Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion

The mannose-specific snowdrop lectin (Galanthus nivalis agglutinin: GNA), when fed to insects, binds to the gut epithelium and passes into the haemolymph. The ability of GNA to act as a carrier protein to deliver an insecticidal spider venom neurotoxin (Segestria florentina toxin 1: SFI1) to the haemolymph of lepidopteran larvae was investigated. Constructs encoding SFI1 and an SFI1/GNA fusion protein were expressed in Pichia pastoris. The insecticidal activity of purified recombinant proteins on injection was found to be comparable to published values for SfI1 purified from spider venom [Toxicon 40 (2002) 125]. Whereas neither GNA nor SFI1 alone showed acute toxicity when fed to larvae of tomato moth (Lacanobia oleracea), feeding SFI1/GNA fusion at 2.5% of dietary proteins was insecticidal to first stadium larvae, causing 100% mortality after 6 days. The protein also showed a significant, dose dependent, toxicity towards fourth and fifth stadium larvae, with growth reduced by up to approximately 90% over a 4-day assay period compared to controls. Delivery of intact SFI1/GNA to the haemolymph in these insects was shown by western blotting; haemolymph samples from fusion-fed larvae contained a GNA-immunoreactive protein of the same molecular weight as the SFI1/GNA fusion. SFI1/GNA and similar fusion proteins offer a novel and effective approach for delivering haemolymph active toxins by oral administration, which could be used in crop protection by expression in transgenic plants.     

Cloning, expression and functional characterisation of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase

Chitinases are vital to moulting in insects, and may also affect gut physiology through their involvement in peritrophic membrane turnover. A cDNA encoding chitinase was cloned from larvae of tomato moth (Lacanobia oleracea), a Lepidopteran pest of crops. The predicted protein contains 553 amino acid residues, with a signal peptide of 20 a.a. Sequence comparison showed 75-80% identity with other Lepidopteran chitinases. L. oleracea chitinase was produced as a functional recombinant enzyme in the yeast Pichia pastoris. A fusion protein containing chitinase joined to the N-terminus of snowdrop lectin (GNA) was also produced, to determine whether GNA could deliver chitinase to the haemolymph of Lepidopteran larvae after oral ingestion. The purified recombinant proteins exhibited similar levels of chitinase activity in vitro. Both proteins were highly toxic to L. oleracea larvae on injection, causing 100% mortality at low dose (2.5 microg/g insect). Injection of chitinase prior to the moult resulted in decreased cuticle thickness. The recombinant proteins caused chronic effects when fed, causing reductions in larval growth and food consumption by up to 60%. The oral toxicity of chitinase was not increased by attaching GNA in the fusion protein, due to degradation in the larval gut, preventing GNA acting as a "carrier".     

The snowdrop lectin Galanthus nivalis agglutinin (GNA) and a fusion protein ButaIT/GNA have a differential affect on a pest noctuid Lacanobia oleracea and the ectoparasitoid Eulophus pennicornis

Fusion proteins have considerable potential as novel insect control agents because they enable the oral delivery of insecticidal peptides to the haemolymph of pests. Transport is achieved via fusion of the toxin to a carrier protein Galanthus nivalis agglutinin (GNA) that, after ingestion, binds to and crosses the insect gut epithelia. A fusion protein comprising a toxin from the South Indian red scorpion (Mesobuthus tamulus) that is fused to a GNA polypeptide (ButaIT/GNA) has a detrimental effect on the development of tomato moth Lacanobia oleracea (L.) (Lepidoptera: Noctuidae) larvae. The present study examines the effects of ButaIT/GNA and GNA, delivered orally or by injection, on the development of L. oleracea larvae, and the subsequent effects on the gregarious ectoparasitoid Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae) developing on ButaIT/GNA‐ and GNA‐treated hosts. The fusion protein, but not GNA, reduces the growth of fifth stadium L. oleracea larvae. The development of E. pennicornis is not affected by the presence of ButaIT/GNA in hosts that ingest the protein, although it is affected when hosts are injected with the protein. This difference is considered to be a result of higher levels of fusion protein being present when the fusion protein is injected. Intact ButaIT/GNA is detected by immunoassay in the haemolymph of L. oleracea larvae after ingestion of the fusion protein. More unexpectedly, negative effects are observed for the growth of E. pennicornis larvae developing on hosts that have either ingested, or been injected with GNA.     

