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Intestinal protein dynamics in the stonefly, Pteronarcys californica
J. fnse
in Great
Bntain.
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INTESTINAL
PROTEIN
Copyright
DYNAMICS
PTERONARCYS
0022.1910!84 $3 00 + 0.00 (’ 1984 Pergamon Press Ltd
IN THE STONEFLY,
CALIFORNICA
DAVID F. GRANT* and G. WAYNE MINSHALL Department of Biology, Idaho State University. Pocatello, Idaho 83209. U.S.A. (Receired 27 Sepfemher
1983; revised 23 November 1983)
Abstract-Characteristics of the intestinal proteins of a detritus-eating aquatic insect, Preronarcys californica were assessed to determine their possible role as a nutrient source during metamorphosis. Total soluble intestinal protein was significantly correlated to larval weight, averaging 3.9”; of dry weight for both males and females. Proteinase activity in the gut was separated into several bands by electrophoresis. Electrophoresis patterns of total intestinal proteins of P. califbrnica larvae of differing instar, sex, and diet were remarkably similar. Larvae incubated with tritium-labelled amino acids produced labelled intestinal proteins. When early instar larvae were removed from label. there was no significant loss of tritium from the intestine after 2 months. Penultimate instars labelled in the same manner however. showed depuration of tritium from the gut according to first order kinetics. This loss was correlated to the 6-8 wk non-feeding stage of metamorphosis, just prior to adult emergence. These results indicate that proteins are accumulated in the intestinal tract of immature P. caljfornica, then are reabsorbed from the gut during metamorphosis. Kev Word Index: Intestinal protein, metamorphosis. digestive enzymes. aquatic detritivore, Pteronarc.w
INTRODUCTION Because metamorphosis is often accompanied by a dramatic change in the habitat of an insect, many proteins essential during immature stages may be useless, as such, to the adult. Digestive enzymes in the intestinal tract of immature insects entering a nonfeeding adult stage would be one such pool of protein. In general, the fate of insect digestive proteins remains largely speculative (Dadd, 1970; House. 1974; Terra and Ferreira, 1981). Conservation and re-use of intestinal enzymes has been suggested in a model given for Rhynchosciara americana larvae (Terra and Ferreira, 1981). This model describes a possible mechanism whereby digestive enzymes are circulated within the endo- and ectoperitrophic spaces of the intestinal tract. This model raises two questions concerning the storage and fate of digestive enzymes. First, in insects which are constantly feeding, do digestive proteins accumulate within the intestinal tract, thereby maintaining constant protein concentration and digestive capabilities as the insect grows? Second, if such proteins are accumulated during feeding stages, what is their fate once the insect becomes nonfeeding? To address these questions. we studied the intestinal protein dynamics of an aquatic detritivore, Ptcronarcy cal~fornicu. P. californica larvae have been shown to possess high proteinase activity in the intestinal tract as well as enzymes which hydroloyze r-l.4 and /?-1,3-glucans (Martin et al.. 1981). Because larvae do not eat for approx 6-8 weeks before they emerge from the stream as non-feeding adults (Poole. 1981; Hynes. 1976), this 6- to 8-week period provides ~~~ _ ___. ~_ .._~ ~~ *To whom correspondence should be addressed. Present address: Department of Entomology, Michigan State University. East Lansing, Michigan 48824. U.S.A.
a good opportunity to monitor the fate of intestinal proteins. This time is also crucial for P. cal~fbrnicu because eggs, wings, and other adult structures are believed to develop fully (Branham, 1975). In this paper we give evidence supporting the hypothesis that a significant amount of soluble protein is synthesized and accumulated in the intestinal tract of P. calijhzica. This intestinal protein is composed of at least 6 digestive enzymes, and is used during the 6- to 8-week non-feeding stage just prior to adult emergence. MATERIALS AND METHODS Animuls Pteronarqs califbrnica (Newport) larvae were collected from Mink Creek, a third-order hardwater stream in Bannock County, Idaho. The larvae were either used immediately or were kept in Mink Creek water in 4-l jars. Those kept in the laboratory were maintained at ambient stream conditions (photoperiod and temperature) in a covered refrigeration unit. Compressed air was used to aerate laboratory insects. Additional P. cal~fhnica larvae were collected from Warm River. a fifth-order hardwater stream in Fremont County, Idaho. Larvae from Warm River were used immediately for a single electrophoresis experiment. Quailtity qf‘ intestinrrl protein
Because of problems measuring total protein from biological samples of unknown composition (Levenbook and Bauer. 1980; Kleg and Hale, 1977). we used a gravimetric technique. Eight P. cal$ornica collected from Mink Creek on 29 November 1981. were transfered to the laboratory, blotted dry. and weighed. The intestinal tracts were removed by cutting off the last abdominal segment. and the head just 441
442
DAVIU F. GRANT and G. WAYW~ MIXSHALL
behind the eyes, then pulling out the intestine from the caudal end of the insect with forceps. The intestinal tract from each insect was broken open and minced by hand in a I-ml conical centrifuge tube using a small metal spatula. We felt this procedure maximized dissolution of lumen contents and minimized protein release from gut tissue. Each homogenate was washed x 3 by centrifugation (40,000~. IO min. 4’ C) in 0.5 ml distilled water. One ml of 60”,, trichloroacetic acid was then added to precipitate the collected supernatant fraction for each of the 8 samples. After I h in an ice bath, each sample was filtered onto a tared. predried glass-fibre filter. The filters were dried overnight at 95 C and reweighed on a Cahn electrobalance. Identical procedures were used for 3 control filters. To convert insect wet weights to dry weights, IO P. cul~$micu larvae were killed (by removing the air supply for 2 h), blotted dry, and weighed. They were dried overnight at 100 C and reweighed. Proteinuse
enynes
To determine if proteinase enzyme activity was represented by more than one protein, slab polyacrylamide gel electrophoresis (S-PAGE) was used to separate proteins prior to enzyme assays, Intestinal proteins were prepared for S-PAGE by homogenizing intestinal tracts in a centrifuge tube, centrifuging the sample (40,000~. IO min. 4’C), and collecting the supernatant fluid. The proteins were then concentrated by lyophilization. Proteins were rehydrated in buffer (IO”,, sucrose in 0.5 M Tris. pH 6.8) and applied to gels in duplicate. One column was sliced and used for proteinase assays, the other column was stained for 30 min in 0.05”, Coomassie blue in 45”,, methanol-lo”,, acetic acid. A densitometer tracing was obtained after destaining overnight in lo”,, methanol-7.5 I,,) acetic acid. Proteins were electrophoresed on 3”,, stacking and either IO or 13”,, resolving, non-denaturing slab gels crosslinked with his-acrylamide using IO”, ammonium persulphate (20 mA per slab, 1 I C, 6 h). The running buffer was 0.3”,, Trisl.4”,, glycine, pH 8.3. Each strip from the sliced column was then placed in a small test tube in 3 ml of 0.5 M Tris buffer. pH 6.8, and eluted overnight at 4 C. The liquid from each tube was added to proteinase substrate (10 mg Azocoll, Sigma) and incubated. Two assays were performed: In one. the proteins eluted from the gels were incubated with substrate at room temperature for 4 h. In the other, the separated proteins were incubated with substrate at 4 C for 7d. The absorbance of each tube was measured according to Martin et (I/. (1980) except that we report relative values rather than absolute activity. The relative proteinase activity in each of the gel sections was plotted with the densitometer tracings to correlate protein bands with enzyme activity. Source of’ intestinal protein Because intestinal proteins may be derived from both food and bacterial sources, it is important to determine the influence of diet on intestinal protein characteristics. We approached this problem in two ways. The first was to compare the electrophoresis patterns of intestinal proteins from P. cul(~brnicu reared on different diets and from different streams.
