1

NOAA Technical Memorandum GLERL-120

RESPIRATION, NITROGEN EXCRETION, AND O:N RATIOS OF THE GREAT

LAKES AMPHIPOD DIPOREIA SP.

Michael A. Quigley, Peter F. Landrum, Wayne S. Gardner,

Christina R. Stubblefield, and Wendy M. Gordon

NOAA, Great Lakes Environmental Research Laboratory, Ann Arbor, MI 48105

Great Lakes Environmental Research Laboratory

Ann Arbor, Michigan

March 2002

UNITED STATES

DEPARTMENT OF COMMERCE

Donald Evans

Secretary

NATIONAL OCEANIC AND

ATMOSPHERIC ADMINISTRATION

D. James Baker

Under Secretary for Oceans

and Atmosphere/Administrator

NAT OI NAL OCEANIC AND ATMOSPHERIC ADMINISTRAT OI N

U.S.DEPARTMENT OF COMMERCE

NATIONAL OCEANIC AND

ATMOSPHERIC ADMINISTRATION

Conrad C. Lautenbacher, Jr.

Administrator
2

NOTICE

Mention of a commercial company or product does not constitute an endorsement

by the NOAA Environmental Research Laboratories. Use of information from

this publication concerning proprietary products or the tests of such products for

publicity or advertising purposes is not authorized. This is GLERL Contribution

No. 704.

This publication is available as a PDF file and can be downloaded from GLERL’s

web site: www.glerl.noaa.gov. Hard copies can be requested from: GLERL

Information Services, 2205 Commonwealth Blvd., Ann Arbor, MI 48105.

pubs.glerl@noaa.gov.
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Table of Contents

ABSTRACT ........................................................................................................................................... 4

INTRODUCTION ................................................................................................................................. 4

MATERIALS AND METHODS ........................................................................................................... 5

Presence/Absence of Sediment Substrate (Exp. No. 5) ............................................................... 6

Animal Acclimation in Test Vessels (Exp. No. 6) ........................................................................ 6

Effects of Decreased Oxygen Availabilility on Respiration......................................................... 6

RESULTS............................................................................................................................................... 7

O:N Experiments (Nos. 1-3) O2 consumption (Exp. No. 5)......................................................... 7

Presence/Absence of Sediment Substrate (Exp. No. 5) ............................................................... 7

Animal Acclimation in Test Vessels (Exp. No. 6) ........................................................................ 7

Effects of Decreased Oxygen Availabilility on Respiration......................................................... 7

DISCUSSION ........................................................................................................................................ 9

Oxygen Consumption................................................................................................................... 9

Ammonia Excretion ................................................................................................................... 11

O:N Ratios .................................................................................................................................. 11

Acknowledgements .............................................................................................................................. 13

REFERENCES .................................................................................................................................... 13

Figures

Figure 1. Effects of acclimation on Diporeia sp. oxygen consumption (mg O2

 mg-1 DW day-1) ........ 8

Figure 2. Eadie-Hofstee plot of regression line calculated for Diporeia sp. oxygen consumption ...... 8

Figure 3. Diporeia sp. O2 clearance (mL H2

O cleared of oxygen g-1 DW h-1) versus ambient

 oxygen concentration (mg O2 L-1) ......................................................................................... 9

Figure 4. Diporeia sp. oxygen consumption (ng-atom O mg-1 DW h-1) versus N excretion

 (ng-atom N mg-1 DW h-1) .................................................................................................... 12

Tables

Table 1. Mean Diporeia sp. oxygen consumption, ammonia-N excretion [+ (SE)] measured

 in six 1986-1989 experiments ............................................................................................... 7
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Respiration, Nitrogen Excretion, and O:N Ratios of the

Great Lakes Amphipod Diporeia sp.

Michael A. Quigley

Peter F. Landrum

Wayne S. Gardner

Christina R. Stubblefield

Wendy M. Gordon

ABSTRACT. Diporeia sp. (formerly Pontoporeia hoyi) Amphipoda, Crustacea obtained

from a 24-29 m deep site in southeastern Lake Michigan were held in stoppered 60 mL

BOD bottles in darkness and at 4o

C during 24-hour intervals. Change in dissolved oxygen concentration of filtered lakewater within test vessels indicated that mean Diporeia

sp. oxygen consumption ranged from 13.9 to 33.9 mg O2

 g-1 DW d-1. Test vessel acclimation studies revealed a minor increase in oxygen uptake early in the incubation interval

that that was not reflected in overall oxygen consumption measured over the entire 24-

hour period. Mean oxygen consumption of amphipods provided with a coarse, ignited

sand substrate was not significantly different (P<0.05) from O2 consumption of animals

in test vessels where no substrate was provided. In a series of 10 other experiments,

where initial ambient oxygen concentration was varied (from 95 to 23% saturation),

