Edible and Medicinal Progress of Cryptotympana atrata (Fabricius) in China.

As an important resource insect, the Cryptotympana atrata is widely distributed in the eastern and central parts of China. The cicada slough is one of the traditional crude drugs in East Asia, and the main component is polysaccharide, which has the functions of anti-convulsion, relieving asthma and improving lipid metabolism. The parasitoid fungus Cordyceps cicadae, which grows inside the cicada nymphs and forms the fruiting bodies on the surface of the host's carcass, is also known as the "cicada flower" in China. The Cordyceps cicadae is another old, traditional Chinese medicine, which has been used as a tonic and medicine to nourish and regulate human immunity for centuries. For the further development and utilization of the golden cicada, this paper summarized the C. atrata from the aspects of their biological characteristics, distribution area, life cycle, history of edible and medicinal use, edible methods and nutritional compositions; emphatically introduced the edible and potential medicinal value of the C. atrata; and specifically expounded the research progress of its application. As one popular insect food, the prospects for the development of C. atrata have also been put forward, especially in artificial breeding technology, food safety risk assessment and medicinal value utilization.

A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms.

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

Molecular characterization of N-methyl-d-aspartate receptor from Bemisia tabaci.

The N-methyl-d-aspartate receptors (NMDARs) are ionotropic ligand gated channels that are highly permeable to calcium ions. In insects, NMDARs are associated with glutamatergic neurotransmission governing diverse physiological and biological processes like vitellogenesis and ovarian development. Therefore, NMDAR may act as attractive target for insect pest control. In present study, we performed structural and functional characterization of NMDARs in Bemisia tabaci, a highly invasive crop pest and potent virus vector. We identified that NMDAR consists of three subunits each encoded by single gene in whiteflies which are highly conserved among different insect orders. Expression analysis suggests that subunit 1 (BtNR1) and subunit 2 (BtNR2) are the main functional units. External supplementation of NMDAR ligand or BtNRs silencing was lethal to insects, which suggested that NMDAR function is highly balanced in whiteflies.

Population structure and genetic differentiation of tea green leafhopper, Empoasca (Matsumurasca) onukii, in China based on microsatellite markers.

The tea green leafhopper, Empoasca (Matsumurasca) onukii Matsuda, is one of the dominant pests in major tea production regions of East Asia. Recent morphological studies have revealed variation in the male genitalic structures within and among populations. However, the genetic structure of this pest remains poorly understood. This study explores the genetic diversity and population structure of this pest in nineteen populations from the four main Chinese tea production areas using microsatellite markers, with one Japanese population also examined. The results show low to moderate levels of genetic differentiation with populations grouped into four clusters, i.e. the Jiangbei group, the Southwest group 1, the Southwest group 2 and the South China group. Populations from China have a close phylogenetic relationship but show significant isolation by distance. Lower genetic diversity and genetic differentiation of E. (M.) onukii were found in the Kagoshima population of Japan. Evidence for genetic bottlenecks was detected in the South China and Jiangnan populations. Population expansion was found in the Southwest, Jiangbei and Kagoshima populations. This is the most extensive study of the population genetics of this species and contributes to our understanding of its origin and evolutionary history.

Reference genes selection for quantitative gene expression studies in tea green leafhoppers, Empoasca onukii Matsuda.

Empoasca onukii Matsuda is one of the most devastating pests of the tea plant (Camellia sinensis). Still, the presumed expression stability of its reference genes (RGs) has not been analyzed. RGs are essential for accurate and reliable gene expression analysis, so this absence has hampered the study of the insect's molecular biology. To find candidate RGs for normalizing gene expression data, we cloned ten common housekeeping genes from E. onukii. Using the ΔCt method, geNorm, NormFinder and BestKeeper, we screened the RGs that were appropriate for quantifying the mRNA transcription of cellular responses under five experimental conditions. We identified the combinations of α-TUB and G6PDH, α-TUB and UBC, two RGs (α-TUB and ß-TUB1) or three RGs (α-TUB, RPL13 and GAPDH), AK and UBC, or RPL13 and α-TUB as the best for analyzing gene expression in E. onukii adults of both sexes in different tissues, nymphs at different developmental stages, nymphs exposed to different temperatures or nymphs exposed to photoperiod stress. Finally, the E. onukii cysteine proteinase (Eocyp) was chosen as the target gene to validate the rationality of the proposed RGs. In conclusion, our study suggests a series of RGs with which to study the gene expression profiles of E. onukii that have been manipulated (biotically or abiotically) using reverse transcription quantitative polymerase chain reaction. The results offer a solid foundation for further studies of the molecular biology of E. onukii.

