Acid phosphatases of the mosquito Culex tarsalis coquillett

Comp. t~iochem. Physiol. Vol. 87B, No. 4, pp. 773-782, 1987 Printed in Great Britain

0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd

ACID PHOSPHATASES OF THE MOSQUITO C U L E X T A R S A L I S COQUILLETT EDWARD J. HOUK and JAMES L. HARDY School of Public Health, Naval Biosciences Laboratory, University of California, Berkeley, CA 94720, USA

(Received 8 September 1986) Abstract--1. Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the acid phosphatases (ACP) of the mosquito, Culex tarsalis, are presented. 2. ACP hydrolysis of P-nitrophenylphosphate (Pnp) was optimal at 37°C, pH 5.25 in the presence of 15 mM MgC12 and 0.1% (w/v) polyvinylpyrollidone (PVP). Vmax and Km values varied significantly between the various mosquito strains examined. 3. Several divalent cations (i.e. Mn 2+, Ca 2+, Ba 2+ and Co 2+), either the chloride or sulphate salts, were stimulatory for ACP. Both Cu 2+ and Fe 2+ (15 mM) were inhibitory. 4. Slight inhibition (i.e. 10%) of ACP activity was observed with dithiothreitol (I00 mM) and 50% inhibition by cysteine (100 mM). 5. ACP activity was cyclic during the 15-day post-adult emergence period of the study. No significant differences were noted between the ACP specific activities of males and females nor between geographic strains. 6. IEF electrophoresis revealed three ct-naphthyl phosphate hydrolytic ACP isozymes within the pH 4.5-5.5 range (i.e. ACP 4's, ACP 52 and ACP55). 7. IEF ACP isozymes were stimulated by PVP, Mg 2+, Zn 2+ and inhibited by cysteine, EDTA (except ACP 5"~) and NaF1. 8. IEF detection of ACP with Pnp revealed an ACP isozyme (ACP43) distinct from those ACP isozymes capable of ct-naphthyl phosphate hydrolysis.

INTRODUCTION Hooper (1976) and Houk and co-workers (1978, 1979, 1980, 1981) have examined the non-specific esterases of two mosquito species, Culex pipiens and Culex tarsalis respectively, through spectrophotometric and electrophoretic methods. The investigators emphasize the necessity for the optimization of incubation conditions for both the enzyme and the sample source, rather than rote adaptation of methods and conditions from the literature. Asakura (1978) and H o u k and Hardy (1984) applied optimized incubation conditions in their analyses of alkaline phosphatases of the mosquitoes. Aedes togoi and C. tarsalis respectively. In addition, Asakura (1978) studied the acid phosphatases of A. togoi. The conclusions from these studies were that the n u m b e r of isozymes detected, the relative intensity of isozyme staining in electrophoretic gels and enzyme kinetics data were all greatly influenced by incubation conditions. Further, enzyme kinetics data are only relevant if optimized incubation conditions are used. The application of incubation conditions established for one biological system (i.e. enzyme and sample source) is simply inadequate when applied to another system, even a closely related system. We have undertaken an analysis of the acid phosphatases (EC3.1.3.1) (ACP) of the mosquito, C. tarsalis, through both electrophoretic and spectroAddress correspondence to: Dr. E J. Houk, Naval Biosciences Laboratory, Naval Supply Center, Oakland, CA 94625, U.S.A.

photometric methods. We optimize relevant enzyme kinetics parameters through an examination of the hydrolysis of the substrate, P-nitrophenylphosphate (Pnp). These optimized incubation conditions are then applied to an electrophoretic-histochemical analysis of the ACP isozymes of this mosquito.