Michaelis-Menten kinetics analysis indicated that Diporeia sp. was able to maintain a

relatively uniform level of oxygen consumption over the upper portion of oxygen concentrations tested. Vmax (predicted O2

 consumption at 100% saturation) was 25.5 mg O2

g-1DW d-1 and fell within the 13.9-33.9 mg O2 g-1DW d-1 range noted in 24-hour oxygen

consumption experiments at near-saturation O2 levels. Km (the predicted O2 concentration

where oxygen consumption had declined to 50% of the Vmax value) was 2.2 mg L-1.

Diporeia sp. nitrogen (ammonia) excretion, simultaneously measured during three of the

oxygen consumption experiments, ranged from 0.45 to 0.77 ng-atom N mg-1 DW h-1.

Calculation of O:N ratios (atoms of oxygen consumed per atom of N excreted) implied

that substrates catabolized by Diporeia sp. consisted primarily of lipids since most (62%)

O:N values were greater than 60. These results complement previously observed high

lipid content of Diporeia sp. and further suggest that the economy of lipids is a prominent aspect of the amphipod’s life history.

INTRODUCTION

The amphipod Diporeia sp. is a prominent macroinvertebrate throughout benthic communities of the

Great Lakes. This organism was previously classified as Pontoporeia hoyi, a species that was believed to

have been evolved from populations of the (sub) arctic marine species Pontoporeia femorata that were

isolated in North American lakes following the retreat of Pleistocene glaciers. However, recent morphological and electrophoretic studies (Bousfield 1989, Vainola and Varvio 1989) have suggested that

Diporeia sp. is more distantly related to P. femorata and, accordingly, Pontoporeia hoyi has now been

assigned to a new genus--Diporeia (Bousfield 1989). Moreover, the identification of distinctly different

mature male forms of Diporeia in North American lakes (Segerstrale 1971) implies that numerous
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species may exist, and at least four may occur in Lake Michigan alone (E.L. Bousfield, National Museums of Canada, P.O. Box 3443, Station D, Ottawa, Ontario, Canada K1P 6P4). In the absence of further

taxonomic clarification of North American Diporeia species, we refer, herein, to our Lake Michigan test

organisms as Diporeia sp.

In Lake Michigan, Diporeia sp. constitutes up to 65% of the resident benthic macroinvertebrate biomass

(Nalepa 1989). Its high abundance in Lake Michigan [a mean abundance of ca. 7000 individuals m-2

(Nalepa et al. 1985)] and its high lipid (and energy) content [up to 50% lipid (dry weight basis) or ca.

5000 cal gm-1, (Green 1971, Gardner et al. 1985)] suggest that further documentation of Diporeia sp.

energetics would provide a clearer understanding of the amphipod’s role in coupling of pelagic and

benthic production. Diporeia sp. provides an important food base for a variety of Great Lakes commercial and sportfish species (Van Oosten and Deason 1938, Wells and Beeton 1963, Anderson and Smith

1971, Wells 1980). In addition, organic contaminant uptake studies (Landrum 1982, 1988, Landrum et

al. 1985) have shown that Diporeia sp. accumulates lipophilic organic contaminants from surrounding

sediments and porewaters and thus may represent an important route in the cycling of such contaminants

within Great Lakes food webs.

At present, considerable evidence indicates that Diporeia sp. production is intimately linked with primary production of pelagic Lake Michigan algal communities (Gardner et al. 1990). Diatom blooms

(and subsequently rich inputs of organic matter to sediments) have recently been associated with increases in Diporeia sp. feeding intensity (Quigley 1988, Evans et al. 1990) and the subsequent and rapid

accumulation of storage lipids (triacylglycerols) (Gardner et al. 1985, Quigley et al. 1989). A similar

linkage between primary production and Diporeia production has also been noted for Lake Ontario

(Dermott and Corning 1988).

Little detailed information is available on Diporeia sp.’s digestive or metabolic physiology. In particular,

the biochemical identity of substrates assimilated and metabolized by Diporeia sp. to support routine

energy demands of growth and reproduction are not presently known. The objective of our series of

studies was to estimate both oxygen consumption and ammonia excretion in Diporeia sp. and to compare these two rates within an O:N ratio (atoms of oxygen consumed per atoms of nitrogen excreted).