Assessing the augmentation of Amblydromalus limonicus with the supplementation of pollen, thread, and substrates to combat greenhouse whitefly populations.

Due to issues with establishment and persistence of natural enemies in biological control, the provision of alternative food sources and oviposition sites are important factors to enhance pest control. In this study, three different supplementation treatments were examined for their ability to increase the populations of the predatory mite Amblydromalus limonicus, and its implications for greenhouse whitefly control on peppers and eggplants. These were: (1) pollen (Typha orientalis), (2) pollen and thread, (3) pollen, thread, and a substrate mixture of buckwheat, gorse, and rice husks, which were compared to a control treatment that had no supplementation. Significant treatment effects were found on pepper for A. limonicus (mite eggs p = 0.008, mobile mites p = <0.0001). The predatory mite successfully established and persisted at high population levels in the pollen-thread, and pollen-thread-substrate treatments. All supplementation treatments were able to control whitefly populations on peppers, while the control treatment failed to. The results obtained were formulated into possible application techniques for greenhouse growers to utilise.

Infection with phytopathogenic bacterium inhibits melatonin biosynthesis, decreases longevity of its vector, and suppresses the free radical-defense.

Vector-borne phytopathogenic bacteria may alter the reproductive fitness, survival, behavior, and metabolism of their vectors. Candidatus Liberibacter asiaticus (CLas) is associated with the Huanglongbing (also known as citrus greening disease), one of the most destructive citrus diseases worldwide, and transmitted by Asian citrus psyllid, Diaphorina citri (Insecta, Hemiptera, Liviidae). The genome sequencing of CLas revealed that it does not have the ability to synthesize tryptophan, the precursor of melatonin, and it must acquire it from its host plant or insect vector to achieve its biologic processes, such as growth and multiplication. Herein, we aimed to develop a GC-MS-SIM-based method to detect the endogenous melatonin from small insects such as D. citri, and to explore the hidden relationship between melatonin content and D. citri-adult survival. Then, we studied the ability of exogenous melatonin supplementation to reverse the negative effects of CLas-infection. Our findings showed that CLas-infection reduced the levels of melatonin and its biosynthetic genes (DcTPHs, DcAAAD, DcSNAT, and DcASMT) of D. citri compared to uninfected insects. In addition, CLas decreased the longevity of its vector, D. citri via the suppression of the free radical-defense associated genes (SODs, GSTs, PODs, and PHGPXs). On the other hand, melatonin supplementation could reverse the negative effects of CLas-infection. Melatonin supplementation enhanced the endogenous melatonin content, melatonin biosynthetic genes, free radical-defense associated genes, and the longevity of both healthy and CLas-infected D. citri. Furthermore, melatonin supplementation decreased the CLas bacterial population within the D. citri psyllids. Based on these findings, we hypothesize that melatonin plays multi-layered defensive roles in D. citri. These roles include acting as a natural antioxidant or as an antibacterial compound.

Expression pattern and pharmacological characterisation of two novel alternative splice variants of the glutamate-gated chloride channel in the small brown planthopper Laodelphax striatellus.

BACKGROUND: Glutamate-gated chloride channels (GluCl) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Although only one GluCl gene was presented in insects, it showed diverse alternative splicing that was speculated could affect channel function and pharmacology. RESULTS: In this study, we isolated GluCl cDNAs from adults of the small brown planthopper (SBPH) Laodelphax striatellus and showed that six L. striatellus GluCl variants (LsGluCl-AS, LsGluCl-BS, LsGluCl-CS, LsGluCl-AL, LsGluCl-BL, LsGluCl-CL) were present in the SBPH. The expression patterns of six variants differed among developmental stages (egg, first- to fifth-instar nymphs, male and female adults) and among the body parts (head, thorax, abdomen, leg) of the female adult SBPH. All the transcripts were abundant in the head of the adult. When expressed in African clawed frog, Xenopus laevis, oocytes, the two functional variants (LsGluCl-AS, LsGluCl-AL) had similar EC50 and IC50 values for L-glutamate and channel blockers picrotoxinin and fipronil. CONCLUSION: This study represents a comprehensive molecular, expression and pharmacological characterisation of GluCl in the SBPH. These findings should be useful in providing more opportunities to discover novel insect control chemicals. © 2016 Society of Chemical Industry.

Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci.

BACKGROUND: The whitefly Bemisia tabaci is an important agricultural pest with global distribution. This phloem-sap feeder harbors a primary symbiont, "Candidatus Portiera aleyrodidarum", which compensates for the deficient nutritional composition of its food sources, and a variety of secondary symbionts. Interestingly, all of these secondary symbionts are found in co-localization with the primary symbiont within the same bacteriocytes, which should favor the evolution of strong interactions between symbionts. RESULTS: In this paper, we analyzed the genome sequences of the primary symbiont Portiera and of the secondary symbiont Hamiltonella in the B. tabaci Mediterranean (MED) species in order to gain insight into the metabolic role of each symbiont in the biology of their host. The genome sequences of the uncultured symbionts Portiera and Hamiltonella were obtained from one single bacteriocyte of MED B. tabaci. As already reported, the genome of Portiera is highly reduced (357 kb), but has kept a number of genes encoding most essential amino-acids and carotenoids. On the other hand, Portiera lacks almost all the genes involved in the synthesis of vitamins and cofactors. Moreover, some pathways are incomplete, notably those involved in the synthesis of some essential amino-acids. Interestingly, the genome of Hamiltonella revealed that this secondary symbiont can not only provide vitamins and cofactors, but also complete the missing steps of some of the pathways of Portiera. In addition, some critical amino-acid biosynthetic genes are missing in the two symbiotic genomes, but analysis of whitefly transcriptome suggests that the missing steps may be performed by the whitefly itself or its microbiota. CONCLUSIONS: These data suggest that Portiera and Hamiltonella are not only complementary but could also be mutually dependent to provide a full complement of nutrients to their host. Altogether, these results illustrate how functional redundancies can lead to gene losses in the genomes of the different symbiotic partners, reinforcing their inter-dependency.

Identification of polymorphisms in Cyrtorhinus lividipennis RDL subunit contributing to fipronil sensitivity.

As one of the most important predatory enemies, the miridbug, Cyrtorhinus lividipennis, plays an important role in rice planthoppers control, such as Nilaparvata lugens (brown planthopper). In order to compare insecticide selectivity between C. lividipennis and N. lugens, the contact acute toxicities of six insecticides (diazoxon, paraoxon, carbaryl, fenobucarb, fipronil and ethofenprox) were monitored. The results showed that all tested insecticides were more toxic to C. lividipennis than to N. lugens and fipronil had the biggest difference. The RDL subunit (Cl-RDL) was cloned from C. lividipennis and a RDL isoform (Cl-RDL-In) was also found with 31 amino acids insertion in RDL intracellular region. In order to understand the role of the insertion on insecticide sensitivities, three subunits (Nl-RDL, Cl-RDL and Cl-RDL-In) were constructed to obtain the functional receptors in Xenopus oocytes and the fipronil sensitivities were detected by the voltage-clamp technique. Nl-RDL (IC50=32.36 ± 4.07 µM) was more insensitive to fipronil than Cl-RDL (IC50=6.47 ± 1.12 µM). The insertion in Cl-RDL significantly reduced fipronil sensitivity with IC50 value in Cl-RDL-In of 16.83 ± 2.30 µM. Interestingly, after the elution of fipronil, the current response of Cl-RDL-In appeared obvious recovery, which were not observed in Cl-RDL and Nl-RDL. It might imply that the insertion played a special role in fipronil sensitivity.

Molecular cloning and characterization of a ryanodine receptor gene in brown planthopper (BPH), Nilaparvata lugens (Stål).