The second approach was to determine whether intestinal proteins were being synthesized from amino acids. or were derived directly from a non-living food source. To determine the effect of diet on intestinal protein patterns, P. calijimicu larvae were reared for I month in the laboratory on four different leaf species: Bctuiu occidentalis, Popuh deltoides, Suli.\r sp. and Acrr negundo. For all of these comparisons. the intestinal tract was homogenized, centrifuged. and the lyophilized supernatant fluid used for nondenaturing electrophoresis as described earlier, except that enzyme activity was not measured. P. cul{fbrnica from Warm River and Mink Creek were used for the between-stream comparison. A tritium-labelled amino acid mixture (ICN Chemical and Radioisotope Division) was used to distinguish between a living and a detrital source of intestinal protein. Three hundred PCi of [‘H]labelled amino acids were added to a 2-l beaker containing I I of Mink Creek water. Two small water-birch logs were added for food. Seven P. cul$ornica larvae were collected on 8 July 1981 and allowed to incorporate the label for 2 months. None of these insects were penultimate instars. One of the insects was then removed (the remaining insects were used as described below) and the soluble intestinal contents were subjected in duplicate to electrophoresis. One column was then sliced into 40 equal sections and the other was stained with Coomassie blue. The sliced sections were eluted overnight and the eluant from each (excluding the marker-dye front) was counted in 10 ml of Insta-Gel (Packard) on a Beckman liquid scintillation counter. The cpm in each section was plotted on the densitometer tracing from the stained gel column. This plot was photocopied, and the densitometer tracing cut out. The tracing was cut into 40 equal sections and each section weighed on a Cahn electrobalance. We then plotted the relative amount of protein in each section ( = weight of paper in that section) versus the cpm in each section. Storuge und reahsorptior~ of’ intestinul protein The 6 P. cu/ifOrnicu remaining from the above labelling experiment were used to determine how quickly label was lost from the intestinal tract. The insects were placed in label-free water changed biweekly. Starting 16 September one insect was removed every 2 weeks. blotted dry, and weighed. The intestinal tract was removed. homogenized, and the supernatants from 3 centrifugation washes (40,OOOb. IO min. 4 C) were collected and weighed. A weighed subsample was then taken. added to scintillation fluid (Insta Gel, Packard) and counted. Using this procedure the total cpm in the intestinal tract per wet weight insect was monitored over time. A similar pulse-chase experiment was begun on 5 December 1981 using penultimate instars as well as earlier ( < nenultimatel instars. Seven randomlv collected penultimate-ins&r females and 7 earlier-&tar female larvae were combined in I 1 of Mink Creek water and labelled using 300 uCi of [‘HIamino acid mixture from 5 December until 22 January 1982. The two groups were then separated and placed in labelfree water (changed bimonthly) until 26 March. the normal emergence time for P. c,ulif;wnicu adults in
Intestinal protein in Prer0narq.s cakfornica
0
38
33
28
23
18
13
443
8
0
Gel section Fig. 1, Densitometer tracing (curves) of proteins separated by S-PAGE (IO:,, resolving. non-denaturmg gels), plotted with the relative proteinase activity (I-Transmittance) in gel sections assayed at 22 and 5 ‘C. Proteins migrated from right to lefty
Idaho. From 22 January until 26 March 1982. one insect from each group was collected and the total radioactivity in the intestinal tract was measured as described above. For the early-instar larvae, no other measurements were taken. For penultimate-instar larvae, developing eggs were collected at each sampling period, dissolved by boiling in loo/;, SDS. and the radioactivity counted. 3H excretion rates for penultimate-instar larvae were measured during the last three, 2-wk intervals between aquarium water changes. These were measured by finding the cpm in a weighed amount of aquarium water every other day (n = 7 for each rate determination), multiplying this by the total amount of water in the aquarium and dividing by the total weight of insects in the aquarium during the 2-wk interval. We also measured the distribution of tritium label from intestinal tract and haemolymph of penultimate instar P. californica. The cpm were subdivided into large molecular weight (cpm retained by dialysis), non-volatile small molecular weight (cpm lost after dialysis, but not after evaporation), and volatile small molecular weight (cpm lost after dialysis and evaporation to dryness). We assumed that volatile small molecular weight cpm was tritiated water. These 3 fractions were expressed as percentages of total cpm by comparing the cpm for dialyzed samples (corrected for dilution) and the cpm of evaporated samples, with untreated gut or haemolymph samples and assumed that volatile cpm represented the difference. RESULTS
The gravimetric protein assays of P. cafijornica soluble intestinal contents gave the following results expressed as percentages ( + 95% CI): Females, X = 3.39% protein ( + 0.99, n = 3); males, X = 4.19% protein ( f 1.24. n = 5); and for males and females
combined, x = 3.89% protein ( f 0.74, n = 8). Using the average of 3.89% intestinal protein per dry weight insect for both males and females and an approximate gut volume for penultimate-instar larvae of 0.2 ml (unpublished data), the concentration of protein in the intestinal tract would be approx 3.4”d. Proteinase enzyme activity was detected in several areas on S-PAGE at both room temperature and at 5°C (Fig. 1). Temperature did not seem to cause a major shift of enzyme activity, but there was a slight decrease in section 21 and an increase in sections 29, 33 and 37 at the 5~C assay. We attempted to repeat these electrophoretic enzyme assays for the a and @glucosidase activity previously found by Martin et al. (1981) but were unable to recover enzyme activity after electrophoresis. Source qf intestinal proteins The electrophoresis patterns of the intestinal protein from P. calijbrnica larvae reared on 4 different diets shows remarkable similarity among all four groups (Fig. 2). There is some indication that insects feeding on willow (lanes 3 and 6) had 1 or 2 qualitative differences in their intestinal protein patterns, however diet is clearly not a major factor influencing these electrophoretic patterns. When the intestinal proteins of P. californica from Warm River are compared to those from Mink Creek (Fig. 3) extremely similar patterns again are shown, Gut analysis showed that P. californica from Warm River were eating only autochthonous detritus (i.e. algae and diatoms) at the time of collection. Poole (personal communication) also has concluded that P. cahf~~rnica in Warm River eat autochthonous detritus as their main food source. This is in contrast to the
allochthonous food base (mostly water-birch logs) for Mink Creek P. californica. This consistency of the electrophoresis patterns is similar for insects collected
444
DAVID F. GRANT and G. WAYNE MINSHALL
from Mink Creek during all seasons and for both sexes (data not shown), strongly suggesting that proteins in the intestinal tract are being synthesized by the insect. Figure 4 shows the relationship between the amount of tritium cpm per gel slice and the relative amount of protein per gel slice after intestinal proteins from P. c&@nicrr reared (for 8 weeks) in water containing ‘H-labelled amino acids were separated on S-PAGE. Although the amount of variation in tritium activity is only partly explained by the amount of protein (r = 0.81) the correlation is very highly significant (P < 0.001). These data suggest that the specific rate of label incorporation was similar for each of the intestinal proteins, although the total counts incorporated were low. These data also strongly indicate that intestinal proteins are being synthesized by the insect and eliminates the possibility that they are originating from a non-living food source such as detritus cell-wall proteins.