O:N ratios have been used extensively to study the underlying metabolism of a variety of crustacean

zooplankton species (see review of Mayzaud and Conover 1988). In general, low O:N ratios (3-16)

imply almost exclusive reliance on proteins as metabolic substrates. Ratios between 16 and 60 are

typical for metabolic use of a mixture of proteins, and lipids and/or carbohydrates, while ratios above 60

reflect a primary dependence on lipids and/or carbohydrates. For amphipods, investigation of O:N ratios

is presently limited to two published studies outlining the metabolism of (1) two brackish-marine

gammarid species (G. duebeni and G. oceanicus, Tedengren et al. 1988), and (2) an arctic sympagic

gammarid (G. wilkitzkii; Aarset and Aunaas 1990).

MATERIALS AND METHODS

 Lake Michigan Diporeia sp., collected with a PONAR grab at depths of 24-29 m from a nearshore area,

4.8 km west of Grand Haven, Michigan, were transported to the laboratory where they were maintained

at 4o

C, in constant darkness, in plexiglas aquaria containing lakewater and a 5-cm layer of sediment

collected from the site.
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In three (O:N) experiments (Experiments Nos. 1-3), both respiration (oxygen consumption) and ammonia-nitrogen excretion rates were determined by maintaining two juvenile animals (mean individual dry

weight = 1.6 mg) within a 60-mL BOD bottle containing filtered (1 μm glass filter) lakewater, in darkness at 4o

C. In a fourth experiment (No. 4), only oxygen consumption was measured. Decline in oxygen

concentration (mgO2

 L-1 over 24 hours) in (7-10 replicate) bottles containing test animals was adjusted

to include any changes noted in blank (animal-free) bottles. Changes in ammonium concentration were

likewise adjusted to blanks. Following 24 hours of incubation, animals were removed from test bottles,

blotted dry, and weighed. Wet weights were then multiplied by 0.269 to obtain dry weight (dry weight

percentage determined for Diporeia at study site; Landrum 1988) or dry weight of animals was directly

determined after 24-hour drying at 60o C. Oxygen concentrations were determined by Winkler titration

(Grasshoff 1983) while ammonium concentrations were determined with a micro-fluorometric method

(Gardner 1978).

In three other experiments, we examined potential effects of conditions in test vessels on Diporeia sp.

respiration. These included the influence of (a) the presence/absence of a sediment substrate on respiration rate (Experiment No. 5), (b) the amount of time provided for animals to acclimate to the test vessels

prior to vessel closure and the subsequent oxygen consumption determination (Experiment No. 6), and

(c) the decline in availability of ambient dissolved oxygen in test vessel water occurring during the

oxygen consumption determination interval (Experiment No. 7).

Presence/Absence of Sediment Substrate (Experiment No. 5)

We evaluated possible effects of the presence/absence of sediments on Diporeia sp. respiration by

providing a 1-cm deep layer of pre-combusted (550oC for 2 hours) 500-550 μm sand to one group of the

test animals while another group’s oxygen consumption was measured without any substrate at the

bottom of test vessesls. Previous studies (Eriksen 1963,1968, Vernberg et al. 1977) have indicated that

oxygen consumption of benthic animals may be significantly affected by the presence and nature of a

substrate into which animals can burrow.

Animal Acclimation in Test Vessels (Experiment No. 6)

 A sixth experiment focused on identifying possible changes in O2

 consumption resulting from handling

stress and/or increased activity of animals following introduction to test bottles. Oxygen consumption of

pre-acclimated animals (animals held, under flow-through conditions, in test vessels for 24 hours prior

to the experiment) was compared with respiration estimates of freshly-introduced animals during respiration measurement intervals of 3.9, 7.9, 24.4, or 48.8 hours.

Effects of Decreased Oxygen Availabilty on Respiration

Because we relied on closed-vessel respirometry to determine Diporeia sp. O2 consumption rates, we

examined potential effects of declining oxygen concentration during the 24-hour incubation period on

corresponding Diporeia sp. respiration. To test for this effect, we measured Diporeia sp. O2 uptake in a

series of 10 experiments where ambient oxygen concentration at the start of the incubation interval was

reduced to approximately 25-75% of control (near-saturation) values by partial stripping with nitrogen.

The ability of test animals to take up and use available oxygen under such O2-limited conditions was

then evaluated in terms of both respiration rate (mg O2 g-1DW day-1) and corresponding oxygen clearance rate (mL of H2

O cleared of oxygen mg-1 DW h-1). Oxygen clearance rates estimated the total volume of water animals were required to “clear” of oxygen to satisfy required metabolic demands thus

providing an assessment of energetic costs imposed on test animals by low dissolved oxygen concentrations.
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 RESULTS

O:N Experiments (Nos. 1 - 3), O2 Consumption (Experiment No. 4)

Mean oxygen consumption rates measured in the three O:N experiments (1-3) ranged from 13.9 to 31.3

mgO2 g-1DW d-1, while ammonia-nitrogen was 0.45 - 0.77 ng-atom N mg-1hr-1 (Tables 1, 2). Corresponding mean O:N ratios ranged from 61.6 to 230.0. Oxygen consumption measured alone in Experiment

No. 4 was 33.9 + 0.37 (SE).

