Hemocytes of Mytilus edulis affected by Prudhoe Bay crude oil emulsion

Marine Ent'ironmental Research 22 (1987) 107-I 22

Hemocytes of Mytilus edulis Affected by Prudhoe Bay Crude Oil Emulsion M. Geraldine M c C o r m i c k - R a y Department of Environmental Sciences. University of Virginia. Charlottesville. Virginia 22903. USA (Received 4 March 1986; revised version accepted 6 December 1986)

A BSTRA CT

Hemocvtes and tissues of Mytilus edulis were examined after 4-5 or 8-9 week's exposure to 390#g/liter or 7401~g/liter Prudhoe Bay crude oil emulsion, ~htring the animals" most metabolically active season. A reduction in hemocytes occurred in animals exposed to 740 ~tg/liter after 4-5 weeks. After 8-9 weeks, hemocvte counts of both test groups increased, ~hw to higher densities of granuloeytes, yet the phagocytic response was reduced. Agramdocyte densities were reduced in animals exposed to 390 #g'liter, due to lower counts of 2-31~m agranaloQ'tes. Further, adipogranular cell percentages hz test animals were retkwed. The results from oil-exposed mussels suggest a stress condition and have implications for monitoring.

INTRODUCTION/BACKGROUND The hemocytes of bivalves carry out a number of important physiological functions, including food and waste transport, internal defense, wound healing and shell repair (Wagge, 1955; Narain, 1973: Bayne et al., 1976; Cheng, 1981). They may be involved in the resorption of gametes following spawning (Moore & gowe, 1977). All of these processes depend upon the phagocytic response, i.e. the cellular engulfment of foreign material. Hemocyte counts vary greatly (IMWW, 1980), and seasonal differences have been noted by Huffman & Tripp (1982). Bayne et al. (1981) suggest that significant sublethal changes may be detected if animals are similar in size, and if seasonal changes related to the gametogenic cycle are considered. 107

Marine En~'iron. Res. 0141-113687/S03"50 England 1987. Printed in Great Britain

(£" Elsevier Applied Science Publishers Ltd,

108

M. Geraldine McCormick-Ray

In southcentral Alaska (60 ~ north latitude), the reproductive cycle and growth of Mvtilus edulis is similar to that of northern European mussels (Feder & Keiser, 1980). The gametogenic cycle begins in late fall. Spawning and feeding commence in spring, and growth occurs between May and September. In summer, following spawning, energy is stored in glycogenrich adipogranular cells, which dominate mantel tissue when gametogenesis commences in the fall (Pieters et al., 1979: Bayne et al., 1982); few of these storage cells remain in mantle tissue by February or early March (Feder & Keiser, 1980). Toxic effects of aromatic hydrocarbons are well known (Silkworth & Loose, 1981; Stegeman, 1981)and high concentrations are found in Prudhoe Bay crude oil (Thompson et al., 197l). Dissolved tractions may cross respiratory epithelial surfaces. In areas such as south-central Alaska, great fluctuations in water temperatures and salinity (Feder & Keiser, 1980) make predictions of solubility difficult (Whitehouse, 1984). Another route of entry into animals is through ingestion of contaminated food or oil particles Shaw, 1977: Stegeman, 1981; Widdows et al., 1982). Mvtilus e&dis was chosen because of the abundant information available Bayne et al., 1976: IMWW, 1980: Widdows et al., 1982), their cosmopolitan (especially northern) distribution, and because of their usefulness in monitoring. An oil spill occurring during these animals" metabolically active season could affect their hemocytes and tissues, and may stress the reproductive potential of the population. The effects could indicate sublethal response important to monitoring.

MATERIALS AND METHODS In June, 1982, 3-4cm (shell length) mussels were detached l¥om intertidal rocks of Resurrection Bay, Alaska. They were transported to nearby Seward Marine Laboratory and acclimated to laboratory conditions for 2 weeks betbre exposure. "Pretest" field animals were collected and examined to establish laboratory procedures and to determine spawning stage. The experimental facility utilized subthermocline water of Resurrection Bay, filtered (~ 10/.~m) and heated to 8-10:C. A mixed culture of algae ([sochr)'sis galhana and Phaeodactvlum tricornutum) in concentrations of 10-12 x 10"~ cells per milliliter was added continuously for food. Fresh Prudhoe Bay crude oil was continuously injected into the top of an emulsifying glass cylinder (5 cm diameter x 1"2 m long tube) at a rate of 2-6/.d/ rain, by a power-driven syringe. A rapidly spinning, non-aerating glass stirrer emulsified the oil with 50 ml/min inflowing seawater. A continuous one-way flow of concentrated emulsion was gravity-fed through a series

