The digestive enzymes of larvae of the black fly, Prosimulium fuscum (Diptera, Simuliidae)

Comp. Biochem. Physiol. Vol. 82B, No. 1, pp. 3239, 1985 Printed in Great Britain

0305-0491/85 $3.00+ 0.00 © 1985PergamonPress Ltd

THE DIGESTIVE ENZYMES OF LARVAE OF THE BLACK FLY, P R O S I M U L I U M F U S C U M (DIPTERA, SIMULIIDAE) M. M. MARTIN,* J. J. KUKOR*, J. S. MARTIN* and R. W. MERRITTt *Division of Biological Sciences, University of Michigan, Ann Arbor, M148109, USA (Tel: 313-764-7376); and tDepartment of Entomology, Michigan State University, East Lansing, MI 48824, USA (Received 31 January 1985)

Abstract--1. The digestive system of larvae of the black fly, Prosimuliumfuscum, resembles those of other aquatic dipteran detritivores, such as mosquito and crane fly larvae. 2. Midgut proteolytic activity is very high, due to the presence of high levels of alkaline proteinases. 3. The only polysaccharides toward which significant enzymatic activity is evident in midgut extracts are ~-1,4- and fl-l,3-glucans. 4. There is negligible activity toward cellulose, carboxymethylcellulose,xylan, locust bean gum, pectin, chitin, Micrococcus lysodeikticus cells, and phenolphthalein mono-fl-glucuronate. 5. The larvae are better adapted to exploit the polysaccharides of fungi and algae than those of vascular plants. INTRODUCTION Black fly larvae are aquatic filter feeders that ingest particulate matter ranging in size from 0.091 # m (colloidal) to 350/~m, with most studies reporting sizes below 100 # m (Wallace and Merritt, 1980). The ingested material includes bacteria (Fredeen, 1964; G16tzel, 1973; Baker and Bradnam, 1976), protozoa (Anderson and Dicke, 1960), spores and hyphae of fungi (Anderson and Dicke, 1960), diatoms and other algae (Carlsson, 1962; Burton, 1973; Carlsson et al., 1977; Schrrder, 1981, 1983), microinvertebrates (Puri, 1925; Maitland and Penny, 1967; Serra-Tosio, 1967), plant detritus (Maitland and Penny, 1967; Maciolek and Tunzi, 1968; Carlsson et aL, 1977) and particles of silt and sand (Anderson and Dicke, 1960). This study is an investigation of the digestive biochemistry of the larvae of Prosimulium f u s c u m Syme and Davies. The larvae of this species are abundant on stream vegetation and rocks throughout the northeastern United States (Merritt et al., 1978). Eggs hatch in mid-November in Michigan and develop slowly during the winter months. Emergence occurs between late March and early May. During the larval stage, this species ingests detritus particles in the 0.45-45 # m size class almost exclusively (Merritt et al., 1982). We have characterized the enzymes of the gut fluid to determine which of the major structural and storage polysaccharides of higher plants, algae, fungi and animals the black fly larvae are able to digest, and to determine whether their gut fluid possesses the high proteolytic capacity typical of other aquatic insect detritivores, such as the nymphs of stoneflies (Plecoptera) (Martin et al., 1981a) and the larvae of caddisflies (Trichoptera) (Martin et al., 1981b) and crane flies (Diptera) (Martin et al., 1980). M E T H O D S AND

MATERIALS

Animals Prosimuliumfuscum larvae were collected in March from Mud Creek, a permanent first-order stream draining a small