Presence/Absence of Sediment Substrate (Expermiment No. 5)

In experiment #5, a t-test (Zar 1984) comparing means of O2

 consumption of animals with, and without,

a sand substrate indicated that oxygen consumption rates were not significantly different (P<0.05).

Mean O2 consumption of both substrate and non-substrate groups was 13.9 mg O2 g-1 DW day-1 and

consequently, the two groups were pooled to give an overall mean of 13.9 + 1.5 (SE) mg O2 g-1 DW

day-1).

Animal Acclimation in Test Vessels (Experiment No. 6)

In Experiment #6, animals held in test vessels under flow-through conditions for 24 hours prior to vessel

closure initially exhibited respiration rates that were significantly lower (T test (P<0.05), Zar 1984) than

rates of non-acclimated animals. Although differences in mean O2 consumption were apparent betwen

pre- and non-acclimated groups after 4.1 and 7.9 hours (Figure 1), the two treatments did not differ after

a 24.4-hour interval. The initial elevation of oxygen consumption rates may have been caused by increased activity as the newly-introduced, non-acclimated animals explored the test container (e.g.

Sutcliffe 1984), but subsequently this activity decreased with no discernible differences in mean oxygen

consumption rates existing over 24 hours. Thus, results of both experiments were pooled within an

overall mean of 17.2 + 0.8 (SE) mg O2

 g-1DW day-1.

Effects of Decreased Ambient O2 on Respiration (Experiment No. 7)

Comparison of Diporeia sp. oxygen consumption versus initial ambient oxygen concentration indicated

that O2 consumption rates remained nearly constant over an interval of 12.4 - 7.5 mg O2 L-1 (or a range

of 95 - 57% O2 saturation at 4o

C) and gradually declined at lower ambient O2 levels. This trend has been

Experiment Date Objective DW (mg) N Excretion O2 Consumption O:N Ratio

 No. ng-atom N mg-1 DW h-1

1 4/25/86 O:N 2.4±0.3 (10) 0.47±0.10 (8) 31.3±1.5 (10) 230.0±56.1 (8)

2 5/8/86 O:N 3.0±0.2 (10) 0.95±0.13 (7) 17.2±1.4 (7) 55.8±10.5 (7)

3 9/22/88 Substrate/O:N 1.6±0.1 (10) 0.45±0.08 (7) 13.9±1.5 (10) 92.23±19.8 (7)

4 3/19/86 O2 1.9±0.2 (21) 33.9±0.37 (20)

4/25/86 Consumption

5 5/8/86 Test vessel 1.5±0.1 (10) 17.2±0.8 (10)

Acclimation

6 10/89 Decreased 2.2±0.1 25.5 (Vmax)

11/89 O2 (n=48)

Table 1. Mean Diporeia sp. oxygen consumption, ammonia-N excretion [+ SE n)] measured

in six 1986-1989 experiments.
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Figure 2. Eadie-Hofstee plot of regression line calculated for Diporeia sp.

oxygen consumption (R) (mg O2 g-1 DW

d-1) versus R divided by ambient oxygen

concentration (D.O.) (mg O2 L-1). R=-

2.1939 (R/D.O.) + 25.4247.

Figure 1. Effects of acclimation on

Diporeia sp. oxygen consumption (mg

O2 mg-1 DW day-1). Striped bars

denote respiration rates of animals

pre-acclimated in test vessels for 24

hours, under flow-through conditions,

while cross-hatched bars represent

respiration of non-acclimated animals

in experiments where determination

of oxygen consumption began

immediately following animal introduction to test vessels. Error bars are

+ SE around a mean.
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previously observed in other amphipod species (Gammarus spp.; Sutcliffe 1984). Since the relationship

between dissolved oxygen concentration and O2 consumption approximated that seen for common

single-substrate enzyme reactions, we replotted the data in Eadie-Hofstee form of the Michaelis-Menten

equation (Lehninger 1975) (Figure 2). This transformed the data to linear form and a regression of O2

consumption (dependent variable) versus oxygen consumption divided by ambient O2 concentration

indicated significant linear correlation (P<0.05, r=0.69, n=10). Vmax, the predicted oxygen consumption

rate at 100% O2 saturation was 25.5 mg O2 g-1 DW d-1 and Km the corresponding ambient O2 concentration where oxygen consumption declined to 50% of the Vmax was 2.2 mg O2

 L-1 .