MATERIALS AND METHODS

Mosquitoes, several geographic strains [i.e. Knight's Landing (KL), Winnipeg (W) Owen's Valley (OV) and Poso Creek (PC)] and western equine encephalomyelitis virus susceptible strain (WS) of C. tarsalis, were maintained in an insectary under constant conditions of temperature, humidity and photoperiod (Houk et al., 1978). Fifty female mosquitoes, unless otherwise specified, were ground in 1 ml of isolation medium (Table 1; Beranek, 1974; Houk et al., 1978; Houk and Hardy, 1984). H20, Tris buffer (0.2M, pH 6.7), Tris/deoxycholate (Beranek, 1974) and Tris/Triton (Houk and Hardy, 1980, 1981, 1984) were compared to determine their respective solubilization efficiencies. The subcellular distribution of ACP activity, in these various media, was determined through differential centrifugation: 600 g, 15 min pellet (cell debris-nuclear fraction); 10,000 g, 15 min pellet (mitochondrial fraction) and 10,000g, 15 min supernatant (soluble fraction). Spectrophotometric assays of ACP activity were performed either at 420 nm (Malaney and Horecker, 1966) or 320nm with Pnp as the substrate. At 420nm and pH 5~25P-nitrophenol (np) has virtually no inherent absorption. However, at 320nm, the molar extinction coefficient at pH 5.25 is 2.1 x 104, comparable to the alkaline extinction coefficient at 420 nm (1.32 x 104). The enzyme sample (25/zl) was preincubated for 10 min at the appropriate temperature in either acetic acid-sodium ace773

774

EDWARD J. HOUK and JAMES L. HARDY

tate (0.1 M) or Tris maleate (0.1 M) buffer, containing inhibitors and other additives, as required. Substrate (0.1ml) was added and the reaction was monitored continuously for 10 min. The pH optimum was determined by monitoring the reaction at 0.5 pH increments from pH 3.0 through 6.5. The temperature (Tmax) and divalent cation requirements were optimized. The divalent cations examined were: Mg 2+, Mn 2+, Ca 2. , Ba 2+, Co 2+, Fe z+, Cu 2+ and Zn 2+" The optimized conditions (±.el temperature, pH, divalent cations) were used to determine the kinetic parameters, Vmax and the Michaelis-Menten constant (K,,). The effect of polyvinylpyrollidone (PVP), reportedly a stabilizing cofactor for phosphatases (Steiner and Joslyn, 1979), was also examined. The effects of known inhibitors of both vertebrate and invertebrate ACP (i.e. NaFI, EDTA, cysteine, phenylalanine and sodium tartrate) were tested against the mosquito enzymes. Protein determinations were based on the method of Bradford (1976) with bovine serum albumin fraction V as the standard. The number and distribution of ACP isozymes was determined through isoelectric focusing (IEF) electrophoresis in polyacrylamide gel (Houk et al., 1979; Houk and Hardy, 1980). ACP isozymes were visualized through the deposition of insoluble Fast Blue RR oxidation products coupled with the hydrolysis of ct-naphthyl ,phosphate (Steiner and Joslyn, 1979), under optimal conditions determined by spectrophotometric assay. The effects of inhibitors and putative cofactors, as delineated above, were examined. The IEF gels were preincubated in Tris maleate buffer (0.1 M, pH 5.25, 37°C), containing cofactors and inhibitors when appropriate. The preincubation buffer was decanted and replaced with fresh buffer containing substrate (i.e. ct-naphthyl phosphate (1% w/v in acetone; final concentration 0.1%) and Fast Blue RR (1 mg/ml). Subjective evaluation determined the duration of staining. The reaction was stopped by replacement of the staining solution with distilled H20. The IEF gels were left in distilled H20 until the background had cleared sufficiently to allow a semiquantitative evaluation of the effects of the various additives as determined through densitometric scanning (610nm). ACP isozymes capable of hydrolyzing Pnp, the substrate used in our spectrophotometric studies, were visualized by preincubating IEF gels in Tris-maleate buffer (0.1M, pH 5.25, 37°C) for 30 min. The preincubation buffer was replaced with fresh buffer containing P n p (100 raM) and incubation was allowed to continue for 30 min. The reaction was stopped by replacement of the incubation medium with distilled H20. After 2-10 min washes in distilled H20, the gels were placed in NaOH (0.01 N). The yellow color of np was immediately apparent and was semiquantitatively examined by gel scanning (420 nm). RESULTS Solubilization efficiency Extensive variability was observed in the solubilization efficiency of the various buffer-detergent c o m b i n a t i o n s tested (Table 1). T r i s / T r i t o n X-100

