Guren Zhang

The first vitellogenin receptor from a Lepidopteran insect: molecular characterization, expression patterns and RNA interference analysis

The vitellogenin receptor (VgR) belongs to the low‐density lipoprotein receptor (LDLR) superfamily, and is an important carrier for the uptake of vitellogenin (Vg) into developing oocytes of all oviparous species. The first full‐length message for a VgR from a Lepidopteran insect was cloned and sequenced from the ovary of Spodoptera litura Fabricius (GenBank accession no. GU983858). The coding region consisted of 5370 bp flanked by a 49 bp 5′‐untranslated region (UTR) and a 177 bp 3′‐UTR, which encoded a 1798‐residue protein with a predicted molecular weight (MW) of 201.69 kDa. S. litura VgR (SlVgR)comprised two ligand binding sites with four LDLR class A repeats in the first domain and seven in the second domain, an epidermal growth factor‐like domain containing an LDLR class B repeat and a YWXD motif, a transmembrane domain and a cytoplasmic domain. A phylogenetic relationship placed SlVgR as a separate group from the other insects. SlVgR messenger RNA (mRNA) was specifically expressed in the ovarian tissues. The developmental expression patterns showed that VgR mRNA was first transcribed in 6th day female pupae and the maximum level of VgR mRNA appeared in 36‐h‐old adults. Immunoblot analysis detected an ovary‐specific VgR protein with a MW of ∼200 kDa, whose development profiles were consistent with VgR mRNA expression patterns. RNA inteference (RNAi) specifically disrupted the VgR gene by injection of 3 or 5 µg VgR double‐stranded RNA per insect in 4th or 6th day pupae. RNAi of SlVgR led to a phenotype characterized by high Vg accumulation in the haemolymph, low Vg deposition in the ovary and the failure of insect spawning. These results mean that VgR is critical for binding Vg and transporting it into the oocytes of the insect ovary, thus playing an important role in insect reproduction.

Molecular characterization and expression pattern of Spodoptera litura (Lepidoptera: Noctuidae) vitellogenin, and its response to lead stress

Vitellogenin (Vg) cDNA from Spodoptera litura Fabricius was cloned and sequenced. The open reading frame (ORF) of Vg cDNA was 5247 nucleotides in length (GenBank Accession no. EU095334), which encoded for a protein of 1748 amino acids. S. litura Vg comprised three conserved regions (Vitellogenin-N domain, DUF1943 and von Willebrand factor type D domain (VWD)), a 17 amino-acid signal peptide and a RXXR cleavage signal (RTIR). The highly conserved GL/ICG motif, the DGXR motif and cysteine residues were found in the C-terminus of the Vg. Vg mRNA was found specifically in the female fat body. Vg expression was first transcribed in 6th day female pupae and levels increased with insect development. The maximum level of Vg mRNA appeared in 24-h-old adults. When S. litura larvae were exposed to lead (Pb) (25-200 mg Pb/kg), there was a significant inhibition in Vg of female adults. The start of Vg expression was advanced ahead by Pb, from 6th day pupae to 3rd day or 4th day pupae. Low levels of Vg in male adults were also induced by low concentrations of Pb (12.5 and 25 mg Pb/kg). These data show that Pb stress elicits an important Vg response in S. litura.

The effect of dietary nickel on the immune responses of Spodoptera litura Fabricius larvae

By exposing Spodoptera litura Fabricius larvae to nickel (Ni) in artificial diets for successive three generations, we investigated the impacts of the dietary Ni on growth and immune response of the fifth and sixth instar larvae at 24 h intervals. The time of newly moulted fifth instar larvae was labelled as 0 h. After exposure to 5 mg/kg Ni for two generations, Ni exposure significantly improved larval phenoloxidase activity and encapsulation grade in fifth instar larvae when compared to controls, except for encapsulation grade at 72-120 h in the second generation. However, higher concentrations of Ni (≥10 mg/kg) only significantly reduced encapsulation grade at 72-120 h. In the third generation, insects given higher dietary levels of Ni (≥10 mg/kg) showed lower immune responses and retarded relative growth rate (RGR) compared to controls, but those exposed to lower Ni levels (≤5 mg/kg) had a significantly improved encapsulation grade at 24-72 h. Larvae at lower Ni level (≤5 mg/kg) treatments had significantly higher RGR in comparison with that in controls. There was no significant difference in food relative consumption rate (RCR) and RGR among any treatment of the fifth instar larvae in three successive generations. These results indicated that the type and extent of effects on growth and immune responses of S. litura varied with the Ni concentrations and exposure periods.

