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Release of Proteases from Larvae of the Brine Shrimp Artemia franciscana and Their Potential Role During the Molting Process
Comp. Biochem. Physiol. Vol. 119B, No. 2, pp. 255–263, 1998 Copyright 1998 Elsevier Science Inc. All rights reserved.
ISSN 0305-0491/98/$19.00 PII S0305-0491(97)00323-4
Release of Proteases from Larvae of the Brine Shrimp Artemia franciscana and Their Potential Role During the Molting Process A. H. Warner and C. Matheson Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada ABSTRACT. In arthropods, molting is required in order for the organism to escape periodically from its exoskeleton (cuticle) to undergo metamorphosis, growth, or reproduction. During molting the cuticle is first released from the epidermis (apolysis) then degraded (ecdysis). This involves a complex set of hydrolases secreted into the exuviate under control of ecdysteroids and several environmental factors. During the first two larval molts in the brine shrimp, Artemia franciscana, large amounts of two serine proteases appear in the incubation medium, concomitant with N-acetyl-β-D-glucosaminidase, a well-described chitin degrading enzyme. These proteases were analyzed by gel filtration, fast protein liquid chromatography (FPLC), electrophoresis on gelatin-polyacrylamide gels, and with various inhibitors, and found to be nearly identical to two serine proteases synthesized during early larval development in Artemia and thought to be digestive enzymes. The appearance or secretion of these serine proteases into the incubation medium during larval development is required for apolysis and ecdysis, and appears to be dependent on cysteine protease activity in the epidermal layer under the cuticle. comp biochem physiol 119B;2:255–263, 1998. 1998 Elsevier Science Inc. KEY WORDS. Artemia, brine shrimp, cysteine proteases, development, molting, serine proteases
INTRODUCTION The process of molting has been the focus of numerous studies on arthropods dating back to the early sixteenth century. Major advances in our understanding of the cellular, biochemical and physiological basis of molting can be found in several excellent reviews (3,8,22,24). In arthropods, molting requires the interaction of a complex set of biochemical and cellular events in order for organisms to escape periodically from the exoskeleton (cuticle) to undergo metamorphosis, growth, or reproduction. During molting, the cuticle, which is composed mainly of chitin and proteins, undergoes cyclical synthesis and degradation under control of ecdysteroids, environmental factors, such as temperature, photoperiod, food supply, and other factors, such as molt-inducing and molt-inhibiting hormones (3,8,24). Among crustaceans molting in larvae and adults appears to be a general process consisting of at least four phases of which the premolt and ecdysis or shedding of the cuticle are the dominant events (7,22). Early in premolt apolysis occurs in which the chitin/proteinaceous cuticle Address reprint requests to: A. H. Warner, Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada. Tel. 519-253-4232, Ext. 2728; Fax 519-971-3609; E-mail: warner1@ucc. uwindsor.ca Received 30 July 1997; revised 22 September 1997; accepted 7 October 1997.
becomes detached from the underlying epidermis. Apolysis is believed to require the secretion of proteases and/or chitinolytic enzymes by epidermal cells (8,19). At the end of the premolt, ecdysis occurs and the cuticle is degraded further by hydrolases secreted into the exuvial fluid. It is not known whether a different complement of hydrolases are released into the exuviate to accomplish both apolysis and ecdysis or whether one set of hydrolases serves both processes. While the degradative pathway of the molt cycle is functioning, the pathways active in cuticle synthesis are activated in the epidermis; the resulting proteins and chitin are secreted to form a new multilayered cuticle (2). In the brine shrimp, Artemia franciscana, molting occurs 16–19 times throughout its life cycle, depending on the nutritional state of its environment and other factors (1,8,32). The first molt is an embryonic event during which time hatching of the embryo occurs from the embryonic cuticle, a chitinoprotein structure of c ⋅ 2 µm thick (4,16). This event is associated with enhanced chitinolytic activity (chitinase and N-acetyl-β-D-glucosaminidase) in both the prenauplius larvae and exuvial fluid, which then appears in the incubation medium (9,10). The next several molts in Artemia are larval molts, with the first and second molts occurring at about 18 and 28 hr, respectively after the embryonic molt (hatching) (6,7). The larval cuticle of Artemia is ,1 µm thick and appears to be less complex than either the embryonic or adult cuticle in Artemia (7).
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From research on several arthropods, including Artemia, considerable evidence has accumulated in support of a role for different classes of proteases in the molting process (5,15,18,19,21,26,30). As well, inhibitors of cysteine proteases have been shown to block apolysis or ecdysis in various organisms, preventing body segment formation and further growth of the organism (15,21,30). These experiments have implicated the cysteine proteases as key hydrolases in the molting process. However, molting is a complex process and the cohort of hydrolases required to sequentially release the cuticle from the epidermis and/or degrade it have not been established in any model system. In the study reported here, we have identified two serine proteases that are either released from or secreted by first and second instar larvae into the incubation medium during the first two larval molts in Artemia. These enzymes appear to be similar to proteases isolated previously from larvae of Artemia and identified as proteases A and B (20). Data are also presented to support the hypothesis that cysteine protease activity may be required for the activation and/or release of serine proteases into the incubation medium during molting in larvae of Artemia.
Culturing of Artemia Larvae Hydrated cysts were incubated in Millipore-filtered (0.45 µm) seawater fortified with penicillin (50 U/ml), streptomycin (50 µg/ml), and amphotericin (0.125 µg/ml) with constant shaking under a white light at 24°–25°C. Nauplius larvae, which hatched between 15 and 18 hr incubation, were collected and adjusted to a concentration of 1000 larvae/ml of incubation medium (seawater). These newly hatched larvae (first instar, or 0-hr larvae) in flasks containing 50,000 each were maintained at 24°–25°C with gentle shaking until needed. In some experiments, Z-PheAla-CH 2F was added at a concentration of 50 or 100 µM to flasks containing larvae at the beginning of incubation. At 6–8 hr intervals larvae were collected on miracloth supported on a Whatman 3 filter disk. The larvae were quick frozen and the filtered incubation medium was concentrated to about 2 ml by ultrafiltration on a YM-10 membrane. Aliquots were taken for protein, protease, and N-acetyl-glucosaminidase (NAGase) assays as described below and the remainder of the sample was concentrated to about 0.5 ml using a Centricon-10 concentrator. All samples prepared in this manner were stored at 215°C for further analyses. Preparation of Aqueous Cytoplasm of Artemia Larvae
MATERIALS AND METHODS Materials Encysted embryos (cysts) of Artemia franciscana (lot #12715) were obtained from Sanders, (Ogden, UT) and stored at 220°C. When needed, cysts were hydrated in 50% seawater for at least 5 hr at 0–4°C, during which time they were dechorionated with antiformin for 30 min as described previously (27). Antibiotics-antimycotics used in the culture of Artemia embryos and larvae were from GibcoBRL (Burlington, ON). Diaflo ultrafiltration membranes (YM10) and Centricon concentrators were purchased from Amicon (Oakville, ON). Chromatographic columns and media, such as MonoQ HR 5/5 and Sephadex G-150 (SF), and protein standards are obtained from Pharmacia (Baie d’Urfe, PQ). Protease inhibitors including pepstatin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), and soybean trypsin inhibitor (STI), p-nitrophenol, p-nitrophenyl-β-Dacetylglucosaminide, electrophoresis grade gelatin, and trinitrobenzene sulfonic acid (TNBS) were from Sigma Chemical Co. (St Louis, MO). Ovomucoid trypsin inhibitor (OTI) was from P-L Biochemicals (Milwaukee, WI) and benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-PheAla-CH2F) was a gift from Dr. D. Rasnick of Prototek Corp. (Dublin, CA). Protein assays were carried out using the bicinchoninic acid (BCA) reagent and protocol supplied by Pierce (Brockville, ON). Acrylamide, sodium dodecylsulfate (SDS), and other electrophoretic supplies were from BioRad (Mississauga, ON). All other chemicals were of ACS grade or better and purchased from either Sigma or Fisher Scientific (Nepean, ON).