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects

Background  

Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake.     

In vivo effects of Manduca sexta allatostatin and allatotropin on larvae of the tomato moth, Lacanobia oleracea

Abstract. Injection of Manduca sexta allatotropin (Manse-AT) into fifth or sixth stadium larval Lacanobia oleracea had no significant effect on larval growth, development or food consumption, compared to control injected insects. In contrast, injection of M. sexta allatostatin (Manse-AS) into fifth stadium larvae resulted in a retardation of growth, reduction in feeding and increased mortality, compared to control injected insects, but had no effect on non-feeding (day 7) sixth instar larvae. Results suggest that Manse-AS is not acting on the corpora allata (CA) to inhibit Juvenile Hormone (JH) synthesis to produce the observed effects, but most likely by its myoinhibiting action on the foregut. Inhibition of foregut peristalsis by Manse-AS in vivo appears to suppress feeding, resulting in increased mortality. Foregut peristalsis may be inhibited by the intact peptide or a deletion peptide produced by cleavage of Manse-AS by haemolymph enzymes, because Manse-AS (5-15) also inhibits muscle contractions in the foregut in vitro .     

In vitro and in vivo binding of snowdrop (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action

When fed in semi-artificial diet the lectins from snowdrop (Galanthus nivalis: GNA: mannose-specific) and jackbean (Canavalia ensiformis: Con A: specific for glucose and mannose) were shown to accumulate in vivo in the guts, malpighian tubules and haemolymph of Lacanobia oleracea (tomato moth) larvae. Con A, but not GNA, also accumulated in the fat bodies of lectin-fed larvae. The presence of glycoproteins which bind to both lectins in vitro was confirmed using labelled lectins to probe blots of polypeptides extracted from larval tissues. Immunolocalisation studies revealed a similar pattern of GNA and Con A binding along the digestive tract with binding concentrated in midgut sections. Binding of lectins to microvilli appeared to lead to transport of the proteins into cells of the gut and malpighian tubules. These results suggested that both lectins are able to exert systemic effects via transport from the gut contents to the haemolymph across the gut epithelium. The delivery of GNA and Con A to the haemolymph was shown to be dependent on their functional integrity by feeding larvae diets containing denatured lectins. Con A, but not GNA, was shown to persist in gut and fat body tissue of lectin-fed larvae chased with control diet for three days. Con A also shows more extensive binding to larval tissues in vitro than GNA, and these two factors are suggested to contribute to the higher levels of toxicity shown by Con A, relative to GNA, in previous long term bioassays.     

Insect resistance of transgenic tobacco expressing an insect chitinase gene

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control     

Freeze–thawing method: a bleeding method from lepidopteran larvae utilizing a spontaneous insect body contraction after a freezing-thawing treatment

Insect haemolymph contains growth promotor(s) for cultured insect cells and is frequently used as an additive to the culture media. Insect haemolymph serves as a pool of a protein product produced by a virus vector–insect host system. Haemolymph collection is an essential step in the above process, which should limit the scale and cost of their performance. In the present study, a simple procedure for bleeding from lepidopteran larvae, Bombyx mori , has been developed which utilized a spontaneous contraction of the insect body after a freezing–thawing treatment. In the case of fifth-instar B. mori , 60 to 80% of the total haemolymph was collected by this method. The authors applied the method to a haemolymph collection from frozen larvae stored at −80°C for longer than 1 month. Preservability of the frozen larvae enabled the development of a system dealing with a huge bulk of insects. The bleeding method was effective under cooled condition at 0°C or 4°C, which was desired for protein handling. Development of a large system would result in a cost reduction for the insect haemolymph products such as insect cell-culture additive. Furthermore, the above bleeding method was applied to the nuclear polyhedrosis virus-infected B. mori larvae and up to 80% of the total haemolymph was collected from the virus-infected larvae. It suggests the bleeding method as an effective means of haemolymph collection in the protein productive system using a virus vector and its insect host.     

Snowdrop lectin (GNA) has no acute toxic effects on a beneficial insect predator,the 2-spot ladybird (Adalia bipunctata L.)

