Yifan Zhai

Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae).

To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

BEHAVIORAL RHYTHMS OF DROSOPHILA SUZUKII AND DROSOPHILA MELANOGASTER

Abstract Drosophila suzukii and Drosophila melanogaster feed on various fruits, causing great economic losses. In order to find the optimum time for controlling D. suzukii and D. melanogaster, the daily rhythms of oviposition, egg hatch, pupation, adult eclosion, copulation, and feeding of these two pests were studied. We found the circadian rhythm of D. suzukii oviposition to have a single pattern with a peak from 20:00–24:00, while the peak oviposition of D. melanogaster was from 16:00–4:00 (the next day). Neither D. suzukii nor D. melanogaster showed a daily pattern of egg hatch; the single peak of egg hatch for D. suzukii occurred 24–32 h after oviposition, while that for D. melanogaster followed a bimodal pattern, with the first peak of egg hatch from 0–4 h after oviposition and the second from 32–36 h after oviposition. Pupation in D. suzukii showed a single peak from 8:00∼16:00, while in D. melanogaster pupation followed a bimodal pattern, with peaks from 4:00–8:00 and 12:00–20:00. Eclosion of of D. suzukii adults followed a unimodal pattern, and generally took place from 0:00–8:00, while that of D. melanogaster also showed a single peak, generally from 0:00–12:00. Meanwhile copulation of D. suzukii, which showed a bimodal pattern, was concentrated from 0:00–12:00 and 20:00–24:00 (the next day), while copulation of D. melanogaster showed a single peak, generally from 0:00–12:00. Both D. suzukii and D. melanogaster had a preference for feeding in light, and in a 24 h photoperiod the percentages of feeding insects were 80.8and 81.1, respectively.

Comparative Developmental Times and Laboratory Life Tables for Drosophlia suzukii and Drosophila melanogaster (Diptera: Drosophilidae)

Abstract Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) and Drosophila melanogaster Meigen were studied in a laboratory at 25 °C, 60% RH and 16:8 h L:D. Stage-specific developmental times, reproduction, stage-specific survival rates, and adult sex ratios were recorded and organized in separate life tables for each species. The intrinsic rate of increase (r), the finite rate of increase (&lgr;), the net reproduction rate (R0) and the mean generation time (T) were 0.12 day-1, 1.13 day-1, 27.57 offspring, and 28.04 days, respectively, for D. suzukii and 0.17 day-1, 1.19 day-1, 38.17 offspring, and 21.27 days, respectively, for D. melanogaster. The use of the age-stage, two-sex life table method to study D. suzukii and D. melanogaster yielded considerably more accurate and useful data than would have been obtained by using the female-only age-specific life table. These life tables can be used for population growth projections, designing mass-rearing programs, and for pest management.

Transcriptome Analysis and Discovery of Genes Relevant to Development in Bradysia odoriphaga at Three Developmental Stages.

Bradysia odoriphaga (Diptera: Sciaridae) is the most important pest of Chinese chive (Allium tuberosum) in Asia; however, the molecular genetics are poorly understood. To explore the molecular biological mechanism of development, Illumina sequencing and de novo assembly were performed in the third-instar, fourth-instar, and pupal B. odoriphaga. The study resulted in 16.2 Gb of clean data and 47,578 unigenes (≥125bp) contained in 7,632,430contigs, 46.21% of which were annotated from non-redundant protein (NR), Gene Ontology (GO), Clusters of Orthologous Groups (COG), Eukaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. It was found that 19.67% of unigenes matched the homologous species mainly, including Aedes aegypti, Culex quinquefasciatus, Ceratitis capitata, and Anopheles gambiae. According to differentially expressed gene (DEG) analysis, 143, 490, and 309 DEGs were annotated as involved in the developmental process in the GO database respectively, in the comparisons of third-instar and fourth-instar larvae, third-instar larvae and pupae, and fourth-instar larvae and pupae. Twenty-five genes were closely related to these processes, including developmental process, reproduction process, and reproductive organs development and programmed cell death (PCD). The information of unigenes assembled in B. odoriphaga through transcriptome and DEG analyses could provide a detailed genetic basis and regulated information for elaborating the developmental mechanism from the larval, pre-pupal to pupal stages of B. odoriphaga.