found for so called storage proteins in the haemolymph of other insects. Haemolymph storage proteins have been shown to be used during metamorphosis for adult development, and have been isolated from Diptera (Munn et al., 1967; Munn and Grevill, 1969; Ueno and Natori, 1982) and from Lepidoptera (Collins, 1975: Wyatt and Pan, 1978; Tojo et al., 1978, 1980: Kramer et ~1.. 1980; Miller and Siehocek, 1982a.b.c). Intestinal proteins found in P. cnlifornica may serve a function during metamorphosis analogous to storage proteins. It is interesting to note that on a percentage basis, the amount of protein accumulated in the gut of P. cal(fornica is similar to the amount of storage protein accumulated in other insects (Kramer et al.. 1980: Levenbook and Bauer. 1980). However, the protein in P. ca/[fornicu is different from haemolymph storage protein not only because it is stored in the intestinal tract, but because some of these proteins are enzymes: The proteinase enzymes found in this study and the r and /I-glucosidase enzymes found by Martin ef ul. (198 1). Storage und reabsorption of‘ intestinal proteirr Our data give no indication that intestinal proteins After P. cchfornicu early-instar larvae were reare used for any one specific function during metamoved from ‘H-labelled amino acids, there was morphosis. Because excretion rates of tritiated water essentially no change in the amount of label in the from the insects were constant, and at the same time, intestinal tract after 73 days during autumn or for 63 depuration of tritium from the gut showed loss days during winter (Fig. 5). Penultimate instars houaccording to first-order kinetics, it is unlikely that ever. showed an intestinal depuration rate of 6”,, per intestinal proteins were used solely for respiration. day (I, = 11.3 d) during the non-feeding stage just When one considers that both male and female P. prior io emergence (Fig. 5). Penultimate instars exc~l~~iwnicohave very similar intestinal proteins in creted tritium label at a very consistent rate from 12 very similar amounts, it seems less surprising that this February until 26 March (Table I). This initially was protein was shown not to be incorporated into develvery surprising since we expected the labelled amino oping eggs. It seems more likely that the protein is acids to be very highly conserved by these insects. being used for a metabolic function similar in both When subsamples of the aquarium water were evapsexes. However, we did not use males in pulse-chase orated however, only S”,, of the radioactivity was experiments and can only assume, based on the retained in the sample (n = 5). suggesting that the electrophoretic and protein quantity data, that deputritium being excreted by the insect was metabolic ration of protein from male intestinal tracts would be water and not amino acids or protein. This hypothsimilar to females. In other insects, haemolymph esis is supported by comparing the form of tritium in storage proteins also were not used for egg prodthe gut and haemolymph of penultimate instars uction (Kramer et (11.. 1980; Levenbook and Bauer. (Table 2). Haemolymph samples contained a very low 1980). percentage of low molecular weight organic tritium We hypothesize that these proteins accumulate in (i.e. amino acids and peptides) whereas 30”,, of the the intestinal tract because they are used for the counts in the haemolymph was lost after evaporation digestion of detritus. Previous studies have shown (i.e. ‘H?O). Just the opposite distribution was present that the digestive enzymes of 3 stream detritivores, P. in the intestinal tract, where almost all of the label pictetti (Martin et al., 1981a). Tip& rrbdominalis was in the form of low molecular weight non-volatile (Martin et (II., 1980). and Pycnopsyche gum”& (Marmolecules or large molecular weight molecules. and tin et d.. 198 1b), and two stream omnivores, Phr_yguonly IO”;, was volatile. These results suggest that tleu sp. and Agrypnio oestitu (Martin et al., 198 I b), all amino acids were used for respiration during the have essentially the same digestive enzyme activities non-feeding period prior to emergence. as those found in P. cakfornica. This strongly suggests that these enzymes serve a similar digestive Because eggs were shown to develop during the function, although the mechanisms by which this 6- to &week period prior to adult emergence (Branoccurs remains unknown. ham, 1975). we considered the possibility that stored Martin et al. (1981) found no significant intestinal intestinal protein could be used for yolk synthesis. enzyme activity in P. californicu using the following This hypothesis was refuted by the finding that eggs substrates: microcrystalline cellulose, carboxy methyl lost label during this time (Fig. 6). cellulose. larchwood xylan, locust-bean gum, potato amylose. laminarin, citrus pectin, and y-chitin. AlDISCUSSION though this list is not exhaustive, it does eliminate many of the enzymes typically thought to be associOur results suggest that P. r&fitmica larvae accuated with detritus digestion. An obvious question mulate soluble protein in the intestinal tract. then use then is: what is the function of the ten or more this protein during metamorphosis. This pattern of proteins isolated by electrophoresis but having no protein accumulation before metamorphosis and dedemonstrable enzymatic activity? We were not able to cline during metamorphosis is analogous to patterns
*
I,
__--------
Fig. 2. S-PAGE (10% resolving, non-denaturing gels) of the intestinal proteins of P. californica reared for 1 month on: 1,4 = water birch leaves (Be&a occident&); 2,5,7,X = poplar leaves (Populus dellodes); 3,6 = willow leaves (Mix sp.); 9 = box elder leaves (Acer negundo).
1
2
3
4
5
6
7
8
9
10
Fig. 3. S-PAGE (13.5’T resolving, non-denaturing gels) of the intestinal proteins of P. californica from Mink Creek and Warm River, Idaho: 1 = late instar female (Warm River); 2 = late instar male (Warm River); 3 = intermediate instar male (Warm River); 4 = early instar male (Warm River); 5 = early instar female (Warm River); 6,10 = Mink Creek; 7 = 0.5 dilution of 1; 8 = 0.5 dilution of 3; 9 = 0.5 dilution of 5.
445
447
Intestinal protein in Pteronarcys cal~fornica Table 1. Excretion Date 12-26 Feb 26 Feb-I2 March 12-26 March
_
I 10
I 20
Fig. 4. Relation between tritium cpm per gel (10% stacking. non-denaturing) slice and relative amount of protein per gel slice after intestinal proteins from P. californica were subjected to electrophoresis. P -C0.001. See Methods for details.
assay for s( and fi-glucoside activity after electrophoresis, however both of these enzymes also may be comprised of a series of isozymes similar to the proteinase activity. In addition, because we used only one type of proteinase substrate, other protein or peptide hydrolyzing enzymes might be present. This would explain the discrepancy between the number of enzymes found in this insect and the number of proteins. There also may be problems with enzyme stability during enzyme assays. Terra et al. (1979) found that the sum of enzyme activities in different midgut sections could be greater or less than activities
E,
I
rates of ‘H for penultimate cpm excreted.g
instar P. californica
insect-“day-’
3514 3487 3615
r’(n = 7) 0.99 0.97 0.96
found in whole midgut homogenates. Various digestive enzymes may be present therefore, yet due to inhibitors and other enzymes in other parts of the intestinal tract, show no activity using typical assay methods. If the bulk of these proteins in P. californica are not digestive enzymes, it becomes very difficult to explain their presence in the intestinal tract. Regardless of the function of these intestinal proteins, an extremely efficient mechanism for protein and amino acid absorption must exist in the intestinal tract of this insect. P. californica consume approx 60% of their dry weight each day at 10°C (Poole, 1981). Our pulse-chase experiments and the excretion data in Table 1 however, indicate that little, if any of the amino acids or proteins are lost from the intestinal tract. Even when the insects were actively feeding, only labelled water was excreted. We considered the possibility that digestive proteins were bound in the plasma membrane covering intestinal cell microvilli as found by Terra et al. (1979, 1982, 1983). However, electrophoresis of solution withdrawn from an intact intestinal tract using a small capillary tube gave results identical to the electrophoresis patterns of proteins from homogenized tissue (data not shown). Our results therefore, support a model analogous to the one proposed by Terra and Ferriera (198 1) for digestive enzyme retention. In addition, we propose that in P. californica, digestive proteins are continually synthesized and accumulated in the gut, leading to a net increase in intestinal protein over time.
