Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae).

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.     

Circadian variation affects the biology and digestive profiles of a nocturnal insect Spodoptera litura (Insecta: Lepidoptera)

The present study was conducted to decipher the impact of circadian rhythm on digestive enzymes of Spodoptera litura under three photoperiods (12L:12D, 0L:24D, and 24L:0D). Longer life cycle, higher developmental traits and significant food utilizing capability were observed in dark conditions (DD), while there was no effect on survival. The activity of lactate dehydrogenase (LDH), α and β-glucosidase depended on complete absence of light (DD) while LL had a significant effect on protease activity. The presence of polypeptides (35, 60 kDa) and lower protease inhibition by PMSF in 0L:24D, and 24L:0D indicated that serine proteases (trypsin) were the main proteases in larval midgut. Overall, zymography profiles suggested that circadian variation, particularly dark period influenced the S. litura development due to fluctuations in the midgut enzymes via food utilization. Although the effect of photoperiod on digestive processes of insects is still unclear, dark regime may underlie the midgut digestive enzymes in S. litura larvae.     

北黄海沉积物可培养产蛋白酶细菌分离鉴定

【目的】揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源。【方法】分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性。【结果】从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas(69.9%)、Sulfitobacter(12.1%)和Salegentibacter(10.6%)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性。【结论】北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶。     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l ‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l ‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.     

The multiple functions of plant serine protease inhibitors: Defense against herbivores and beyond

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development.Key words: plant protease inhibitors, plant defense, Solanum nigrum, neo-functionalization     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Bacillus thuringiensis proteases: Production and role in growth,sporulation and synergism

Bacillus thuringiensis (Bt) subspecies produces metalloproteases and serine alkaline proteases (endogenous) which affect sporulation and entomotoxicity against different insect orders. The production of Bt proteases is investigated in conventional medium and alternative substrates with future repercussions on Bt formulations and larval mortality. Relationship between protease activity and total cell count during Bt fermentation has been discussed while protease activity as a potential indicator of entomotoxicity has also been explored. In general, the proteases influence entomotoxicity in two divergent ways—processing of inactive protoxins to active toxin fractions (by endogenous Bt as well as exogenous larval midgut proteases) and degradation of protoxins to fragments which sometimes lack insecticidal activity (usually by Bt proteases). In fact, the function of endogenous (intra and extracellular) proteases is ambiguous and has been raising serious questions on their role in larval mortality. The review explores various schools of thoughts (traditional as well as advanced) to solve the enigma of protease interactions with crystal toxins at different levels (sporulation and insecticidal action).     

Gene expression and activity of digestive proteases in <Emphasis Type="Italic">Daphnia</Emphasis>: effects of cyanobacterial protease inhibitors

Background  

The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia 's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels.     

Characterization and expression analysis of a trypsin-like serine protease from planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

Trypsin-like serine proteases are involved in large number of processes, especially in digestive degradation and immune responses. Here, we identify the characterization of a trypsin-like serine protease in planarian, Djtry, which interestingly has the incompletely conserved catalytic triad (K, D, and S). Phylogenetic analysis suggests that Djtry is an ancient type of trypsin-like serine proteases. The spatial and temporal expression patterns of Djtry are shown during regenerating and embryonic development by whole-mount in situ hybridization. Djtry is found to display a tissue specific expression pattern, with a predominant expression detected in whole gut region of intact and regenerating planarian. While the tissue- and stage-specific expression patterns during the embryonic development imply the roles of Djtry involve in yolk degradation and gut formation. Quantitative real-time PCR was carried out to analyze the function of this protease in vivo after planarians were stimulated to a bacterial challenge and food. The results showed that Djtry increased after a bacterial challenge and was basically stable for food. Therefore, the trypsin-like serine protease might be involved in the innate defense reactions against bacterial infection.     