Involvement of Jasmonate­ signaling pathway in the herbivore-induced rice plant defense

The expression patterns of eight defenserelated genes in the herbivore-infested and jasmonatetreated (jasmonic acid, JA and its derivative MeJA) rice leaves were analyzed using RT-PCR. The results showed thatSpodoptera litura Fabricius (Lepidoptera: Noctuidae) herbivory induced the expression of lipoxygenase (LOX) and allene oxide synthase (AOS) genes that are involved in the jasmonate-signaling pathway. Moreover,S. litura damage resulted in the expression of farnesyl pyrophosphate synthase (FPS), Bowman-birk proteinase inhibitor (BBPI), phenylalanine ammonia-lyase (PAL) and other rice defenserelated genes that were also induced by aqueous JA treatment or gaseous MeJA treatment. These indicated that in rice leaves, the JA-related signaling pathway was involved in theS. litura-induced chemical defense. Mechanical damage and brown planthopper (BPH),Nilaparvata lugens (Stål) (Homoptera: Delphacidae) damage induced the expression ofLOX gene, but both treatments did not induce the expression ofAOS gene. However, BPH damage induced the expression of acidic pathogen-related protein 1 (PR1a), Chitinase (PR-3), andPAL genes, which is involved in the salicylatesignaling pathway. It was suggested that salicylate-related signaling pathway or other pathways, rather than jasmonate-signaling pathway was involved in the BPH-induced rice plant defense.

Apoptosis of Spodoptera litura larval hemocytes induced by heavy metal zinc

By adding different amount of zinc into the artificial medium of the insect larvae, the zinc-induced apoptosis of the larvae haemocytes of the herbivorous insectSpodoptera litura Fabricius was investigated with flow cytometer. The results showed that the increase of zinc dose in the artificial feed led to the accumulations of zinc in the larval hemolymph and fat body, and more zinc was accumulated in fat body than in hemolymph. The apoptosis of hemocytes was significantly induced at high zinc concentration (1000 mg·kg−1) in the insect diet, and the apoptosis rate was 63.63%, which was remarkably higher than that at control and lower concentrations (50-500 mg·kg−1). This suggests that the high dose of zinc in the artificial diet ofS. litura larvae could induce the apoptosis of the larval hemocytes ofS. litura.

Structure and expression of β-1,3-glucan recognition proteins from the ghost moth, Thitarodes pui (Hepialidae), and their response to Beauveria bassiana infection.

Beta-1,3-glucan recognition proteins (βGRPs), as a class of pattern recognition receptors, are involved in activation of the immune response in invertebrates. We cloned two cDNAs encoding putative βGRPs from larvae of Thitarodes pui, a host species of Ophiocordyceps sinensis with great economic importance in the Tibetan Plateau. The two putative βGRPs were phylogenetically classified into a novel clade 4, and designated TpβGRP-4a and TpβGRP-4b, respectively, with calculated molecular masses of 53,265 and 43,991 Da. Both TpβGRPs contained a C-terminal domain with sequence similarity to β-1,3-glucanases but without the glucanase active site. TpβGRP-4b markedly differed from other family members including TpβGRP-4a in the N-terminal region by a large deletion of ∼80 amino acid residues. Homology modelings revealed an eight-stranded β-sandwich fold (β1-β8) and two β-strands (only β1 and β2), respectively, in the N-terminal domains of TpβGRP-4a and -4b. TpβGRPs showed similar developmental expression patterns in fat body. TpβGRP-4a and -4b transcripts were induced highest 313- and 16-fold, respectively, in resistant 8th instar larvae challenged with conidia of entomopathogenic fungus Beauveria bassiana. By contrast, significant reductions in TpβGRPs expression were observed in conidia-injected susceptible 6th instar larvae (compared with saline-injected controls), accompanied by production of hyphal bodies in hemolymph. These results suggest that TpβGRPs might contribute to host defense against fungal infection, and TpβGRP-4b with the unusual deletion of the N-terminal region might have evolved new functions for βGRP family proteins.

Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300-750mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch.

Lead exposure improves the tolerance of Spodoptera litura (Lepidoptera: Noctuidae) to cypermethrin

Many ecological factors such as heavy metals can affect the tolerance of herbivorous insects to chemical insecticide. Spodoptera litura larvae exposed to lead (Pb) (0-100 mg kg(-1) in artificial diet) did not inhibit their growth. After 96 h of Pb (0-100 mg kg(-1)) exposure, topical application and feeding of cypermethrin to S. litura decreased their mortality and increased weight gain. Moreover, the mortality of S. litura treated with 25 and 50 mg kg(-1) of Pb for five generations was significantly lower than control. In addition, Pb accumulation was detected in midgut, fat body, brain and hemolymph, and its highest level was in the midgut. Furthermore, there was a significant negative correlation between Pb accumulation in fat body and mortality after topical application of cypermethrin. After 96 h of Pb exposure, there was increase expression of detoxification enzymes (CYP9A39 and CYP6B47) in midgut and fat body of S. litura. Therefore, the tolerance of S. litura to cypermethrin is increased by Pb exposure at certain concentrations through Pb accumulation in body and the increase of CYP9A39 and CYP6B47 expression.

QUANTIFYING PREDATION BY UMMELIATA INSEC‐TICEPS BOES. ET STR. (ARANEAE: LINYPHIIDAE) ON RICE PLANTHOPPERS USING ELISA*

Abstract  An enzyme‐linked immunosorbent assay (ELISA) has been developed to quantify predation by Ummeliata insecticeps Boes. et Str. on rice planthoppers in paddy fields in Dasha Township, Guangdong Province. The assay was completely specific for rice planthopper materials. The detection periods for antigens after an adult of U. insecticeps had fed on three 3–5th instar nymphs of white‐back planthopper (WBPH), Sogatella furcifera (Horvath), and brown planthopper (BPH), Nilaparvata lugens (Stal), at room temperature were 96 h and 120 h, respectively. The proportion of individual species of predators scoring positive ranged from 19. 05% to 47. 34% for WBPH, and from 9. 25% to 66. 67% for BPH in the early rice season. Although the numbers of the rice planthoppers consumed by U. insecticeps increased with increasing prey densities, the predation rates declined. When rice planthopper densities were low, the predation rates were relatively high. This kind of predation can explain why the rice planthoppers have never reached outbreak levels in rice fields in Dasha Township since 1973.

Characterization of a coelomic gregarine parasite from Thitarodes pui (Lepidoptera: Hepialidae) in the Tibetan Plateau.

Thitarodes pui, one of the host species of entomopathogenic fungus Ophiocordyceps sinensis, has great economic importance in the Tibetan Plateau. We report here, for the first time, a gregarine parasite found in the coelom of 7th instar and adults of T. pui. Gregarine gamonts (ovoid, ~15×8 μm) underwent syzygy to produce reproductive gametocysts in T. pui larval hemolymph. All infected T. pui carried 2-17 mature gametocysts filled with numerous oocysts (lemon-shaped, 17.17±0.73×6.49±0.4 μm). Transmission electron microscopy showed that these oocysts contained vacuoles of various sizes and amylopectin granules in the cytoplasm; scanning electron microscopy revealed a number of small bumps all over the surface of these oocysts. Small subunit ribosomal DNA sequence analysis showed a close relationship between the gregarine and the species of Ascogregarina (Eugregarinorida: Lecudinidae). Internal transcribed spacers and 5.8S ribosomal DNA from this gregarine exhibited 76% highest sequence identity with that from Ascogregarina culicis Ross.