A plot of oxygen clearance rates versus ambient O2 concentration (Figure 3) showed that animals responded to decreased oxygen availabilty by processing progressively larger volumes of water to satisfy

metabolic demands.

DISCUSSION

Oxygen Consumption

Diporeia sp. mean oxygen consumption estimated in our four experiments ranged from 13.9 to 33.9 mg

O2

 mg-1DW day-1. To compare these rates with data from existing published data on Diporeia sp. respiration (Johnson 1970; Johnson and Brinkhurst 1971; Bay of Quinte, Lake Ontario, Johannsson et al. 1985)

we entered the test temperature (4o

C) and the overall mean dry weight (1.6 mg) of our test animals into a

regression equation used to describe O2 consumption of juvenile Lake Ontario Diporeia sp. where;

 ln R = 1.48 + 0.014 T - 0.3 ln W (Johnson and Brinkhurst 1971) and;

 T = Temperature (o

C)

 W = Ash-free dry weight (AFDW)(mg) = 0.87 x dry weight (Landrum 1988)

 R = Respiration (ug O2

 mg-1 AFDW day-1)

When dry weight of our test animals was converted to AFDW by multiplying by 0.87 (Landrum 1988)

for a temperature (T) of 4o

C, this equation gave a respiration rate R (corrected back to a dry weight

Figure 3. Diporeia sp. O2 clearance

(mL H2O cleared of oxygen g-1 DW h-1)

versus ambient oxygen concentration

(mg O2 L-1). Solid line denotes calculated regression line where r=0.82

(significant (P<0.05, n=10) and O2

Clearance = -11.51 (Ambient O2) +

208.78.
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basis) of 4.0 mg O2 g-1 DW day-1 that was only 12 to 29% of our values. Johnson and Brinkhurst (1971)

measured oxygen consumption of juvenile animals using closed vessels, but measured oxygen with an

electrode equipped with an oscillating stirrer. One would expect, a priori, that O2

 consumption might be

elevated if the constant agitation used, produced increased activity and higher oxygen uptake in test

animals. In subsequent studies of Lake Ontario macroinvertebrates, Chua and Brinkhurst (1973) and

Brinkhurst et al. (1972) showed that oligochaete oxygen consumption (also measured by Johnson and

Brinkhurst in the 1971 study) was higher when measured with the electrode/oscillating stirrer device

compared to an electrode/rotary stirrer. Brinkhurst et al. (1972) suggested that excessive vibration

produced by the oscillating stirrer may have led to higher activity and elevated O2

 consumption of test

animals. If Diporeia sp. respiration measured in the 1971 study was similarly affected, O2

 consumption

would be expected to be larger than rates measured with our Winkler method where no water movement

or disturbance occurred.

Sutcliffe (1984) extensively reviewed a variety of factors influencing oxygen uptake in both freshwater

and brackish-marine Gammarus amphipod species and concluded that among numerous studies, estimates of active metabolism (O2

 consumption measured in animals held in constantly-stirred water) are

generally 2-4 times the standard or resting rates (where O2

 consumption was measured in unstirred,

undisturbed water). Given such results, Johnson and Brinkhurst’s data should reflect higher activity and

oxygen consumption compared to our estimates. However, our estimates were greater than rates predicted by the Lake Ontario equation for animals with weights comparable to those tested in our study.

Aside from the minor increase in activity we noted following introduction of animals to test vessels (see

results) Diporeia sp. activity was likely not higher than the activity level of Lake Ontario animals.

Cederwall (1979) noted increased activity and higher oxygen consumption associated with swimming in

overlying water, during dark portions of a laboratory photoperiod in two Baltic pontoporeiids, and such

differences in activity and respiration rates might occur in Diporeia sp. We did not evaluate such possible diurnal changes in activity and oxygen consumption. However, our observations of Diporeia sp.

during periods when animals were held in sediments and constant darkness have indicated that animals

generally adopt a consistent level of activity under such conditions, and we believe that such activity

may may approximate normal levels for individuals in natural conditions.

A second study of Diporeia sp. respiration at three Lake Ontario locations (Johannsson et al. 1985)

provided a regression equation where;

R = exp [ -7.19 + 0.036 (T) - 0.219 ln (W) ] and;

R = Respiration (mg O2

 mg-1 DW hr-1)

T = Temperature (o

C)

W = Dry Weight (mg)

At 4oC and a mean dry weight of 1.6 mg this equation predicts O2 consumption of 18.9 mg O2 g-1DW d-1.