Hemocytes of Mytilis affected by crude oil

[09

G R A V I T Y FEED CRUDE OIL EXPOSURE

[ Sea Water = Sea Water

=i Filter ~ - ~

Reservoir Food Added

~

Water Heated

E1 r

,,

Oi( Emulsifying Chamber

Col" trol Chal rbber

Discard

Medium Oil Mussels

High Oil Mussels

No Oil Mussels

Fig. I. Schema for oil emulsion dilution and distribution to mussels. Water from Resurrection Bay was filtered, heated, and food added. Part of this water flowed to the control group, part to the emulsion chamber, and part to diluting chambers. Oil was pumped into the emulsifying chamber then channeled to a chamber and diluted to high-oil concentration. This water was sent to mussels and to a second chamber, diluted to medium-oil concentration and sent to a second group of mussels.

of dilution cylinders (Fig. 1). Nominal exposure levels were 500nl/liter (medium oil) and 1000 nl/liter (high oil). Clement & Shaw (1982) determined that the concentration of hydrocarbons in seawater that eluted between naphthalene and dotriacontane during gas chromatography was 390/~g/ liter for medium exposure and 740 #g/liter for high-oil exposure. Emulsion samples were collected and analyzed for maintenance of hydrocarbon concentrations. Flow was regulated by teflon and glass stopcocks, and flow rates were checked and adjusted daily. Oil flow was continuous for 9 weeks except for a 3-day interruption in the beginning of the third week. Ninety mussels were randomly assigned to three groups: medium-oil, high-oil and controls. Field samples indicated this population was in mid or earlier stages of spawning at time of collection. Study groups were sampled after 4-5 or 8-9 weeks of exposure. Twenty control, 23 medium-oil and 9 high-oiled animals were examined in the 9-week period. Animals were sampled randomly without replacement. The byssal threads were cut for each animal examined. The animal was held in its exposure water until it was filtering. Fresh hemolymph was extracted from the posterior adductor muscle, between the 3rd and 4th

! I0

M. Geraldine .WcCorrnick-Ray

growth rings, with a 25-gage sterile hypodermic syringe. A notch was made on the dorsal edge of the shell, posterior to the heart, where the needle was inserted. The syringe barrel contained freshly filtered (0-2~m Millipore) seawater into which the hemolymph was drawn and diluted 50%. After gentle mixing, the first two drops of diluted hemolymph were discarded and both chambers of a hemocytometer filled. Hemocytes were allowed to settle for 5 min prior to counting. Counting time ranged from 30 to 60 min. Any sample contaminated with sperm cells or eggs was not counted. All hemocytes in all squares of both chambers of the hemocytometer were differentially counted and corrected for dilution according to standard hematological technique (Miale, 1982). Counts were expressed as total hemocyte (cell) counts per microliter of hemolymph or as the total for the different cell types. Two basic cell types were recognized: granulocytes and agranulocytes. (Foley & Cheng, 1972: Takatsuki, 1934). The granulocytes were relatively larger (9-12 pm), generally motile and pleomorphic cells with granules, occurring as singlets, doublets, or in groups of three or more and counted as one because of the difficulty of identifying individual cells. Agranulocytes were recognized as opaque and stationary cells, with two sizes differentiated: smaller 2-3 pm cells and larger 4-7 pm cells. The phagocytic response of hemocytes to yeast was measured by placing four drops of fresh, diluted hemolymph on each of two precleaned glass slides. Heat-killed baker's yeast cells, diluted in filtered (0"2 pm Millipore) seawater to equal approximately a 2 : 1 yeast-to-hemocyte ratio, were added to the hemolymph. These prepared slides were incubated in a moist, covered chamber at room temperature (18~C) for 55 or 65 rain before counting. Each slide was rinsed with Millipore-filtered seawater to remove hemolymph and unphagocytized yeast cells (Bayne et al., 1979). One thousand hemocytes on each of two separate slides were counted, and the percentage of hemocytes with yeast determined (those with at least one phagocytized yeast cell and those without). Immediately after hemocyte enumerations, the adductor muscle was severed. After several rinses the animal was fixed in Baker's formol-calcium (Lowe et al., 1982), and stored for 2 weeks. A section of the posterior mantle and two transverse sections of the mid and anterior regions were prepared for histological examination using standard techniques (Lowe et al., 1982; Luna, 19681. Tissue sections were cut (6-7/~m) on a rotary microtome. Half of the prepared slides were stained with Papanicolaou (Lowe et al., 1982) and half with hemotoxylin and eosin (Luna, 1968). Four to six consecutive sections of cut tissues were placed on two separate slides. Skipping 20-60 sections, six more consecutive tissue sections were added to different slides. In total, 4-6 sets of slides were made for each animal. The percentage of storage tissue and follicles in the mantle established