BACKGROUND: Ryanodine receptors (RyRs) are a distinct class of intracellular calcium (Ca(2+)) release channel. The recent discovery of diamide insecticides has prompted studies on insect RyRs. However, information about the structure and function of insect RyRs is still limited. In this study, we isolated and characterized a full-length RyR cDNA (named NlRyR) from the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae), a serious rice pest throughout Asia. RESULTS: The composite NlRyR gene contains an open reading frame of 15 423 bp encoding a protein of 5140 amino acid residues, which shares high sequence identity (78-81%) with other insect homologues, except for two regions (IDR1: 4379-4732; IDR2: 1307-1529) with markedly low identity (44-48 and 38-41%, respectively). All hallmarks of the RyR proteins are conserved in the NlRyR protein, including the RyR domain as well as mannosyltransferase, IP3 R and RyR (pfam02815) (MIR) and RyR and IP3 R homology (pfam01365) (RIH) domains. Expression analysis of NlRyR revealed significant differences in mRNA expression levels among N. lugens developmental stages. Furthermore, three alternative splicing sites were identified in NlRyR, one of which forms the mutually exclusive exons A/B and is conserved in various insect species. Diagnostic PCR assays showed that the splice variant containing exon A was predominantly detected in all developmental stages. CONCLUSION: NlRyR may play an important role in the control of developmental processes of N. lugens. Alternative splicing may generate the functional diversity of NlRyR. The results provided the basis for further structural and functional characterization of NlRyR.

Transporters involved in glucose and water absorption in the Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) anterior midgut.

Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut. Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects.

Some biological features of cotton whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) on various host plants.

Development and reproduction of the cotton whitefly, Bemisia tabaci (Gennadius) were studied on aubergine, tomato and potato under laboratory conditions (30 degrees C and 55% RH). Total life cycle from egg to adult was 14.9, 20.0 and 14.2 days on aubergine, tomato and potato, respectively. Immature mortality were 12.9, 18.1 and 12.3% at the same three host plants. Females of B. tabaci oviposited means of 51.8, 60.1 and 67.5 eggs on aubergine, tomato and potato, respectively and had a mean longevity of 8, 14 and 12.9 days on the same three host plants. The net reproductive rate was 18.12, 15.06 and 27.63 and the daily intrinsic rate of increase was 0.141, 0.092 and 0.165 on aubergine, tomato and potato, respectively.

Consequences of nitrogen and phosphorus limitation for the performance of two planthoppers with divergent life-history strategies.

Phytophagous insects have a much higher nitrogen and phosphorus content than their host plants, an elemental mismatch that places inherent constraints on meeting nutritional requirements. Although nitrogen limitation is well documented in insect herbivores, phosphorus limitation is poorly studied. Using factorial experiments in the laboratory and field, in which levels of soil nitrogen and phosphorus were manipulated, we studied the relative consequences of macronutrient limitation for two herbivores, namely the phloem-feeding planthoppers Prokelisia dolus and P. marginata. These planthoppers inhabit the salt marshes of North America where large stands of their Spartina host plant are found. Notably, these congeners differ in their dispersal abilities; P. marginata is dispersive whereas P. dolus is sedentary. Both nitrogen and phosphorus subsidies enhanced the nitrogen and phosphorus content of Spartina. When P. dolus and P. marginata were raised on plants with an enriched nitrogen signature, they exhibited greater survival, grew to a larger size, developed more rapidly, and achieved higher densities than on nitrogen-deficient plants. However, P. marginata experienced greater fitness penalties than P. dolus on nitrogen-deficient plants. Phosphorus limitation and associated fitness penalties were not as severe as nitrogen limitation for P. marginata, and were not detected in P. dolus. The tempered response of P. dolus to N- and P-deficient Spartina is probably due to its greater investment in feeding musculature and hence ability to compensate for nutrient deficiencies with increased ingestion. To cope with deteriorating plant quality, P. dolus employs compensatory feeding, whereas P. marginata disperses to higher quality Spartina. When its option of dispersal is eliminated and P. marginata is confined on nutrient-deficient plants, its performance is drastically reduced compared with P. dolus. This research highlights the importance of interfacing herbivore life-history strategies with ecological stoichiometry in order to interpret the consequences of macronutrient limitation on herbivore performance and population dynamics.

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests.