Frozen larvae were homogenized directly in 5 vol of a buffer containing 150 mM sorbitol, 70 mM potassium gluconate, 5 mM monobasic potassium phosphate, and 3 mM Hepes, adjusted to pH 7.2. The homogenate was centrifuged at 1630 g to sediment the nuclei and yolk platelets, then at 15,000 g for 30 min (4°C) to obtain the aqueous cytoplasm as described previously (31). The 1630 g pellet was re-extracted with homogenization buffer and treated as described above. The aqueous cytoplasms were combined and passed through a Sephadex G-25 column to remove low molecular weight components, then concentrated to approximately 1 ml using a Centricon 3 filter. All concentrated samples were stored at 215°C for subsequent analyses. Enzyme Assays Aliquots of concentrated incubation medium, aqueous cytoplasm of larvae, or column fractions were assayed for protease activity using the trinitrobenzene sulfonic acid (TNBS) reagent described previously (28). The protease substrates were either protamine sulfate or gelatin at 2.5–4.0 mg/ml. Assays for the serine-type ‘‘neutral’’ proteases were carried out at pH 7.4 and 37°C, whereas assays for cysteine protease activity were carried out at pH 4 and 40°C. One milliunit (mEU) of ‘‘neutral’’ protease activity is defined as the release of 1 nmole per minute of amino peptide from the substrate (protamine sulfate or gelatin) at pH 7.4 and 37°C. Assays for N-acetyl-glucosaminidase (NAGase) activity were carried out as described previously using p-nitrophenyl-β-D-acetylglucosaminide as substrate in 0.1 M sodium
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acetate, pH 5.0 at 37°C (13). Aliquots of a 200 µl reaction mix were taken at 15-min intervals, diluted to 1 ml with 0.2 M Na 2CO 3 and the absorbance was read at 400 nm. The amount of p-nitrophenol released from the substrate was measured against a standard curve of p-nitrophenol. One milliunit of NAGase activity released 1 nmole/min of p-nitrophenol from the substrate. The effect of various inhibitors on protease or N-acetylglucosaminidase activity was determined by including the inhibitor in the reaction vessel at the desired concentrations. Control assays contained equivalent amounts of the solvents (dimethylsulfoxide or ethanol) used in the preparation of the inhibitor stock solutions. Chromatography of the Incubation Medium and Aqueous Cytoplasm of Larvae Concentrated proteins from the incubation medium or aqueous cytoplasm, equivalent to 35,000–40,000 Artemia larvae, were applied to a Sephadex G-150 (SF) column (75 3 1 cm) equilibrated with a buffer containing 15 mM potassium phosphate, 25 mM KCL, and 10% glycerol, pH 7.2. The proteins were eluted from the column with the equilibration buffer and the effluent monitored for protein by measuring the absorbance of each fraction at 280 nm. Aliquots of each column fraction were also assayed for protease activity at pH 7.4 using the TNBS reagent (28). The concentrated incubation medium and aqueous cytoplasm of larvae were also fractionated by fast protein liquid chromatography (FPLC) on a MonoQ column (HR 5/5) using a Beckman Altex solvent delivery system. Approximately 1000 mEU of neutral protease activity from the concentrated incubation medium or aqueous cytoplasm of larvae were applied to the column equilibrated with the buffer used in gel filtration. Following the sample application and a 2-min wash with the starting buffer, the proteins were eluted from the column using a gradient of KCl to 0.75 M over 30 min at a flow rate of 1.0 ml/min. The column eluate was monitored at 280 nm and fractions were collected for protease assays at pH 7.4 and 4.0. Electrophoresis on Gelatin-SDS-Polyacrylamide Gels Serine proteases in the incubation medium and larval preparations were analyzed on 12% polyacrylamide gels containing 0.1% SDS and 0.2% gelatin using the Bio-Rad mini-protean apparatus as described previously (17). Approximately 1.5 mEU of serine protease activity from each fraction to be analyzed were mixed with standard SDSmercaptoethanol loading buffer, then applied to the gel without heating. The loading and running buffers were as described previously (14). Following electrophoresis at 125 V (2 hr) and 4°C, the gel was soaked in 2.5% Triton X100 for 30 min then incubated for 1.5 hr in 50 mM sodium phosphate, pH 7.4, to allow the serine proteases in the gel
FIG. 1. Release of neutral proteases and N-acetyl-glucosaminidase into the incubation medium of Artemia larvae. The incubation medium was collected at 6-hr intervals, concentrated and aliquots taken for protease assays at pH 7.4 (h, e, n) and N-acetyl-glucosaminidase (NAGase) activity (open bars). The solid line represents a computer-generated trend curve from the average of protease measurements from three experiments, while the bars represent the average of two NAGase experiments. M1 and M2 indicate the times of the first and second larval molt, respectively.
to hydrolyze the gelatin, identifying their migration position. The gel was then stained for 1 hr with a solution containing 2.5% Coomassie Brilliant Blue, 50% methanol, and 7.5% acetic acid, then destained overnight in a solution of 5% methanol and 7.5% acetic acid and photographed. RESULTS Appearance of Protease Activity in the Incubation Medium of Artemia Nauplii In a previous study we observed neutral protease activity in the incubation medium (seawater) of swimming Artemia larvae during the larval molt cycle (30). We have quantified this protease activity during the first 42 hr of larval development, and compared it to N-acetyl-glucosaminidase (a chitinolytic enzyme), which also appears in the incubation medium during larval molting. The results in Fig. 1 show that the largest increase in protease activity occurred between the first and second larval molts, reaching about 300 mEU of enzyme activity per 10,000 larvae. Beyond 40–42 hr incubation, no increase in protease activity was observed in the incubation medium, and in some cases the activity actually declined. The protease activity profile in the incubation medium followed closely the N-acetyl-glucosaminidase (NAGase) activity, a well-known indicator of molting.
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FIG. 2. Gel filtration of proteases in the incubation medium
and aqueous cytoplasm of third instar larvae of Artemia. Concentrated samples of cytoplasm (panel A) and incubation medium (panel B) from third instar larvae (40 hr posthatch) were filtered through a calibrated column of Sephadex G-150 as described under Materials and Methods. Column fractions were assayed for protease activity at pH 7.4 (e, absorbance at 420 nm) and protein content (■, absorbance at 280 nm). The numbers at the top of each panel indicate the elution position of standard proteins.
Analyses were not conducted on Artemia larvae beyond the third instar stage of development (40–45 hr after hatching) because larvae begin to die after the second larval molt (third instar stage) unless fed appropriately. Characterization of the Proteases in Incubation Medium of Second and Third Instar Larvae of Artemia To characterize the proteases released from developing larvae of Artemia, aliquots of the incubation medium were collected after the first and second larval molt, concentrated and analyzed by gel filtration, FPLC, and gel electrophoresis. The results in Fig. 2 (panel B) show that all protease activity in the incubation medium of 40 hr post-hatch Artemia larvae eluted from a Sephadex G-150 column as a single peak with a molecular mass of about 28.5 kDa. The elution position of this peak corresponded with a peak of ‘‘neutral’’ protease activity in the concentrated cytoplasm of 40 hr Artemia larvae (Fig. 2, panel A). A similar elution profile was seen when 24 and 30 hr post-hatch samples were analyzed (data not shown). The incubation medium did not contain any acidic protease activity, although the cytoplasm of larvae is known to have cysteine protease activity with an acidic pH optimum [see below; (31)].
A. H. Warner and C. Matheson
FIG. 3. Fast protein liquid chromatography (FPLC) of proteases in the aqueous cytoplasm and incubation medium of third instar larvae of Artemia. Concentrated samples of aqueous cytoplasm (panel A) and incubation medium (panel B) of third instar larvae were applied to a Mono Q column, then eluted with a linear gradient of KCl in the starting buffer. The solid line represents the absorbance at 280 nm of material eluting from the column. Column fractions were assayed for protease activity at pH 7.4 (■) and pH 4.0 (e) with the results shown as absorbance at 420 nm. CP represents the elution position of the major cysteine protease in larvae, whereas E-1, E-2, and E-3 represent the elution positions of the serine proteases. No cysteine protease activity was detected in the concentrated incubation medium (30); therefore, no CP assays were carried out on column fractions of the incubation medium. The dash line (—) represents the KCl gradient.