Hemocytes of Mytilis affected by crude oil

I 11

tO0 M~d

8O

60 Adv

40 End

2O Crop

0

i

0

i

20

i

i

40

i

i

60

80

100

Percent Storage

Fig. 2. Percentages of follicles and storage cells that define spawning stages: Mid --- mid spawning;Adv = advance spawning;End = end spawning;and Crop = completed spawning. spawning stage (Lowe et al., 1982). Volumetric percentages of follicles and storage tissue cells (both adipogranular and vesicular) were determined by stereology (Lowe et al., 1982), using a Weibel eyepiece graticle (Graticules, Ltd) with statistically derived line-points. The ocular grid of the graticle was positioned over a tranverse section of mantle. Enumeration procedure closely followed that of Weibel et al. (1966) and Briarty (1975). Point-counts of three mantle sections from two different slides have been determined by Lowe et al. (1982) to be adequate for determining statistically significant changes in mantle tissue. The counts from six separate mantle sections were averaged and percentages of follicles and storage tissue determined for each animal (adipogranular and vesicular cells separately counted). Four spawning stages were defined (Fig. 2). The first stage for this study began with mid spawning (Mid), when follicles were at maximum ripeness (Bayne et al., 1982) and represented 70% or greater of the total mantle. The last stage, completed spawning (Crop) (the reproductively inactive stage), was established when 70% or greater of the mantle was storage tissue (Lowe et al., 1982). The spawning stages advance (Adv.) and end (End) were arbitrarily defined as approximate dividers between mid and completed spawners. This was 35-70% follicles and 18-49% storage tissue for Adv. spawners and 15-35% follicles and 50-70% storage tissue for End spawners. Percentages which overlapped any two class categories were labeled intermediate (Int). Statistical evaluation included Student t-tests for determining differences between means and confidence limits (Zar, 1984), and two-way analysis of variance using the SPSS ANOVA computer program (Hull & Nie, 1981) to determine differences due to oil exposure, time and time/oil interaction. Percentages were arcsin transformed before analysis (Zar, 1984). The Fisher

112

M. Geraldine McCormick-Ray

Exact Test was used to determine proportional differences between groups (Zar, 1984).

RESULTS Exposure to 740/~g/liter ofoil emulsion for 4-5 weeks reduced the density of circulating hemocytes (Table 1). The number of high-oiled animals with hemocyte counts less than 1500 cells/#liter was significantly different from controls (Fisher Exact Test: P = 0"006 99). Alter 8-9 weeks, however, total cell counts increased in all groups but were greater in test groups exposed to oil due to an increase in granulocytes (Table 2). An A N O V A on total cell counts revealed a significant time effect (FI2..~6) = 9"29, P < 0-004). From 4-5 to 8-9 weeks, total cell counts increased only 8% in the control group (two-tailed tils) = 0"527, P > 0"05), 49% in the medium-oiled group (one-tailed t(_~1)=2-371, P<0-05) and 180% (twotailed t(7)= 2-124, P > 0-05) in the high-oiled group. After 8-9 weeks, test groups had higher cell counts, but no statistical differences were found between control and medium-oiled groups (two-tailed t(zt)= 1-007, P > 0-05), or between control and high-oiled groups (two-tailed t(t4) = 0" 14, P > 0-05). Total cells and granulocytes were highly positively correlated (r = 0"98, P < 0-001), but there was no relationship between total cells and all agranulocytes (r = 0-14) or with the smaller agranulocyte form (r = 0"16). Granulocyte counts in the high-oiled group were lower than controls (two-tailed t(~l)= 2"2433, P < 0-05) after 4-5 weeks, but not lower in the medium-oiled group (two-tailed 621) = 0-599, P > 0-05)(Table 2). Then, I¥om 4-5 to 8-9 weeks the granulocytes of the control group decreased 4% (twotailed ttt s) = 0-189, P > 0-05), yet they increased in the medium-oiled group by 46% (two-tailed t(2t)= 2"204, P_< 0-05) and in the high-oiled group by 187% (two-tailed t(7)= 1.9864, P>0-05). Nevertheless, after 8-9 weeks granulocyte densities of the controls were comparable to the medium-oiled group (two-tailed t(ta)= 1-564, P > 0"05) and to the high-oiled group (twot a i l e d t(t6) = 0"78, P > 0"05). Agranulocyte counts of the control group were comparable to the medium-oiled group (two-tailed t(ls) = 1-6746, P > 0-05), and to the highoiled group (two-tailed t(t 1) = 0"57, P > 0-05) after 4-5 weeks. From 4-5 to 8-9 weeks, agranulocytes increased in the control group by 262% (twot a i l e d t(ts) = 3-09, P < 0-05), in the medium-oiled group by 150% (two-tailed t(zt)=3"261, P_<0-05), in the high-oiled group by 106% (two-tailed t(7)=0"59, P>0"05). Although agranulocytes increased, the 8-9 week control group had higher cell densities than the medium-oiled group (onetailed t121)= 3-4615, P < 0-05).

