive pest in North America and Europe since its accidental introductions in the 1990s and 2000s, respectively. Like other hemipteran insects, H. halys releases defensive chemicals against natural enemies, including (E)-2-decenal, which is an aldehyde associated with alarm pheromones. In this study, a series of electrophysiological and behavioral tests were conducted to characterize the alarm functions of (E)-2-decenal among H. halys adults and nymphs. An antennal transcriptome was obtained from a Chinese H. halys population, and 44 odorant-binding protein (OBP) genes were annotated. Among them, five putative alarm pheromone-binding proteins were screened and were extremely consistent with their homologs from US populations. These five OBPs were later expressed in a heterologous expression system, harvested, and then challenged with (E)-2-decenal in a binding assay. All five OBPs showed high binding activities to (E)-2-decenal, which demonstrated its behavioral significance as a defensive component in H. halys, as well as being the first report of its olfactory reception. These findings will help develop behavioral-mediating tools as part of integrated pest management approaches to control this invasive pest.

Effects of Wolbachia on mitochondrial DNA variation in populations of Athetis lepigone (Lepidoptera: Noctuidae) in China.

Wolbachia are endosymbiotic bacteria that infect arthropods and incompatibility among strains can affect gene flow within host insect populations that can significantly result in host mitochondrial DNA (MtDNA) variation within or among populations. The effects of Wolbachia infection on Athetis lepigone MtDNA variation were studied across 12 geographical populations sampled from North China. Mitochondrial COI gene and Wolbachia wsp gene were both obtained from 423 individuals, including 46 Wolbachia infected and 377 uninfected individuals. Average A. lepigone infection rate was 11% (range: 0 to 26%), and wsp sequence variation defined three distinct Wolbachia strains; wLep1, wLep2, and wLep3, respectively. A haplotype network showed no relationship between A. lepigone MtDNA haplotype and Wolbachia infection. Furthermore, haplotype variation mainly exists within populations, although the proportion of variation is higher within uninfected compared to infected individuals. The lack of correlation between A. lepigone haplotype and Wolbachia strain, suggests symbiont infection may not affect mating compatibility or gene flow within host populations in China.

Does Bt maize expressing Cry1Ac protein have adverse effects on the parasitoid Macrocentrus cingulum (Hymenoptera: Braconidae)?

The potential effects of insect-resistant, genetically engineered (GE) crops on non-target organisms, especially on predators and parasitoids, must be evaluated before their commercial cultivation. The effects of GE maize that produces Cry1Ac toxin on the parasitoid Macrocentrus cingulum were assessed by direct bioassay and indirect bioassay. In the indirect bioassay, parasitism rate, cocoon weight and the number of M. cingulum progeny produced per host were significantly reduced when M. cingulum-parasitized Cry1Ac-susceptible Ostrinia furnacalis were fed a diet containing purified Cry1Ac; however, life-table parameters of M. cingulum were not adversely affected when the same assay was performed with Cry1Ac-resistant O. furnacalis. These results indicated that the detrimental effects detected with a Cry1Ac-susceptible host were mediated by poor host quality. In a direct bioassay, no difference in life-table parameters were detected when M. cingulum adults were directly fed a 20% honey solution with or without Cry1Ac; however, survival and longevity were significantly reduced when M. cingulum adults were fed a honey solution containing potassium arsenate, which was used as a positive control. The stability and bioactivity of Cry1Ac toxin in the food sources and Cry1Ac toxin uptake by the host insect and parasitoid were confirmed by enzyme-linked immunosorbent assay and sensitive-insect bioassays. Our results demonstrate that M. cingulum is not sensitive to Cry1Ac toxin at concentrations exceeding those encountered in Bacillus thuringiensis maize fields. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms and will be useful for assessing other non-target impacts.

Parallel evolution of behaviour during independent host-shifts following maize introduction into Asia and Europe.