20--

E o

15-

IO=o = t3~

5E ::L 4.0

510

i 6.0

I

zO

pH

Fig. 1. ACP reaction rate vs pH. Intervals are __+2 SEM (n = 4). (0.2M, pH6.7/l%v/v) was the m o s t efficient b u f f e r ~ l e t e r g e n t c o m b i n a t i o n tested; 84% of the total A C P activity f o u n d in the soluble phase. Tris buffer was the least effective; 38% in the soluble fraction a n d 52% in the cellular debris pellet. The distribution o f A C P activity in the various centrifugation fractions is totally d e p e n d e n t u p o n the solubilization medium, perhaps indicative of the form a t i o n a n d / o r release of various size classes of m e m b r a n e delimited vesicles in response to the composition of the solubilization medium. Since A C P activity was apparently unaffected by the T r i s / T r i t o n X-100 ( 0 . 2 M , p H 6 . 7 / l % v / v ) c o m b i n a t i o n , this buffer-detergent c o m b i n a t i o n was used routinely in all subsequent s p e e t r o p h o t o m e t r i c a n d electrophoretic analyses.

Spectrophotometry The p H of m a x i m a l activity for A C P was 5.25 (Fig. 1). Substantial variability was observed at all of the p H values examined. This was p r o b a b l y a consequence o f the variable precipitation o f m o s q u i t o proteins at these low p H values, which c o n t r i b u t e d a considerable a m o u n t of light scattering to the a t t e m p t e d collection of s p e c t r o p h o t o m e t r i c data. H o m o g e n i z a t i o n of mosquitoes at the p H to be used for A C P assay did n o t alleviate the problem. The t e m p e r a t u r e of m a x i m a l activity for A C P was 37°C (Fig. 2). The s t a n d a r d error o f the m e a n (SEM) values are quite large at each m e a s u r e d temperature. This variability can be partially explained o n the basis of the use of different m o s q u i t o strains as independent samples in the collection o f these data. In addition, the use of different m o s q u i t o strains also adds to the variability n o t e d in establishing the p H

Table 1. Solubilization efficiencyof various isolation media for acid phosphatases of Culex tarsalis Tris/deoxycholate Tris/Triton Tris/Triton H20 Tris (0.5% w/v) (0.5% v/v) (1.0%v/v) Crude homogenate 100 100 100 100 100 Nuclei/cell debris 20 ± 6ad¢ 52 + 8bcae 25 ± 3ade 12 ± 1~bc 7 ± 6abe (600g pellet) Mitochondria 31 ± 3acd¢ 13 ± 3bd 15 + 2~ 24 ± 1ab¢~ 16 ± 3abed ( 10,000g pellet) Soluble 55 ± 3a°~¢ 38 ± 11~d¢ 63 ± 2~bac 76 ± Iabo~ 84 ± 3abed Statistical significance (P 0.05) between various solvents is indicated by: (a) H20, (b) Tris, (c) Tris/deoxycholate (0.5%w/v), (d) Tris/Triton (0.5%v/v) and (e) Tris/Triton (1.0%)v/v). Mean + SD (N = 7).

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(°C)

Fig. 2. ACP reaction rate vs temperature. Intervals are + 2 SEM (n = 6).

nn Fe Cu

Sulphate Salts

Fig. 4. Comparison of the stimulatory effects of both chloride and sulphate salts of various cations on the ACP reaction rate. Intervals are +2 SEM (n = 3). The kinetics data from three different strains of C.

optimum (Fig. 1). It will subsequently become apparent that these different mosquito strains are quite distinct in some of their kinetic parameters. Thus, the treatment of these mosquito strains as samples from the same population lead to increased variability (i.e. large SEM). ACP activity was maximally stimulated by the addition of 15 mM MgC12 (Fig. 3). The chloride salts of other divalent cations (i.e. Ba 2÷, Ca ~÷ and Mn 2÷) were slightly less stimulatory than Mg 2÷ (Fig. 4). In addition, the sulphate salts of Mg 2÷ and other divalent cations (i.e. Co 2÷, Cu 2÷ and Fe 2÷) were compared in their ability to stimulate ACP activity in P n p hydrolysis. Cu 2÷ and Fe 2÷ were inhibitory, while Co 2÷ was significantly less stimulatory than MgSO4 (Fig. 4). Relative kinetics data collection is based on the assumption that substrate hydrolysis is zero-order. To insure that our data were appropriate, we examined the effects of variable sample size (i.e. pl mosquito homogenate) on the linearity of P n p hydrolysis (Fig. 5). To further insure that the extended time of our kinetics assay (i.e. 10 min) did not result in a deviation from linearity during the latter stages of the reaction, a first derivative of the reaction rate between 10 and 14 min was examined (Fig. 5, inset). ACP hydrolysis of P n p is linear well beyond the established period of data collection in our standard reaction, containing 25 #1 of the soluble fraction of the mosquito homogenate.

