Identification, expression analysis, and functional verification of three opsin genes related to the phototactic behaviour of Ostrinia furnacalis.

Ostrinia furnacalis is a disreputable herbivorous pest that poses a serious threat to corn crops. Phototaxis in nocturnal moths plays a crucial role in pest prediction and control. Insect opsins are the main component of insect visual system. However, the inherent molecular relationship between phototactic behaviour and vision of insects remains a mystery. Herein, three opsin genes were identified and cloned from O. furnacalis (OfLW, OfBL, and OfUV). Bioinformatics analysis revealed that all opsin genes had visual pigment (opsin) retinal binding sites and seven transmembrane domains. Opsin genes were distributed across different developmental stages and tissues, with the highest expression in adults and compound eyes. The photoperiod-induced assay elucidated that the expression of three opsin genes in females were higher during daytime, while their expression in males tended to increase at night. Under the sustained darkness, the expression of opsin genes increased circularly, although the increasing amplitude in males was lower when compared with females. Furthermore, the expression of OfLW, OfBL, and OfUV was upregulated under green, blue, and ultraviolet light, respectively. The results of RNA interference showed that the knockout of opsin genes decreased the phototaxis efficiency of female and male moths to green, blue, and ultraviolet light. Our results reveal that opsin genes are involved in the phototactic behaviour of moths, providing a potential target gene for pest control and a basis for further investigation on the phototactic behaviour of Lepidoptera insects.

Integrated transcriptomic and proteomic analyses reveal the molecular mechanism underlying the thermotolerant response of Spodoptera frugiperda.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a highly destructive invasive pest with remarkable adaptability to extreme climatic conditions, posing a substantial global threat. Although the effects of temperature stress on the biological and ecological properties of S. frugiperda have been elucidated, the molecular mechanisms underlying its responses remain unclear. Herein, we combined transcriptomic and proteomic analyses to explore the key genes and proteins involved in thermotolerance regulation in S. frugiperda larvae at 42 °C. Overall, 1528 differentially expressed genes (DEGs) and 154 differentially expressed proteins (DEPs) were identified in S. frugiperda larvae under heat stress, including antioxidant enzymes, heat shock proteins (Hsps), cytochrome P450s, starch and sucrose metabolism genes, and insulin signaling pathway genes, indicating their involvement in heat tolerance regulation. Correlation analysis of DEGs and DEPs revealed that seven and eight had the same and opposite expression profiles, respectively. After nanocarrier-mediated RNA interference knockdown of SfHsp29, SfHsp20.4, SfCAT, and SfGST, the body weight and mortality of S. frugiperda larvae significantly decreased and increased under heat stress, respectively. This indicates that SfHsp29, SfHsp20.4, SfCAT, and SfGST play a crucial role in the thermotolerance of S. frugiperda larvae. These results provide insight into the mechanism of heat tolerance in S. frugiperda.

Enzymatic activity and development of Frankliniella occidentalis (Thysanoptera: Thripidae) in response to exogenous calcium treatments of kidney bean plants.

Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) is an agricultural pest threatening various horticultural crops worldwide. Inducing plant resistance is an ecologically beneficial and potentially effective method for controlling F. occidentalis. As an essential nutrient element, exogenous calcium enhances plant-induced resistance. This study investigated the effects of CaCl2 on the secondary metabolites of kidney bean plants and detoxifying and digestive enzymes in F. occidentalis. We found that treatment of plants and treatment time and also the interactions of the 2 factors significantly affected secondary metabolites contents (tannin, flavonoids, total phenol, alkaloid, and lignin) of kidney bean leaves, which indicated that that the effect of treatment of plants on secondary metabolites varied with treatment time. Moreover, when thrips fed on CaCl2-treated plants, the activities of detoxifying enzymes, enzymes glutathione S-transferase and cytochrome P450 substantially increased compared to those in which thrips fed on control plants. However, the activity of carboxylesterase significantly decreased. The detoxifying enzyme genes CL992.contig6, CYP4PN1, and CYP4PJ2 were significantly upregulated at 24 and 48 h. The activities of digestive enzymes (α-amylase, chymotrypsin, and lipase) increased substantially in F. occidentalis. The digestive enzyme gene, FoAMY-1, was significantly upregulated at 24 and 48 h after treatment. The pupation rate and pupal weight of F. occidentalis were significantly reduced. The results indicated that exogenous CaCl2-induced metabolic changes in kidney bean plants and altered the enzymatic activity and development of F. occidentalis that fed upon them.