Hemocytes o f Mytilis affected by crude oil TABLE

l 13

l

2 x 2 Contingency Table of the Number of Control and High-oiled Animals with Hemocyte Counts Greater Than ( > ) and Less Than ( < I t 500 Hemocytes per Microtiter After 4-5 Weeks" Exposure < 1500

> 15o0

4 1

0 8

Control High-oiled

F u r t h e r e x a m i n a t i o n o f a g r a n u l o c y t e s revealed an increase in small (2-3 pro) a g r a n u l o c y t e s in the c o n t r o l g r o u p t h a t did n o t o c c u r in test g r o u p s ( T a b l e 3). A n A N O V A o n arcsin t r a n s f o r m e d p e r c e n t a g e s d e m o n s t r a t e d a significant t r e a t m e n t effect (Ft2..,6~ = 7.87, P < 0-001) a n d t r e a t m e n t / t i m e i n t e r a c t i o n (FI2.,~6~= 5-24, P < 0 - 0 0 1 ) . F r o m 4 - 5 to 8 - 9 weeks, small a g r a n u l o c y t e s i n c r e a s e d by 1 9 8 % in the c o n t r o l g r o u p ( t w o - t a i l e d t~ts~ = 2.257, P < 0-05), b y o n l y 8 % in the m e d i u m - o i l e d g r o u p ( t w o - t a i l e d t~2t~= 1-25, P > 0 " 0 5 ) a n d 7 2 % in the h i g h - o i l e d g r o u p ( t w o - t a i l e d TABLE

2

Total and Differential Hemocytc Counts of the Control, Medium-oiled and High-oiled Groups after 4-5 and 8-9 Weeks" Exposure 4 - 5 Weeks n~ Controls

Granulocytes Agranulocytes Total

SU

2 124 105 2 229

348 35 364

1 880 50 I 930

237 10 240

896 97 993

187 31 212

n

Mean

SE

2 031 380 2 411

340 75 333

-4 262 *~ 8

2 744 125 2 869

306 19 309

46* 150" 49*

2 578 200 2778

794 152 725

187* 106 180"

12

4

5

--- number of mussels analyzed. Mean number of cells per microliter. " Standard error. Per cent change between sampling times. Asterisk indicates significant differences between sampling times. n

%a

lI

11

Granulocytes Agranulocytes Total High-oiled

Mean ~

9

Granulocytes Agranulocytes Total Medium-oiled

8-9 Weeks

114

,t4. Geraldine McCormick-Ray TABLE 3 Counts of Small Agranulocytes (Symbols as in Table 2) 4-5 weeks

Controls Medium-oiled High-oiled

8-9 weeks

n

Mean

SE

n

Mean

SE

%

9

88 39 79

33 8 32

11 12 5

262 54 136

64 8 176

198" 38 72

1 4

/(7) = 0-618, P > 0-05). After 8-9 weeks, the control group had higher cell

densities than the medium-oiled group (two-tailed t(2 ~)= 3.3548, P < 0-05), but comparable densities to the high-oiled group (two-tailed t(t,t)= 0-98, P > 0-05). Oil exposure caused proportional differences in hemocytes (Fig. 3). An A N O V A on arcsin transformed percentages for the different cell types and treatment groups demonstrated a treatment effect (F(2.,~6~= 6.87, P < 0-002) and a treatment/time interaction (F~2.,~6) = 6-93, P < 0-002) on granulocytes, and a treatment effect (F(2.~6)= 6.94, P < 0 - 0 0 2 ) and a treatment/time interaction (F~2.,61 = 6.997, P < 0 - 0 0 2 ) on agranulocytes. Although no differences were found among groups in the 4-5 week period, after 8-9 Controls Medium Oil Higtl Od

N : 9. 11 N : TI, 12 N : 4,

100

100

"i!

80

8O

il '

6O

6o

E L~

o..

40

4O

T

L T

r

20

-

20

0

8-9

4-5

weeks

4-5

8"9 weeks

8-9

4-5

weeks

Gr anulocytes

Agranulocytes

Phagocytic

(a)

(b)

(c}

Fig. 3. Proportional differences in hemocytes of the control, medium-oiled, and high-oiled groups after 4-5 and 8-9 weeks. Mean percentages of total cells counted, with 95% confidence limits (back transformed from arcsin transformed data) for (a) granulocytes, (b) agranulocytes and (c) the phagocytic response of yeast.

Hernocytes of Mytilis affected by crude oil

1l 5

)(...)

z

i,i ---I c~ i,i

ii

(a)

iI

FI. Z

L.I I (._J i,i

I.

H

C~_

i

C H MID

INT

AOV

i

CM.M

C H

CMH

[NT

ENO

[NT

m

CM

CMP

SPAWNING STAGE - 4 TO 5 WEEKS

~--

80'

(b)

Z H eY u..

5~.

z

40.

