This study aims to explore the relationship between the resistance of maize (Zea mays) to Spodoptera frugiperda and the content of secondary metabolites (ferulic acid, caffeic acid, chlorogenic acid and DIMBOA) in leaves, as well as the gene expression of key enzymes ZmPALs and ZmBXs in their synthesis pathways, so as to provide theoretical guidance for the breeding of insect-resistant maize varieties.

The resistance difference of six maize varieties cultivated in Shaanxi Province to S. frugiperda was evaluated by using artificial insect inoculation at maize whorl stage and leaf-feeding methods, with plant damage ratings, and development and reproduction parameters of S. frugiperda after feeding as indicators. Contents of three phenolic acids and DIMBOA were determined by colorimetry and high-performance liquid chromatography (HPLC), respectively. Real-time quantitative PCR (RT-qPCR) was used to determine mRNA expression levels of ZmPAL6, ZmPAL7, ZmBX10, and ZmBX11 in maize leaves of different varieties at 0, 6, and 18 h after infestation. And then, the relationships between secondary metabolite contents and resistance indicators measured were studied by correlation analysis.

Among the maize varieties tested, Zhengdan958, Qinlong14 and Shanke16 showed less damage, with mean ratings lower than 4.20 at 7-14 d after S. frugiperda infestation, while Zhengda12, Jiyuan1 and Dedan123 suffered the heaviest damage, with ratings from 4.31 to 7.22. Moreover, S. frugiperda larvae and pupae had the fastest development and significantly greater weight; adults had significantly longer lifespan and oviposition duration, as well as higher fecundity on Zhengda12 and Jiyuan1 compared to other varieties. By contrast, the slowest larvae development, significantly lower larval and pupal weight, longer adult preoviposition period lifespan, shorter oviposition duration and lower fecundity occurred on Zhengdan958, followed by Qinlong14 and Shanke16. Correlation analysis results showed that contents of chlorogenic acid, ferulic acid and caffeic acid in maize leaves showed significantly negative correlation with plant damage rating and larval weight of S. frugiperda, and significantly positive correlation with larval developmental period. In addition, chlorogenic acid content exhibited significantly negative correlation with pupal weight. DIMBOA content had significantly negative correlation with plant damage rating. The expressions of ZmPAL6, ZmPAL7, ZmBX10 and ZmBX11 were significantly induced by larval feeding. Whether non-infested or 6 and 18 h after infested plants, the expression levels of these four genes in Zhengdan958 and Qinlong14 were significantly higher than those in Jiyuan1 and Zhengda12.

Maize varieties Zhengdan958, Qinlong14 and Shanke16 planted in Shaanxi Province exhibit certain resistance to damage and growth and development of S. frugiperda, and their resistance is closely related to the contents of three phenolic acids and DIMBOA as well as the expression levels of ZmPALs and ZmBXs in leaves.

The wheat blossom midge Sitodiplosis mosellana (Diptera: Cecidomyiidae), one of the most important wheat pests, undergoes obligatory larval diapause to survive adverse temperature extremes during hot summers and cold winters. This study aims to explore the potential roles of small heat shock protein (sHsp) gene Hsp21.9 in diapause process of S. mosellana.

RACE and RT-PCR technologies were used to clone the full-length cDNA of Hsp21.9 from S. mosellana pre-diapause larvae. Bioinformatics programs were used to characterize the nucleotide and amino acid sequence of cloned Hsp21.9. Real-time quantitative PCR (RT-qPCR) was used to determine the mRNA expression level of Hsp21.9 in pre-diapause, diapause, post-diapause quiescent and developing larvae of S. mosellana, as well as over-summering larvae exposed to short-term (≤120 min) heat stress (35-50℃) and over-wintering larvae exposed to short-term (≤120 min) cold stress (0 to -15℃). The recombinant Hsp21.9 protein was expressed by Escherichia coli prokaryotic expression technology, and then purified. The activity of bacterially expressed recombinant proteins to suppress thermal aggregation of pig heart mitochondrial malate dehydrogenase (MDH) was determined by colorimetry.

The full-length cDNA of S. mosellana Hsp21.9 (SmHsp21.9) obtained was 1 087 bp (GenBank accession number: KT749988), which contained a 582 bp open reading frame (ORF). The predicted ORF encoded a protein of 193 amino acids of which the content of glutamic acid (12.4%) was the most, and the content of cysteine (0.5%) was the least. The estimated molecular weight and isoelectric point were 21.9 kD and 5.67, respectively. The amino acid sequence of SmHsp21.9 contains typical α-crystallin domain of the sHsp family. The domain consists of six β-sheets, which forms a β-sandwich structure. Sequence alignment and phylogenetic analysis suggested that SmHsp21.9 displayed the highest amino acid identity and the closest relationship to Hsp27 from the Nematocera Chironomus riparius. RT-qPCR indicated that SmHsp21.9 expression differed significantly among different diapause stages. The expression level was decreased after the initiation of diapause, gradually increased in October, and peaked in early-to-mid phase of post-diapause (December and January). Compared with the untreated control, the expression level of SmHsp21.9 was significantly induced in over-summering larvae exposed to heat stress (35-45℃) or over-wintering larvae exposed to cold stressed (-5 to -10℃), but temperature extremes i.e. as high as 50℃ or as low as -15℃ failed to do so. The treatment duration also affected transcript levels of SmHsp21.9, with the maximum value at 30-60 min. Recombinant SmHsp21.9 proteins obtained significantly prevented heat-induced (43℃) aggregation of MDH, suggesting its significant molecular chaperone functionality.

The expression of SmHsp21.9 is regulated not only by diapause development, but also by environmental temperature. SmHsp21.9 might be involved in initiation and termination of diapause, and heat/cold tolerance during diapause in S. mosellana.