The digestive system of human lice: current advances and potential applications

Abstract. Haematophagy has evolved independently in different insect groups. This has resulted in the development of different kinds of adaptations (e.g. suitable mouthparts as well as alterations in the gut anatomy and in digestive enzyme composition). At the same time, insects have acquired different levels of adaptation to blood‐sucking habits. One of the most interesting examples is the co‐evolution of sucking lice (Anoplura) with their hosts. Human head and body lice, Pediculus humanus capitis De Geer, 1767 and Pediculus humanus humanus Linnaeus, 1758, are highly host‐ and anatomic‐region‐specific ectoparasites. They spend their entire lives on the host, ingesting small blood meals, several times per day. To date, several genes encoding digestive protease and proteolytic activities have been identified. The blood is stored in the anterior distensible region of the midgut, where genes encoding serine endopeptidases are highly expressed. Likewise, in the narrow posterior region of the midgut, the high‐level expression of genes encoding exopeptidases has been documented. Symbiotic microorganisms inhabiting the mycetome complete the deficient nutrition of human lice. This review focuses on the digestion habits of the best‐investigated taxon of human lice P. h. humanus in relation to other blood‐sucking insects.     

Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet,Brassica species,and protease inhibitor

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one‐dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease‐encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin‐like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin‐like gene McSP34. The expression of the trypsin‐like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources. Published 2010 Wiley Periodicals, Inc.     

绿盲蝽丝氨酸蛋白酶基因AlSP4的克隆及取食不同寄主植物后的表达谱分析

胰蛋白酶类和胰凝乳蛋白酶类丝氨酸蛋白酶是盲蝽科昆虫消化系统内重要的消化酶。为了更好地了解丝氨酸类蛋白酶在绿盲蝽Apolygus lucorum消化系统中的作用, 本研究首次克隆了绿盲蝽丝氨酸蛋白酶基因AlSP4 (GenBank登录号为JQ609682)。序列分析结果表明, 该基因开放阅读框长999 bp, 编码332个氨基酸, 预测分子量为36.84 kDa, 理论等电点为5.35, N末端疏水区包含有16个氨基酸组成的信号肽。蛋白特征分析表明, 该基因翻译后的蛋白质具有丝氨酸蛋白酶的典型特征, 即氨基酸序列中具有组氨酸(His)、 天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件; 该基因翻译后还具有明显的胰蛋白酶前体的特征, 即此基因具有信号肽、 激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。利用荧光定量PCR技术对绿盲蝽雌、 雄成虫取食不同寄主植物后AlSP4的表达谱进行分析, 结果表明: 相对于其他寄主植物, 雌成虫取食Bt棉后AlSP4的表达量最高, 并显著高于取食常规棉后的表达量(P＜0.01)。雄成虫取食茼蒿后AlSP4的表达量最高; 雄成虫取食Bt棉后, AlSP4的表达水平仅次于取食茼蒿后的表达量, 也显著高于取食常规棉后的表达量(P＜0.01)。由此可见, AlSP4是绿盲蝽取食Bt棉后的重要消化酶基因, 对绿盲蝽适应Bt棉取食具有重要作用。     

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP intracellular serine protease - ISP-A-Bsu A-50 and ISP-B-Bsu A-50 molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively - SDS sodium dodecyl sulfate - PMSF phenylmethyl sulfonylfluoride - pNA p-nitroanilide - Buffer A 50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl₂ adjusted to pH 8.5 with HCl     

Effect of jasmonic acid and salicylic acid induced resistance in groundnut on Helicoverpa armigera

Induced resistance in plants affects insect growth and development as a result of the up‐regulation of defence‐related secondary metabolites or enzyme‐binding proteins. In the present study, the effects of jasmonic acid (JA) and salicylic acid (SA) induced resistance in groundnut on Helicoverpa armigera (Hübner) are examined. Larval survival, larval weights and the activities of digestive enzymes (total serine protease and trypsin) and of detoxifying enzymes [glutathione S‐transferase (GST) and esterase (EST)] are studied in insects fed on four groundnut genotypes with moderate levels of resistance to H. armigera (ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697) and a susceptible genotype (JL 24). The plants are pre‐ and/or simultaneously treated with JA and SA, and then infested with H. armigera, which are allowed to feed for 6 days. Significantly lower serine protease and trypsin activities are observed in H. armigera fed on plants treated with JA. Greater GST activity is recorded in insects fed on JA and SA treated plants, whereas EST activity is low in H. armigera larvae fed on plants treated with JA and SA. Serine proteases, trypsin and GST activities and larval weights (r = 0.74–0.95) and larval survival (r = 0.77–0.93) are positively correlated, whereas EST activity and larval weight (r = ?0.55) and larval survival (r = ?0.65) are negatively correlated. The results suggest that midgut digestive and detoxifying enzymes can be used as indicators of the adverse effects of constitutive and/or induced resistance in crop plants on the insect pests and the role of JA and SA in insect pest management.     

