Functional roles of two novel P450 genes in the adaptability of Conogethes punctiferalis to three commonly used pesticides.

Introduction: Insect cytochrome P450 (CYP450) genes play important roles in the detoxification and metabolism of xenobiotics, such as plant allelochemicals, mycotoxins and pesticides. The polyphagous Conogethes punctiferalis is a serious economic pest of fruit trees and agricultural crops, and it shows high adaptability to different living environments. Methods: The two novel P450 genes CYP6CV1 and CYP6AB51 were identified and characterized. Quantitative real-time PCR (qRT-PCR) technology was used to study the expression patterns of the two target genes in different larval developmental stages and tissues of C. punctiferalis. Furthermore, RNA interference (RNAi) technology was used to study the potential functions of the two P450 genes by treating RNAi-silenced larvae with three commonly used pesticides. Results: The CYP6CV1 and CYP6AB51 genes were expressed throughout various C. punctiferalis larval stages and in different tissues. Their expression levels increased along with larval development, and expression levels of the two target genes in the midgut were significantly higher than in other tissues. The toxicity bioassay results showed that the LC50 values of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin on C. punctiferalis larvae were 0.2028 µg/g, 0.0683 µg/g and 0.6110 mg/L, respectively. After treating with different concentrations of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin (LC10, LC30, LC50), independently, the relative expressions of the two genes CYP6CV1 and CYP6AB51 were significantly induced. After the dsRNA injection, the expression profiles of the two CYP genes were reduced 72.91% and 70.94%, respectively, and the mortality rates of the larvae significantly increased when treated with the three insecticides independently at LC10 values. Discussion: In the summary, after interfering with the CYP6CV1 and CYP6AB51 in C. punctiferalis, respectively, the sensitivity of C. punctiferalis to chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin was significantly increased, indicating that the two CYP6 genes were responsible for the adaptability of C. punctiferalis to the three chemical insecticides in C. punctiferalis. The results from this study demonstrated that CYP6CV1 and CYP6AB51 in C. punctiferalis play crucial roles in the detoxification of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin.

The Role of Insect Symbiotic Bacteria in Metabolizing Phytochemicals and Agrochemicals.

The diversity and high adaptability of insects are heavily associated with their symbiotic microbes, which include bacteria, fungi, viruses, protozoa, and archaea. These microbes play important roles in many aspects of the biology and physiology of insects, such as helping the host insects with food digestion, nutrition absorption, strengthening immunity and confronting plant defenses. To maintain normal development and population reproduction, herbivorous insects have developed strategies to detoxify the substances to which they may be exposed in the living habitat, such as the detoxifying enzymes carboxylesterase, glutathione-S-transferases (GSTs), and cytochrome P450 monooxygenases (CYP450s). Additionally, insect symbiotic bacteria can act as an important factor to modulate the adaptability of insects to the exposed detrimental substances. This review summarizes the current research progress on the role of insect symbiotic bacteria in metabolizing phytochemicals and agrochemicals (insecticides and herbicides). Given the importance of insect microbiota, more functional symbiotic bacteria that modulate the adaptability of insects to the detrimental substances to which they are exposed should be identified, and the underlying mechanisms should also be further studied, facilitating the development of microbial-resource-based pest control approaches or protective methods for beneficial insects.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization in Athetis dissimilis (Lepidoptera: Noctuidae) Under Different Conditions.