\ \
P
Fig. 5. Depuration plots of intestinal tritium from early instar (0) and penultimate instar (0) P. californica after larvae were labelled with a tritiated amino acid mixture. Two experiments are shown. In the first, early instar larvae were labelled from 8 July until 16 September and depuration was followed from 16 September until 27 November. In the second, both early and penultimate-instar females were labelled from 5 December until 22 January (break in abcissa) and depuration was followed for both groups until 26 March. Each point represents a single insect. For (0). rz = 0.86, P < 0.005.
Fig. 6. Tritium cpm in eggs taken from penultimate instar experiment described in Fig. 5. Each point represents the cpm of eggs taken from a single insect. r’ = 0.78. p < 0.02. P. californica larvae during the depuration
Table 2. Percentage distribution of lH cpm in the gut and haemolymph of penultimate instar P. calz~ornica sampled on 26 Feb, 12 March, and 26 March during depuration experiment R”,, + SE (n = 3) Sample Intestine Haemolymph
Lost after evaporation 8 f 1.45 30 * 6.06
Lost after dialysis but not after evap.
Remaining after dialysis
48 f 5.77 4 + 4.33
44 & 4.67 66 f 3.79
448
DAVID F. GRANT and G. WAYNE MINS~AL~
Because intestinal protein incorporated tritium labelled amino acids. these proteins are being synthesized either by the insect or by microorganisms. Sharma er al. (1984) have shown that proteinases in Tipula abclnminalis are not acquired from ingested microbes, but are produced by the insect. The intestinal tract of P. cul(jbrnica is essentially a straight tube and would give little opportunity for the establishment of a permanent gut flora. The consistency of the electrophoresis patterns of intestinal proteins from P. cal@rnica eating different leaf species and detritus types suggests that the insect. and not gut bacteria are synthesizing these proteins. Microbial synthesis cannot be totally disregarded since bacterial production of extracellular proteins is common (Glenn. 1976). Furthermore, there are some obvious problems with conclusions based on electrophoretic similarity (Johnson, 1977). The method we used to label protein gives no indication whether intestinal proteins can traverse the gut wall during early instars. Rapid exchange of label could occur and yet show the no net change in intestinal cpm that we observed for early instars. However. from electrophoresis and enzyme assays 01 haemolymph samples from early instars (data not shown), it seems unlikely that exchange of protein between the gut and haemolymph is occuring except during the 6-9 weeks during metamorphosis. We also have no indication of how proteins are processed as they leave the gut. The percentage distribution data used to compile Table 2 indicates that no particular size fraction of label was preferentially lost or was accumulated in the haemolymph during depuration. Additional studies will be required to determine how proteins are processed and where they are used once they leave the intestinal tract. There are several other questions that are posed by our results. For instance. how are proteinase enzymes prevented from digesting other soluble proteins in the intestinal tract during storage? There are several known protein inhibitors of proteinases (Leshowski and Kato. 1980). Whether any of these are present in P. cabfornica is unknown. It is also unknown how long intestinal enzymes can retain activity. Larvae of P. ca/@nica have a 3-year life span. It is doubtful that first instar enzymes remain active for 3 years. It seems more likely that digestive enzyme synthesis must account for both dilution and denaturation during growth. Another problem is the question of whether ingested microbes can use insect intestinal protein as a nutrient source. It is difficult to explain how P. cal$ornica can store protein in a form and place vulnerable to constant microbial attack. unless most of the protein was stored in the ectoperitrophic space. It also would be interesting to know if the quantity of intestinal protein remains constant during periods of food shortage when the insects would be forced to use metabolic reserves. From our results. we would speculate that if intestinal protein is used for metabolism prior to metamorphosis. larval survival will be enhanced at the expense of adult fitness. Ackno~cledRemunts-Earlier drafts of this manuscript were improved by comments from J. E. Anderson. D. A. Bruns, R. W. McCune, and J. R. Miller. This work was supported by a Grant-in-Aid of Research Idaho State University.
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by
REFERENCES Branham J. M. and Hathaway R. R. (1975) Sexual differences in the growth of Preronarc.vs californica (Newport) and Pteronarcella hndia (Hagen) (Plecoptera). Gun. .f. 2001. 53, 501-506. Collins J. V. (1975) Secretion and uptake of “C proteins by fat body of Culipodes ethlius Stall. (Lepidoptera, Hesperiidae). Dijerentiarion 3, 143-148. Dadd R. H. (1970) Digestion in insects. In The Ph_vsiolog> qf lnsectu (Ed. by Rockstein M.), 2nd edn. Vol. 5. pp. 63-117. Academic Press, New York. Ferreira C. and Terra W. R. (1982) Function of midgut caeca and ventriculus: microvilli bound enzymes from cells of different midgut regions of starving and feeding Rhynchosciura umericanu larvae. lnsrct Biochetn. 12, 251--16X Glenn A. R. (1976) Production of extracellular proteins by bacteria. .4. Rec. Microhiol. 30, 41-62. House H. L. (1974) Digestion. In The Physiology qf‘lnsecru (Ed. by Rockstein M.). 2nd edn. Vol. 5. pp. 63-117. Academtc Press, New York. Hynes H. B. N. (1976) Biology of Plecoptera. A. Rer. Em. 21, 1355153. Johnson G. B. (1977) Assessing electrophoretic similarity: the problem of hidden heterogeneity. Ann. Rera. Ecol. Sysr. 8, 309-328. Kley H. V. and Hale S. (1977) Assay for protein by dye binding. An&t. Biochem. 81, 485-487. Kramer S. J.. Mundall E. C. and Law J. H. (1980) Purification and properties of manducin, an amino acid storage protein of the haemolymph of larval and pupal Manducu SC.UU.Insect Biochem. 10, 279-288. Laskowski M. Jr and Kato I. (1980) Protein inhibitors of proteinases. A. Rev. Biochem. 49, 593-626. Levenbook L. and Bauer A. C. (1980) Calliphorin and soluble protein of haemolymph and tissues during larval growth and adult development of Calliphoriu tkino. ft~cr Biochem. 10, 693-701. Martin M. M.. Kukor J. J., Martin J. S.. Lawson D. L. and Merritt R. W. (198la) Digestive enzymes of larvae 01 three species of caddisflies (Trichoptera). Insect Biochem. 11, 501-505. Martin M. M.. Martin J. S.. Kukor J. J. and Merritt R. W. f I98 I b) The digestive enzymes of detritus-feeding stonefly nymphs (Plecoptera; Pteronarcidae). Con. J. Zool. 59, 1947-1951. Martin M. M., Martin J. S.. Kukor J. J. and Merritt R. W. ( 1980) The digestion of protein and carbohydrate by the stream detritivore. Tipulu abdominalis (Diptera. Tipulidae). Orcologiu (Berlin) 46, 360-364. Miller S. G. and Silhacek D. L. (1982a) Identification and purification of storage proteins in tissues of the greater wax moth Gulleriu mellonella (L.). Insect Biochem. 12, 117-192. Miller S. G. and Silhacek D. L. (1982b) The synthesis and uptake of haemolymph storage proteins by the fat body of the greater wax moth Gulleriu mellonellu (L.). Insecr Biochem. 12, 293-300. Miller S. G. and Silhacek D. L. (1982~) The effects of tunicamycin on the synthesis and export of fat body proteins and glycoproteins in larvae of the greater wax moth Galleria mellonellu(L.). tnsect Biochem. 12, 301-309. Munn E. A., Feinstein A. and Greville G. D. (1967) A major protein constituent of pupae of the blowfly, Culliphoru erylhrocrphalu (Diptera). Biochem. J. 102, 5-6. Munn E. A. and Grevill G. D. (1969) The soluble proteins of developing Calliphora erythrocephula and similar proteins in other insects. J. Insect Phvsiol. 15. 1935-1950. Poole W. C. (1981) The ecological bioenergetics of Pteronurc:vs culifornicu (Newport). Ph.D. Thesis. Idaho State University. 165 pp.