Two-spot ladybird (Adalia bipunctata L.) larvae were fed on aphids (Myzus persicae (Sulz.)) which had been loaded with snowdrop lectin (Galanthus nivalis agglutinin; GNA) by feeding on artificial diet containing the protein. Treatment with GNA significantly decreased the growth of aphids. No acute toxicity of GNA-containing aphids towards the ladybird larvae was observed, although there were small effects on development. When fed a fixed number of aphids, larvae exposed to GNA spent longer in the 4th instar, taking 6 extra days to reach pupation; however, retardation of development was not observed in ladybird larvae fed equal weights of aphids. Ladybird larvae fed GNA-containing aphids were found to be 8-15% smaller than controls, but ate a significantly greater number of aphids (approx. 40% to pupation). GNA was shown to be present on the microvilli of the midgut brush border membrane and within gut epithelial cells in ladybird larvae fed on GNA-dosed aphids, although disruption of the brush border was not observed. It is hypothesised that GNA does not have significant direct toxic or adverse effects on developing ladybird larvae, but that the effects observed may be due to the fact that the aphids fed on GNA are compromised and are thus a suboptimal food.     

A comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (Lacanobia oleracea)

When fed in semi-artificial diet in short- and long-term bioassays, the lectins from snowdrop (Galanthus nivalis; GNA) and jackbean (Canavalia ensiformis; Con A) affected the activities of soluble and brush border membrane (BBM) enzymes in the midgut of Lacanobia oleracea larvae. In the short term both lectins increased gut protein levels and BBM aminopeptidase activity. The lectins also increased trypsin activity, both in the gut (Con A) and in the faeces (GNA). GNA also increased the activity of alpha-glucosidase, but neither lectin had a significant effect on alkaline phosphatase activity. Trypsin mRNA levels were similar in lectin-fed and control larvae in the short term, showing that there is no direct effect on expression of the encoding genes. Larvae chronically exposed to GNA and Con A showed reductions in weight of 50-60%, and exhibited a significant reduction in alpha-glucosidase activity, but little change in other enzyme activities. Con A bound to many BBM and peritrophic matrix (PM) proteins in vitro, whereas GNA showed more specific binding, with strongest binding to a 94kDa uncharacterised BBM protein. Both lectins accumulated in gut tissues of insects after chronic exposure in vivo, but Con A was present at higher levels than GNA.     

Direct effects of snowdrop lectin (GNA) on larvae of three aphid predators and fate of GNA after ingestion

Plants genetically modified to express Galanthus nivalis agglutinin (GNA) have been found to confer partial resistance to homopteran pests. Laboratory experiments were conducted to investigate direct effects of GNA on larvae of three species of aphid predators that differ in their feeding and digestive physiology, i.e. Chrysoperla carnea, Adalia bipunctata and Coccinella septempunctata. Longevity of all three predator species was directly affected by GNA, when they were fed a sucrose solution containing 1% GNA. However, a difference in sensitivity towards GNA was observed when comparing the first and last larval stage of the three species. In vitro studies revealed that gut enzymes from none of the three species were able to break down GNA. In vivo feed-chase studies demonstrated accumulation of GNA in the larvae. After the larvae had been transferred to a diet devoid of GNA, the protein stayed present in the body of C. carnea, but decreased over time in both ladybirds. Binding studies showed that GNA binds to glycoproteins that can be found in the guts of larvae of all three predator species. Immunoassay by Western blotting of haemolymph samples only occasionally showed the presence of GNA. Fluorescence microscopy confirmed GNA accumulation in the midgut of C. carnea larvae. Implications of these findings for non-target risk assessment of GNA-transgenic crops are discussed.     

Transgenic potato plants with enhanced resistance to the tomato moth,Lacanobia oleracea: growth room trials

Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat -amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI). Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced. All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene. GNA and BCH were accumulated at levels up to 2.0% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing. Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes.Resistance of the transgenic plants to insect attack was assayed by exposing the plants to larvae of the tomato moth, Lacanobia oleracea. All the plants tested which were expressing GNA showed an enhanced level of resistance. Leaf damage was reduced by more than 50% compared to controls; total insect biomass per plant was reduced by 45-65%, but larval survival was only slightly reduced (20%). These results support the hypothesis that GNA has a significant antifeedant effect on insects. Expression of BCH had no protective effect against this insect. Expression of CpTI in transgenic potatoes had similar effects to expression of GNA on total insect biomass and survival, but did not afford protection against insect damage to the plant.     