Identification and characterization of midgut proteases in <Emphasis Type="Italic">Achaea janata</Emphasis> and their implications

Insect midgut proteases are excellent targets for insecticidal agents such as Bacillus thuringiensis Cry toxins and protease inhibitors. The midgut proteases of Achaea janata have been characterized and Casein zymograms indicated at least five distinct activities corresponding to approx 17, 20, 29 and 80, and 90 kDa. Using a combination of synthetic substrates and specific inhibitors in casein zymograms, photometric assays and activity blots, three trypsin-like and one elastase-like serine proteases were identified but no chymotrypsin-like activity. Various proteinase inhibitors displayed differential inhibitory effects towards the midgut proteases.     

Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say)

Quantitative and qualitative changes in digestive proteolytic activities were monitored in fourth-instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say) subjected to three different leaf diets. Depending on the diet, the larvae exhibited variable growth rates, similar for potato (Solanum tuberosum) and eggplant (Solanum melongena) diets but lower for the tomato (Lycopersicon esculentum) diet. Interestingly, these growth rates were not associated with total protease activity in the midgut. While growth of tomato-fed insects was negligible, midgut protease activity in these insects was 1.5 and 4.2 times higher than that measured for potato- and eggplant-fed insects, respectively. As seen on gelatin-containing polyacrylamide gels, midgut extracts from insects that ingested eggplant leaves contained only a few proteinase forms, while numerous forms were observed in extracts of potato- and tomato-fed larvae. Although several forms were common to the three diets, their relative importance in the insect midgut varied. This diet-related plasticity of the digestive proteolytic system in Colorado potato beetle larvae leads one to question the potential for control approaches based on the inhibition of digestive proteases. Arch. Insect Biochem. Physiol. 36:241–250, 1997. © 1997 Wiley-Liss, Inc.     

Proteases and nucleases involved in the biphasic digestion process of the brown marmorated stink bug,Halyomorpha halys (Hemiptera: Pentatomidae)

Management of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), an invasive, agricultural pest in the United States, has presented significant challenges. This polyphagous insect uses both extra‐oral and gut‐based digestion thwarting protein‐ or nucleotide‐based control strategies. The objective of this study was to biochemically characterize the digestive enzymes (proteases and nucleases) from the saliva, salivary gland and the gut of H. halys. Enzyme profiles for the two tissues and saliva radically differ: The pH optimum for proteases in the gut was six, with cysteine proteases predominant. In contrast, the alkaline pH optima for protease activity in the salivary gland (8–10) and saliva (7) reflected abundant serine protease and cathepsin activities. RNase enzymes were most abundant in saliva, while dsRNase and DNase activities were higher in the salivary gland and saliva compared to those in the gut. These very different enzyme profiles highlight the biphasic digestive system used by this invasive species for efficient processing of plant nutrients. Knowledge of H. halys digestive physiology will allow for counteractive measures targeting digestive enzymes or for appropriate protection of protein‐ or nucleotide‐based management options targeting this pest.     

A serine protease in the midgut of the silkworm,Bombyx mori: Protein sequencing,identification of cDNA,demonstration of its synthesis as zymogen form and activation during midgut remodeling

We identified a serine protease with a molecular mass of 37 kDa in the midgut of the silkworm, Bombyx mori. The activity of this protease (37-kDa protease: p37k) appears after pupation, when the metamorphic remodeling of the midgut is under progress. The sequence analysis of the purified protease and its cDNA revealed that p37k is a trypsin-type serine protease, which is highly similar to serine proteases of other insects, including CG4386 of Drosophila melanogaster. In our molecular phylogenetic analysis, these proteases are grouped together with CG4386-like serine proteases of other insects to form an isolated cluster. The p37k protein and its putative orthologs present in this cluster have two unique sequence motifs, CxxCxC and FIDWLxxLLG, in the N-terminal side of the catalytic region. The gene for p37k is expressed in the midgut on day 2 of the silk-spinning larva, and the p37k polypeptide becomes detectable with a specific antibody at this stage of the midgut. On the other hand, p37k activity is not detectable until pupation, indicating that p37k is present in the larval midgut as an inactive precursor, which then is activated after pupation. A recombinant p37k produced using a baculovirus system is also inactive in its intact form. However, the recombinant p37k can be converted to an active protease when incubated in the homogenate of the midgut, suggesting that some unidentified midgut factor(s) are involved in the activation of p37k.