~::

M H ~o-

~ MH

C MID

C~I INT

^{Iv

H INT

H ENO

H INT

CMP

SPAWNING STAGE - B TO g WEEKS Fig. 4. Percentages of animals in spawning stages for the control (C), medium-oiled (M) and high-oiled (HI groups after (a) 4-5 and (b) 8-9 weeks. (See Fig. 2 for spawning definitions.) weeks' exposure granulocyte percentage of the medium-oiled group was higher than the control group, and consequently the agranulocyte percentage was lower. Thus, the ratio of granulocytes to agranulocytes of the control grouF decreased due to increased densities in small agranulocytes, while the ratio for the test groups increased due to increased densities of granulocytes. Increased densities ofgranulocytes of test animals in the 8-9 week period did not increase the phagocytic response (Fig. 3(c)). An A N O V A demonstrated a treatment effect (F(2,~vl= 14-732, P < 0 - 0 0 1 ) , a time effect (F(~,~7)=41"029, P < 0 - 0 0 1 ) , and a treatment/time interaction (F(2.~7) = 5-057, P < 0-01) on arcsin transformed percentages of cells that phagocytized yeast cells• In the 4 - 5 week period, the lower percentages in test

I 16

M. Geraldine McCormick- Ray

groups were not significant: 89% for the control group, 80% for the medium-oiled group and 73% for the high-oiled group. After 8-9 weeks, however, the phagocytic response was reduced in all groups, but the 19% response of the high-oiled group was significantly lowest. Control animals moved from mid-spawning in the 4-5 week period to the more completed stages in the 8-9 week period (Fig.4). An A N O V A of arcsin transformed percentages of mantle storage tissue demonstrated a time effect (F(1.,;5~ = 7"989, P<0-007), and a treatment/time interaction (F(2.45~ = 4.12, P < 0-02). Although no treatment effect was found, some test animals in the 4-5 week period, unlike controls, were in End and Crop. stages of spawning, and after 8-9 weeks some test animals, also unlike controls, remained in Mid spawning. Oil emulsion reduced adipogranular cells in the mantle of test animals. An A N O V A of arcsin transformed percentages of adipogranular cells demonstrated a significant treatment effect (F~2.,~s~= 4.286, P < 0-02), and time effect (F~t.~s~=9-964, P<0-003). Figure 5 indicates that control animals' adipogranular cell percentages increased with advancement of spawning stage. The percentages for the medium-oiled animals increased with spawning stage, but were lower relative to controls, except in the last stage of spawning. On the other hand, high-oiled animals had almost no adipogranular cell development with completion of spawning. Histological examination revealed those adipogranular cells of medium-oiled animals to be thinner than those of the control group. Vesicular cells often appeared smaller and irregularly formed. 40 ¢n

-¢J

30 Jl

(3

l

o o

D c

20

o

o o

io

•

j

o

o i

~,

8

8

.H

C

o

o

~

M

Mid.

n

H

C

M

Adv.

H

C

ld

End

H

C

M Cmo.

Fig. 5. Percentages ofadipogranular cells in mantles of animals classified by spawning stage (see Fig. 2 for definitions) to illustrate increasing percentages with time for the control (C) group in comparison to the medium-oiled (M) and high-oiled groups [H).

Hemoo'tes of Mytilis affected by crude oil

117

DISCUSSION This study demonstrated Prudhoe Bay crude oil emulsion has significant sublethal effects on blue mussels during their most metabolically active season. Lower densities of circulating hemocytes after 4-5 weeks may have been due to hemocyte lysing. Hemocytes contain an abundance of lysosomes (Cheng et al., 1969: Sminia, 1981), and polycyclic aromatic hydrocarbons have been shown to induce cytolysis in lysosome-enriched ceils (Moore et aL, 1978). Fries & Tripp (1980) reported phenol-induced hemocyte lysing in Mercenaria mercenaria.

Accumulated aliphatic and aromatic hydrocarbons (Stegeman & Teal, 1973) may have affected hemocytes. Clement & Shaw (1982) determined tissue concentrations of hydrocarbons in cohorts of the present study after 8 weeks of exposure. They found aliphatic and aromatic hydrocarbon concentrations both to be approximately 150/~g/g wet weight in animals exposed to 390 ~Lg/liter, and 210 ~lg/g for aromatic and 280 ,ug/g for aliphatic hydrocarbons in animals exposed to 740 ~g/liter. Control animals had less than 40~zg/g aliphatic hydrocarbons and less than 10Fzg/g aromatic hydrocarbons. Observations during this study indicated that some ingested oil was discarded in pseudofeces (along with a high number of food organisms), some entered the anterior gut chambers, and little or none reached the mid gut region. Clement and Shaw also reported significant reductions in growth, scope for growth, and weight, results that agreed with those of Widdows et al. (1982). Widdows et al. (1985) have stated that the degree of stress (e.g. a measurable physiological response) in mussels is a simple function of toxicant concentration in the tissues. The reduction and subsequent increases in circulating granulocytes and in total ceils, the reduced phagocytic response, the lack of increase in 2-3 ~m agranulocytes in test animals, and the lower numbers of adipogranular cells suggest indirect effects and metabolic responses to tissue-accumulated oil. Time and oil exposure were shown to be significant factors in the changes occurring in hemocytes. The lack of significant differences between control and test groups" granulocyte and total cell counts in the 8-9 week period relates to several factors. High densities of granulocytes and total cells were found in test animals in the 8-9 week period. The high cell concentrations increased clumping, and each clump was counted as one cell because individual ceils could not be delineated. Variability in hemocyte densities was due to several factors (Feng, 1965; Narain, 1973; Huffman & Tripp, 1982; Miale, 1982), including diversity of cell types associated with hemocyte function and to