Expression and function of opsin genes associated with phototaxis in Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae).

BACKGROUND: Zeugodacus cucuribitae is a major agricultural pest that causes significant damage to varieties of plants. Vision plays a critical role in phototactic behavior of herbivorous insects. However, the effect of opsin on the phototactic behavior in Z. cucuribitae remains unknown. The aim of this research is to explore the key opsin genes that associate with phototaxis behavior of Z. cucurbitae. RESULTS: Five opsin genes were identified and their expression patterns were analyzed. The relative expression levels of ZcRh1, ZcRh4 and ZcRh6 were highest in 4-day-old larvae, ZcRh2 and ZcRh3 were highest in 3rd-instar larvae and 5-day-old pupae, respectively. Furthermore, five opsin genes had the highest expression levels in compound eyes, followed by the antennae and head, whereas the lower occurred in other tissues. The expression of the long-wavelength-sensitive (LW) opsins first decreased and then increased under green light exposure. In contrast, the expression of ultraviolet-sensitive (UV) opsins first increased and then decreased with the duration of UV exposure. Silencing of LW opsin (dsZcRh1, dsZcRh2, and dsZcRh6) and UV opsin (dsZcRh3 and dsZcRh4) reduced the phototactic efficiency of Z. cucurbitae to green light by 52.27%, 60.72%, and 67.89%, and to UV light by 68.59% and 61.73%, respectively. CONCLUSION: The results indicate that RNAi inhibited the expression of opsin, thereby inhibiting the phototaxis of Z. cucurbitae. This result provides theoretical support for the physical control of Z. cucurbitae and lays the foundation for further exploration of the mechanism of insect phototaxis. © 2023 Society of Chemical Industry.

Identification of Six Small Heat Shock Protein Genes in Ostrinia furnacalis (Lepidoptera: Pyralidae) and Analysis of Their Expression Patterns in Response to Environmental Stressors.

Ostrinia furnacalis (Guenée) is a major insect pest in maize production that is highly adaptable to the environment. Small heat shock proteins (sHsps) are a class of chaperone proteins that play an important role in insect responses to various environmental stresses. The present study aimed to clarify the responses of six O. furnacalis sHsps to environmental stressors. In particular, we cloned six sHsp genes, namely, OfHsp24.2, OfHsp21.3, OfHsp20.7, OfHsp21.8, OfHsp29.7, and OfHsp19.9, from O. furnacalis. The putative proteins encoded by these genes contained a typical α-crystallin domain. Real-time quantitative polymerase chain reaction was used to analyze the differences in the expression of these genes at different developmental stages, in different tissues of male and female adults, and in O. furnacalis under UV-A and extreme temperature stresses. The six OfsHsp genes were expressed at significantly different levels based on the developmental stage and tissue type in male and female adults. Furthermore, all OfsHsp genes were significantly upregulated in both male and female adults under extreme temperature and UV-A stresses. Thus, O. furnacalis OfsHsp genes play important and unique regulatory roles in the developmental stages of the insect and in response to various environmental stressors.

Molecular characterization of six heat shock protein 70 genes from Arma chinensis and their expression patterns in response to temperature stress.