Maize was introduced into opposite sides of Eurasia 500 years ago, in Western Europe and in Asia. This caused two host-shifts in the phytophagous genus Ostrinia; O. nubilalis (the European corn borer; ECB) and O. furnacalis (the Asian corn borer; ACB) are now major pests of maize worldwide. They originated independently from Dicot-feeding ancestors, similar to O. scapulalis (the Adzuki bean borer; ABB). Unlike other host-plants, maize is yearly harvested, and harvesting practices impose severe mortality on larvae found above the cut-off line. Positive geotaxis in the ECB has been proposed as a behavioural adaptation to harvesting practices, allowing larvae to move below the cut-off line and thus escape harvest mortality. Here, we test whether the same behavioural adaptation evolved independently in Europe and in Asia. We sampled eight genetically differentiated ECB, ACB and ABB populations in France and China and monitored geotaxis through the entire larval development in artificial stacks mimicking maize stems. We find that all ECB and ACB populations show a similar tendency to move down during the latest larval stages, a behaviour not observed in any European or Asian ABB population. The behaviour is robustly expressed regardless of larval density, development mode or environmental conditions. Our results indicate that maize introduction triggered parallel behavioural adaptations in Europe and Asia, harvest selection presumably being the main driver.

Identification of differentially expressed microRNAs between Bacillus thuringiensis Cry1Ab-resistant and -susceptible strains of Ostrinia furnacalis.

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), can develop strong resistance to Cry1Ab, the most widely commercialized Cry toxin for Bt maize worldwide. It is essential to understand the mechanism of resistance for management of this species, but information on the post-transcriptional regulation of Bt resistance in this target insect is limited. In the present study, RNA was extracted from the ACB in various larval stages (1-5 instar) from Cry1Ab-sensitive (ACB-BtS) and -resistant (ACB-AbR) strains, each of which included two biological replicates. Using Illumina sequencing, a total of 23,809,890 high-quality reads were collected from the four ACB libraries. The numbers of known microRNAs (miRNAs) were 302 and 395 for ACB-BtS and 268 and 287 for ACB-AbR. Using Mireap software, we identified 32 and 16 potential novel miRNAs for ACB-BtS and 18 and 22 for ACB-AbR. Among them, 21 known and 1 novel miRNAs had significantly different expression between ACB-BtS and ACB-AbR. Several miRNAs were observed to target potential Bt receptor genes, such as aminopeptidase N and cadherin-like protein. The glycosylphosphatidylinositol-anchor biosynthetic process and ABC transporters pathway were identified through Gene Ontology and KEGG pathway analysis of target genes of the differentially expressed miRNAs.

Seasonal and geographical variation in diapause and cold hardiness of the Asian corn borer, Ostrinia furnacalis.

Asian corn borer, Ostrinia furnacalis (Guenée), is a key corn pest in the Asian-Western Pacific countries. It overwinters as full-grown larvae in plant stalks or in a spun-silk covering located in the plant debris in the temperate regions of China. Supercooling point (SCP) and survival rate after low sub-zero temperature treatment were assessed for field-collected populations in the laboratory using a cool bath with a 1°C/min cooling rate until -40°C. Mean SCPs were varied among geographical populations, with a significant decline from -22.7°C of Haikou, the multivoltine tropical population in the south, to -28.5°C of Gongzhuling, the univoltine temperate population in the northeast of China. In addition, there was more than 1°C difference in SCP between Gongzhuling univoltine and bivoltine populations that were from the same geographic origin. Mean SCPs of the Guangzhou population fluctuated over the year, with significantly lower SCPs in winter than in other seasons, which correlated with a significantly higher proportion of diapausing larvae in winter than in other seasons. Over 41% of overwintering larvae from the northeast population could withstand to be supercooled for a few minutes to the low sub-zero temperature of -40°C, but only 6.7% of their southern counterparts did so. The findings from this study suggest that O. furnacalis mostly takes advantage of freeze avoidance as diapausing larvae for overwintering in the southern region, whereas it exhibits freeze tolerance in diapause in the northeastern region.

Effects of photoperiod and temperature on diapause induction in Conogethes punctiferalis (Lepidoptera: Pyralidae).