Intestinal protein in Pteronarcys culifornica Sharma B. R., Martin M. M. and Shafer J. A. (1984) Alkaline proteases from the gut fluids of detritus-feeding larvae of the crane fly, Tipula abdominalis (Say) (Diptera, Tipulidae). Insect Biochem. 14, 37-44. Terra W. R. and Ferreira C. (1981) The physiological role of the peritrophic membrane and trehalase: digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara. .I. Insect Physiol. 27, 325-331.
Terra W. R. and Ferreira C. (1983) Further evidence that enzymes involved in the &al stages of digestion by Rhynchosciaru do not enter the endoperitrophic space. Insect Biochem. 13, 143-150.
Terra W. R., Ferreira C. and De Bianchi A. G. (1979)
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Distribution of digestive enzymes among the endo- and ectoperitrophic spaces and midgut cells of Rhynchosciura and its physiological significance. J. Insect Physiol. 25, 487-494.
Tojo S., Nagata M. and Lobayashi M. (1980) Storage proteins in the silkworm, Eombyx mori. Insect Biochem. 10, 289-303.
Ueno K. and Natori S. (1982) Activation of fat body by 20-hvdroxvecdvsone for the selective incoporation of . storage protein in Sarcophaga peregrina larvae. Insect Biochem. 12, 185-191. Wyatt G. R. and Pan M. L. (1978) Insect plasma proteins. A. Rev. Biochem. 47, 779-817.
in Great
Bntain.
All rights reserved
INTESTINAL
PROTEIN
Copyright
DYNAMICS
PTERONARCYS
0022.1910!84 $3 00 + 0.00 (’ 1984 Pergamon Press Ltd
IN THE STONEFLY,
CALIFORNICA
DAVID F. GRANT* and G. WAYNE MINSHALL Department of Biology, Idaho State University. Pocatello, Idaho 83209. U.S.A. (Receired 27 Sepfemher
1983; revised 23 November 1983)
Abstract-Characteristics of the intestinal proteins of a detritus-eating aquatic insect, Preronarcys californica were assessed to determine their possible role as a nutrient source during metamorphosis. Total soluble intestinal protein was significantly correlated to larval weight, averaging 3.9”; of dry weight for both males and females. Proteinase activity in the gut was separated into several bands by electrophoresis. Electrophoresis patterns of total intestinal proteins of P. califbrnica larvae of differing instar, sex, and diet were remarkably similar. Larvae incubated with tritium-labelled amino acids produced labelled intestinal proteins. When early instar larvae were removed from label. there was no significant loss of tritium from the intestine after 2 months. Penultimate instars labelled in the same manner however. showed depuration of tritium from the gut according to first order kinetics. This loss was correlated to the 6-8 wk non-feeding stage of metamorphosis, just prior to adult emergence. These results indicate that proteins are accumulated in the intestinal tract of immature P. caljfornica, then are reabsorbed from the gut during metamorphosis. Kev Word Index: Intestinal protein, metamorphosis. digestive enzymes. aquatic detritivore, Pteronarc.w
INTRODUCTION Because metamorphosis is often accompanied by a dramatic change in the habitat of an insect, many proteins essential during immature stages may be useless, as such, to the adult. Digestive enzymes in the intestinal tract of immature insects entering a nonfeeding adult stage would be one such pool of protein. In general, the fate of insect digestive proteins remains largely speculative (Dadd, 1970; House. 1974; Terra and Ferreira, 1981). Conservation and re-use of intestinal enzymes has been suggested in a model given for Rhynchosciara americana larvae (Terra and Ferreira, 1981). This model describes a possible mechanism whereby digestive enzymes are circulated within the endo- and ectoperitrophic spaces of the intestinal tract. This model raises two questions concerning the storage and fate of digestive enzymes. First, in insects which are constantly feeding, do digestive proteins accumulate within the intestinal tract, thereby maintaining constant protein concentration and digestive capabilities as the insect grows? Second, if such proteins are accumulated during feeding stages, what is their fate once the insect becomes nonfeeding? To address these questions. we studied the intestinal protein dynamics of an aquatic detritivore, Ptcronarcy cal~fornicu. P. californica larvae have been shown to possess high proteinase activity in the intestinal tract as well as enzymes which hydroloyze r-l.4 and /?-1,3-glucans (Martin et al.. 1981). Because larvae do not eat for approx 6-8 weeks before they emerge from the stream as non-feeding adults (Poole. 1981; Hynes. 1976), this 6- to 8-week period provides ~~~ _ ___. ~_ .._~ ~~ *To whom correspondence should be addressed. Present address: Department of Entomology, Michigan State University. East Lansing, Michigan 48824. U.S.A.