Arma chinensis is an important predatory enemy of many agricultural and forest pests. Heat shock protein 70 (Hsp70) plays an essential role in insect adaptation to various stress factors. To explore the functions of Hsp70s in relation to thermal tolerance of A. chinensis, full-length cDNAs of six Hsp70 genes (AcHsp70Ba, AcHsp70-4, AcHsp68a, AcHsp68b, AcHsp70-2, and AcHsc70-4) were cloned. Their open reading frames (ORFs) were 1902, 2454, 1884, 1905, 1872, and 1947 bp, respectively. Developmental expression profiles showed that AcHsp70Ba, AcHsp70-4, and AcHsc70-4 were extremely highly expressed in adult stages. AcHsp68a and AcHsp70-2 showed the highest level of expression in nymph stages, and AcHsp68b was mainly expressed in male adults. Tissue distribution analysis demonstrated that the AcHsp70s were ubiquitously expressed but showing gene-specific and sex-driven patterns of expression. High temperature induced the expression of the six AcHsp70s. Among them, AcHsp70Ba, AcHsp70-4, AcHsp68a, and AcHsc70-4 were significantly induced at 38 °C for 6 h, while all six AcHsp70s were significantly induced at 38 °C for 24 h. There were differences in responses of the six AcHsp70s to low-temperature stress. The expressions of AcHsp70-4, AcHsp68a, and AcHsp68b in male adults were significantly repressed at 4 °C for 6 h, whereas AcHsp70Ba and AcHsp70-2 were significantly induced. The levels of AcHsp70Ba, AcHsp68b, and AcHsp70-2 in female adults were significantly repressed at 4 °C for 24 h, whereas AcHsc70-4 was significantly induced. These results suggested that AcHsp70s play important roles in various developmental stages and tissue function, and contribute to the tolerance of A. chinensis to extreme temperatures.

Induced heat shock protein 70 confers biological tolerance in UV-B stress-adapted Myzus persicae (Hemiptera).

As an environmental stress factor, ultraviolet-B (UV-B) radiation directly affects insect growth, development, and reproduction. Heat shock protein 70s kDa (Hsp70s) plays an important role in the environmental adaptation of insects. To determine the role of MpHsp70s in the UV-B tolerance of Myzus persicae (Sulzer), we identified the complete complementary DNA sequences of seven MpHsp70s. They were found to be ubiquitously expressed during different developmental stages and were highly expressed in second-instar nymphs and wingless adults. The expression levels of the MpHsp70s were significantly upregulated when exposed to different durations of UV-B stress. Nanocarrier-mediated dsMpHsp70 suppressed the expression of the MpHsp70s and reduced the body length, weight, survival rate, and fecundity of M. persicae under UV-B exposure. When the combinational RNAi approach was adopted, the effects on the survival rate and fecundity were greater under UV-B stress, except for MpHsc70-4. These results suggest that MpHsp70s are essential for the resistance of M. persicae to UV-B stress.

Expression stability of candidate RT-qPCR housekeeping genes in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is commonly used to quantify gene expression. For normalization, the expression of each gene is compared with a reference "housekeeping" gene that is stably expressed under relevant stress. Unfortunately, there have been no reports on the stability of such reference genes under various treatments of the Spodoptera frugiperda. In this study, we used five tools (RefFinder, GeNorm, NormFinder, BestKeeper, and ΔCt methods) to evaluate the stability of 12 candidate reference genes (RPS18, ß-tubulin, GAPDH, RPS7, RPS15, RPL7, RPL32, Actin-5C, EF1-α, EF1-γ, RPL27, and ACE) in different instars, tissues, and treatments (high and low temperature, UV-A, and emamectin benzoate). Several ribosomal proteins (RPS7, RPS15, RPL32, RPS18, and RPL7), GAPDH, Actin-5C, and ß-tubulin, were relatively stable, suggesting that they are ideal housekeeping genes for various treatments. ACE was extremely unstable under various experimental treatments, rendering it unsuitable as an internal reference. This study identified the reference housekeeping genes stably expressed by S. frugiperda under different treatments, thus setting a foundation for further exploration of the physiological and biochemical mechanisms.

Transcriptome Analysis of Myzus persicae to UV-B Stress.

As an environmental stress factor, ultraviolet-B (UV-B) radiation directly affects the growth and development of Myzus persicae (Sulzer) (Homoptera: Aphididae). How M. persicae responds to UV-B stress and the molecular mechanisms underlying this adaptation remain unknown. Here, we analyzed transcriptome data for M. persicae following exposure to UV-B radiation for 30 min. We identified 758 significant differentially expressed genes (DEGs) following exposure to UV-B stress, including 423 upregulated and 335 downregulated genes. In addition, enrichment analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases illustrated that these DEGs are associated with antioxidation and detoxification, metabolic and protein turnover, immune response, and stress signal transduction. Simultaneously, these DEGs are closely related to the adaptability to UV-B stress. Our research can raise awareness of the mechanisms of insect responses to UV-B stress.

Identification of five small heat shock protein genes in Spodoptera frugiperda and expression analysis in response to different environmental stressors.