When the incubation medium containing second or third instar larvae (i.e., 30 or 42 hr samples) was applied to a Mono Q column, the protease activity was resolved into two fractions (E-1 and E-2) with elution characteristics identical with two ‘‘neutral’’ proteases in the cytoplasm of third instar (40 hr) larvae. These results are shown in Fig. 3 (panels A and B). Another neutral protease (E-3) in the cytoplasm of third instar larvae, which eluted from the Mono Q column at 0.7 M KCl, was barely detectable in the incubation medium even after 50-fold concentration of the medium. Cysteine protease (CP) activity was present in the cytoplasm of third instar larvae, but not in the incubation medium, although a small amount of the large subunit of the CP was detected by Western blotting in the incubation medium in an earlier study (30). Thus the main proteases present in the incubation medium after 36–42 hr incubation (i.e., E-1 and E-2) appear to be identical to two ‘‘neutral’’ proteases present in the cytoplasm of third instar lar-
Release of Proteases During Molting in Artemia
FIG. 4. Electrophoresis of the serine proteases in the incuba-
tion medium and aqueous cytoplasm from Artemia larvae on a gelatin-SDS-polyacrylamide gel. The E-1 and E-2 fractions from the Mono Q column (see Fig. 3) were concentrated and 1.5 mEU of each were applied to a 12% polyacrylamide gel containing 0.1% SDS and 0.2% gelatin. The clear areas show the migration positions of the proteases, which hydrolyzed gelatin in the gel at pH 7.4. M and L refer to incubation medium and larvae, respectively, as the source of the E-1 and E-2 proteases. The sizes (kDa) and migration positions of standard proteins are shown at the left, while the sizes (kDa) of the largest and smallest active proteases in the E-1 and E-2 fractions are shown at the right.
vae, and in similar proportion to the E-1 and E-2 proteases in the cytoplasm of second and third instar larvae. To further characterize the proteases in the incubation medium, the E-1 and E-2 fractions from the Mono Q column were subjected to electrophoresis on a gelatin-SDSpolyacrylamide gel, and their migration positions in the gel were compared with the proteases in the E-1 and E-2 fractions from the cytoplasm of second instar larvae. The results in Fig. 4 show that incubation of the gelatin gel in phosphate buffer, pH 7.4, following electrophoresis revealed several bands of protease activity in each of the E-1 and E-2 fractions from both the incubation medium and larvae. The E-2 fraction from the incubation medium contained eight bands of protease activity, ranging in molecular mass from 26.5 kDa to 15.5 kDa, and with electrophoretic mobilities identical to eight bands of protease activity in the E-2 fraction from larvae. In contrast, the protease composition of the E-1 fraction from the incubation medium and larvae was different. The E-1 fraction from the larvae and incubation medium both contained proteases of 26.5, 26, 24, and 23.5 kDa, whereas the proteases of 19.5, 19, 17, and 15.5 kDa were either absent or in reduced amounts in the E-1 fraction of third instar larvae. Otherwise, both the E-1 and E-2 fractions from the Mono Q column contained proteases of very similar sizes after electrophoresis in the gelatin gel, irrespec-
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tive of their origin (i.e., incubation medium or larvae). Electrophoresis of the E-1 and E-2 proteases on native gels (i.e., lacking SDS) did not yield distinct bands of protease activity, but only a smear about 2 cm in length near the top of the gel. (Note: since multiple forms of the E-1 and E-2 proteases were not found in earlier studies of these enzymes, the lower molecular weight forms of these enzymes may be the results of autolysis in the presence of SDS). Also, no protease activity was observed in gelatin gels incubated in sodium acetate buffer at pH 5 (data not shown). The unfractionated proteases in the incubation medium were also assayed between pH 5.5 and 8.5 in phosphate buffer, and found to have optimal activity at pH 7.5 (data not shown), which is similar to that reported previously for the major serine proteases in the aqueous cytoplasm of second and third instar larvae (20). The results of the chromatographic, electrophoretic, and pH measurements suggest that the proteases that appear in the incubation medium during the first two molt cycles of Artemia have physical characteristics similar to at least two of the ‘‘neutral’’ proteases in the cytoplasm of Artemia larvae. Specificity of the Neutral Proteases in the Incubation Medium and Cytoplasm of Young Larvae of Artemia to Inhibitors The proteases released into the incubation medium during the molt cycle of Artemia (i.e., fractions E-1 and E-2) were assayed in the presence of various well-known inhibitors of proteases, and their activities were compared to protease assays on the E-1 and E-2 fractions of second instar larvae of Artemia carried out under identical conditions. The results of these experiments are summarized in Table 1. The proteases in the E-1 fraction from the incubation medium and larvae were differentially sensitive to PMSF and TLCK, inhibitors of serine proteases. Specifically, the E-1 proteases in the incubation medium were more sensitive to PMSF and TLCK than the larval E-1 proteases. Differences in sensitivity to PMSF and TLCK may be due to the fact that the E1 fraction from the medium has a slightly different protease composition than the E-1 fraction from larvae (see Fig. 4). The different responses to these inhibitors could also be due to posttranslational modifications to the E-1 proteases before their secretion/release to the medium. The proteases in the E-2 fraction from the incubation medium and larvae represent the largest amount of neutral protease activity in each of these sources, comprising about 80% of the total neutral protease activity in each case. However, unlike the proteases in the E-1 fraction, proteases in the E-2 fraction are mostly resistant to PMSF and TLCK, but sensitive to STI, OTI and leupeptin, suggesting that the proteases in the E-2 fractions are also serine-type proteases, but different from the E-1 fraction proteases. None of the proteases in the incubation medium was af-
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TABLE 1. Effect of various inhibitors on incubation medium and larval neutral proteases from Artemia
Residual activity (%) E-1
E-2
Inhibitor
Concentration
Medium
Larvae
Medium
Larvae
STI OTI PMSF
10 µg/ml 10 µg/ml 50 µM 100 µM 100 µM 50 µM 50 µM 100 µM 100 µM 2 mM 0.1% 0.5% (60°C, 30 min)
86 100 72 28 62 86 — 100 98 106 64 0 0
66 100 100 66 96 113 — 100 87 94 95 0 0
12 23 — 95 93 119 92 108 20 90 91 48 0
20 37 — 85 96 139 103 99 31 102 99 18 0
TLCK Pepstatin Z-Phe-Ala-CH2F Leupeptin EDTA SDS* Heating*
*In these assays the substrate was gelatin; all other assays were carried out using protamine sulfate as substrate.
fected by inhibitors of cysteine, aspartic, and metalloproteases. Thus, Z-Phe-Ala-CH 2F, pepstatin, and EDTA, respectively, had no inhibitory effect on the proteases in the E-1 and E-2 fractions. However, Z-Phe-Ala-CH 2F (a synthetic inhibitor of cysteine protease) had a marked inhibitory effect on the release of E-1 and E-2 proteases into the medium (see below), although it did not inhibit the activity of the E-1 or E-2 proteases in the medium or larvae. Finally, it should be noted that the E-1 and E-2 proteases from both the larvae and incubation medium were generally resistant to treatment with 0.1% SDS, whereas 0.5% SDS in the protease assays completely inhibited the E-1 proteases and partially inhibited the E-2 proteases. These results support the conclusion that the proteases released into the incubation medium during development of first and second instar larvae of Artemia appear to be slightly modified forms of serine proteases originating in the cytoplasm of these larvae.
the first and second larval molt by SDS-polyacrylamide gel electrophoresis suggested that appearance of the proteases in the medium is not the results of larvae decomposing during the incubation (data not shown). Also, visual inspection of the culture flasks did not reveal any dead larvae during the sampling times in these experiments. Finally, it
The Developmental Profile of the Serine Proteases in Post-Hatch Larvae of Artemia Compared to Their Appearance in the Incubation Medium Developing embryos of the brine shrimp, Artemia, have very little, if any, active serine, aspartic, or metalloproteases (20,29). However, beginning about 8–10 hr after hatching of the nauplius larvae, there is considerable synthesis and/ or activation of at least three serine-type proteases (20). The appearance of serine proteases in the incubation medium follows by 8–10 hr their appearance in the larvae. These results are shown in Fig. 5. Near the end of the second molt cycle (24–30 hr after hatching), the specific activity of the proteases in the medium is 17–18 times higher than it is in the cytoplasm of larvae, suggesting that the appearance of the proteases in the medium during this period of development may be the results of a secretory process. Moreover, analysis of the proteins in the medium after
FIG. 5. Appearance of neutral protease activity in the aque-
ous cytoplasm and incubation medium of first instar larvae of Artemia at various times after hatching. Each point represents the average of two experiments. The numbers above several points represent the specific activity (mEU/ mg protein) of the proteases in the cytoplasm or incubation medium of Artemia larvae at the incubation times indicated.
