不同水稻品种对稻纵卷叶螟生长发育、存活、生殖及飞行能力的影响

系统研究了稻纵卷叶螟在6种不同水稻品种(常规粳稻武育粳3号,杂交粳稻宁粳1号,常规籼稻TN1,杂交籼稻汕优63,超级杂交籼稻两优培九,超级杂交籼粳稻甬优9号)上取食后的发育历期、存活率、卵巢发育进度、繁殖力和飞行能力.结果表明,武育粳3号和宁粳1号能显著延长稻纵卷叶螟未成熟期的发育历期,降低其存活率,延缓卵巢发育进度,降低成虫繁殖率,并提高成虫的飞行能力;不同水稻品种间的影响存在显著差异,其影响从大到小排列为杂交粳稻＞常规粳稻＞常规籼稻＞杂交籼稻＞超级杂交稻.这说明,幼虫期营养对稻纵卷叶螟的生长发育、存活、生殖和飞行能力具有显著影响.     

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut

In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.     

Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae.     

Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.     

Resistance of transgenic rice events (rbcS:cry1Ac) against three lepidopteran rice pests

The transgenic rice expressing cry1Ac gene, which is linked to the rice rbcS promoter and its transit peptide sequence (tp), was highly resistant against all instars of Cnaphalocrocis medinalis (Guenetée) (Lepidoptera: Crambidae). In this study, we evaluated the larval mortality, behavior change, and field occurrence of three main rice pests, C. medinalis, Naranga aenescens (Moore) (Lepidoptera: Noctuidae), and Parnara guttata (Bremer & Grey) (Lepidoptera: Hesperiidae) in T4 generations of three Bt rice events (rbcS3:cry1Ac; 608102, 608104 and 608107) and non-Bt rice. All of the three Bt rice events were resistant to C. medinalis which showed significantly higher mortality for all instars compared to non-Bt rice. The resistance of Bt rice events against the larvae decreased gradually as the larvae developed. However, the survived larvae which ingested Bt rice events died eventually without further development. The resistance of three Bt rice events was investigated in the pot test, which was conducted with 3rd instars of C. medinalis, N. aenescens, and P. guttata, showed mortalities of over 70%. In behavioral assay, C. medinalis fed on the Bt rice events showed feeding avoidance and less leaf rolling behavior compared to that of the larvae fed on non-Bt rice. A 2-yr field survey conducted with larvae of C. medinalis and P. guttata also showed that the three Bt rice events significantly had lower damaged on leaves compared to that of non-Bt rice. Overall, the three Bt rice events were highly resistant to the larvae of lepidopteran target rice pests.     

Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene

Chitin synthases, that catalyze the formation of chitin the major component of cell walls in most filamentous fungi, play crucial roles in the growth and morphogenesis. To investigate the roles of chitin synthase in Penicillium chrysogenum, we developed an RNAi system to silence the class III chitin synthase gene chs4. After transformation, mutants had a slow growth rate and shorter but highly branched hyphae. All transformants either were unable to form conidia or could form only a few. Changes in chs4 expression could lead to a completely different morphology and eventually cause distinct penicillin yields. In particular, the yield of one transformant was 41 % higher than that of the original strain.     

Isolation of a class IV chitin synthase gene from a zygomycete fungus, Rhizopus oligosporus

We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes. We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3. The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase. Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation. This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.     

Characterization of a Chitin Synthase Encoding Gene and Effect of Diflubenzuron in Soybean Aphid, Aphis Glycines

Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®), a chitin-synthesis inhibitor, on A. glycines'' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.     

Larval RNAi in <Emphasis Type="Italic">Tribolium</Emphasis> (Coleoptera) for analyzing adult development

We report here on the use of RNA interference (RNAi) to create pupal and adult loss-of-function phenotypes in the red flour beetle, Tribolium castaneum, by injection of double-stranded RNA (dsRNA) into late instar larvae (we refer to this method as larval RNAi). RNAi is well-established as a useful method to mimic loss-of-function phenotypes in many organisms including insects. However, with a few exceptions (such as in the fruit fly Drosophila melanogaster), RNAi analysis has usually been limited to studies of embryogenesis. Here we demonstrate that injection of green fluorescent protein (GFP) dsRNA into the larval body cavity can inhibit GFP expression beginning shortly after injection and continuing through pupal and adult stages. RNAi analysis of the Tc-achaete-scute-homolog (Tc-ASH) revealed that larval RNAi can induce morphological defects in adult beetles, and also that larval RNAi affects the entire body rather than being localized near the site of injection. The larval RNAi technique will be useful to analyze gene functions in post-embryonic development, giving us the opportunity to study the molecular basis of adult morphological diversity in various organisms.Edited by D. Tautz     

Characterization of hostplant innate immunity from Kharif-II rice cultivars against rice leaf folder (Cnaphalocrocis medinalis Guen.) via in-situ screening and physicochemical assays

