Biochemical characterization of digestive proteases and carbohydrases of the carob moth,Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

Alkaline proteases in the midgut tissue and digestive fluid of the silkworm,Bombyx mori

In larvae of Bombyx mori proteolytic activity was found in the midgut tissue and digestive fluid. The pH-activity curves of both proteases were very similar and optimal activity was about pH 11.2. The reduction in activity at 50°C, for 10 min was about 80% in digestive fluid protease, and about 40% in tissue protease. HgCl₂ and DFP strongly inhibited protease activity, and the influence was greater in digestive fluid than in the midgut tissue. Most of the tissue proteases was found to be membrane bound enzyme from results of differential centrifugation and column experiments.     

Characterization and distribution of digestive proteases of the stalk corn borer,Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae)

Larval midgut extracts from the noctuid Sesamia nonagrioides Lef. were assayed for protease activity. Total proteolytic activity, as measured by azocasein hydrolysis, showed a pH optimum in the range 10.0 to 11.5, suggesting a digestive system based largely on serine-like proteases. The ability of midgut extracts to hydrolyze specific synthetic substrates, the elucidation of the pH at which maximal hydrolysis occurs, and their sensitivity to protease inhibitors confirmed the presence of the serine endoproteases: trypsin, chymotrypsin, and elastase; and the exopeptidases: carboxypeptidase A, carboxypeptidase B, and leucine aminopeptidase. The distribution of these digestive proteases along the gut sections and among the different midgut regions was examined. All types of endoproteases and exopeptidases were mainly located in the midgut, with less than 5% of the activity in the foregut and hindgut. When the two halves of the midgut were compared, all proteolytic activities were higher in the anterior portion of the midgut. Trypsin, chymotrypsin, elastase, and carboxypeptidase B activities were mainly located in the endoperitrophic space of the midgut, with some activity in the ectoperitrophic space, whereas aminopeptidase and carboxypeptidase A activities were preferentially located in the midgut epithelium. © 1996 Wiley-Liss, Inc.     

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF PROTEASES IN LARVAL PREPARATIONS OF THE CEREAL LEAF BEETLE (Oulema melanopus,CHRYSOMELIDAE, COLEOPTERA)

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole‐larval preparations, permitting studies of the very small whole‐larval preparations. Protein preparations were analyzed using FITC–casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0–4.0). Cysteine protease activity increased at acidic pH and in the presence of β‐mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe²⁺, Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ (10 mM). Fe²⁺ and Zn²⁺ significantly decreased enzyme activity at all pH values, Ca²⁺ and Mg²⁺ at pH 6.2 and Mg²⁺ at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, N_α‐tosyl‐l‐lysine chloromethyl ketone hydrochloride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone, trans‐epoxysuccinyl‐l‐leucylamido‐(4‐guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin‐like) proteases and metalloproteases act in cereal leaf beetle digestion.     

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.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.     

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory

Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l ‐BApNA and l ‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l ‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.     

12a-羟基鱼藤酮对斜纹夜蛾生殖力的影响及其作用机理

为探讨12a-羟基鱼藤酮对斜纹夜蛾Prodenia litura (Fabricius)生殖力的影响及其作用机理, 本文测定了该虫取食12a-羟基鱼藤酮后的产卵量。在用高效液相色谱仪确定其卵巢组织中存在12a-羟基鱼藤酮后, 通过MTT法测定该化合物对斜纹夜蛾卵巢细胞（PL细胞）的细胞毒性,并利用流式细胞仪检测该化合物对PL细胞细胞周期、膜电位、胞内钙离子浓度、线粒体膜电位的影响。结果表明:斜纹夜蛾幼虫取食12a-羟基鱼藤酮后产卵量下降80％以上, 并从卵巢组织中检测出该化合物。12a-羟基鱼藤酮对PL细胞增殖具有抑制活性, 其IC₅₀为6.6 mg/L。该化合物将PL细胞周期阻滞于S期, 导致PL细胞膜电位、线粒体膜电位和细胞内游离钙离子浓度均显著升高。该化合物导致斜纹夜蛾繁殖力降低。12a-羟基鱼藤酮将PL细胞增殖阻滞于细胞周期的S期。同时, 该化合物对卵巢细胞具有较弱的毒杀活性, 导致一些卵巢细胞死亡。由于上述原因, 卵巢细胞数量逐渐减少而导致卵巢管萎缩。卵巢管的萎缩使卵巢管内的卵母细胞不能发育成卵细胞, 故其生殖力下降。     

Midgut cysteine protease-inhibiting activity in Trichoplusia ni protects the peritrophic membrane from degradation by plant cysteine proteases

The action of plant cysteine proteases on the midgut peritrophic membrane (PM) of a polyphagous herbivorous lepidopteran, Trichoplusia ni, was studied. Proteins in PMs isolated from T. ni larvae were confirmed to be highly resistant to the serine proteinases trypsin and chymotrypsin, but were susceptible to degradation by plant cysteine proteases, which is consistent with the known molecular and biochemical characteristics of the T. ni PM proteins. However, the PM proteins were not degraded by plant cysteine proteases in larvae or in the presence of larval midgut fluid in vitro. With further biochemical analysis, cysteine protease-inhibiting activity was identified in the midgut fluid of T. ni larvae. The cysteine protease-inhibiting activity was heat resistant and active in the tested pH range from 6.0 to 10.0, but could be suppressed by thiol reducing reagents or reduced by treatment with catalase. In addition to T. ni, cysteine protease-inhibiting activity was also identified from two other polyphagous Lepidoptera species, Helicoverpa zea and Heliothis virescens. In conclusion, results from this study uncovered that herbivorous insects may counteract the attack of plant cysteine proteases on the PM by inhibiting the potentially insecticidal cysteine proteases from plants in the digestive tract. However, the biochemical identity of the cysteine protease-inhibiting activity in midgut fluid has yet to be identified.     