Release of Proteases During Molting in Artemia
FIG. 6. Effect of Z-Phe-Ala-CH2F on release of neutral prote-
ases into the incubation medium of Artemia larvae. Z-PheAla-CH2F was added to flasks containing incubation medium and newly hatched larvae, and samples were collected at 8-hr intervals, beginning at 16 hr incubation, for neutral protease measurements. Untreated larvae (solid bars), 50 mM (stippled bars) or 100 mM (hatched bars) concentrations of the drug.
should be noted that during the first two larval molts, the serine proteases released/secreted into the incubation medium represent only 10–15% of the total serine protease content of the larvae. Effect of the Cysteine Protease Inhibitor Z-Phe-Ala-CH2F on Hydrolases Released into the Incubation Medium of Artemia Larvae In a previous study, we demonstrated that addition of ZPhe-Ala-CH 2F (a cysteine protease inhibitor) to the incubation medium containing newly hatched Artemia larvae
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inhibited apolysis, and subsequently ecdysis, during the first two larval molt cycles in Artemia (30). Therefore, we tested the effect of Z-Phe-Ala-CH 2F on the release of serine proteases and N-acetyl-glucosaminidase into the incubation medium of newly hatched Artemia larvae. The results in Fig. 6 and Table 2 show that addition of Z-Phe-Ala-CH 2F to the incubation medium had a marked inhibitory effect on the synthesis/activation of the serine proteases in the larvae, and their subsequent release or secretion into the incubation medium. The reduced level of the serine proteases (40–50% of controls) appearing in the incubation medium during the first two larval molts appears to reflect the marked reduction (23–25% of controls) in these enzymes in the aqueous cytoplasm of second and third instar larvae. In contrast to its effect on serine protease release to the incubation medium, Z-Phe-Ala-CH 2F had very little effect on the release of NAGase into the medium, although the level of NAGase in the larvae was reduced 23–32% by this drug (see Table 2). Since Z-Phe-Ala-CH 2F had no inhibitory effect (in vitro) on the serine proteases in the E-1 and E-2 fractions at 50–100 µM (see Table 1), these results suggest that the major cysteine proteases in larvae of Artemia may play a role in the processing and/or release of the serine proteases to the medium during the larval molt cycles. DISCUSSION Among the crustaceans, the brine shrimp Artemia has provided information of potential importance to our understanding of the molting process in embryos and larvae. In a previous study, three serine proteases were shown to increase markedly during development of first and second instar larvae, and since these enzymes were thought to be digestive enzymes, their role in development was not determined experimentally (20). The results of the present study suggest that at least two of these proteases play a role in the larval molting process. The serine proteases appearing in the incubation medium during molting of second and third instar larvae (E-1 and E-2) have chromatographic and electrophoretic properties identical with proteases A and B isolated from larvae of Artemia as described previously (20). Their release into the exuviate during the first two
TABLE 2. Effect of the cysteine protease inhibitor Z-Phe-Ala-CH2F on various hydrolase activity levels in the incubation me-
dium and larvae of Artemia franciscana Milliunits enzyme in larvae† Enzyme Serine proteases Serine proteases NAGase NAGase
Milliunits enzyme in incubation medium†
Treatment*
Second instar larvae
Third instar larvae
Second instar larvae
Third instar larvae
Control Z-Phe-Ala-CH2F Control Z-Phe-Ala-CH2F
2140 532 (25%)‡ 132 90 (68%)
2443 567 (23%) 125 95 (77%)
45.8 18.5 (40%) 11.3 10.4 (92%)
283.1 141.5 (50%) 37.0 35.4 (96%)
*Flasks containing newly hatched larvae were given Z-Phe-Ala-CH2F at 100 µM final concentration compared to control flasks lacking this drug. †The milliunits of enzymes are based on 10,000 larvae and the average of two samples at each larval stage shown. ‡The numbers in parentheses represent the activity remaining in Z-Phe-Ala-CH2F treated samples compared to controls.
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larval molts also corresponds closely with the release of the chitinolytic enzyme N-acetyl-glucosaminidase into the incubation medium. Therefore, it appears reasonable to conclude that two of the serine proteases, which first appear in first instar larvae, are among the cohort of hydrolases required to accomplish apolysis and ecdysis in larvae of Artemia. The results of this study also suggest that a different cohort of proteases function in larval molting than in embryonic molting, at least in Artemia. Thus while the release of Artemia embryos (hatching) from the embryonic cuticle occurs at the time of maximal ecdysteroid synthesis and release of chitinolytic enzymes (i.e., chitinase and N-acetylglucosaminidase) into the incubation medium (9), serine proteases of the type active during larval molting are not synthesized in Artemia embryos until several hours after hatching (20,29). Therefore, these proteases cannot play a role in the embryonic molt. Recent studies have indicated that cysteine proteases are released into the exuviate during the embryonic molt in Artemia, and these proteases may function as molting (hatching) enzymes in Artemia (23,30). The release of the serine proteases into the incubation medium containing second and third instar larvae of Artemia, concomitant with the occurrence of apolysis and ecdysis, appears to be dependent on cysteine protease activity (probably in the epidermis), since inhibition of larval cysteine proteases with Z-Phe-Ala-CH 2F had a marked inhibitory effect on both the release of serine proteases into the exuviate and molting [this study; (30)]. Support for the cysteine proteases as key enzymes during premolt also comes from immunocytochemical studies of first and second instar larvae of Artemia, which showed high concentrations of cysteine protease in the apical cytoplasm of epidermal cells of second instar larvae prior to molting (30). Z-Phe-Ala-CH 2F such as used in our experiments, and other fluoromethyl ketones block molting in nematodes suggesting a cysteine protease requirement in the molting process in other invertebrates as well (15,21). From studies on crabs, crayfish, lobsters, and shrimp there is good evidence to support the hypothesis that the epidermis plays a central role in the cyclical events of molting (2,8,9,12,18,19,30). During intermolt, chitin can be detected in vesicles near the apical surface of epidermal cells and in the cuticle, confirming a role for these cells in cuticle formation (12). As well, N-acetyl-β-D-glucosaminidase and chitinase, enzymes required for chitin degradation, and proteases required for chitinoprotein degradation are synthesized and/or localized in epidermal cells (25,30). However, the mechanisms by which the ecdysteroids promote or control the secretion of both chitin and degradative enzymes at distinct phases of the molt cycle are not known. Perhaps serine proteases are synthesized in epidermal cells as proenzymes, which are then activated by certain cysteine proteases in epidermal cells in response to ecdysteroid signalling. The fact that cysteine proteases have been shown to acti-
vate latent proteases in embryos of Artemia suggests a key role for the cysteine proteases in activation of latent serine proteases required in the molting process in crustaceans (29). In Artemia, the hindgut and foregut of second instar larvae are lined with a cuticle that must be removed before the gut becomes fully functional following the second larval molt (11). To what extent the serine proteases and N-acetyl-glucosaminidase appearing in the incubation medium arise from cuticle destruction in the gut is not known. Midgut glands in other shrimp are known to secrete chitinolytic enzymes, so it is possible that some of the serine proteases and N-acetyl-β-D-glucosaminidase appearing in the incubation medium are derived from the gut epithelium prior to the gut becoming fully functional (11,25). Whether these enzymes also have a digestive function following gut cuticle degradation remains to be ascertained. In summary, this study has demonstrated that at least two serine proteases are released into the incubation medium concomitant with the appearance of N-acetyl-β-D-glucosaminidase during the first two molt cycles in larvae of the brine shrimp, Artemia franciscana. Their potential role in apolysis and ecdysis in Artemia appears to depend on the activity of other enzymes in the apical cytoplasm of epidermal cells, probably cysteine proteases, which are abundant in these cells prior to molting. We wish to thank Dr. M. L. Petras for his critical reading of the manuscript and the Natural Sciences and Engineering Research Council of Canada for their financial support of this study.