Does host‐feeding on GNA‐intoxicated aphids by Aphelinus abdominalis affect their longevity and/or fecundity?

Transgenic potatoes have been transformed with a gene coding the snowdrop lectin Galanthus nivalis agglutinin (GNA) and they have been shown to be partially resistant to aphids. GNA binds to insect gut cells, including those of aphids, consequently inducing disruption of nutrient assimilation. Aphid parasitoids are important natural biocontrol agents of aphids and some species such as Aphelinus abdominalis are commercially available. Aphid parasitoids are endoparasitoids during their larval stages and free-living insects as adults. They could be directly or indirectly affected by GNA during both these stages. In this work, we present data on the potential direct and indirect effects of GNA on adult A. abdominalis. Aphelinus abdominalis is a synovigenic species (eggs are matured throughout the adult life) which needs a diet relatively rich in proteins and amino-acids to produce anhydropic eggs (large, yolk-rich eggs that do not expand in the host during embryonic stages). Adult A. abdominalis females feed on aphid haemolymph and they may be directly exposed to the entomotoxin or indirectly affected by a change in aphid haemolymph quality due to intoxication by GNA. We conducted a first tier experiment to investigate this potential risk. A. abdominalis females were offered either aphids reared on control diet or aphids reared on GNA 0.1% diet as hosts (i.e., as food and oviposition sites). No trace of GNA was found in females fed with GNA-aphids but no GNA could be detected in the haemolymph of aphids fed a 0.1% GNA diet. Longevity and fecundity were not affected suggesting that the quality of the haemolymph necessary for A. abdominalis egg maturation and production was not significantly altered.     

Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees

Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a ‘carrier’ protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a ‘carrier’, is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD₅₀ > 100 µg bee⁻¹) or injection of fusion protein. Bees fed acute (100 µg bee⁻¹) or chronic (0.35 mg ml⁻¹) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.     

Phylloplane bacteria increase the negative impact of food limitation on insect fitness

1. When populations of herbivorous insects increase in density, they can alter the quantity or quality of their food. The impacts of diet‐related stressors on insect fitness have been investigated singly, but not simultaneously. 2. Foliage quantity and quality of red alder, Alnus rubra, were manipulated together with the presence of non‐entomopathogenic phylloplane bacteria to investigate their impacts, singly and in combination, on survival, pupal mass, growth rate, fecundity and egg quality of a cyclic forest insect, the western tent caterpillar, Malacosoma californicum pluviale. 3. Food limitation (half food) had strong negative impacts on all life‐history traits. When the larvae were fed continuously, however, neither ingesting phylloplane bacteria nor eating leaves from damaged branches (induced foliage) affected survival. In the half‐food treatment, ingesting bacteria further increased mortality, while feeding on induced foliage improved survival. 4. Growth rate and pupal mass of both sexes were reduced for larvae with food limitation compared with continuously fed insects and this was exacerbated when the larvae also ate bacteria‐treated leaves. A combination of bacteria and induced foliage also reduced larval growth rate by 5% in the full‐food treatment. 5. Fecundity (eggs per egg mass) was 2.7 times greater in full‐food than in food‐limited treatments but neither phylloplane bacteria nor plant induction had an effect. Insects fed induced foliage produced smaller eggs. Overall, there was no evidence of a three‐way interaction between the three stressors, although there were negative synergistic effects, primarily between food limitation and the ingestion of phylloplane bacteria.     

Effects of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on the development of tomato moth (Lacanobia oleracea) larvae in laboratory and glasshouse trials

The effects of snowdrop lectin (Galanthus nivalis agglutinin, GNA) on Lacanobia oleracea larval growth, development, consumption, and survival, were examined by 3 distinct bioassay methods. Larvae were reared on artificial diet containing GNA at 2% (w/w) dietary protein; on excised leaves of transgenic potato expressing GNA at approx. 0.07% of total soluble proteins; and on transgenic potato plants expressing GNA at approx. 0.6% of total soluble proteins in glasshouse trials. Significant effects on larval growth were observed with all three treatments. At 21days after hatch mean larval biomass was reduced by 32 and 23%, in the artificial diet and excised leaf bioassays respectively. In glasshouse trials a 48% reduction in insect biomass per plant was observed after 35days. The artificial diet and excised leaf assays also showed that GNA significantly slowed larval development as assessed by instar duration. GNA caused a 59% overall reduction in mean daily consumption in the artificial diet assay, and a significant reduction in leaf damage in glasshouse trials. However, prolonged compensatory feeding by larvae in the excised leaf assay resulted in their consuming 15% more total leaf material than the control group. Adaptation to low levels of GNA, in terms of biomass recovery and compensatory feeding, was observed within one larval generation in the detached leaf assay. No significant effects of GNA on larval survival were observed in the artificial diet and detached leaf bioassays, whereas survival was decreased by approx. 40% in the glasshouse bioassay. The assays show that the insecticidal effects of GNA can be observed both in vitro when fed in artificial diet and in planta, and can be demonstrated in the glasshouse as well as under growth cabinet conditions.     