a good opportunity to monitor the fate of intestinal proteins. This time is also crucial for P. cal~fbrnicu because eggs, wings, and other adult structures are believed to develop fully (Branham, 1975). In this paper we give evidence supporting the hypothesis that a significant amount of soluble protein is synthesized and accumulated in the intestinal tract of P. calijhzica. This intestinal protein is composed of at least 6 digestive enzymes, and is used during the 6- to 8-week non-feeding stage just prior to adult emergence. MATERIALS AND METHODS Animuls Pteronarqs califbrnica (Newport) larvae were collected from Mink Creek, a third-order hardwater stream in Bannock County, Idaho. The larvae were either used immediately or were kept in Mink Creek water in 4-l jars. Those kept in the laboratory were maintained at ambient stream conditions (photoperiod and temperature) in a covered refrigeration unit. Compressed air was used to aerate laboratory insects. Additional P. cal~fhnica larvae were collected from Warm River. a fifth-order hardwater stream in Fremont County, Idaho. Larvae from Warm River were used immediately for a single electrophoresis experiment. Quailtity qf‘ intestinrrl protein
Because of problems measuring total protein from biological samples of unknown composition (Levenbook and Bauer. 1980; Kleg and Hale, 1977). we used a gravimetric technique. Eight P. cal$ornica collected from Mink Creek on 29 November 1981. were transfered to the laboratory, blotted dry. and weighed. The intestinal tracts were removed by cutting off the last abdominal segment. and the head just 441
442
DAVIU F. GRANT and G. WAYW~ MIXSHALL
behind the eyes, then pulling out the intestine from the caudal end of the insect with forceps. The intestinal tract from each insect was broken open and minced by hand in a I-ml conical centrifuge tube using a small metal spatula. We felt this procedure maximized dissolution of lumen contents and minimized protein release from gut tissue. Each homogenate was washed x 3 by centrifugation (40,000~. IO min. 4’ C) in 0.5 ml distilled water. One ml of 60”,, trichloroacetic acid was then added to precipitate the collected supernatant fraction for each of the 8 samples. After I h in an ice bath, each sample was filtered onto a tared. predried glass-fibre filter. The filters were dried overnight at 95 C and reweighed on a Cahn electrobalance. Identical procedures were used for 3 control filters. To convert insect wet weights to dry weights, IO P. cul~$micu larvae were killed (by removing the air supply for 2 h), blotted dry, and weighed. They were dried overnight at 100 C and reweighed. Proteinuse
enynes
To determine if proteinase enzyme activity was represented by more than one protein, slab polyacrylamide gel electrophoresis (S-PAGE) was used to separate proteins prior to enzyme assays, Intestinal proteins were prepared for S-PAGE by homogenizing intestinal tracts in a centrifuge tube, centrifuging the sample (40,000~. IO min. 4’C), and collecting the supernatant fluid. The proteins were then concentrated by lyophilization. Proteins were rehydrated in buffer (IO”,, sucrose in 0.5 M Tris. pH 6.8) and applied to gels in duplicate. One column was sliced and used for proteinase assays, the other column was stained for 30 min in 0.05”, Coomassie blue in 45”,, methanol-lo”,, acetic acid. A densitometer tracing was obtained after destaining overnight in lo”,, methanol-7.5 I,,) acetic acid. Proteins were electrophoresed on 3”,, stacking and either IO or 13”,, resolving, non-denaturing slab gels crosslinked with his-acrylamide using IO”, ammonium persulphate (20 mA per slab, 1 I C, 6 h). The running buffer was 0.3”,, Trisl.4”,, glycine, pH 8.3. Each strip from the sliced column was then placed in a small test tube in 3 ml of 0.5 M Tris buffer. pH 6.8, and eluted overnight at 4 C. The liquid from each tube was added to proteinase substrate (10 mg Azocoll, Sigma) and incubated. Two assays were performed: In one. the proteins eluted from the gels were incubated with substrate at room temperature for 4 h. In the other, the separated proteins were incubated with substrate at 4 C for 7d. The absorbance of each tube was measured according to Martin et (I/. (1980) except that we report relative values rather than absolute activity. The relative proteinase activity in each of the gel sections was plotted with the densitometer tracings to correlate protein bands with enzyme activity. Source of’ intestinal protein Because intestinal proteins may be derived from both food and bacterial sources, it is important to determine the influence of diet on intestinal protein characteristics. We approached this problem in two ways. The first was to compare the electrophoresis patterns of intestinal proteins from P. cul(~brnicu reared on different diets and from different streams.
The second approach was to determine whether intestinal proteins were being synthesized from amino acids. or were derived directly from a non-living food source. To determine the effect of diet on intestinal protein patterns, P. calijimicu larvae were reared for I month in the laboratory on four different leaf species: Bctuiu occidentalis, Popuh deltoides, Suli.\r sp. and Acrr negundo. For all of these comparisons. the intestinal tract was homogenized, centrifuged. and the lyophilized supernatant fluid used for nondenaturing electrophoresis as described earlier, except that enzyme activity was not measured. P. cul{fbrnica from Warm River and Mink Creek were used for the between-stream comparison. A tritium-labelled amino acid mixture (ICN Chemical and Radioisotope Division) was used to distinguish between a living and a detrital source of intestinal protein. Three hundred PCi of [‘H]labelled amino acids were added to a 2-l beaker containing I I of Mink Creek water. Two small water-birch logs were added for food. Seven P. cul$ornica larvae were collected on 8 July 1981 and allowed to incorporate the label for 2 months. None of these insects were penultimate instars. One of the insects was then removed (the remaining insects were used as described below) and the soluble intestinal contents were subjected in duplicate to electrophoresis. One column was then sliced into 40 equal sections and the other was stained with Coomassie blue. The sliced sections were eluted overnight and the eluant from each (excluding the marker-dye front) was counted in 10 ml of Insta-Gel (Packard) on a Beckman liquid scintillation counter. The cpm in each section was plotted on the densitometer tracing from the stained gel column. This plot was photocopied, and the densitometer tracing cut out. The tracing was cut into 40 equal sections and each section weighed on a Cahn electrobalance. We then plotted the relative amount of protein in each section ( = weight of paper in that section) versus the cpm in each section. Storuge und reahsorptior~ of’ intestinul protein The 6 P. cu/ifOrnicu remaining from the above labelling experiment were used to determine how quickly label was lost from the intestinal tract. The insects were placed in label-free water changed biweekly. Starting 16 September one insect was removed every 2 weeks. blotted dry, and weighed. The intestinal tract was removed. homogenized, and the supernatants from 3 centrifugation washes (40,OOOb. IO min. 4 C) were collected and weighed. A weighed subsample was then taken. added to scintillation fluid (Insta Gel, Packard) and counted. Using this procedure the total cpm in the intestinal tract per wet weight insect was monitored over time. A similar pulse-chase experiment was begun on 5 December 1981 using penultimate instars as well as earlier ( < nenultimatel instars. Seven randomlv collected penultimate-ins&r females and 7 earlier-&tar female larvae were combined in I 1 of Mink Creek water and labelled using 300 uCi of [‘HIamino acid mixture from 5 December until 22 January 1982. The two groups were then separated and placed in labelfree water (changed bimonthly) until 26 March. the normal emergence time for P. c,ulif;wnicu adults in
Intestinal protein in Prer0narq.s cakfornica
0
38
33
28
23
18
13
443
8
0
Gel section Fig. 1, Densitometer tracing (curves) of proteins separated by S-PAGE (IO:,, resolving. non-denaturmg gels), plotted with the relative proteinase activity (I-Transmittance) in gel sections assayed at 22 and 5 ‘C. Proteins migrated from right to lefty
Idaho. From 22 January until 26 March 1982. one insect from each group was collected and the total radioactivity in the intestinal tract was measured as described above. For the early-instar larvae, no other measurements were taken. For penultimate-instar larvae, developing eggs were collected at each sampling period, dissolved by boiling in loo/;, SDS. and the radioactivity counted. 3H excretion rates for penultimate-instar larvae were measured during the last three, 2-wk intervals between aquarium water changes. These were measured by finding the cpm in a weighed amount of aquarium water every other day (n = 7 for each rate determination), multiplying this by the total amount of water in the aquarium and dividing by the total weight of insects in the aquarium during the 2-wk interval. We also measured the distribution of tritium label from intestinal tract and haemolymph of penultimate instar P. californica. The cpm were subdivided into large molecular weight (cpm retained by dialysis), non-volatile small molecular weight (cpm lost after dialysis, but not after evaporation), and volatile small molecular weight (cpm lost after dialysis and evaporation to dryness). We assumed that volatile small molecular weight cpm was tritiated water. These 3 fractions were expressed as percentages of total cpm by comparing the cpm for dialyzed samples (corrected for dilution) and the cpm of evaporated samples, with untreated gut or haemolymph samples and assumed that volatile cpm represented the difference. RESULTS