References 1. Anderson, D.T. Larval development and segment formation in the branchiopod crustaceans Limnadia stanlejana (Conchostraca) and Artemia salina. Aust. J. Zool. 15:47–91; 1967. 2. Cariolou, M.A.; Flytzanis, C.N. Differential expression of cuticle-epidermis proteins in the shrimp Penaeus vannemei during molting. Comp. Biochem. Physiol. 108B:367–373; 1994. 3. Chang, E.S. Physiological and biochemical changes during the molt cycle in decapod crustaceans: an overview. J. Exp. Mar. Biol. Ecol. 193:1–14;1995. 4. Clegg, J.S.; Conte, F.P. A review of the cellular and developmental biology of Artemia. In: Persoone, G.; Sorgeloos, P.; Roels, O.; Jaspers, E. (eds). The Brine Shrimp Artemia, Vol. 2. Wetteren, Belgium: Universa Press; 1980:12–54. 5. Dwivedi, J.; Agrawal, O.P. Degradation of cuticle during larval-pupal and pupal-adult development of the housefly, Musca domestica. Physiol. Entomol. 20:318–322;1995. 6. Freeman, J.A. Epidermal cell proliferation during thoracic development in larvae of Artemia. J. Crustacean Biol. 6:37–48; 1986. 7. Freeman, J.A. The integument of Artemia during early development. In: MacRae, T.H.; Bagshaw, J.C.; Warner, A.H. (eds). Biochemistry and Cell Biology of Artemia. Boca Raton, FL: CRC Press; 1989:233–256. 8. Freeman, J.A. The crustacean epidermis during larval devel-
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ISSN 0305-0491/98/$19.00 PII S0305-0491(97)00323-4
Release of Proteases from Larvae of the Brine Shrimp Artemia franciscana and Their Potential Role During the Molting Process A. H. Warner and C. Matheson Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada ABSTRACT. In arthropods, molting is required in order for the organism to escape periodically from its exoskeleton (cuticle) to undergo metamorphosis, growth, or reproduction. During molting the cuticle is first released from the epidermis (apolysis) then degraded (ecdysis). This involves a complex set of hydrolases secreted into the exuviate under control of ecdysteroids and several environmental factors. During the first two larval molts in the brine shrimp, Artemia franciscana, large amounts of two serine proteases appear in the incubation medium, concomitant with N-acetyl-β-D-glucosaminidase, a well-described chitin degrading enzyme. These proteases were analyzed by gel filtration, fast protein liquid chromatography (FPLC), electrophoresis on gelatin-polyacrylamide gels, and with various inhibitors, and found to be nearly identical to two serine proteases synthesized during early larval development in Artemia and thought to be digestive enzymes. The appearance or secretion of these serine proteases into the incubation medium during larval development is required for apolysis and ecdysis, and appears to be dependent on cysteine protease activity in the epidermal layer under the cuticle. comp biochem physiol 119B;2:255–263, 1998. 1998 Elsevier Science Inc. KEY WORDS. Artemia, brine shrimp, cysteine proteases, development, molting, serine proteases
INTRODUCTION The process of molting has been the focus of numerous studies on arthropods dating back to the early sixteenth century. Major advances in our understanding of the cellular, biochemical and physiological basis of molting can be found in several excellent reviews (3,8,22,24). In arthropods, molting requires the interaction of a complex set of biochemical and cellular events in order for organisms to escape periodically from the exoskeleton (cuticle) to undergo metamorphosis, growth, or reproduction. During molting, the cuticle, which is composed mainly of chitin and proteins, undergoes cyclical synthesis and degradation under control of ecdysteroids, environmental factors, such as temperature, photoperiod, food supply, and other factors, such as molt-inducing and molt-inhibiting hormones (3,8,24). Among crustaceans molting in larvae and adults appears to be a general process consisting of at least four phases of which the premolt and ecdysis or shedding of the cuticle are the dominant events (7,22). Early in premolt apolysis occurs in which the chitin/proteinaceous cuticle Address reprint requests to: A. H. Warner, Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada. Tel. 519-253-4232, Ext. 2728; Fax 519-971-3609; E-mail: warner1@ucc. uwindsor.ca Received 30 July 1997; revised 22 September 1997; accepted 7 October 1997.
becomes detached from the underlying epidermis. Apolysis is believed to require the secretion of proteases and/or chitinolytic enzymes by epidermal cells (8,19). At the end of the premolt, ecdysis occurs and the cuticle is degraded further by hydrolases secreted into the exuvial fluid. It is not known whether a different complement of hydrolases are released into the exuviate to accomplish both apolysis and ecdysis or whether one set of hydrolases serves both processes. While the degradative pathway of the molt cycle is functioning, the pathways active in cuticle synthesis are activated in the epidermis; the resulting proteins and chitin are secreted to form a new multilayered cuticle (2). In the brine shrimp, Artemia franciscana, molting occurs 16–19 times throughout its life cycle, depending on the nutritional state of its environment and other factors (1,8,32). The first molt is an embryonic event during which time hatching of the embryo occurs from the embryonic cuticle, a chitinoprotein structure of c ⋅ 2 µm thick (4,16). This event is associated with enhanced chitinolytic activity (chitinase and N-acetyl-β-D-glucosaminidase) in both the prenauplius larvae and exuvial fluid, which then appears in the incubation medium (9,10). The next several molts in Artemia are larval molts, with the first and second molts occurring at about 18 and 28 hr, respectively after the embryonic molt (hatching) (6,7). The larval cuticle of Artemia is ,1 µm thick and appears to be less complex than either the embryonic or adult cuticle in Artemia (7).
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From research on several arthropods, including Artemia, considerable evidence has accumulated in support of a role for different classes of proteases in the molting process (5,15,18,19,21,26,30). As well, inhibitors of cysteine proteases have been shown to block apolysis or ecdysis in various organisms, preventing body segment formation and further growth of the organism (15,21,30). These experiments have implicated the cysteine proteases as key hydrolases in the molting process. However, molting is a complex process and the cohort of hydrolases required to sequentially release the cuticle from the epidermis and/or degrade it have not been established in any model system. In the study reported here, we have identified two serine proteases that are either released from or secreted by first and second instar larvae into the incubation medium during the first two larval molts in Artemia. These enzymes appear to be similar to proteases isolated previously from larvae of Artemia and identified as proteases A and B (20). Data are also presented to support the hypothesis that cysteine protease activity may be required for the activation and/or release of serine proteases into the incubation medium during molting in larvae of Artemia.
Culturing of Artemia Larvae Hydrated cysts were incubated in Millipore-filtered (0.45 µm) seawater fortified with penicillin (50 U/ml), streptomycin (50 µg/ml), and amphotericin (0.125 µg/ml) with constant shaking under a white light at 24°–25°C. Nauplius larvae, which hatched between 15 and 18 hr incubation, were collected and adjusted to a concentration of 1000 larvae/ml of incubation medium (seawater). These newly hatched larvae (first instar, or 0-hr larvae) in flasks containing 50,000 each were maintained at 24°–25°C with gentle shaking until needed. In some experiments, Z-PheAla-CH 2F was added at a concentration of 50 or 100 µM to flasks containing larvae at the beginning of incubation. At 6–8 hr intervals larvae were collected on miracloth supported on a Whatman 3 filter disk. The larvae were quick frozen and the filtered incubation medium was concentrated to about 2 ml by ultrafiltration on a YM-10 membrane. Aliquots were taken for protein, protease, and N-acetyl-glucosaminidase (NAGase) assays as described below and the remainder of the sample was concentrated to about 0.5 ml using a Centricon-10 concentrator. All samples prepared in this manner were stored at 215°C for further analyses. Preparation of Aqueous Cytoplasm of Artemia Larvae
MATERIALS AND METHODS Materials Encysted embryos (cysts) of Artemia franciscana (lot #12715) were obtained from Sanders, (Ogden, UT) and stored at 220°C. When needed, cysts were hydrated in 50% seawater for at least 5 hr at 0–4°C, during which time they were dechorionated with antiformin for 30 min as described previously (27). Antibiotics-antimycotics used in the culture of Artemia embryos and larvae were from GibcoBRL (Burlington, ON). Diaflo ultrafiltration membranes (YM10) and Centricon concentrators were purchased from Amicon (Oakville, ON). Chromatographic columns and media, such as MonoQ HR 5/5 and Sephadex G-150 (SF), and protein standards are obtained from Pharmacia (Baie d’Urfe, PQ). Protease inhibitors including pepstatin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), and soybean trypsin inhibitor (STI), p-nitrophenol, p-nitrophenyl-β-Dacetylglucosaminide, electrophoresis grade gelatin, and trinitrobenzene sulfonic acid (TNBS) were from Sigma Chemical Co. (St Louis, MO). Ovomucoid trypsin inhibitor (OTI) was from P-L Biochemicals (Milwaukee, WI) and benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-PheAla-CH2F) was a gift from Dr. D. Rasnick of Prototek Corp. (Dublin, CA). Protein assays were carried out using the bicinchoninic acid (BCA) reagent and protocol supplied by Pierce (Brockville, ON). Acrylamide, sodium dodecylsulfate (SDS), and other electrophoretic supplies were from BioRad (Mississauga, ON). All other chemicals were of ACS grade or better and purchased from either Sigma or Fisher Scientific (Nepean, ON).