Nymphal Diapause in Laodelphax striatellus (Hemiptera: Delphacidae)1

Abstract Diapause, an important developmental process in insects, is a physiological adaptation for avoiding adverse environmental conditions. Laodelphax striatellus Fallén is an important graminaceous crop pest in East Asia, and there are few reports regarding the nymphal diapause from fields with rice–wheat rotation. In the present study, we determined the fourth-instar nymph as the main diapause stage through investigation under field and laboratory conditions. Developmental duration of the third and fourth instars was longer during the short day-length (10 h light:14 h dark [10L:14D]) at 20°C than during other photoperiods. The third-instar nymph is the most sensitive stage to changes in the photoperiod. The optimal environmental factor for diapause termination was long day-length (16L:8D) at 25 to 28°C. The supercooling point was significantly reduced in diapause nymphs, and activities of trehalase, pyruvate kinase, and sorbitol dehydrogenase were significantly decreased (46.46, 37.90, and 17.64%, respectively). The information obtained in this study may be beneficial to the development of control strategies for this pest.

Ecological Niche Difference Associated with Varied Ethanol Tolerance between Drosophila suzukii and Drosophila melanogaster (Diptera: Drosophilidae)

Abstract Drosophila suzukii (Matsumura) (Diptera: Drosphilidae) is an important pest that causes damage to fruits of over 60 plant species. Drosophila suzukii oviposits on ripe fruit, while D. melanogaster oviposits on decaying fruit. Therefore, these species occupy separate ecological niches. To provide a better understanding of the alcohol tolerance between these 2 species and explore the relationship of ecological niche differences and alcohol tolerance, ethanol and acetaldehyde content was examined in red grapes infested by D. melanogaster and D. suzukii. We assessed mortality and alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activity levels for 2 Drosophila species exposed to ethanol. The study results showed that ethanol content gradually increased as the fruit decayed while being infested by Drosophila. The ethanol content was higher in the presence of D. melanogaster than in the presence of D. suzukii. In the mortality experiment, the LC50 of D. melanogaster adults was approximately 8.0% following exposure to ethanol for more than 6 h, while it was only 2.7% in D. suzukii. Moreover, D. melanogaster adults and larvae all had higher ADH and ALDH activity than D. suzukii exposed to ethanol. Our results suggest that D. melanogaster and D. suzukii may occupy different ecological niches due to their discrepancy in tolerance to environmental ethanol, which is mainly regulated by ADH and ALDH.

Hormone Signaling Regulates Nymphal Diapause in Laodelphax striatellus (Hemiptera: Delphacidae)

Diapause is a physiological adaptation that allows an organism to survive adverse environmental conditions. Diapause occurs at a specific developmental stage in each species. There are few reports regarding the molecular regulatory mechanism of nymphal diapause in Laodelphax striatellus, which is an important graminaceous crop pest. Our previous studies identified the conditions for nymphal diapause in this species. Here, we combined RNA sequencing transcriptomics and quantitative proteomic analyses to identify nymphal diapause-related genes and proteins. The analysis of differentially regulated genes identified four gene/protein pairs that were synchronously up-regulated, and six gene/protein pairs that were synchronously down-regulated, suggesting that these genes may regulate nymphal diapause. The up-regulated gene juvenile hormone acid methyl transferase (JHAMT) and the down-regulated gene cytochrome P450 monooxygenase (CYP314A1, Shd) were chosen for further functional studies. After knocking-down of LsJHAMT and LsShd in vivo by RNA interference, the titer of JH III and 20E decreased significantly, and the duration of the nymphal development period was severely altered. Thus LsJHAMT and LsShd regulated JH III and 20E titers in the hemolymph to control the nymphal diapause status. This study may lead to new information on the regulation nymphal diapause of this important agricultural insect pest.