Reference genes are the key to study gene expression patterns using quantitative real-time PCR (qRT-PCR). No studies on the reference genes of Athetis dissimilis, an important agricultural pest, have been reported. In order to determine the reference genes for qRT-PCR normalization in A. dissimilis under different conditions, 10 candidate genes [18S ribosomal protein (18S), 28S ribosomal protein (28S), arginine kinase (AK), elongation factor 1 alpha (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L32 (RPL32), ribosomal protein L40 (RPL40), alpha-tubulin (α-TUB), beta-actin (ß-ACT), and beta-tubulin (ß-TUB)] of A. dissimilis were selected to evaluate their stability as reference genes under different biotic and abiotic conditions by using five tools, geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. Furthermore, CSP1 and superoxide dismutase (SOD) were used as target genes to validate the candidate reference genes. The results showed that different reference genes were needed under different experimental conditions, among which, EF-1α, RPL40, and 18S are most suitable reference genes for studying genes related development stages of A. dissimilis, RPL40 and α-TUB for larval tissues, α-TUB and 28S for adult tissues, EF-1α and ß-ACT for insecticidal treatments, ß-ACT and 28S for temperature treatments, EF-1α and ß-ACT for starvation treatments, RPL40 and 18S for dietary treatments, and 18S, 28S, and α-TUB for all the samples. These results provide suitable reference genes for studying gene expression in A. dissimilis under different experimental conditions, and also lay the foundation for further research into the function of related genes in A. dissimilis.

Identification of the differences in olfactory system between male and female oriental tobacco budworm Helicoverpa assulta.

The olfactory system of insects facilitates their search for host and mates, hence it plays an essential role for insect survival and reproduction. Insects recognize odor substances through olfactory neurons and olfactory genes. Previous studies showed that there are significant sex-specific differences in how insects identify odorant substances, especially sex pheromones. However, whether the sex-specific recognition of odorant substances is caused by differences in the expression of olfaction-related genes between males and females remains unclear. To clarify this problem, the whole transcriptome sequence of the adult Helicoverpa assulta, an important agricultural pest of tobacco and other Solanaceae plants, was obtained using Pacbio sequencing. RNA-seq analysis showed that there were 27 odorant binding proteins (OBPs), 24 chemosensory proteins, 4 pheromone-binding proteins (PBPs), 68 odorant receptors and 2 sensory neuron membrane proteins (SNMPs) genes, that were expressed in the antennae of male and female H. assulta. Females had significantly higher expression of General odorant-binding protein 1-like, OBP, OBP3, PBP3 and SNMP1 than males, while males had significantly higher expression of GOBP1, OBP7, OBP13, PBP2 and SNMP2. These results improve our understanding of mate search and host differentiation in H. assulta.

Nonanal modulates oviposition preference in female Helicoverpa assulta (Lepidoptera: Noctuidae) via the activation of peripheral neurons.

BACKGROUND: The insect olfactory system can recognize odorants for feeding, courtship, oviposition and avoiding natural enemies. Odorant cues from host plants play important roles in insect behaviours. Tobacco (Nicotiana tabacum) is the main cultivated host of the oriental tobacco budworm Helicoverpa assult. Volatiles of tobacco plants attract and stimulate oviposition in female moths. However, it is still not known how female H. assulta recognize tobacco volatiles and which odorant compounds are used as oviposition cues. RESULTS: We detected 14 volatile compounds emitted from a tobacco plant during vegetative growth, using gas chromatography-mass spectrometry. Electroantennogram tests indicated that eight of the 14 compounds induced responses in female H. assulta. Among these eight volatiles, nonanal greatly increased oviposition preference. Single-sensillum recording (SSR) results showed that many neurons housed in three types of short basiconic sensilla and four types of long basiconic sensilla responded to nonanal and heptanal as its structural analogue. The responses to nonanal were significantly stronger than those to the other compounds. Nonanal was the main ligand of OR67, an odorant receptor from H. assulta. This was demonstrated using an in vitro Xenopus oocytes expression system that supported the SSR results. CONCLUSION: Nonanal is a key signal volatile of tobacco plants that attracts female H. assulta moths to oviposit. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Flight and Walking Performance of Dark Black Chafer Beetle Holotrichia parallela (Coleoptera: Scarabaeidae) in the Presence of Known Hosts and Attractive Nonhost Plants.