This value was toward the lower end of our Lake Michigan range of 13.9 - 33.9 mg O2 g-1DW d-1 and the

calculated Michaelis-Menten Vmax of 25.5 mg O2 g-1DW d-1.

Overall, our estimates of Lake Michigan Diporeia sp. respiration rates appear to be equal to, or greater

than, rates noted for Lake Ontario animals. It is possible that marked physiological differences exist

between Diporeia sp. populations of the two lakes. Although the underlying reasons for such differences

are not presently known, the two populations may possess distinct and separate life history strategies in

response to differing environments of the two lakes. Moreover, given the apparent existence of numer-
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ous Diporeia species in the Great Lakes and other large lakes of North America (see Introduction), the

oxygen consumption differences we noted between our Lake Michigan data and Lake Ontario results

may be due to species differences between resident populations of the two lakes.

Results of Michaelis-Menten analysis of Diporeia sp. oxygen consumption at decreased ambient O2

concentrations demonstrated that the amphipod is able to maintain a fairly uniform uptake of O2 , despite

declines in available oxygen. Thus, Lake Michigan Diporeia sp. are partial respiratory “regulators” and

have at least some capability of tolerating low-oxygen concentrations. In addition, these results suggest

that the approximate 10% drop in dissolved O2

 concentrations that occurred during our 24-hour incubations had minimal influence on the corresponding Diporeia sp. oxygen consumption rates. The 24-hour

incubation interval was also sufficiently long to offset effects of increased O2

 consumption noted during

the early portion of the incubation period. Thus, our 24-hour measurement interval was sufficiently long

to ensure reliable O2

 consumption estimates, yet short enough to avoid effects of decreased oxygen

availabilty occurring in test vessels.

We were not able to compare our Michaelis-Menten / oxygen clearance results from Lake Michigan

Diporeia sp. directly with either of the two Lake Ontario studies because effects of decreased ambient

O2

 concentrations were not examined. Moreover, Michaelis-Menten analysis of of data for two Baltic

Sea pontoporeiids Monoporeia affinis (formerly Pontoporeia affinis) and Pontoporeia femorata did not

show any significant linear relationship (P<0.05) existing for a Eadie-Hofstee transform (O2

 consumption versus O2

 consumption divided by O2 concentration) (raw data provided by Hans Cederwall, Dept.

Systems Ecology, Univ. Stockholm, S-106 91, Stockholm, Sweden).

Ammonia Excretion

Mean ammonia excretion determined for Diporeia sp. in the present study varied between 0.45 and 0.77

ng-atom N mg-1 DW hr-1. This range was lower than a previously reported 3-5 ng-atom N mg-1 DW hr-1

range for Lake Michigan Diporeia sp. obtained at 45 and 100 m depths and held at 4oC (Gardner et al.

1987), but overlapped with a 0.5-1.3 ng-atom N mg-1 DW hr-1 range recently reported for animals collected at our 29 m Lake Michigan station (4o

C test temperature; Gauvin et al. 1989). Nitrogen excretion

rates for Diporeia sp. are generally lower than published rates of other amphipod species. Nitrogen

excretion of two Baltic brackish-marine gammarid species (10o

C) G. duebeni and G. oceanicus ranged

from 2.6-2.8 ng-atom N mg-1 DW h-1 (Tedengren et al. 1988) and mean nitrogen excretion of the marine

amphipod Corophium volutator was 5.7 ng-atom N mg-1 DW h-1 at 22o C (Hawkins and Keizer 1982).

Although the rates of these species are higher than those we noted for Diporeia sp., this difference may

be partly explained by differences in experimental temperatures employed.

O:N Ratios

O:N ratios (atoms of oxygen consumed per atom of nitrogen excreted) (Figure 4) were generally greater

than 60 (i.e. 62% of all data points exceeded this value). These results suggest that most of the animals

in the experiments were relying primarily on lipids as metabolic substrates with protein catabolism

playing a secondary role. These conclusions are based on a recent, extensive review of the use of O:N

atomic ratios to assess nutritional status and underlying metabolism (anabolism/catabolism) in crustacean zooplankton populations (Mayzaud and Conover 1988). In general, zooplankton O:N ratios were

characterized as being strongly influenced by the disposition of an animal’s free amino acid pool with

regard to tissue growth (anabolism) or tissue degradation (catabolism). Depending on an animal’s

internal energy requirements, nutritional status and trophic history, and quantity and quality of food

supply, free amino acids may be incorporated within tissues as protein, or catabolized to produce energy
12

(primarily through transdeamination via the glutamate dehydrogenase pathway (GDH) in crustaceans).