tarsalis were combined to generate the data for the

response of ACP activity to increasing substrate concentration (Fig. 6). An elongated hyperbolic kinetics curve, exhibiting considerable variability at each point, was obtained. The transformation of these data into a form for the graphic solution of values for K m and for limax revealed a kinetic basis for this variability (Fig. 7). The Vmax for the three mosquito strains examined varied as much as 25%: 9.7/~moles/10 rain (PC), 10.6 #moles/10 rain (W) and 12.8 #moles/10 rain (KL). The/
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Fig. 3. ACP reaction rate vs MgC12 concentration. Intervals are __+2SEM (n = 7).

Fig. 5. ACP reaction rate vs enzyme homogenate added. Inset is first derivative scan of 50/d enzyme for the time interval 10-14rain. Intervals are +2 SEM (n = 3).

776

EDWARD J. HOUK and JAMESL. HARDY 10.0.

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Fig. 6. Response of ACP reaction rate to increasing concentrations of Pnp. Intervals are + 2 SEM (n = 4). ACP activity was compared between the sexes and between the geographic strains used in this study. The sexes were compared at random through 19 days posteelosion with no significant differences observed (Table 2). There were no significant differences in total specific activity of ACP when the KL, OV, PC and W strains of C. tarsalis were compared (Fig. 9). There was considerable variation in ACP activity on a temporal basis (Fig. 10). ACP activity was highest during the initial 24 hr posteclosion period, with diminishing peaks of activity at 3-4 days, 8-9 days and 12-14 days posteclosion. There were significant differences in ACP activity, when the 3-4 day peak was compared to the low points of activity on either side of this peak, but not between any of the other points along the curve.

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The additives that appeared to enhance ACP hydrolytic activity in the spectrophotometric assay were

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days posteclosion. Intervals are +2 SEM (n = 3).

applied individually, and in combination, to determine effects on ACP isozymes separated by IEF electrophoresis (Fig. 11). Three ACP isozymes pIs of 4.8, 5.3 and 5.5, were detected by incubating the IEF gels in Tris-maleate buffer (0.1 M, pH 5.25) with no additives [Fig. I I(A)]. The addition of 15 mM MgC12 significantly stimulated the hydrolytic activity of all three ACP isozymes [Fig. 1 I(B)]. ACP 55 was stimulated approx. 8-fold, while ACP 53 and ACW 8 were stimulated 2.5- and 2-fold respectively. Zn 2+ stimulated ACP activity but to a lesser extent than Mg 2+ [Fig. 1I(C)]. The addition of PVP (0.1% w/v) stimulated ACP, approx. 3-fold ACP 55, 2.5-fold for ACP 53 and 1.7-fold for ACP 4s [Fig. 11(D)]. The combination of both MgCI2 (15raM) and PVP (0.1% w/v) resulted in an additive stimulation of ACP 53 (5-fold) and ACP 48 (3.2-fold) [Fig. 1 I(E)]. However, ACP 55 did not respond in an additive manner. The stimulation observed in the presence of both MgC12 and PVP (i.e. 4.3-fold) was greater than that observed for PVP [i.e. 3-fold; Fig. 1 I(D)] but much less than that observed for MgCI2 [i.e. 8-fold; Fig. l l(C)]. We examined the effects of inhibitors from the spectrophotometry assay and other inhibitors of potential importance in the establishment of the enzyme activity as ACP. EDTA (30mM) demonstrated Table 2. Comparison of acid phosphatase specific activity between sexes Days post Emergence 4 5 11 12 18 19