The gravimetric protein assays of P. cafijornica soluble intestinal contents gave the following results expressed as percentages ( + 95% CI): Females, X = 3.39% protein ( + 0.99, n = 3); males, X = 4.19% protein ( f 1.24. n = 5); and for males and females
combined, x = 3.89% protein ( f 0.74, n = 8). Using the average of 3.89% intestinal protein per dry weight insect for both males and females and an approximate gut volume for penultimate-instar larvae of 0.2 ml (unpublished data), the concentration of protein in the intestinal tract would be approx 3.4”d. Proteinase enzyme activity was detected in several areas on S-PAGE at both room temperature and at 5°C (Fig. 1). Temperature did not seem to cause a major shift of enzyme activity, but there was a slight decrease in section 21 and an increase in sections 29, 33 and 37 at the 5~C assay. We attempted to repeat these electrophoretic enzyme assays for the a and @glucosidase activity previously found by Martin et al. (1981) but were unable to recover enzyme activity after electrophoresis. Source qf intestinal proteins The electrophoresis patterns of the intestinal protein from P. calijbrnica larvae reared on 4 different diets shows remarkable similarity among all four groups (Fig. 2). There is some indication that insects feeding on willow (lanes 3 and 6) had 1 or 2 qualitative differences in their intestinal protein patterns, however diet is clearly not a major factor influencing these electrophoretic patterns. When the intestinal proteins of P. californica from Warm River are compared to those from Mink Creek (Fig. 3) extremely similar patterns again are shown, Gut analysis showed that P. californica from Warm River were eating only autochthonous detritus (i.e. algae and diatoms) at the time of collection. Poole (personal communication) also has concluded that P. cahf~~rnica in Warm River eat autochthonous detritus as their main food source. This is in contrast to the
allochthonous food base (mostly water-birch logs) for Mink Creek P. californica. This consistency of the electrophoresis patterns is similar for insects collected
444
DAVID F. GRANT and G. WAYNE MINSHALL
from Mink Creek during all seasons and for both sexes (data not shown), strongly suggesting that proteins in the intestinal tract are being synthesized by the insect. Figure 4 shows the relationship between the amount of tritium cpm per gel slice and the relative amount of protein per gel slice after intestinal proteins from P. c&@nicrr reared (for 8 weeks) in water containing ‘H-labelled amino acids were separated on S-PAGE. Although the amount of variation in tritium activity is only partly explained by the amount of protein (r = 0.81) the correlation is very highly significant (P < 0.001). These data suggest that the specific rate of label incorporation was similar for each of the intestinal proteins, although the total counts incorporated were low. These data also strongly indicate that intestinal proteins are being synthesized by the insect and eliminates the possibility that they are originating from a non-living food source such as detritus cell-wall proteins.
found for so called storage proteins in the haemolymph of other insects. Haemolymph storage proteins have been shown to be used during metamorphosis for adult development, and have been isolated from Diptera (Munn et al., 1967; Munn and Grevill, 1969; Ueno and Natori, 1982) and from Lepidoptera (Collins, 1975: Wyatt and Pan, 1978; Tojo et al., 1978, 1980: Kramer et ~1.. 1980; Miller and Siehocek, 1982a.b.c). Intestinal proteins found in P. cnlifornica may serve a function during metamorphosis analogous to storage proteins. It is interesting to note that on a percentage basis, the amount of protein accumulated in the gut of P. cal(fornica is similar to the amount of storage protein accumulated in other insects (Kramer et al.. 1980: Levenbook and Bauer. 1980). However, the protein in P. ca/[fornicu is different from haemolymph storage protein not only because it is stored in the intestinal tract, but because some of these proteins are enzymes: The proteinase enzymes found in this study and the r and /I-glucosidase enzymes found by Martin ef ul. (198 1). Storage und reabsorption of‘ intestinal proteirr Our data give no indication that intestinal proteins After P. cchfornicu early-instar larvae were reare used for any one specific function during metamoved from ‘H-labelled amino acids, there was morphosis. Because excretion rates of tritiated water essentially no change in the amount of label in the from the insects were constant, and at the same time, intestinal tract after 73 days during autumn or for 63 depuration of tritium from the gut showed loss days during winter (Fig. 5). Penultimate instars houaccording to first-order kinetics, it is unlikely that ever. showed an intestinal depuration rate of 6”,, per intestinal proteins were used solely for respiration. day (I, = 11.3 d) during the non-feeding stage just When one considers that both male and female P. prior io emergence (Fig. 5). Penultimate instars exc~l~~iwnicohave very similar intestinal proteins in creted tritium label at a very consistent rate from 12 very similar amounts, it seems less surprising that this February until 26 March (Table I). This initially was protein was shown not to be incorporated into develvery surprising since we expected the labelled amino oping eggs. It seems more likely that the protein is acids to be very highly conserved by these insects. being used for a metabolic function similar in both When subsamples of the aquarium water were evapsexes. However, we did not use males in pulse-chase orated however, only S”,, of the radioactivity was experiments and can only assume, based on the retained in the sample (n = 5). suggesting that the electrophoretic and protein quantity data, that deputritium being excreted by the insect was metabolic ration of protein from male intestinal tracts would be water and not amino acids or protein. This hypothsimilar to females. In other insects, haemolymph esis is supported by comparing the form of tritium in storage proteins also were not used for egg prodthe gut and haemolymph of penultimate instars uction (Kramer et (11.. 1980; Levenbook and Bauer. (Table 2). Haemolymph samples contained a very low 1980). percentage of low molecular weight organic tritium We hypothesize that these proteins accumulate in (i.e. amino acids and peptides) whereas 30”,, of the the intestinal tract because they are used for the counts in the haemolymph was lost after evaporation digestion of detritus. Previous studies have shown (i.e. ‘H?O). Just the opposite distribution was present that the digestive enzymes of 3 stream detritivores, P. in the intestinal tract, where almost all of the label pictetti (Martin et al., 1981a). Tip& rrbdominalis was in the form of low molecular weight non-volatile (Martin et (II., 1980). and Pycnopsyche gum”& (Marmolecules or large molecular weight molecules. and tin et d.. 198 1b), and two stream omnivores, Phr_yguonly IO”;, was volatile. These results suggest that tleu sp. and Agrypnio oestitu (Martin et al., 198 I b), all amino acids were used for respiration during the have essentially the same digestive enzyme activities non-feeding period prior to emergence. as those found in P. cakfornica. This strongly suggests that these enzymes serve a similar digestive Because eggs were shown to develop during the function, although the mechanisms by which this 6- to &week period prior to adult emergence (Branoccurs remains unknown. ham, 1975). we considered the possibility that stored Martin et al. (1981) found no significant intestinal intestinal protein could be used for yolk synthesis. enzyme activity in P. californicu using the following This hypothesis was refuted by the finding that eggs substrates: microcrystalline cellulose, carboxy methyl lost label during this time (Fig. 6). cellulose. larchwood xylan, locust-bean gum, potato amylose. laminarin, citrus pectin, and y-chitin. AlDISCUSSION though this list is not exhaustive, it does eliminate many of the enzymes typically thought to be associOur results suggest that P. r&fitmica larvae accuated with detritus digestion. An obvious question mulate soluble protein in the intestinal tract. then use then is: what is the function of the ten or more this protein during metamorphosis. This pattern of proteins isolated by electrophoresis but having no protein accumulation before metamorphosis and dedemonstrable enzymatic activity? We were not able to cline during metamorphosis is analogous to patterns
*
I,
__--------
Fig. 2. S-PAGE (10% resolving, non-denaturing gels) of the intestinal proteins of P. californica reared for 1 month on: 1,4 = water birch leaves (Be&a occident&); 2,5,7,X = poplar leaves (Populus dellodes); 3,6 = willow leaves (Mix sp.); 9 = box elder leaves (Acer negundo).