Holotrichia parallela damages seriously on peanut (Arachis hypogaea) pods. Elucidation of its flight and walking performance in the presence of different plants may provide an insight in its host selection process and an explanation to its strong olfactory preference to an attractive nonhost, castor bean (Ricinus communis). We determined the relationships among flight performance, mate choice, and body weight of H. parallela beetles, and then investigated their flight and walking patterns in the presence of known hosts and attractive nonhost plants using a flight mill and a locomotion compensator, respectively. Body weights were not related to mating success, regardless of sex. The flight proportion of selected females drastically decreased compared with nonselected females, nonselected males, and selected males. Within mated males, heavier individuals exhibited poorer flight performance than lighter ones. In flight bioassay, peanut showed an arrestment effect on virgin females. For walking activity factors (distance, time, and speed), the host plants velvetleaf (Abutilon theophrasti) and Siberian elm (Ulmus pumila) elicited the strongest responses in females and males, respectively. Interestingly, the most preferred adult host, Siberian elm, and the nonhost, castor bean, elicited the highest values of two orientation factors (orientation and upwind length) in females. The chemical similarity hypothesis, which states that feeding or oviposition of insects mistakenly on nonhost can be traced to their chemical similarity to actual hosts, could explain the attraction of H. parallela to castor bean.

Fatal Attraction: Ricinus communis Provides an Attractive but Risky Mating Site for Holotrichia parallela Beetles.

The castor bean, Ricinus communis L., is a non-host plant for the large black chafer, Holotrichia parallela Motschulsky (Coleoptera: Scarabaeidae). In laboratory bioassays we found that this plant was no less attractive than the main host plant (peanut, Arachis hypogaea) and three food plant species: velvetleaf (Abutilon theophrasti), the glossy privet (Ligustrum lucidum), and the Siberian elm (Ulmus pumila). In field trapping experiments a Soxhlet extract of castor bean leaves caught more beetles than the optimal sex lure blend [(R)-(-)-linalool and (L)-isoleucine methyl ester blended in a ratio of 1:4], compared at equal doses (500 µl), and laboratory bioassays indicated that a castor bean plant could enhance the attractiveness of different blend ratios of sex lures. Olfactometer bioassays showed that males prefer volatiles emitted from different combinations of castor bean plant extracts and a signaling female over a female alone. In the presence of castor bean plants copulation rates of H. parallela were highest among all test environments both in laboratory bioassays (60%) and in field tests (70%). This study, combined with our previous observation of the feeding behavior of H. parallela adults on castor bean leaves, suggests that castor bean plants may provide an attractive but risky mating site for H. parallela beetles. The enhancement of male mate-location and copulation rate in the presence of castor bean plants can balance its paralytic effects on H. parallela after intake of potential toxins contained in its leaves.

Repellence of Common Tobacco Flavorants on Lasioderma serricorne (Coleoptera: Anobiidae).

The cigarette beetle, Lasioderma serricorne (F.) (Coleoptera: Anobiidae) is a destructive pest species of tobacco. Olfactory repellents derived from permitted tobacco flavorants have the advantage of not adversely effecting tobacco flavor. Among 12 test compounds, neral exhibited the strongest repellent effect. Among six binary blends prepared, three blends (neral + ethyl cinnamate, neral + cinnamaldehyde, and neral + methyl cinnamate) evoked the strongest repellent response. The interactions between neral and any one of the cinnamic acid derivatives were additive, and the interactions between neral and the cinnamic acid derivatives were antagonistic. In a 32-d tobacco barn bioassay, neral + cinnamaldehyde (embedded in 0.5% agaropectin) showed the strongest repellent effect with a persistence of at least 30 d. The binary blend of two tobacco additives (neral and cinnamaldehyde) appears promising as a repellent for controlling cigarette beetles in tobacco barns.

Experience-based mediation of feeding and oviposition behaviors in the cotton bollworm: Helicoverpa armigera (Lepidoptera: Noctuidae).

Experience is well known to affect sensory-guided behaviors in many herbivorous insects. Here, we investigated the effects of natural feeding experiences of Helicoverpa armigera larvae on subsequent preferences of larval approaching and feeding, as well as the effect of host-contacting experiences of mated females on subsequent ovipositional preference. The results show that the extent of experience-induced preference, expressed by statistical analysis, depended on the plant species paired with the experienced host plant. Larval feeding preference was much easier to be induced by natural feeding experience than larval approaching preference. Naïve larvae, reared on artificial diet, exhibited clear host-ranking order as follows: tobacco ≥ cotton > tomato > hot pepper. Feeding experiences on hot pepper and tobacco could always induce positive feeding preference, while those on cotton often induced negative effect, suggesting that the direction of host plant experience-induced preference is not related to innate feeding preference. Inexperienced female adults ranked tobacco as the most preferred ovipositional host plant, and this innate preference could be masked or weakened but could not be reversed by host-contacting experience after emergence.