Oxidative deamination of amino acids through the GDH pathway, in turn, produces ammonia which is

subsequently excreted. When a variety of amino acids and possible catabolic pathways were considered

with respect to NH3 excretion and oxygen consumption, Mayzaud and Conover concluded that animals

catabolizing protein exclusively would exhibit O:N ratios between 3 and 16. When theoretical considerations of both protein and lipids were considered, catabolism of approximately equal amounts of these

two substrates were expected to produce O:N ratios between 50 and 60. Above 60, O:N ratios were

interpreted as reflecting a lipid-dominant catabolism. Overall, the apparent lipid-dominant catabolism

we observed in Diporeia sp. was far different than results noted in the two other existing studies of O:N

ratios of amphipods. Tedengren et al. (1988) found a protein-dominant catabolism (O:N ratios of 33-38)

in two brackish marine gammmarids and Aarset and Aunaas (1990) noted utilization of a protein/lipid

substrate by an arctic sympagic gammarid (O:N ratio of 21). In two other related studies, however,

measurement of the respiratory quotient (CO2

 produced/O2 consumed) indicated lipid-dominant catabolism (R.Q. of 0.72-0.85) in Gammarus pulex (Wright and Wright 1976) and catabolism of both lipid and

protein in G. fossarum (R.Q.=0.64) (Orzechowski 1974).

While the above results indicate that the nature of the substrates catabolized by different amphipod

species may vary, other indirect evidence suggests that Diporeia sp. has a lipid-dominant catabolism,

given its biochemical composition and life history. Total carbon content (more specifically lipid content)

is closely associated with variation in the O:N ratio of aquatic invertebrates (Mayzaud and Conover

1988). The unusually high lipid content of Diporeia sp. is well-known (Green 1971, Gardner et al.

1985). Lipids can constitute up to half of the amphipod’s dry weight (Gardner et al 1985) and lipid

content steadily increases with size throughout juvenile life (Quigley et al. 1989). Thus, it is not surprising that the O:N ratios we observed show a catabolism that is largely lipid-dominant.

Diporeia sp.’s ability to store and use lipids appears to be part of an energetic strategy that allows the

organism to rapidly exploit food (primarily phytoplankton or detritus generated from the Lake Michigan

Figure 4. Diporeia sp.

oxygen consumption

(ng-atom O mg-1 DW h1) versus N excretion

(ng-atom N mg-1 DW

h-1). Regions in figure

correspond to (1)

Exclusive catabolism

of protein (O:N<16),

(2) Catabolism dominated by protein (16

>O:N<50), (3) Catabolism of equal amounts

of protein and lipid

(50>O:N<60), and (4)

Catabolism dominated

by lipids (O:N>60).
13

spring diatom bloom) that is only seasonally available (Gardner et al. 1990). This strategy is similar to

one typical of crustacean zooplankton species which store large lipid supplies, enabling them to maintain

metabolic homeostasis for considerable periods when food is limited or unavailable. For such species,

O:N ratios will primarily reflect seasonal changes in biochemical composition associated with storage

and use of energy reserves (Mayzaud and Conover 1988). Given Diporeia sp.’s high lipid content (most

notably triacylglycerols; Gardner et al. 1985, Quigley et al. 1989), this amphipod should also show a

close relationship between O:N ratio and biochemical composition. Though Diporeia sp. lipid content is

known to change seasonally (Gardner et al. 1985), its relationship to O:N ratios has not yet been examined. Although we did not document lipid content of test animals in our study, the clumping of September O:N data points in the upper oxygen consumption range (Figure 4) suggests some seasonal difference, compared to April and May results. Additional assessment of such trends relative to Diporeia sp.

biochemical composition is clearly needed and may yield further detailed understanding of the energetic

and biochemical changes occurring throughout the animal’s life history.

ACKNOWLEDGMENTS. We thank Catherine Hill, Tom Nalepa, and Henry Vanderploeg for the

valuable comments they provided in review of our manuscript. We also extend special thanks to Hans

Cederwall for graciously providing his raw data and for his review of the manuscript.

REFERENCES

Aarset, A.V., and T. Aunaas. Effects of osmotic stress on oxygen consumption and ammonia excretion of

the Arctic sympagic amphipod Gammarus wilkitzkii. Marine Ecology Progress Series 58:217-224

(1990).

Anderson, E.D., and L.L. Smith. A synoptic study of food habits of 30 fish species from western Lake

Superior. Univ. Minn. Agric. Exp. Stn. Tech. Bull. no. 279 (1971).

Bousfield, E.L. Revised morphological relationships within the amphipod genera Pontoporeia and

Gammaracanthus and the “glacial relict” significance of their postglacial distributions. Canadian Journal of Fisheries and Aquatic Sciences 46:1714-1725 (1989).