Midgut proteases of the cardamom shoot and capsule borer Conogethes punctiferalis (Lepidoptera: Pyralidae) and their interaction with aprotinin

Protease inhibitors cause mortality in a range of insects, and transgenic plants expressing protease inhibitors have been protected against pest attack, particularly internal feeders that are not amenable to control by conventional means. A study of luminal proteases in Conogethes punctiferalis Guenée was performed to identify potential targets for proteinaceous biopesticides, such as protease inhibitors. The midgut protease profile of the gut lumen from C. punctiferalis was studied to determine the conditions for optimal protein hydrolysis. Optimum conditions for peptidase activity were found to be in 50 mm Tris-HCl, pH 10 containing 20 mm CaCl2; incubation for 30 min at 40 degrees C. Four synthetic substrates, i.e. benzoyl-arg-p-nitroanilide, benzoyl-tyr-p-nitroanilide, succinyl-ala-ala-pro-leu-p-nitroanilide (SAAPLpNA) and leu-p-nitroanilide were hydrolysed by C. punctiferalis gut proteases in Tris-HCl buffer pH 10. Trypsin and elastase-like chymotrypsin were the prominent digestive proteases, and age-related modulation of midgut proteases existed for trypsin, chymotrypsin, elastase-like chymotrypsin and leucine aminopeptidase. Serine protease inhibitors such as aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride inhibited peptidase activity. Some metal ions such as Ca(2+), Mg(2+), Pb(2+) and Co(2+) enhanced BApNA-ase activity whereas others like Mn(2+), Zn(2+), Cu(2+), Fe(2+) and Hg(2+) were inhibitory at 6 mm concentration. Trypsin and elastase-like chymotrypsin were significantly inhibited by 94% and 29%, respectively, by aprotinin (150 nm) under in vitro conditions. A possible incorporation of protease inhibitors into transgenic plants is discussed.     

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.     

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.     

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.     

Inhibition of Helicoverpa armigera gut pro‐proteinase activation in response to synthetic protease inhibitors

Protease inhibitors play an important role in host plant defence against herbivores. However, insects have the ability to elevate the production of proteinases or resort to production of a diverse array of proteinases to offset the effect of proteinase inhibitors. Therefore, we studied the inhibition of pro‐proteinase(s) activation in the midgut of the polyphagous pest Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in response to protease inhibitors to develop appropriate strategies for the control of this pest. Gelatin coating present on X‐ray film was used as a substrate to detect electrophoretically separated pro‐proteinases and proteinases of H. armigera gut extract on native‐ and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Six activated pro‐proteinase bands were detected in H. armigera gut lumen, which were partially purified and characterized using substrate assays. Activated H. armigera midgut pro‐proteinase(s) showed activity maxima at pH 8 and 10, and exhibited optimal activity at 40 °C. The activation of H. armigera gut pro‐proteinase isoforms was observed in the fraction eluted on benzamidine‐sepharose 4B column. Purification and substrate assay studies revealed that 23–70 kDa polypeptides were likely the trypsin/chymotrypsin‐like pro‐proteinases. Larvae of H. armigera fed on a cocktail of synthetic inhibitors (antipain, aprotinin, leupeptin, and pefabloc) showed maximum activation of pro‐proteinases compared with the larvae fed on individual inhibitors. The implications of these results for developing plants expressing proteinase inhibitors for conferring resistance to H. armigera are discussed.     

Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae)

The tarnished plant bug, Lygus hesperus Knight, is a pest that causes considerable economic losses to vegetables, cotton, canola, and alfalfa. Detailed knowledge of its digestive physiology will provide new opportunities for a sustainable pest management approach to control this insect. Little is known about the different protease class contributions to the overall digestion of a specific protein. To this end, the proteolytic activities in female adult L. hesperus salivary gland and midgut homogenates were quantified over a range of pH's and time points, and the contribution of different classes of proteases to the degradation of FITC-casein was determined. In the salivary gland, serine proteases were the predominant class responsible for caseinolytic activity, with the rate of activity increasing with increasing pH. In contrast, both aspartic and serine proteases contributed to caseinolytic activity in the midgut. Aspartic protease activity predominated at pH 5.0 and occurred immediately after incubation, whereas serine protease activity predominated at pH 7.5 after a 9h delay and was resistant to aprotinin. The salivary serine proteases were distinctly different from midgut serine proteases, based on the tissue-specific differential susceptibility to aprotinin and differing pH optima. Collectively, the caseinolytic activities complement one another, expanding the location and pH range over which digestion can occur.     

Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.     

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 and comparison of digestive proteinases of the Cortez swimming crab, Callinectes bellicosus, and the arched swimming crab, Callinectes arcuatus

Abstract. Protease activity in the midgut gland, gastric chamber, and gastric juice from the crabs Callinectes bellicosus and Callinectes arcuatus was characterized by several methods, confirming that the composition of digestive proteases is the same in the gastric juice and the midgut gland. Gastric juice was suitable for the identification and characterization of the proteinases trypsin and chymotrypsin. Such enzymes were presented as isotrypsins and isochymotrypsins. Proteinase composition evaluated by SDS-PAGE and substrate-SDS-PAGE showed differences between species, but not between gender. Proteinases were thermostable at 40°–50°C for 1 h and showed maximum activity at pH 6–8, making the use of digestive proteinases for evaluations of protein digestibility by the pHstat method possible. We propose using gastric juice as a source of digestive enzymes for in vitro studies of enzymes in digestibility assays and characterization procedures.