118

M. Geraldine McCormick-Ray

ontological stages of hemocyte development IMix, 1976; Moore & Lowe, 1977: Cheng, 1981). Increases in small (2-3 #m) agranulocytes in 8-9 week control animals were unrelated to sex and coincident with late spawning stage. As oil accumulated in tissues, an interference with the production of this cell type, or a rapid differentiation of this cell into another cell type, occurred. The effect, however, was not unanimous for all test animals. One high-oiled animal in this period had small agranulocyte densities comparable to those of control animals. Granulocytes are normally more phagocytic than agranutocytes (Foley & Cheng, 1975), yet increases in granulocytes of test animals in the 8-9 week period did not increase the phagocytic response. The decreased phagocytic response of the control group may have been due to increases in agranulocytes; the decrease in the high-oiled group suggests that oil may have affected hemocyte motility or membrane recognition. Fries & Tripp (1980) found a reduced phagocytic response in hemocytes of the hard clam Mercenaria mercenaria exposed to 10 ppb (and higher) of phenol; however, membrane structure appeared undisturbed. Nott et al. (1985) demonstrated pathological alterations induced by polynuclear aromatic hydrocarbons (PNAH) in the lysosomal membrane. Moore & Farrar (1985) showed that effects of PNAH on digestive cell lysosomal stability was variable. The effect, nonetheless, of a reduced phagocytic response may encourage microbial infections by opportunistic microbes proliferating in oil of contaminated waters, as well as affecting food and waste transport, wound and shell repair, and glycogen storage within animals. Further, decreased phagocytosis may increase mortality due to secondary effects such as disease or harsh environmental conditions. The role of hemocytes in energy storage needs further investigation. Data from this study suggest that an 8-9 week exposure to oil affected storage tissue of test animals. High-oiled animals exposed for 8-9 weeks did not develop adipogranular cells even though they were found to be in advanced to completed stages of spawning. Control animals had higher percentages of adipogranular cells. Lowe & Pipe (1985) have shown reductions in adipogranular cells in animals exposed to 28 and 127 ppb concentrations of emulsified diesel oil, but after depuration of animals exposed to the lower concentration, these cells significantly increased. A reduction in stored energy in the mantle may affect fall gametogenesis and reduce population recruitment the following spring (Myint & Tyler, 1982). The role circulating hemocytes play in transporting energy to the mantle, or their relationship to storage cells during post-spawning stages, is also uncertain. The intimate relationship observed between granulocytes and storage cells of the mantle tissue has been cited by several authors (Liebman,

Hernocytes of Mytilis affected by crude oil

119

1946; Moore & Lowe, 1977). Lubet et al. (1976) believe hemocytes give rise to mantle storage tissue, while Cheng (1981) and Wagge (1955) suggest mantle cells may give rise to circulating cells. In conclusion, the initial reduction in granulocytes, their increases with time, and the reduced densities of agranulocytes in mussels exposed to emulsion during their most metabolically active season may be important indicators of a general adaptive response to stress. Feng et al. (197 I) reported increased percentages of granulocytes when bacteria were experimentally injected into Crassostrea virginica. Mix & Sparks (1980) examined ratios between agranulocytes and granulocytes, and correlated an increase in the percentage of granulocytes of tanner crabs (Chinonocetes bairdi, Rathbun) infected with the fungus, Trichomaris invadens. Differences in cell densities and cell types may indicate stress. ACKNOWLEDGEMENTS Very special thanks to Dr D Shaw and Mr L. Clement (University of Alaska, Fairbanks) for guidance and use of the oil facility: to Dr A. Bulgur, Mr J. Porter, Dr G. C. Ray (University of Virginia); Dr M. Tripp (University of Delaware) and Dr A. Sparks (N.M.F.S., Seattle, Wa.); to the University of Alaska and Institute of Marine Science (UAF) for financial assistance, and to all others who helped. This work was completed in partial fulfillment for the MS degree at the University of Alaska, Institute of Marine Science.