Spodoptera frugiperda (J. E. Smith) is a highly adaptable polyphagous migratory pest in tropical and subtropical regions. Small heat shock proteins (sHsps) are molecular chaperones that play important roles in the adaptation to various environment stressors. The present study aimed to clarify the response mechanisms of S. frugiperda to various environmental stressors. We obtained five S. furcifera sHsp genes (SfsHsp21.3, SfsHsp20, SfsHsp20.1, SfsHsp19.3, and SfsHsp29) via cloning. The putative proteins encoded by these genes contained a typical α-crystallin domain. The expression patterns of these genes during different developmental stages, in various tissues of male and female adults, as well as in response to extreme temperatures and UV-A stress were studied via real-time quantitative polymerase chain reaction. The results showed that the expression levels of all five SfsHsp genes differed among the developmental stages as well as among the different tissues of male and female adults. The expression levels of most SfsHsp genes under extreme temperatures and UV-A-induced stress were significantly upregulated in both male and female adults. In contrast, those of SfsHsp20.1 and SfsHsp19.3 were significantly downregulated under cold stress in male adults. Therefore, the different SfsHsp genes of S. frugiperda play unique regulatory roles during development as well as in response to various environmental stressors.

Molecular Characterization and Expression of OfJNK and Ofp38 in Ostrinia furnacalis (Guenée) Under Different Environmental Stressors.

Ostrinia furnacalis, an important pest of corn, has substantial detrimental effects on corn production. The mitogen-activated protein kinase (MAPK) signaling pathway plays a pivotal role in an insect's resistance to environmental stress. The expression levels of JNK and p38 have been well recorded in several insects under different environmental stressors, at different developmental stages, and in various tissue types; however, there is limited information on JNK and p38 in agricultural insects. To clarify the mechanism whereby O. furnacalis responds to environmental stress, we cloned JNK and p38 from O. furnacalis and subsequently named them OfJNK and Ofp38, respectively. Further, we examined the expression levels of OfJNK and Ofp38 under different environmental stressors. In this study, we obtained full-length sequences of OfJNK and Ofp38, and RT-qPCR results showed that these genes were expressed at all developmental stages, in various tissues (head, chest, abdomen, leg, wing, antennae, compound eye, midgut, and ovary) and under different environmental stressors (4°C and ultraviolet A treatment for 0, 30, 60, 90, and 120 min). The expression levels of OfJNK and Ofp38 were relatively higher in eggs and 3-day-old adult females than in other developmental stages. Moreover, the expression level of OfJNK was higher in the wings than in other tissues, whereas that of Ofp38 was significantly higher in the ovaries than in other tissues. OfJNK and Ofp38 showed high expression 90 min after being subjected to treatment at 4°C and ultraviolet A irradiation; the expression of Ofp38 peaked at 30 min, whereas that of OfJNK peaked at 60 min. These results indicate that O. furnacalis differs in terms of its response under different environmental stressors. In summary, our results will provide a foundation for additional research needed to determine the role of the MAPK signaling pathway and the underlying mechanisms by which it shows resistance to environmental stresses in insects.

A proteomic analysis of Helicoverpa armigera adults after exposure to UV light irradiation.

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400nm, has been used worldwide in light trapping of insect pests. To gain a better understanding of the response of Helicoverpa armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in phototactic insects under UV light irradiation conditions and gives new insights into the adaptation mechanisms responsive to UV light irradiation stress.

Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults.

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. In this article, we test the hypothesis that one of the effects of UV light irradiation is to increase oxidative stress on insects. The effects of UV light irradiation on total antioxidant capacity, malondialdehyde (MDA) and protein carbonyl contents and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidases (POX) and glutathione-S-transferase (GST)were investigated in Helicoverpa armigera adults. The adults were exposed to UV light for various time periods (0, 30, 60 and 90 min). We found that exposure to UV light for 30 min resulted in increased total antioxidant capacity, protein carbonyl content and activities of SOD, CAT, POX and GST. When the exposure time lasted for 60 and 90 min, the protein carbonyl content and activities of CAT and GST remained significantly higher than the control. However, the antioxidant capacity and SOD activity returned to control levels, and POX activity decreased at 60 and 90 min. Our results confirm the hypothesis that UV light irradiation increases the level of oxidative stress in H. armigera adults.