In 2019 and 2020, ten rice (Oryza sativa) cultivars including check commonly grown in the Kharif-II season (June-November) were in-situ screened against rice leaf folder (RLF, Cnaphalocrocis medinalis) in RLF-prone experimental plots in Bagerhat, Bangladesh, following RCBD (Randomized Complete Block Design) with three replicates and physicochemical analyses in the lab to evaluate their innate immunity to the pest. Of ten cultivars, only BRRI (Bangladesh Rice Research Institute) rice 72 and 87 tended to be moderately resistant (damage scale 3.00–4.99), while BRRI rice 73, 80, and 90 were susceptible to RLF (damage scale 7.00–8.99) and none appearing to be highly resistant or susceptible. In-field evaluation of Kharif-II rice cultivars against RLF, principal component analysis identified 14 key variables of 30 contributing maximum variance of PCA1 and PCA2. Cluster analysis grouped studied rice cultivars into four clades with clade II of BRRI rice 72 and 87 belonging to resistant club with high tannin and phenol contents (mg/g) assuring relatively higher yield (MT/ha) over others. A 3-D surface plot demonstrated, upsurge of larvae/ hill speeded up RLF incidence and severity (%) while bubble plot depicted, tiller infection (%) and RLF-fold length (cm) enhanced by leaf width (cm) limited the grain yield (MT/ha) in rice, well-supportive to correlation analysis. As per regression analysis, unit increment of tannin and phenol content (mg/g) declined tiller infection by 11 and 14%. Identified these two resistant cultivars could be used as potential parents in further breeding programs to develop an elite high yielding Kharif-II rice line by combining their novel resistance through hybridization.     

Development of leaffolder resistant transgenic rice expressing <Emphasis Type="Italic">cry2AX1</Emphasis> gene driven by green tissue-specific <Emphasis Type="Italic">rbcS</Emphasis> promoter

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T₀ transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33 %. Stable inheritance and expression of cry2AX1 gene in T₁ progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T₁ plants showed 83.33–90.00 % mortality against C. medinalis. The whole plant bioassay for T₁ plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.     

Transgenic Bt Rice Does Not Challenge Host Preference of the Target Pest of Rice Leaffolder,Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Background

Transgenic Bt rice line T2A-1 expresses a synthesized cry2A gene that shows high resistance to Lepidoptera pests, including Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae). Plant volatile orientation cues and the physical characteristics of the leaf surface play key roles in host location or host-plant acceptance of phytophagous insects. These volatile compounds and physical traits may become altered in Bt rice and it is not known whether this influences the behavior of C. medinalis when searching for oviposition sites.

Results

The results of electronic nose analysis showed that the Radar map of Bt rice cultivars was analogous to the non- Bt rice cultivars at each growing stage. PCA analysis was able to partly discriminate between some of the Bt vs. non-Bt rice sensors, but could not to separate Bt cultivars from non-Bt cultivars. The total ion chromatogram between Bt and non-Bt rice cultivars at the seedling, booting and tillering stages were similar and 25 main compounds were identified by GC-MS. For most compounds, there was no significant difference in compound quantities between Bt and non-Bt rice cultivars at equivalent growth stages. The densities of the tubercle papicles and the trichomes on the upper and lower surfaces were statistically equal in Bt and non-Bt rice. The target pest, C. medinalis, was attracted to host rice plants, but it could not distinguish between the transgenic and the isogenic rice lines.

Conclusions

There were no significant differences between the Bt rice line, T2A-1 and the non-Bt rice for volatiles produced or in its physical characteristics and there were no negative impacts on C. medinalis oviposition behavior. These results add to the mounting evidence that Bt rice has no negative impact on the target insect oviposition behavior.     

The chitin synthase genes chs-1 and chs-2 are essential for C. elegans development and responsible for chitin deposition in the eggshell and pharynx, respectively

It is widely accepted that chitin is present in nematodes. However, its precise role in embryogenesis is unclear and it is unknown if chitin is necessary in other nematode tissues. Here, we determined the roles of chitin and the two predicted chitin synthase genes in Caenorhabditis elegans by chitin localization and gene disruption. Using a novel probe, we detected chitin in the eggshell and discovered elaborate chitin localization patterns in the pharyngeal lumen walls. Chitin deposition in these two sites is likely regulated by the activities of chs-1 (T25G3.2) and chs-2 (F48A11.1), respectively. Reducing chs-1 gene activity by RNAi led to eggs that were fragile and permeable to small molecules, and in the most severe case, absence of embryonic cell division. Complete loss of function in a chs-1 deletion resulted in embryos that lacked chitin in their eggshells and failed to divide. These results showed that eggshell chitin provides both mechanical support and chemical impermeability essential to developing embryos. Knocking down chs-2 by RNAi caused a defect in the pharynx and led to L1 larval arrest, indicating that chitin is involved in the development and function of the pharynx.