Frozen larvae were homogenized directly in 5 vol of a buffer containing 150 mM sorbitol, 70 mM potassium gluconate, 5 mM monobasic potassium phosphate, and 3 mM Hepes, adjusted to pH 7.2. The homogenate was centrifuged at 1630 g to sediment the nuclei and yolk platelets, then at 15,000 g for 30 min (4°C) to obtain the aqueous cytoplasm as described previously (31). The 1630 g pellet was re-extracted with homogenization buffer and treated as described above. The aqueous cytoplasms were combined and passed through a Sephadex G-25 column to remove low molecular weight components, then concentrated to approximately 1 ml using a Centricon 3 filter. All concentrated samples were stored at 215°C for subsequent analyses. Enzyme Assays Aliquots of concentrated incubation medium, aqueous cytoplasm of larvae, or column fractions were assayed for protease activity using the trinitrobenzene sulfonic acid (TNBS) reagent described previously (28). The protease substrates were either protamine sulfate or gelatin at 2.5–4.0 mg/ml. Assays for the serine-type ‘‘neutral’’ proteases were carried out at pH 7.4 and 37°C, whereas assays for cysteine protease activity were carried out at pH 4 and 40°C. One milliunit (mEU) of ‘‘neutral’’ protease activity is defined as the release of 1 nmole per minute of amino peptide from the substrate (protamine sulfate or gelatin) at pH 7.4 and 37°C. Assays for N-acetyl-glucosaminidase (NAGase) activity were carried out as described previously using p-nitrophenyl-β-D-acetylglucosaminide as substrate in 0.1 M sodium
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acetate, pH 5.0 at 37°C (13). Aliquots of a 200 µl reaction mix were taken at 15-min intervals, diluted to 1 ml with 0.2 M Na 2CO 3 and the absorbance was read at 400 nm. The amount of p-nitrophenol released from the substrate was measured against a standard curve of p-nitrophenol. One milliunit of NAGase activity released 1 nmole/min of p-nitrophenol from the substrate. The effect of various inhibitors on protease or N-acetylglucosaminidase activity was determined by including the inhibitor in the reaction vessel at the desired concentrations. Control assays contained equivalent amounts of the solvents (dimethylsulfoxide or ethanol) used in the preparation of the inhibitor stock solutions. Chromatography of the Incubation Medium and Aqueous Cytoplasm of Larvae Concentrated proteins from the incubation medium or aqueous cytoplasm, equivalent to 35,000–40,000 Artemia larvae, were applied to a Sephadex G-150 (SF) column (75 3 1 cm) equilibrated with a buffer containing 15 mM potassium phosphate, 25 mM KCL, and 10% glycerol, pH 7.2. The proteins were eluted from the column with the equilibration buffer and the effluent monitored for protein by measuring the absorbance of each fraction at 280 nm. Aliquots of each column fraction were also assayed for protease activity at pH 7.4 using the TNBS reagent (28). The concentrated incubation medium and aqueous cytoplasm of larvae were also fractionated by fast protein liquid chromatography (FPLC) on a MonoQ column (HR 5/5) using a Beckman Altex solvent delivery system. Approximately 1000 mEU of neutral protease activity from the concentrated incubation medium or aqueous cytoplasm of larvae were applied to the column equilibrated with the buffer used in gel filtration. Following the sample application and a 2-min wash with the starting buffer, the proteins were eluted from the column using a gradient of KCl to 0.75 M over 30 min at a flow rate of 1.0 ml/min. The column eluate was monitored at 280 nm and fractions were collected for protease assays at pH 7.4 and 4.0. Electrophoresis on Gelatin-SDS-Polyacrylamide Gels Serine proteases in the incubation medium and larval preparations were analyzed on 12% polyacrylamide gels containing 0.1% SDS and 0.2% gelatin using the Bio-Rad mini-protean apparatus as described previously (17). Approximately 1.5 mEU of serine protease activity from each fraction to be analyzed were mixed with standard SDSmercaptoethanol loading buffer, then applied to the gel without heating. The loading and running buffers were as described previously (14). Following electrophoresis at 125 V (2 hr) and 4°C, the gel was soaked in 2.5% Triton X100 for 30 min then incubated for 1.5 hr in 50 mM sodium phosphate, pH 7.4, to allow the serine proteases in the gel
FIG. 1. Release of neutral proteases and N-acetyl-glucosaminidase into the incubation medium of Artemia larvae. The incubation medium was collected at 6-hr intervals, concentrated and aliquots taken for protease assays at pH 7.4 (h, e, n) and N-acetyl-glucosaminidase (NAGase) activity (open bars). The solid line represents a computer-generated trend curve from the average of protease measurements from three experiments, while the bars represent the average of two NAGase experiments. M1 and M2 indicate the times of the first and second larval molt, respectively.
to hydrolyze the gelatin, identifying their migration position. The gel was then stained for 1 hr with a solution containing 2.5% Coomassie Brilliant Blue, 50% methanol, and 7.5% acetic acid, then destained overnight in a solution of 5% methanol and 7.5% acetic acid and photographed. RESULTS Appearance of Protease Activity in the Incubation Medium of Artemia Nauplii In a previous study we observed neutral protease activity in the incubation medium (seawater) of swimming Artemia larvae during the larval molt cycle (30). We have quantified this protease activity during the first 42 hr of larval development, and compared it to N-acetyl-glucosaminidase (a chitinolytic enzyme), which also appears in the incubation medium during larval molting. The results in Fig. 1 show that the largest increase in protease activity occurred between the first and second larval molts, reaching about 300 mEU of enzyme activity per 10,000 larvae. Beyond 40–42 hr incubation, no increase in protease activity was observed in the incubation medium, and in some cases the activity actually declined. The protease activity profile in the incubation medium followed closely the N-acetyl-glucosaminidase (NAGase) activity, a well-known indicator of molting.
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FIG. 2. Gel filtration of proteases in the incubation medium
and aqueous cytoplasm of third instar larvae of Artemia. Concentrated samples of cytoplasm (panel A) and incubation medium (panel B) from third instar larvae (40 hr posthatch) were filtered through a calibrated column of Sephadex G-150 as described under Materials and Methods. Column fractions were assayed for protease activity at pH 7.4 (e, absorbance at 420 nm) and protein content (■, absorbance at 280 nm). The numbers at the top of each panel indicate the elution position of standard proteins.
Analyses were not conducted on Artemia larvae beyond the third instar stage of development (40–45 hr after hatching) because larvae begin to die after the second larval molt (third instar stage) unless fed appropriately. Characterization of the Proteases in Incubation Medium of Second and Third Instar Larvae of Artemia To characterize the proteases released from developing larvae of Artemia, aliquots of the incubation medium were collected after the first and second larval molt, concentrated and analyzed by gel filtration, FPLC, and gel electrophoresis. The results in Fig. 2 (panel B) show that all protease activity in the incubation medium of 40 hr post-hatch Artemia larvae eluted from a Sephadex G-150 column as a single peak with a molecular mass of about 28.5 kDa. The elution position of this peak corresponded with a peak of ‘‘neutral’’ protease activity in the concentrated cytoplasm of 40 hr Artemia larvae (Fig. 2, panel A). A similar elution profile was seen when 24 and 30 hr post-hatch samples were analyzed (data not shown). The incubation medium did not contain any acidic protease activity, although the cytoplasm of larvae is known to have cysteine protease activity with an acidic pH optimum [see below; (31)].
A. H. Warner and C. Matheson
FIG. 3. Fast protein liquid chromatography (FPLC) of proteases in the aqueous cytoplasm and incubation medium of third instar larvae of Artemia. Concentrated samples of aqueous cytoplasm (panel A) and incubation medium (panel B) of third instar larvae were applied to a Mono Q column, then eluted with a linear gradient of KCl in the starting buffer. The solid line represents the absorbance at 280 nm of material eluting from the column. Column fractions were assayed for protease activity at pH 7.4 (■) and pH 4.0 (e) with the results shown as absorbance at 420 nm. CP represents the elution position of the major cysteine protease in larvae, whereas E-1, E-2, and E-3 represent the elution positions of the serine proteases. No cysteine protease activity was detected in the concentrated incubation medium (30); therefore, no CP assays were carried out on column fractions of the incubation medium. The dash line (—) represents the KCl gradient.