A General Asymmetric Synthesis of (R)-Matsutakeol and Flavored Analogs.

An efficient and practical synthetic route toward chiral matsutakeol and analogs was developed by asymmetric addition of terminal alkyne to aldehydes. (R)-matsutakeol and other flavored substances were feasibly synthesized from various alkylaldehydes in high yield (up to 49.5%, in three steps) and excellent enantiomeric excess (up to >99%). The protocols may serve as an alternative asymmetric synthetic method for active small-molecule library of natural fatty acid metabolites and analogs. These chiral allyl alcohols are prepared for food analysis and screening insect attractants.

Comparative host selection responses of specialist (Helicoverpa assulta) and generalist (Helicoverpa armigera) moths in complex plant environments.

We tested the behavioral responses of ovipositing females and natal larvae of two sibling species, a generalist Helicoverpa armigera (Hübner) and a specialist Helicoverpa assulta (Guenée), to odor sources emitted from different combinations of six plant species (tobacco, Nicotiana tabacum; hot pepper, Capsicum annuum; tomato, Solanum esculentum; cotton, Gossypium hirsutum; peanut, Arachis hypogaea; maize, Zea mays). Under the conditions of plant materials versus corresponding controls, both stages of both species could find their corresponding host plants. However, H. assulta females and larvae exhibited a supersensitive and an insensitive response, respectively. Under the conditions of tobacco paired with each plant species, H. assulta females exhibited more specialized ovipositional response to tobacco than its sibling. When each plant species were combined with tobacco and tested against tobacco reference, peanut played an opposite role in the two species in their ovipositional responses to tobacco, and cotton can enhance the approaching response of H. armigera larvae when combined with tobacco. It seems that two attractive host plants also can act antagonistically with respect to host selection of the generalist via volatile exchange. Tomato should better be excluded from host list of H. assulta.

A global-wide search for sexual dimorphism of glomeruli in the antennal lobe of female and male Helicoverpa armigera.

By using immunostaining and three-dimensional reconstruction, the anatomical organization of the antennal lobe glomeruli of the female cotton bollworm Helicoverpa armigera was investigated. Eighty-one glomeruli were identified, 15 of which were not previously discovered. The general anatomical organization of the AL of female is similar to that of male and all glomeruli were classified into four sub-groups, including the female-specific glomerular complex, posterior complex, labial-palp pit organ glomerulus, and ordinary glomeruli. A global-wide comparison on the complete glomerular map of female and male was performed and for the first time the quantitative difference in volume for each individual homologous glomerulus was analyzed. We found that the sexual dimorphism includes not only the sex-specific glomeruli but also some of the other glomeruli. The findings in the present study may provide a reference to examine the antennal-lobe organization more in detail and to identify new glomeruli in other moth species. In addition, the complete identification and global-wide comparison of the sexes provide an important basis for mapping the function of distinct glomeruli and for understanding neural mechanisms underlying sexually dimorphic olfactory behaviors.

Glomerular identification in the antennal lobe of the male moth Helicoverpa armigera.

This study investigates anatomical organization of the antennal lobe (AL) glomeruli of the male cotton bollworm Helicoverpa armigera by synaptic antibody staining combined with three-dimensional reconstruction. To identify all glomeruli, their boundaries were accurately determined by means of several additional staining techniques visualizing the neuron categories forming the characteristic spherical neuropils. In total, 78-80 glomeruli were identified in the male H. armigera. The number of glomeruli was considerably larger than that previously reported in this species. Thus, compared with previous studies, we identified 15 new glomeruli, G63-G77. Most of them are located in the posterior part of the AL, which was previously considered to be a part of the protocerebrum. From the general anatomical organization of the AL glomeruli of H. armigera, we classified these neuropil structures into four groups, the macroglomerular complex, posterior complex, labial-palp pit organ glomerulus, and ordinary glomeruli. The complete identification of glomeruli is important for future studies seeking to explore further the coding mechanisms residing within the primary olfactory center of the moth brain. J. Comp. Neurol. 524:2993-3013, 2016. © 2016 Wiley Periodicals, Inc.