The yellow peach moth, Conogethes punctiferalis (Guenée), a multivoltine species that overwinters as diapausing larvae, is one of the most serious insect pests on maize in China. Effect of photoperiod and temperature on larval diapause was examined under empirical laboratory conditions. Short-day treatments caused larval diapause at 25°C, and the critical photoperiod was between 12 and 13 h (or 12 h 51 min) light per day. No sensitive instar was identified for diapause induction under alternated short- (L : D 11 : 13 h) and long-day (L : D 14 : 10 h) treatments at different larval stages. However, accumulative treatment of three instars and 10 d under short-day treatment was required for the induction of 50% larval diapause. All larvae entered diapause at 20°C, whereas less than 3% did so at 30°C, irrespective of the long- or short-day treatment. Furthermore, under the short-day treatment, more than 90% of larvae went into diapause with temperatures ≤ 25°C, but less than 17% did so at 28°C. In contrast, under the long-day treatment, less than 19% of larvae went into diapause with temperatures ≥ 23°C. The forward shift (5°C) of critical temperature under the long-day regime demonstrated the compensatory effect of temperature and photoperiod on diapause induction. In conclusion, C. punctiferalis had a temperature-dependent type I photoperiodic diapause response; there was no sensitive instar for diapause determination, but the photoperiodic accumulation time countermeasures both of the short-day cycles and the number of instars exposed, and the photoperiodic diapause response, was a temperature-compensated phenomenon.

Three amino acid residues bind corn odorants to McinOBP1 in the polyembryonic endoparasitoid of Macrocentrus cingulum Brischke.

Odorant binding proteins (OBPs) play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-1-ol>trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.

[Effects of elevated atmospheric CO2 on plant, herbivorous insect, and its natural enemy: a review].

Since the industrial revolution, the huge consumption of fossil fuels and unduly destruction of natural habitats by human activities have led to the ever-increasing concentration of atmospheric CO2. To study the adaptation mechanisms of plant, herbivorous insect, and its natural enemy within agricultural ecosystems to the elevated atmospheric CO2 concentration is of significance in deciphering the damage pattern of agricultural pest occurrence and controlling the pest occurrence and in mitigating the CO2 emission from agricultural ecosystems. This paper reviewed the research progress on the effects of elevated atmospheric CO2 on the host plant, herbivorous insect, and its natural enemy in agro-ecosystem, with the focuses on the improvement of related research methods, the variation patterns of host plant primary and secondary metabolites induced by elevated atmospheric CO2, the effects of the elevated CO2 on the growth and development, population density, and behaviors of herbivorous insect, and the biology and predation and/or parasitism rates of natural enemy. The future research frontiers in this research area were also discussed.

Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions.

Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis and the European corn borer, Ostrinia nubilalis. To better understand the host location mechanism, we examined the external morphology and ultrastructure of the antennal sensilla of this parasitoid by using scanning and transmission electron microscopy. Antennae of male and female of the M. cingulum are filiform in shape, 5.90-6.64mm in length and consist of scape, pedicel, and flagellum with 39 and 40 flagellomeres, respectively. Cuticular pore and nine types of morphologically distinct sensilla were identified in both sexes, including two types of sensilla chaetica (nonporous), s. trichodea (nonporous), s. basiconica I (nonporous blunt tip), s. basiconica II (porous wall) and basiconica III (nonporous wall) with branched blunt tip, s. coeloconica with finger-like projections, protruded s. campaniform with central tip pore, and plate-like s. placodea (porous). We compared number, morphology, and distribution of sensilla between sexes. S. campaniform and non-porous basiconica type I may play a role in gustatory functions whereas type II, and s. placodea may play a function in detecting odor stimuli due to their pores wall. The sensilla chaetica and s. trichodea may be involved in mechnosensation. S. coeloconica probably plays a role as thermo-hygro receptor, whilst cuticular pores may detect odor stimuli. No differences in antenna shape and basic structure in the males and females, but male antennae length and width were significantly greater than those of females. Furthermore, males had more placodea than females. The sensilla types, morphology, and structure of both sexes were compared to those found in other parasitic hymenoptera, especially braconid wasps.

[Effects of transgenic corn expressing Bacillus thuringiensis cry1Ab toxin on population increase of Rhopalosiphum maidis Fitch].

A laboratory study was made on the effects of transgenic corn expressing cry1Ab toxin from the bacterium Bacillus thuringiensis (Bt) on the life table of an experimental population of corn leaf aphid Rhopalosiphum maidis Fitch. The results showed that no obvious negative effects of two Bt corn hybrids DK647BTY (event MON810) and NX4777 (event Bt11) were observed on the net reproductive rate, average generation time, innate capacity for increase, and finite rate of increase of R. maidis, and there were no significant differences in the rate of alate, mortality of nymph, and longevity and fecundity duration of corn leaf aphid when feeding on Bt and non-Bt corn hybrids, suggesting that Bt corn expressing cry1Ab toxin had no side-effects on the development and reproduction of R. maidis.

Expression of a modified Cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer.

The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.