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marsh lake in Ingham County in the southcentral portion of Michigan's lower peninsula. During the 1-3 day interval between collection and analysis, the insects were maintained in the laboratory at 5-10°C in an aerated aquarium containing water collected from the stream of origin. There was no significant mortality during this period. Preparation of extracts Live insects were dissected under distilled water by removing the head capsule with forceps and disconnecting the foregut by pinching with forceps. After clearing connections of the anterior end of the gut with other tissues, the gut was pulled out gently, severed at the point of insertion of the Malpighian tubules, removed, and placed in a small homogenizing tube in an ice bath. Midguts, with contents intact, from 30 to 100 individuals were homogenized by hand in about 1 ml of distilled water. Following centrifugation (10,000 g, 20 rain, 5°C), the pellet was resuspended in about 1 ml of water, centrifuged as before, and the supernatant solution combined with the original extract. The extract was made up to a volume which was an integral multiple of 2.5ml, and 2.5ml aliquots were placed on a Sephadex G-25M column (1.7 x 5.0 cm, Pharmacia Fine Chemicals, PD-10 Columns). Proteins were eluted in 3.5 ml of water. Enzyme assays Proteolytic activity was assayed using Azocoll (Calbiochem 19493) as the substrate (Martin et al., 1981a,b). Activities toward microcrystalline cellulose (Polyscience 4853), potato amylose (Calbiochem 17681), carboxymethylcellulose (Sigma C-8758), laminarin (Calbiochem 428001), larchwood xylan (United States Biochemicals 12077), locust bean gum (Sigma G-0753), citrus pectin (Sigma P-9135) and ),-chitin (Calbiochem 220463) were assayed by measuring the rate of liberation of reducing groups (maltose equivalents) using the 3,5,-dinitrosalicylicacid reagent (Martin et al., 1981c). Lysozyme activity was assayed by measuring the decrease in turbidity of a suspension of Micrococcus lysodeikticus cells (Sigma M-0128), by following the change in A4~0 in the incubation mixture, as described in the Sigma technical bulletin supplied with lysozyme (Sigma L-6876). fl-Glucuronidase activity was assayed by measuring the rate of liberation of phenolphthalein from the sodium salt of phenolphthalein mono-fl-glucuronic acid (Sigma P4262), by following the change in A~0 in the incubation

M. M. MARTINet aL

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Table I. Activity of midgut homogenates from larvae of Prosimulium fuscum toward Azocoll, potato amylose and laminarin Activity* T (~C)

pH

37 37 37 8 8

7.5 9.0 11.5 9.0 11.5

Proteinase (U/mg dry wt) 280 + 14 (2) 471 + 2 2 ( 3 ) 777_+85(3) NDI 7 (2)

Amylase (U x 103/mg dry wt) 18.2 (1) 13.1 + 1.4(3) 0.9 (1) 2.7 (1) ND

Laminarinase (U × 103/mg dry wt) 24.4 (1) 11.2 + 0.6 (3) 3.2 (1)

l.O

(1) ND

*One unit of activity toward Azocoll is the amount of enzyme required to bring about a change in absorbance at 520nm of 0.001 absorbance unit per minute under the conditions of the assay. One unit of activity toward amylose or laminarin is the amount of enzyme required to liberate one micromole of maltose equivalents per minute under the conditions of the assay. Each entry is the mean + SEM of determinations made on the number of separate extracts indicated in parentheses. t N D , not determined.

mixture as described in the Sigma technical bulletin supplied with fl-glucuronidase (Sigma G-0751). Controls in all of the above enzyme assays were run using aliquots of extracts inactivated by heating. The buffers used routinely in these assays were 0.1 M acetate (pH 5.0), 0.066M phosphate (pH 6.24), 0.1M phosphate (pH 7.5, 11.5), and 0.1 M Tris (pH 9.0). RESULTS AND DISCUSSION