Diapause induction in Apolygus lucorum and Adelphocoris suturalis (Hemiptera: Miridae) in northern China.

With widespread planting of Bt cotton and an associated reduction in the use of broad-spectrum insecticides, the mirid bugs Apolygus lucorum (Meyer-Dür) and Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) become major pests of cotton in northern China in recent years. Both species overwinter as diapausing eggs that may enhance ability to survive the cold winter. The effects of photoperiod and temperature on diapause induction in A. lucorum and A. suturalis were investigated under laboratory conditions. Egg diapause was induced primarily by short photoperiod. Temperatures ranging between 17 and 26°C had little effect on diapause induction for both species. The impact of photoperiod (x) on diapause incidence (y) was described with modified Michaelis-Menten models y = 1.1(14 - x)/(14.82 - x) for A. lucorum and y = 1.07(14 - x)/(14.79 - x) for A. suturalis. The critical photoperiod for diapause induction in the first instars of parental A. lucorum and A. suturalis calculated from the models above was almost identical, at 13.3 h (13 h, 18 min) in a 24-h cycle. The parental nymphal stage was sensitive to short photoperiod for diapause induction in both species, with the first-instar nymphs the most sensitive. The sensitivity to short photoperiod decreased gradually as the A. suturalis nymphs developed, whereas the sensitivity dropped sharply at the second instar of A. lucorum. The adults of both species exhibited little sensitivity to photoperiod for diapause induction.

Inheritance and linkage analysis of co-dominant SSR markers on the Z chromosome of the silkworm (Bombyx mori L.).

Microsatellites or simple sequence repeats (SSRs) are co-dominant molecular markers. When we used fluorescent SSR markers to construct a linkage map for the female heterogametic silkworm (Bombyx mori, ZW), we found that some loci did not segregate in a Mendelian ratio of 1:1 in a backcross population. These loci segregated in a 3:1 ratio of single bands compared with double bands. Further examination of band patterns indicated that three types of SSR bands were present: two homozygotes and one heterozygote. In the beginning, we considered to discard these markers. By scoring male and female F1 individuals, we confirmed that these loci were located on the Z chromosome. Using the sex-linked visible mutation sch (K05) and its wild-type (C108), we constructed an F1 male backcross (BC1M) mapping population. The combination of sch backcross and SSR data enabled us to map the SSR markers to the Z chromosome. By adjusting input parameters based on these data, we were able to use Mapmaker software to construct a linkage map. This strategy takes advantage of co-dominant markers for positional cloning of genes on the Z chromosome. We localized sch to the Z chromosome relative to six SSR markers and one PCR marker, covering a total of 76.1 cM. The sch mutation is an important sex-linked visible mutation widely used in breeding of commercial silkworms (e.g. male silkworm selection rearing). Localization of the sch gene may prove helpful in cloning the gene and developing strains for marker-assisted selection in silkworm breeding.

[Chemical components of volatiles form withered black poplar leaves with different physiological age].

By the methods of steam distillation and GC-MS, this paper analyzed the chemical components of the volatiles from withered black poplar leaves. The main components of the volatiles from young leaves were(Z)-3-hexenol (44.81% ),4-methyl-1-pentanol (21.85%) and 2-hydroxy-benzaldehyde (15.19%), those from matured leaves were(Z)-3-hexenol (28.71%) and 2-hydroxy-benzaldehyde (10.35%), while in the volatiles from senescent leaves, the main components were 2-hydroxy-benzaldehyde (28.81%) and benzyl alcohol (15.06%). The (Z)-3-hexenol content in the volatiles was evidently decreased with increasing leaf age, while the species and contents of aromatic compounds were in adverse.