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be approximately 12.1%+/-0.351 (mean +/- SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P < 0.01), 32% (P < 0.05) and 40.5, 29.5% (P < 0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

Insecticidal spider venom toxin fused to snowdrop lectin is toxic to the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) and the rice brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The SFI1/GNA fusion protein, comprising of snowdrop lectin (Galanthus nivalis agglutinin, GNA) fused to an insecticidal spider venom neurotoxin (Segestria florentina toxin 1, SFI1) was tested for toxicity against the rice brown planthopper Nilaparvata lugens (Stål) and the peach-potato aphid Myzus persicae (Sulzer) by incorporation into artificial diets. Significant effects on the mortality of N. lugens were observed, with 100% of the insects fed on the SFI1/GNA fusion protein diet dead by day 7. The survival of the aphid M. persicae was also reduced when fed on the SFI1/GNA fusion protein. After 14 days, only 49% of the aphids that were fed on the fusion protein were still alive compared with approximately 90% of the aphids fed on the control diet or on diet containing GNA only. The SFI1/GNA fusion protein also slowed the development of M. persicae, and the reproductive capacity of the aphids fed on the SFI1/GNA fusion protein was severely reduced. The ability of GNA to act as a carrier protein, and deliver the SFI1 neurotoxin to the haemolymph of N. lugens, following oral ingestion, was investigated. The successful delivery of intact SFI1/GNA fusion protein to the haemolymph of these insects was shown by western blotting. Haemolymph taken from the insects that were fed on the fusion protein contained two GNA-immunoreactive proteins of molecular weights corresponding to GNA and to the SFI1/GNA fusion protein.

Molecular dynamics of detoxification and toxin-tolerance genes in brown planthopper (Nilaparvata lugens Stål., Homoptera: Delphacidae) feeding on resistant rice plants.

To investigate the molecular response of brown planthopper, Nilaparvata lugens (BPH) to BPH-resistant rice plants, we isolated cDNA fragments of the genes encoding for carboxylesterase (CAR), trypsin (TRY), cytochrome P450 monooxygenase (P450), NADH-quinone oxidoreductase (NQO), acetylcholinesterase (ACE), and Glutathione S-transferase (GST). Expression profiles of the genes were monitored on fourth instar nymphs feeding on rice varieties with different resistance levels. Northern blot hybridization showed that, compared with BPH reared on susceptible rice TN1, expression of the genes for P450 and CAR was apparently up-regulated and TRY mRNA decreased in BPH feeding on a highly resistant rice line B5 and a moderately resistant rice variety MH63, respectively. Two transcripts of GST increased in BPH feeding on B5; but in BPH feeding on MH63, this gene was inducible and its expression reached a maximum level at 24 h, and then decreased slightly. The expression of NQO gene was enhanced in BPH on B5 plants but showed a constant expression in BPH on MH63 plants. No difference in ACE gene expression among BPH on different rice plants was detected by the RT-PCR method. The results suggest these genes may play important roles in the defense response of BPH to resistant rice.

Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice.

Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage.

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant.

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.

A juvenile hormone-repressible transferrin-like protein from the bean bug, Riptortus clavatus: cDNA sequence analysis and protein identification during diapause and vitellogenesis.

We found several juvenile hormone-responsive cDNAs in the bean bug, Riptortus clavatus, by using mRNA differential display (Hirai et al., 1998). One of them, a juvenile hormone-repressible cDNA, JR-3, was cloned, sequenced, characterized and identified as a transferrin (RcTf). RcTf cDNA encoded 652 amino acids with a calculated molecular weight of 71,453 Da. The deduced amino acid sequence showed significant homology with the transferin genes of several insects, Manduca sexta (43% identity), Blaberus discoidalis (43%), Aedes aegypti (43%), Drosophila melanogaster (36%), Sarcophaga peregrina (36%) and the human (25%). Antiserum was prepared by using recombinant RcTf protein expressed in Escherichia coli as an antigen. The antiserum reacted specifically with both the recombinant protein and the native protein from the bugs, with sizes of 70 and 75 kDa, respectively. The 75 kDa protein was partially purified from hemolymph of diapausing female bugs and the first ten amino acids were found to be identical to that of RcTf cDNA, indicating that the 75 kDa protein is RcTf. The tissue distribution of RcTf in the bug was examined by Western blot analysis. In diapausing animals, RcTf was detected in the fat body, hemolymph and ovary but not in the gut. In the post-diapause stage, RcTf was also detected in eggs, in addition to the fat body and ovary. These results indicate that RcTf is incorporated into the oocytes during vitellogenesis, and suggest that it may provide iron for the developing embryos.