Specific activity Male Female 41.5 33.8 34.1 39.0 20.8 27.2 12.1 X = 29.8

32.0 25.5 34.0 38.7 17.0 21.2 16.8 26.4

Difference between means is insignificant:

t~ = 1.74. C.B.P. 87/4B--1

777

clearly the dependence of ACP activity on divalent cations [Fig. 1 I(F)], inhibiting ACP 4s activity 100% and approx. 80% for both ACP 55 and ACP 53. Phenylalanine (30 mM) and cysteine (30 mM) were examined with no apparent effects for the former [Fig. II(G)], while the latter was virtually 100% effective in the inhibition of all ACP activity [Fig. l l(H)]. NaFI (30 mM) reduced the apparent activity of ACP by approx. 75% [Fig. 11(I)]. Sodium tartrate (30 mM) was an effective inhibitor of ACP 4s and ACP 55 but had little effect on ACP 53 [Fig. l l(J)]. The IEF gel distribution of ACP isozymes capable of Pnp hydrolysis was also examined. The ACP activity, whether the mosquitoes were homogenized in Tris/Triton X-100 [Fig. 12(A)] or Tris/CHAPS [Fig. 12(B)], exhibited a single peak of activity (pI = 6.3). This appears to represent an ACP isozyme with substrate specificity for Pnp, unique from the ACP isozymes cabable of ~t-naphthyl phosphate hydrolysis. A small peak of ct-naphthyl phosphate hydrolyzing ACP activity [Fig. 1 I(E)] is found at the same pI (i.e. 6.3) and the Pnp hydrolyzing ACP activity (Fig. 12). However, ACP activity exhibiting Pnp hydrolysis in the pH 4.5-5.5 range (i.e. ct-naphthyl phosphate hydrolyzing ACP) was virtually nil. DISCUSSION

ACP is associated with lysosomes and as such has been designated as one of a number of marker enzymes for this population of subcellular vacuoles (DeDuve and Wattiaux, 1966). Enzymes, such as ACP, that are found within vacuoles and intimately associated with the inner membrane leaflet are difficult to study. These enzymes must be solubilized with nondenaturing detergents or the substrates used must be able to freely traverse the vacuolar membrane. Our studies, a combination of spectrophotometric and electrophoretic analyses, require that the enzyme(s) of interest be rendered soluble. The solubilization of ACP makes optimization of incubation conditions, to include examination of necessary co-factors, a much easier task. ACP is an example o f ' a vacuolar enzyme that requires fairly rigorous solubilization conditions. Triton X-100 (1% w/v) in Tris buffer (0.2M, pH 6.7) yielded 84% of the total measured ACP activity in the soluble fraction (i.e., 10,000g, 20min supernatant). Homogenization media containing lesser amounts of Triton, deoxycholate or no detergent additives were significantly less effective in solubilizing ACP activity. Hegdekar and Smallman (1967), in their study of Musca domestica, recognized the efficacy of detergents in solubilizing ACP. However, they added Triton X-100 (0.05% w/v) to the pellets obtained from differential centrifugation and not to the homogenization medium (i.e. distilled H20). Based on the results presented herein, the concentration of Triton X-100 used by Hegdekar and Smallman (0.05% w/v; 1967) would have been only marginally effective. A number of other studies of insect ACP have also been based on tissue or whole insect homogenization in H20 (Lambremont, 1959; Couch and Mills, 1968; Beadle and Gahan, 1969; Asakura, 1978) or buffer (Barker and Alexander, 1958; Bowen, 1967; Schin and Clever, 1965;

778

EDWARD J. HOUK and JAMESL. HARDY

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Fig. 11. IEF electropherograms of ACP isozymes: (A) no additives to Tris-maleate buffer (0.1 M, pH 5.25); (B) 15 mM MgC12; (C) 15 mM ZnC12; (D) polyvinylpyrollidone (0.1% w/v); (E) combination of 15 mM MgCI2 and 0.1% PVP; insets include representative acrylamide gel staining pattern and pH profile; (F) 30 mM EDTA; (G) 30 mM phenylalanine; (H) 30 mM cysteine; (I) 30 mM NaF1 and (J) 30 mM sodium tartrate.