The midgut extract from P. fuscum exhibits high activity toward the general protease substrate, Azocoll (Table 1). The level of activity falls in the same high range recorded in other aquatic detritivores, including larvae of the crane fly, Tipula abdominalis (Martin et al., 1980), larvae of the caddisffies, Pycnopsyche guttifer, Agrypnia vestita and Phryganea sp. (Martin et al., 1981b), and nymphs of the stoneflies, Pteronarcys pictetii and P. californicus (Martin et al., 1981a). Maximum activity of the proteinases occurs at very high pH. Since the midguts of black fly larvae are highly alkaline, with pH's above 11 over 40% of their length (Lacey and Federici, 1979), it is clear that the conditions which prevail in the digestive tract are highly conducive to protein digestion. Even at 8°C, a temperature that the larvae normally encounter in natural streams, proteolytic activity is at a functionally significant level. Similar alkaline proteinases, adapted to function in highly alkaline guts, have been detected in other dipteran detritivores, including mosquito larvae (Dadd, 1975) and crane fly larvae (Martin et al., 1980; Sharma et al., 1984). We have tested the activity of midgut extracts of P. fuscum toward a variety of polysaccharide substrates, representative of major classes of polysaccharides that occur naturally in vascular plants, algae, fungi, bacteria, and animals. The pattern of activity we have observed is very similar to the one observed earlier in the crane fly, T. abdominalis (Martin et al., 1980). T. abdominalis is also a detritus-feeder, but it is a shredder, not a filter-feeder, ct-Amylose (an ~-l,4glucan) and laminarin (a fl-l,3-glucan) are the only two substrates toward which significant activity was observed (Table 1). Because of the virtual inactivity of these enzymes at very high pH, their participation in digestion would be restricted to the anterior and posterior sections of the midgut where pH values are below 10 (Lacey and Federici, i979). Although neither ~-1,4- nor fl-l,3-glucans are quantitatively significant constituents of dead tissues derived from

vascular plants, both of these classes of polysaccharides are present in algae and fungi. Glycogen, the major storage polysaccharide of animals, is also an ct-l,4-glucan. Thus, to the extent that fungal tissue, diatoms, fragments of filamentous algae, and microinvertebrates are ingested, digestive ~-I,4and r-1,3-glucanases are of adaptive value. No activity could be detected toward chitin, which is a major component of fungal cell walls and arthropod integument. The midgut fluid of P. fuscum does not possess significant activity toward cellulose, hemicellulose or pectin, which are the three major structural polysaccharides of vascular plants. No activity could be measured toward microcrystalline cellulose at 37°C and pH 9.0, and only traces of activity were detected toward carboxymethylcellulose, larchwood xylan (representative of the hemicelluloses containing a fl-l,4-xylan backbone), locust bean gum (representative of the hemicelluloses containing a galacto-fl- 1,4mannan backbone), or citrus pectin. Bacteria are assimilated with high efficiency by black fly larvae (Baker and Bradnam, 1976), and can be used as the exclusive food for several simuliid species in laboratory cultures (Fredeen, 1964). Two enzymes that have been associated with bacterial lysis and digestion are lysozyme and fl-glucuronidase, which attack cell wall peptidoglycans and cell surface mucopolysaccharides, respectively. We could detect no activity in the P. fuscurn gut fluid attributable to either of these classes of enzymes. No activity toward suspended cells of Micrococcus lysodeikticus was evident at 8 or 22°C at pH 6.24 or 9.0 and no activity toward monosodium phenolphthalein mono-fl-glucuronate was detected at 8 or 37°C at pH 5.0. We presume that the lysis and digestion of ingested bacteria would be achieved by the active proteolytic enzymes of the gut fluid. Even though bacteria may be efficiently digested and assimilated, they probably make only a minor quantitative contribution to black fly nutrition under natural conditions (Baker and Bradnam, 1976; Frenchel and Jorgensen, 1977; Martin and Kukor, 1984). In summary, we conclude that aquatic dipteran detritivores have digestive systems characterized by highproteolytic capacity, owing to high midgut pH and the presence of high levels of alkaline proteinases. By contrast, the capacity for polysaccharide digestion is more limited. The only classes of polysaccharides

Digestive enzymes of black fly larvae against which significant enzymatic activity is evident in the gut fluid are the ct-l,4- and #-l,3-glucans. Thus, the larvae are better adapted to exploit the polysaccharides of fungal and algal tissue than tissues derived from vascular plants. Acknowledgements--This research was supported by grants from the National Science Foundation to M. M. Martin (PCM 78-22733) and R. W. Merritt (DEB-80-22634). Partial support for R. W. Merritt also came from Regional Black Fly Research Project NE-118. REFERENCES

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