REFERENCES Bayne, B. L. (Ed) (1976). Marine mussels: Their ecology and pILvsiology. Cambridge, Cambridge University Press. Bayne, B. L., Clark, K. R. & Moore, M. N. (1981). Some practical considerations in the measurement of pollution effects on bivalve molluscs, and some possible consequences. Aqua. Toxi. 1, 159-74. Bayne, B. L., Widdows, J. & Thompson. R. J. (1976). Physiological integrations. In: Marine mussels. (Bayne, B. L. (Ed)), Cambridge, Cambridge University Press, 207-60. Bayne, B. L., Bubel, A., Gabbott, P. A., Livingstone, D. F., Lowe, D. M. & Moore, M. N. (1982). Glycogen utilization and gametogenesis in Mvtilus edulis L. Mat'. Biol. Lett., 3, 89-105. Bayne, C. J., Moore, N. M., Carefoot, T. H. & Thompson, R. J. (1979). Hemolymph functions in Mvtih~s californianus: The cytochemistry of hemocytes and their responses to foreign implants and hemolymph factors in phagocytosis. J. lnvertebr. PathoL, 34, 1-20. Briarty, L. G. (1975). Stereology: Methods for quantitative light and electron microscopy. Sci. Prog., Oxf, 62, 1-32.

120

M. Geraldine McCormick-Ray

Cheng, Y. C. [ 1981). Bivalve. In: Invertebrate blood cells (Ratcliff, N. A. & Rowley, A. F. [Edsl}, New York, Academic Press, 233-300. Cheng, T. C., Thakur, A. S. & Rifkin, E. (1969). Phagocytosis as an internal defense mechanism in the mollusca: With an experimental study of the role of leucocytes in the removal of ink particles in Lillorina scabra (Linn). In: Proceedings of the Syrnposium on the Mollusca. Part II. Marine Biological Association of India, 546-63. Clement, L. & Shaw, D. t 1982). Biological indicators ofoil pollution: A comparative study. Manuscript. University of Alaska, Institute of Marine Science. (Unpublished.) Feder. H. M. & Keiser, G. E. (1980). Port Valdex, Alaska: Enrironmental studies 1976-1979. Chapter 8, Intertidal biology. Occasional Publication No. 5, Fairbanks, Institute of Marine Science, Univ. Alaska, 145-233. Feng, S. Y. (1965). Heart rate and leucocyte circulation in Crassostrea t'irginica. Biol. Bull., Mar. Biol. Lab., Woods Hole, 128, 198-210. Feng, S. Y., Feng, J. S., Burke, C. N. & Khairallah, L. H. (1971). Light and electron microscopy of the leucocytes of Crassostrea virginica [Mollusca: Petecypodaj. Z. Zellforsch., 120, 222-45. Foley, D. A. & Cheng, T. C. (1972). Interaction of molluscs and foreign substances: The morphology and behavior of hemolymph cells of the American oyster, Crassostrea virginica in tritro. J. Invertehr. Pathol., 19, 383-94. Foley, D. A. & Cheng. T. C. 11975). A quantitative study of phagocytosis by hemolymph cells of the pelecypod Crassostrea virginica and Mercenaria mercenaria. J. lncertehr. Pathol., 25, 189-97. Fries, C. R. & Tripp, M. R. (1980). Depression of phagocytosis in Mercenaria following chemical stress. Det'elop. Comparat. lmmuno, 4, 233-44. H uffman, J. E. & Tripp. M. R. (I 982). Cell types and hydrolytic enzymes of soft shell clam (Mya arenaria) hemocytes. J. lnvertehra. Pathol., 40, 68-74. Hull, C. H. & Nie, N. H. (Eds). (I 981). SPSSupdate 7-9. New procedures and facilities Jot release. New York, McGraw-Hill. International Mussel Watch Workshop {IMWW) (1980). The international mussel watch. Washington, DC, National Academy of Sciences. Liebman, M. {1946). On trephocytes and trephocytosis: A study on the role of leucocytes in nutrition and growth. Growth, 10, 291-330. Lowe, D. M. & Pipe, R. K. (1985). Cellular responses in the mussel Mvtihls edulis following exposure to diesel oil emulsions: Reproductive and nutrient storage cells. Mar. Era:. Res., 17, 234-7. Loire, D. M., Moore, M. N. & Bayne, B. L. (1982). Aspects of gametogenesis in the marine mussel Mytih~s edulis L. Mar. Biol. Assoc. U.K., 62, 133-45. Lubet, P., Herlin, P., Mathieu, M. & Collin, F. {19761. Tissu de r6serve et cycle sexual chez les lamellibranches. Haliotis, 1, 59-62. L una, L. G. (Ed.)(1968). Manual ofhistologic stainhtg methods of the Armed Forces Institute of Pathology. (3rd edn), New York, The Blakiston Division, McGrawHill Book Co. Miale, J. (1982). Laboratot 3" medicine hematology (6th edn), St. Louis, The C.V. Mosby Co., 1098 pp. Mix, M. C. [1976). A general model for leucocyte cell renewal in bivalve molluscs. Mar. Fish. Re~'., 38, 37-41. Mix, M. C. & Sparks, A. K. {1980). Tanner crab Chionocetes bairdi Rathbun