A

t

B

E

o o

25

50

75

Oistonce (mm)

,&o

,~s

2~

5'o

A

do

,~s

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Fig. 12. IEF gels of ACP hydrolyzing Pnp for two different strains of C. tarsalis (WS and KL) homogenized in two different detergents (1% w/v): (A) Triton X-100 and (B) CHAPS.

Mosquito acid phosphatases Trebatoski and Haynes, 1969; Steiner and Joslyn, 1979) without the benefit of nonionic detergent additives. According to our data, this could result in as little as 50% of the total ACP activity being available for assay. Although the effects of low or no detergent homogenization media on the relative activity and/or differential solubilization of different ACP species has not been examined, this could be a significant problem for both spectrophotometric and electrophoretic studies. The pH for optimal hydrolysis of P n p was determined to be 5.25 for C. tarsalis. Asakura (1978) assayed A. togoi ACP activity at the unusually low pH optimum of 3.0. Lambremont (1959) reported that optimal activity for A. aegypti ACP was in the range of pH 4.6-5.2. The remainder of the insect ACP studies surveyed did not report an optimal pH but all assayed ACP activity in the range of 4.6-5.3 (Hegdekar and Smallman, 1967; Couch and Mills, 1968; Trebatoski and Haynes, 1969; Steiner and Joslyn, 1979). The Tmax of C. tarsalis ACP activity was 37°C. Many of the other studies of insect ACP also used this, or a close approximation of this temperature (Barker and Alexander, 1958; Hegdekar and Smallman, 1967; Couch and Mills, 1968; Beadle and Gahan, 1969) but without the benefit of attempting to determine an optimum. In those studies that attempted to determine a Tmax (Lambremont, 1959; Asakura, 1978), no sharp temperature optima were noted. Lambremont (1959) reported an optimum of 45°C for A. aegypti and Asakura (1978) reported little difference in A. togoi ACP activity between 28°C and 37°C. The data presented herein and the results of Lambremont (1959) and Asakura (1978) point to a very broad range of ACP activity vs temperature. Most of the studies of insect ACP do not add divalent cations to the incubation medium (Barker and Alexander, 1958; Hegdekar and Smallman, 1967; Trebatoski and Haynes, 1969; Asakura, 1978; Steiner and Joslyn, 1979). Couch and Mills (1968) report that MgCI2 is stimulatory but not necessary for the spectrophotometric determination and cytochemical detection of Periplaneta americana ACP. Lambremont (1959) demonstrated quite clearly that MgCI2 activated A. aegypti ACP activity, as did a number of other divalent cations. Further, Lambremont (1959) demonstrated that FeC12 was inhibitory for A. aegypti ACP. The observations of Lambremont (1959) with regard to divalent cations corroborates our own study with regard to their effects on the stimulation and inhibition of ACP activity. This study of C. tarsalis ACP is an example of the problems one can encounter in an attempt to combine spectrophotometric and electrophoretic studies of enzyme systems. The major problem with these types of studies is that often the most sensitive substrates for spectrophotometric detection of enzyme activity may not be the most efficacious for electrophoretic detection. In this study, we optimize incubation conditions for the mosquito ACP hydrolysis of Pnp. We follow this with an electrophoretic study of mosquito ACP isozymes using ~-naphthyl phosphate as the substrate. In the end, we discover through electrophoretic analysis that those enzymes capable of ~-naphthyl phosphate hydrolysis are

781

unique from those capable of P n p hydrolysis. However, we demonstrate that the two different enzyme-substrate pairs share some cofactor requirements and inhibition characteristics. First, both pairs require Mg 2+ for optimal activity. Second, both respond to the addition of PVP (0.1% w/v) to the incubation medium. Third, both are inhibited by the amino acid cysteine. Although, the electrophoretic isozymes were inhibited I00%, while the spectrophotometric enzymes(s) were inhibited only 50%. Acknowledgements--This research was supported by a

U.S. Army Contract/Grant (DAMD 17-77-C-7018), U.S. Army Research and Development Command, Washington, D.C. and by the Office of Naval Research, Microbiology Program, Naval Biology Project (N00014-81-C-0570/ Nr205-001). We recognize the assistance of Ms. Sharon Lynn in the grahic and photographic arts. REFERENCES

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