Exoproteinases of the type A in pathogenesis of insect bacterial diseases

Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects.     

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R₀) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R₀ plants (designated R₁) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.     

TAT-Cloan-DH-EGFP融合蛋白的原核表达及其在舞毒蛾幼虫体内的跨膜转导

【目的】为了探究滞育激素经昆虫取食的生物效应, 需设计一种能够加速跨膜运输的融合蛋白, 克服经昆虫消化道的降解作用。【方法】本研究以分月扇舟蛾Clostera anastomosis滞育激素基因Cloan-DH和TAT穿膜肽编码序列为基础, 构建TAT-Cloan-DH融合基因; 然后以增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）为标签, 构建在pET-22b载体中。在0.2 mmol/L IPTG诱导下, 在大肠杆菌Escherichia coli BL21 Rosetta(DE3)中表达出TAT-Cloan-DH-EGFP融合蛋白。 再分别将表达有TAT-Cloan-DH-EGFP和EGFP融合蛋白的大肠杆菌BL21菌体拌入人工饲料,经舞毒蛾Lymantria dispar幼虫持续取食0, 8, 24, 48, 72和90 h后, 随机挑选试虫进行组织固定并制作石蜡切片, 选取中肠所在体段切片进行组织荧光分析。【结果】37℃ 0.2 mmol/L IPTG诱导条件下, 表达的TAT-Cloan-DH-EGFP融合蛋白的分子量为61 kD, 主要以包涵体形式表达, 融合蛋白约占细胞总蛋白含量的18.1%; 随着试虫取食含TAT-Cloan-DH-EGFP融合蛋白饲料的增加, 虫体组织的绿色荧光强度逐渐增加, 取食72 h后, 组织荧光强度达到最高。【结论】实验结果说明融合蛋白TAT-Cloan-DH-EGFP可以通过消化道横向跨膜运输, 到达虫体其他组织, 且该过程与蛋白的摄入量成比例。     

Parasitism enhances the induction of glucogenesis by the insect, Manduca sexta L

Metabolic alterations that accompany parasitism of invertebrate animals can play an important role in parasite development. Employing 13C NMR, this study examined pyruvate cycling from (2-(13)C)pyruvate in the lepidopteran insect Manduca sexta, and the effects of parasitism by the hymenopteran Cotesia congregata on the gluconeogenic formation of trehalose, the haemolymph or blood sugar of insects. Larvae were maintained on a semi-synthetic sucrose-free diet, or on the same diet with sucrose at 8.5 g/l. Pyruvate cycling was evident from the 13C enrichment in C3 of alanine, derived following carboxylation to oxaloacetate, and was similar in parasitized and normal insects regardless of diet. Trehalose was formed following de novo synthesis of glucose, and net synthesis was estimated from the 13C distribution in trehalose and alanine. The 13C-enrichment ratio [2trehalose C6/alanine C3] is an indicator of the level of gluconeogenesis relative to glycolysis, both enrichments were derived from (2-(13)C)pyruvate in the same manner. The ratio was greater than unity in all insects, regardless of diet, but was significantly greater in parasitized larvae, demonstrating an enhanced level of gluconeogenesis. This was confirmed by analysis of the 13C distribution in trehalose and glutamine derived from (3-(13)C)alanine. Despite enhanced de novo trehalose formation in parasitized insects, the haemolymph sugar level was similar to that of normal larvae. Because haemolymph trehalose regulates dietary carbohydrate intake, but not gluconeogenesis, the results suggest that accelerated induction of gluconeogenesis is an adaptive response to parasitism that provides increased carbohydrate for parasite growth and simultaneously maintains nutrient intake.