When the incubation medium containing second or third instar larvae (i.e., 30 or 42 hr samples) was applied to a Mono Q column, the protease activity was resolved into two fractions (E-1 and E-2) with elution characteristics identical with two ‘‘neutral’’ proteases in the cytoplasm of third instar (40 hr) larvae. These results are shown in Fig. 3 (panels A and B). Another neutral protease (E-3) in the cytoplasm of third instar larvae, which eluted from the Mono Q column at 0.7 M KCl, was barely detectable in the incubation medium even after 50-fold concentration of the medium. Cysteine protease (CP) activity was present in the cytoplasm of third instar larvae, but not in the incubation medium, although a small amount of the large subunit of the CP was detected by Western blotting in the incubation medium in an earlier study (30). Thus the main proteases present in the incubation medium after 36–42 hr incubation (i.e., E-1 and E-2) appear to be identical to two ‘‘neutral’’ proteases present in the cytoplasm of third instar lar-
Release of Proteases During Molting in Artemia
FIG. 4. Electrophoresis of the serine proteases in the incuba-
tion medium and aqueous cytoplasm from Artemia larvae on a gelatin-SDS-polyacrylamide gel. The E-1 and E-2 fractions from the Mono Q column (see Fig. 3) were concentrated and 1.5 mEU of each were applied to a 12% polyacrylamide gel containing 0.1% SDS and 0.2% gelatin. The clear areas show the migration positions of the proteases, which hydrolyzed gelatin in the gel at pH 7.4. M and L refer to incubation medium and larvae, respectively, as the source of the E-1 and E-2 proteases. The sizes (kDa) and migration positions of standard proteins are shown at the left, while the sizes (kDa) of the largest and smallest active proteases in the E-1 and E-2 fractions are shown at the right.
vae, and in similar proportion to the E-1 and E-2 proteases in the cytoplasm of second and third instar larvae. To further characterize the proteases in the incubation medium, the E-1 and E-2 fractions from the Mono Q column were subjected to electrophoresis on a gelatin-SDSpolyacrylamide gel, and their migration positions in the gel were compared with the proteases in the E-1 and E-2 fractions from the cytoplasm of second instar larvae. The results in Fig. 4 show that incubation of the gelatin gel in phosphate buffer, pH 7.4, following electrophoresis revealed several bands of protease activity in each of the E-1 and E-2 fractions from both the incubation medium and larvae. The E-2 fraction from the incubation medium contained eight bands of protease activity, ranging in molecular mass from 26.5 kDa to 15.5 kDa, and with electrophoretic mobilities identical to eight bands of protease activity in the E-2 fraction from larvae. In contrast, the protease composition of the E-1 fraction from the incubation medium and larvae was different. The E-1 fraction from the larvae and incubation medium both contained proteases of 26.5, 26, 24, and 23.5 kDa, whereas the proteases of 19.5, 19, 17, and 15.5 kDa were either absent or in reduced amounts in the E-1 fraction of third instar larvae. Otherwise, both the E-1 and E-2 fractions from the Mono Q column contained proteases of very similar sizes after electrophoresis in the gelatin gel, irrespec-
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tive of their origin (i.e., incubation medium or larvae). Electrophoresis of the E-1 and E-2 proteases on native gels (i.e., lacking SDS) did not yield distinct bands of protease activity, but only a smear about 2 cm in length near the top of the gel. (Note: since multiple forms of the E-1 and E-2 proteases were not found in earlier studies of these enzymes, the lower molecular weight forms of these enzymes may be the results of autolysis in the presence of SDS). Also, no protease activity was observed in gelatin gels incubated in sodium acetate buffer at pH 5 (data not shown). The unfractionated proteases in the incubation medium were also assayed between pH 5.5 and 8.5 in phosphate buffer, and found to have optimal activity at pH 7.5 (data not shown), which is similar to that reported previously for the major serine proteases in the aqueous cytoplasm of second and third instar larvae (20). The results of the chromatographic, electrophoretic, and pH measurements suggest that the proteases that appear in the incubation medium during the first two molt cycles of Artemia have physical characteristics similar to at least two of the ‘‘neutral’’ proteases in the cytoplasm of Artemia larvae. Specificity of the Neutral Proteases in the Incubation Medium and Cytoplasm of Young Larvae of Artemia to Inhibitors The proteases released into the incubation medium during the molt cycle of Artemia (i.e., fractions E-1 and E-2) were assayed in the presence of various well-known inhibitors of proteases, and their activities were compared to protease assays on the E-1 and E-2 fractions of second instar larvae of Artemia carried out under identical conditions. The results of these experiments are summarized in Table 1. The proteases in the E-1 fraction from the incubation medium and larvae were differentially sensitive to PMSF and TLCK, inhibitors of serine proteases. Specifically, the E-1 proteases in the incubation medium were more sensitive to PMSF and TLCK than the larval E-1 proteases. Differences in sensitivity to PMSF and TLCK may be due to the fact that the E1 fraction from the medium has a slightly different protease composition than the E-1 fraction from larvae (see Fig. 4). The different responses to these inhibitors could also be due to posttranslational modifications to the E-1 proteases before their secretion/release to the medium. The proteases in the E-2 fraction from the incubation medium and larvae represent the largest amount of neutral protease activity in each of these sources, comprising about 80% of the total neutral protease activity in each case. However, unlike the proteases in the E-1 fraction, proteases in the E-2 fraction are mostly resistant to PMSF and TLCK, but sensitive to STI, OTI and leupeptin, suggesting that the proteases in the E-2 fractions are also serine-type proteases, but different from the E-1 fraction proteases. None of the proteases in the incubation medium was af-
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TABLE 1. Effect of various inhibitors on incubation medium and larval neutral proteases from Artemia
Residual activity (%) E-1
E-2
Inhibitor
Concentration
Medium
Larvae
Medium
Larvae
STI OTI PMSF
10 µg/ml 10 µg/ml 50 µM 100 µM 100 µM 50 µM 50 µM 100 µM 100 µM 2 mM 0.1% 0.5% (60°C, 30 min)
86 100 72 28 62 86 — 100 98 106 64 0 0
66 100 100 66 96 113 — 100 87 94 95 0 0
12 23 — 95 93 119 92 108 20 90 91 48 0
20 37 — 85 96 139 103 99 31 102 99 18 0
TLCK Pepstatin Z-Phe-Ala-CH2F Leupeptin EDTA SDS* Heating*
*In these assays the substrate was gelatin; all other assays were carried out using protamine sulfate as substrate.
fected by inhibitors of cysteine, aspartic, and metalloproteases. Thus, Z-Phe-Ala-CH 2F, pepstatin, and EDTA, respectively, had no inhibitory effect on the proteases in the E-1 and E-2 fractions. However, Z-Phe-Ala-CH 2F (a synthetic inhibitor of cysteine protease) had a marked inhibitory effect on the release of E-1 and E-2 proteases into the medium (see below), although it did not inhibit the activity of the E-1 or E-2 proteases in the medium or larvae. Finally, it should be noted that the E-1 and E-2 proteases from both the larvae and incubation medium were generally resistant to treatment with 0.1% SDS, whereas 0.5% SDS in the protease assays completely inhibited the E-1 proteases and partially inhibited the E-2 proteases. These results support the conclusion that the proteases released into the incubation medium during development of first and second instar larvae of Artemia appear to be slightly modified forms of serine proteases originating in the cytoplasm of these larvae.
the first and second larval molt by SDS-polyacrylamide gel electrophoresis suggested that appearance of the proteases in the medium is not the results of larvae decomposing during the incubation (data not shown). Also, visual inspection of the culture flasks did not reveal any dead larvae during the sampling times in these experiments. Finally, it
The Developmental Profile of the Serine Proteases in Post-Hatch Larvae of Artemia Compared to Their Appearance in the Incubation Medium Developing embryos of the brine shrimp, Artemia, have very little, if any, active serine, aspartic, or metalloproteases (20,29). However, beginning about 8–10 hr after hatching of the nauplius larvae, there is considerable synthesis and/ or activation of at least three serine-type proteases (20). The appearance of serine proteases in the incubation medium follows by 8–10 hr their appearance in the larvae. These results are shown in Fig. 5. Near the end of the second molt cycle (24–30 hr after hatching), the specific activity of the proteases in the medium is 17–18 times higher than it is in the cytoplasm of larvae, suggesting that the appearance of the proteases in the medium during this period of development may be the results of a secretory process. Moreover, analysis of the proteins in the medium after
FIG. 5. Appearance of neutral protease activity in the aque-
ous cytoplasm and incubation medium of first instar larvae of Artemia at various times after hatching. Each point represents the average of two experiments. The numbers above several points represent the specific activity (mEU/ mg protein) of the proteases in the cytoplasm or incubation medium of Artemia larvae at the incubation times indicated.