Hemocytes of Mytilis affected by crude oil

121

haemocyte classification and an evaluation of using differential counts to measure infection with a fungal disease. J. Fish. Dis., 3, 285-93. Moore, M. N. & Farrar, S. V. (1985). Effects of polynuclear aromatic hydrocarbons on lysosomal membranes in molluscs. Mar. Env Res., 17, 222-5. Moore, M. N. & Lowe, D. M. (1977). The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles. J. lnvertebr. Pathol., 29, 18-30. Moore, M. N., Lowe, D. M. & Fieth, P. E. M. (1978). Lysosomal responses experimentally injected anthracene in the digestive cells of Mytilus edulis. Mar. Biol., 66, 209-23. Myint, U. M. & Tyler, P. A. (1982). Effects of temperature, nutritive and metal stressors on the reproductive biology of Mytilus edulis. Mar. Biol., 66, 209-23. Narain, A. S. (1973). The amoebocytes of lamellibranch molluscs, with special reference to the circulating amoebocytes. Matacol. Rev., 6, 1-12. Nott, J. A., Moore, M. N., Mavin, L. J. & Ryan, K. P. (1985). The fine structure of lysosomal membranes and endoplasmic reticulum in the digestive cells of Mytilus edulis exposed to anthracene and phenanthrene. Mar. Env. Res., 17, 226-9. Pieters, H., Kluytmans, J. H., Zurburg, W. & Zandee, D. I. (1979). The influence of seasonal changes on energy metabolism in Mytilus edulis (L). I. Growth rate and biochemical composition in relation to environmental parameters and spawning. In: Cyclic phenomena in marine plants and animals. Proceedings of the Thirteenth European Marine Biology Symposium. (Naylor, E. & Hartnoll, R. G. (Eds)), Oxford, Pergamon Press, 285-92. Shaw, D. G. (1977). Hydrocarbons in the water column. In: Fate and effects of petroleum hydrocarbons in marine organisms and ecosystems. (Wolfe, D. A. (Ed.)) Oxford, Pergamon Press, 8-18. Silkworth, J. B. & Loose, L. D. (1981). Assessment of environmental contaminantinduced lymphocyte dysfunction. Environ. Health Perspect., 39, 105-28. Sminia, T. (1981). Phagocytic cells in molluscs. In: Aspects of developmental comparative immunology. (Solomon, J. B. (Ed.)), Oxford, Pergamon Press, 125-32. Stegeman, John J. (198 I). Polynuclear aromatic hydrocarbons and their metabolism in the marine environment. In: Polycyclic hydrocarbons and cancer. Vol. 3 (Gilboin, H. V. & Ts'O, P. O. P. (Eds)), New York, Academic Press, 1-60. Stegeman, J. J. & Teal, J. M. (1973). Accumulation, release and retention of petroleum hydrocarbons by the oyster Crassostrea uirginica. Mar. Biol., 22, 37-44. Takatsuki, S. (1934). On the nature and functions of amoebocytes of Ostrea cdulis. Quart. J. Microsc. Sci., 76, 379-431. Thompson, C, J., Coleman, H. J., Dooley, J. E. & Hirsch, D. E. (1971). Bumines analysis shows characteristics of Prudhoe Bay crude oil. Arctic Oil and Gas, 43, 112-20. Wagge, L. E. (1955). Amoebocytes. In: International review of cytology, Vol. 4. (Bourne, G. & Danielli, J, F. (Eds)), New York, Academic Press, 31-78. Weibel, E. R., Kistler, G. S. & Scherle, W. F. (1966). Practical stereological methods for morphometric cytology. J. Cell Bioll., 30, 23-38. Whitehouse, B. G. (1984). The effect of temperature and salinity on the aqueous solubility of polynuclear aromatic hydrocarbons. Mar. Chem., 14, 319-32.

122

M. Geraldine .klcCormick- Ray

Widdows, J., Bakke, T., Bayne, B. L., Donkin, P.. Livingstone, D. R., Lowe, D. M., Moore, M. N., Evans, S. V. & Moore, S. L. (19821. Responses of Mytilus edulis on exposure to the water-accommodated fraction of North Sea oil. Mar. Biol., 67, 15-3l. Widdows, J., Donkin, P. & Evan, S. V. (1985). Recovery of Mytilus edulis L. from chronic oil exposure. Mar. Env. Res., 17, 250-3. Zar, J. H. [1984). Biostatistical analysis. Englewood Cliffs, New Jersey.