1
2
3
4
5
6
7
8
9
10
Fig. 3. S-PAGE (13.5’T resolving, non-denaturing gels) of the intestinal proteins of P. californica from Mink Creek and Warm River, Idaho: 1 = late instar female (Warm River); 2 = late instar male (Warm River); 3 = intermediate instar male (Warm River); 4 = early instar male (Warm River); 5 = early instar female (Warm River); 6,10 = Mink Creek; 7 = 0.5 dilution of 1; 8 = 0.5 dilution of 3; 9 = 0.5 dilution of 5.
445
447
Intestinal protein in Pteronarcys cal~fornica Table 1. Excretion Date 12-26 Feb 26 Feb-I2 March 12-26 March
_
I 10
I 20
Fig. 4. Relation between tritium cpm per gel (10% stacking. non-denaturing) slice and relative amount of protein per gel slice after intestinal proteins from P. californica were subjected to electrophoresis. P -C0.001. See Methods for details.
assay for s( and fi-glucoside activity after electrophoresis, however both of these enzymes also may be comprised of a series of isozymes similar to the proteinase activity. In addition, because we used only one type of proteinase substrate, other protein or peptide hydrolyzing enzymes might be present. This would explain the discrepancy between the number of enzymes found in this insect and the number of proteins. There also may be problems with enzyme stability during enzyme assays. Terra et al. (1979) found that the sum of enzyme activities in different midgut sections could be greater or less than activities
E,
I
rates of ‘H for penultimate cpm excreted.g
instar P. californica
insect-“day-’
3514 3487 3615
r’(n = 7) 0.99 0.97 0.96
found in whole midgut homogenates. Various digestive enzymes may be present therefore, yet due to inhibitors and other enzymes in other parts of the intestinal tract, show no activity using typical assay methods. If the bulk of these proteins in P. californica are not digestive enzymes, it becomes very difficult to explain their presence in the intestinal tract. Regardless of the function of these intestinal proteins, an extremely efficient mechanism for protein and amino acid absorption must exist in the intestinal tract of this insect. P. californica consume approx 60% of their dry weight each day at 10°C (Poole, 1981). Our pulse-chase experiments and the excretion data in Table 1 however, indicate that little, if any of the amino acids or proteins are lost from the intestinal tract. Even when the insects were actively feeding, only labelled water was excreted. We considered the possibility that digestive proteins were bound in the plasma membrane covering intestinal cell microvilli as found by Terra et al. (1979, 1982, 1983). However, electrophoresis of solution withdrawn from an intact intestinal tract using a small capillary tube gave results identical to the electrophoresis patterns of proteins from homogenized tissue (data not shown). Our results therefore, support a model analogous to the one proposed by Terra and Ferriera (198 1) for digestive enzyme retention. In addition, we propose that in P. californica, digestive proteins are continually synthesized and accumulated in the gut, leading to a net increase in intestinal protein over time.
\ \
P
Fig. 5. Depuration plots of intestinal tritium from early instar (0) and penultimate instar (0) P. californica after larvae were labelled with a tritiated amino acid mixture. Two experiments are shown. In the first, early instar larvae were labelled from 8 July until 16 September and depuration was followed from 16 September until 27 November. In the second, both early and penultimate-instar females were labelled from 5 December until 22 January (break in abcissa) and depuration was followed for both groups until 26 March. Each point represents a single insect. For (0). rz = 0.86, P < 0.005.
Fig. 6. Tritium cpm in eggs taken from penultimate instar experiment described in Fig. 5. Each point represents the cpm of eggs taken from a single insect. r’ = 0.78. p < 0.02. P. californica larvae during the depuration
Table 2. Percentage distribution of lH cpm in the gut and haemolymph of penultimate instar P. calz~ornica sampled on 26 Feb, 12 March, and 26 March during depuration experiment R”,, + SE (n = 3) Sample Intestine Haemolymph
Lost after evaporation 8 f 1.45 30 * 6.06
Lost after dialysis but not after evap.
Remaining after dialysis
48 f 5.77 4 + 4.33
44 & 4.67 66 f 3.79
448
DAVID F. GRANT and G. WAYNE MINS~AL~
Because intestinal protein incorporated tritium labelled amino acids. these proteins are being synthesized either by the insect or by microorganisms. Sharma er al. (1984) have shown that proteinases in Tipula abclnminalis are not acquired from ingested microbes, but are produced by the insect. The intestinal tract of P. cul(jbrnica is essentially a straight tube and would give little opportunity for the establishment of a permanent gut flora. The consistency of the electrophoresis patterns of intestinal proteins from P. cal@rnica eating different leaf species and detritus types suggests that the insect. and not gut bacteria are synthesizing these proteins. Microbial synthesis cannot be totally disregarded since bacterial production of extracellular proteins is common (Glenn. 1976). Furthermore, there are some obvious problems with conclusions based on electrophoretic similarity (Johnson, 1977). The method we used to label protein gives no indication whether intestinal proteins can traverse the gut wall during early instars. Rapid exchange of label could occur and yet show the no net change in intestinal cpm that we observed for early instars. However. from electrophoresis and enzyme assays 01 haemolymph samples from early instars (data not shown), it seems unlikely that exchange of protein between the gut and haemolymph is occuring except during the 6-9 weeks during metamorphosis. We also have no indication of how proteins are processed as they leave the gut. The percentage distribution data used to compile Table 2 indicates that no particular size fraction of label was preferentially lost or was accumulated in the haemolymph during depuration. Additional studies will be required to determine how proteins are processed and where they are used once they leave the intestinal tract. There are several other questions that are posed by our results. For instance. how are proteinase enzymes prevented from digesting other soluble proteins in the intestinal tract during storage? There are several known protein inhibitors of proteinases (Leshowski and Kato. 1980). Whether any of these are present in P. cabfornica is unknown. It is also unknown how long intestinal enzymes can retain activity. Larvae of P. ca/@nica have a 3-year life span. It is doubtful that first instar enzymes remain active for 3 years. It seems more likely that digestive enzyme synthesis must account for both dilution and denaturation during growth. Another problem is the question of whether ingested microbes can use insect intestinal protein as a nutrient source. It is difficult to explain how P. cal$ornica can store protein in a form and place vulnerable to constant microbial attack. unless most of the protein was stored in the ectoperitrophic space. It also would be interesting to know if the quantity of intestinal protein remains constant during periods of food shortage when the insects would be forced to use metabolic reserves. From our results. we would speculate that if intestinal protein is used for metabolism prior to metamorphosis. larval survival will be enhanced at the expense of adult fitness. Ackno~cledRemunts-Earlier drafts of this manuscript were improved by comments from J. E. Anderson. D. A. Bruns, R. W. McCune, and J. R. Miller. This work was supported by a Grant-in-Aid of Research Idaho State University.
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