Release of Proteases During Molting in Artemia
FIG. 6. Effect of Z-Phe-Ala-CH2F on release of neutral prote-
ases into the incubation medium of Artemia larvae. Z-PheAla-CH2F was added to flasks containing incubation medium and newly hatched larvae, and samples were collected at 8-hr intervals, beginning at 16 hr incubation, for neutral protease measurements. Untreated larvae (solid bars), 50 mM (stippled bars) or 100 mM (hatched bars) concentrations of the drug.
should be noted that during the first two larval molts, the serine proteases released/secreted into the incubation medium represent only 10–15% of the total serine protease content of the larvae. Effect of the Cysteine Protease Inhibitor Z-Phe-Ala-CH2F on Hydrolases Released into the Incubation Medium of Artemia Larvae In a previous study, we demonstrated that addition of ZPhe-Ala-CH 2F (a cysteine protease inhibitor) to the incubation medium containing newly hatched Artemia larvae
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inhibited apolysis, and subsequently ecdysis, during the first two larval molt cycles in Artemia (30). Therefore, we tested the effect of Z-Phe-Ala-CH 2F on the release of serine proteases and N-acetyl-glucosaminidase into the incubation medium of newly hatched Artemia larvae. The results in Fig. 6 and Table 2 show that addition of Z-Phe-Ala-CH 2F to the incubation medium had a marked inhibitory effect on the synthesis/activation of the serine proteases in the larvae, and their subsequent release or secretion into the incubation medium. The reduced level of the serine proteases (40–50% of controls) appearing in the incubation medium during the first two larval molts appears to reflect the marked reduction (23–25% of controls) in these enzymes in the aqueous cytoplasm of second and third instar larvae. In contrast to its effect on serine protease release to the incubation medium, Z-Phe-Ala-CH 2F had very little effect on the release of NAGase into the medium, although the level of NAGase in the larvae was reduced 23–32% by this drug (see Table 2). Since Z-Phe-Ala-CH 2F had no inhibitory effect (in vitro) on the serine proteases in the E-1 and E-2 fractions at 50–100 µM (see Table 1), these results suggest that the major cysteine proteases in larvae of Artemia may play a role in the processing and/or release of the serine proteases to the medium during the larval molt cycles. DISCUSSION Among the crustaceans, the brine shrimp Artemia has provided information of potential importance to our understanding of the molting process in embryos and larvae. In a previous study, three serine proteases were shown to increase markedly during development of first and second instar larvae, and since these enzymes were thought to be digestive enzymes, their role in development was not determined experimentally (20). The results of the present study suggest that at least two of these proteases play a role in the larval molting process. The serine proteases appearing in the incubation medium during molting of second and third instar larvae (E-1 and E-2) have chromatographic and electrophoretic properties identical with proteases A and B isolated from larvae of Artemia as described previously (20). Their release into the exuviate during the first two
TABLE 2. Effect of the cysteine protease inhibitor Z-Phe-Ala-CH2F on various hydrolase activity levels in the incubation me-
dium and larvae of Artemia franciscana Milliunits enzyme in larvae† Enzyme Serine proteases Serine proteases NAGase NAGase
Milliunits enzyme in incubation medium†
Treatment*
Second instar larvae
Third instar larvae
Second instar larvae
Third instar larvae
Control Z-Phe-Ala-CH2F Control Z-Phe-Ala-CH2F
2140 532 (25%)‡ 132 90 (68%)
2443 567 (23%) 125 95 (77%)
45.8 18.5 (40%) 11.3 10.4 (92%)
283.1 141.5 (50%) 37.0 35.4 (96%)
*Flasks containing newly hatched larvae were given Z-Phe-Ala-CH2F at 100 µM final concentration compared to control flasks lacking this drug. †The milliunits of enzymes are based on 10,000 larvae and the average of two samples at each larval stage shown. ‡The numbers in parentheses represent the activity remaining in Z-Phe-Ala-CH2F treated samples compared to controls.
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larval molts also corresponds closely with the release of the chitinolytic enzyme N-acetyl-glucosaminidase into the incubation medium. Therefore, it appears reasonable to conclude that two of the serine proteases, which first appear in first instar larvae, are among the cohort of hydrolases required to accomplish apolysis and ecdysis in larvae of Artemia. The results of this study also suggest that a different cohort of proteases function in larval molting than in embryonic molting, at least in Artemia. Thus while the release of Artemia embryos (hatching) from the embryonic cuticle occurs at the time of maximal ecdysteroid synthesis and release of chitinolytic enzymes (i.e., chitinase and N-acetylglucosaminidase) into the incubation medium (9), serine proteases of the type active during larval molting are not synthesized in Artemia embryos until several hours after hatching (20,29). Therefore, these proteases cannot play a role in the embryonic molt. Recent studies have indicated that cysteine proteases are released into the exuviate during the embryonic molt in Artemia, and these proteases may function as molting (hatching) enzymes in Artemia (23,30). The release of the serine proteases into the incubation medium containing second and third instar larvae of Artemia, concomitant with the occurrence of apolysis and ecdysis, appears to be dependent on cysteine protease activity (probably in the epidermis), since inhibition of larval cysteine proteases with Z-Phe-Ala-CH 2F had a marked inhibitory effect on both the release of serine proteases into the exuviate and molting [this study; (30)]. Support for the cysteine proteases as key enzymes during premolt also comes from immunocytochemical studies of first and second instar larvae of Artemia, which showed high concentrations of cysteine protease in the apical cytoplasm of epidermal cells of second instar larvae prior to molting (30). Z-Phe-Ala-CH 2F such as used in our experiments, and other fluoromethyl ketones block molting in nematodes suggesting a cysteine protease requirement in the molting process in other invertebrates as well (15,21). From studies on crabs, crayfish, lobsters, and shrimp there is good evidence to support the hypothesis that the epidermis plays a central role in the cyclical events of molting (2,8,9,12,18,19,30). During intermolt, chitin can be detected in vesicles near the apical surface of epidermal cells and in the cuticle, confirming a role for these cells in cuticle formation (12). As well, N-acetyl-β-D-glucosaminidase and chitinase, enzymes required for chitin degradation, and proteases required for chitinoprotein degradation are synthesized and/or localized in epidermal cells (25,30). However, the mechanisms by which the ecdysteroids promote or control the secretion of both chitin and degradative enzymes at distinct phases of the molt cycle are not known. Perhaps serine proteases are synthesized in epidermal cells as proenzymes, which are then activated by certain cysteine proteases in epidermal cells in response to ecdysteroid signalling. The fact that cysteine proteases have been shown to acti-
vate latent proteases in embryos of Artemia suggests a key role for the cysteine proteases in activation of latent serine proteases required in the molting process in crustaceans (29). In Artemia, the hindgut and foregut of second instar larvae are lined with a cuticle that must be removed before the gut becomes fully functional following the second larval molt (11). To what extent the serine proteases and N-acetyl-glucosaminidase appearing in the incubation medium arise from cuticle destruction in the gut is not known. Midgut glands in other shrimp are known to secrete chitinolytic enzymes, so it is possible that some of the serine proteases and N-acetyl-β-D-glucosaminidase appearing in the incubation medium are derived from the gut epithelium prior to the gut becoming fully functional (11,25). Whether these enzymes also have a digestive function following gut cuticle degradation remains to be ascertained. In summary, this study has demonstrated that at least two serine proteases are released into the incubation medium concomitant with the appearance of N-acetyl-β-D-glucosaminidase during the first two molt cycles in larvae of the brine shrimp, Artemia franciscana. Their potential role in apolysis and ecdysis in Artemia appears to depend on the activity of other enzymes in the apical cytoplasm of epidermal cells, probably cysteine proteases, which are abundant in these cells prior to molting. We wish to thank Dr. M. L. Petras for his critical reading of the manuscript and the Natural Sciences and Engineering Research Council of Canada for their financial support of this study.
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