Brinkhurst, R.O., K.I. Chua, and N.K. Kaushik. Interspecific interactions and selective feeding by

tubificid oligochaetes. Limnolology Oceanography 17:122-133 (1972).

Cederwall, H. Diurnal oxygen consumption and activity of two Pontoporeia (Amphipoda, Crustacea)

Species, In Cyclic Phenomena in Marine Plants and Animals 13th European Marine Biology Symposium, E. Naylor and R. G Hartnoll, Eds. Pergamon, Oxford, England, pp. 309-316 (1979).

Chua, K.E., and R.O. Brinkhurst. Evidence of interspecific interactions in the respiration of tubificid

oligochaetes. Journal of Fisheries Research Board of Canada 30:617-622 (1973).

Dermott, R., and K. Corning. Seasonal Ingestion Rates of Pontoporeia hoyi (Amphipoda) in Lake

Ontario. Canadian Journal of Fisheries and Aquatic Sciences 45:1886-1895 (1988).

Eriksen, C.H. The relation of oxygen consumption to substrate particle size in two burrowing mayflies.

Journal of Exp. Biology 40:447-453 (1963).
14

Eriksen, C.H. Ecological significance of respiration and substrate for burrowing Ephemeroptera. Canadian Journal of Zoology 46:93-103 (1968).

Evans, M.S., M.A. Quigley, and J.A. Wojcik. Comparative ecology of Pontoporeia hoyi populations in

southern Lake Michigan: the profundal versus the slope and shelf regions. Journal of Great Lakes

Research 16:27-40 (1990).

Gardner, W.S. Microfluorometric method to measure ammonium in natural waters. Limnolology Oceanography 23:1069-1072 (1978).

Gardner, W.S., T.F. Nalepa, W.A. Frez, E.A. Cichocki, and P.F. Landrum. Seasonal patterns in lipid

content of Lake Michigan macroinvertebrates. Canadian Journal of Fisheries and Aquatic Sciences

42:1827-1832 (1985).

Gardner, W.S. , T.F. Nalepa, and J.M. Malczyk. Nitrogen mineralization and denitrification in Lake

Michigan sediments. Limnolology Oceanography 32:1226-1238 (1987).

Gardner, W.S. , M.A. Quigley, G.L. Fahnenstiel, D. Scavia, and W.A. Frez. Pontoporeia hoyi an apparent direct link between spring diatoms and fish in Lake Michigan. In Functional and Structural Properties of Large Lakes, M. Tilzer, Ed. Heidelberg, Springer-Verlag, pp 632-644 (1990).

Gauvin, J.M., W.S. Gardner, and M.A. Quigley. Effects of food-removal on nutrient-release rates, lipid

content and survival time of Lake Michigan Pontoporeia hoyi. Canadian Journal of Fisheries and

Aquatic Sciences 46:1125-1130 (1989).

Grasshoff, K. Determination of oxygen. In Methods of Seawater Analysis. K. Grasshoff, M. Ehrhardt,

and K. Kremling, Eds. Verlag Chemie, Wheinheim, Federal Republic of Germany, Chapter 4, pp. 61-72

(1983).

Green, R.H. Lipid and caloric contents of the relict amphipod Pontoporeia affinis in Cayuga Lake, New

York. Journal of Fisheries Research Board of Canada 28:776-777 (1971).

Hawkins, C.M., and P.D. Keizer. Ammonia excretion in Corophium volutator: Using an automated

method. Canadian Journal of Fisheries and Aquatic Sciences 39:640-643 (1982).

Hill, C., and R. Elmgren. Vertical distribution in the sediment in the co-occurring benthic amphipods

Pontoporeia affinis and P. femorata. Oikos 49:221-229 (1987).

Johannsson, O.E., R.M. Dermott, R. Feldkamp, and J.E. Moore. Lake Ontario Long Term Biological

Monitoring Program. Report for 1981 and 1982. Can. Tech. Rep. Fish. Aquat. Sci. No. 1414 (1985).

Johnson, M.G. Production, energy flow and structure in benthic macroinvertebrate communities of Lake

Ontario. Doctoral Dissertation, Univ. Toronto, Ontario, Canada (1970).

Johnson, M.G., and R.O. Brinkhurst. Production of benthic macroinvertebrates of Bay of Quinte and

Lake Ontario. Journal of Fisheries Research Board of Canada 28:1699-1714 (1971).
15

Landrum, P.F. Uptake, depuration and biotransformation of anthracene by the scud, Pontoporeia hoyi.

Chemosphere 11:1049-1057 (1982).

Landrum, P.F. Toxicokinetics of organic xenobiotics in the amphipod, Pontoporeia hoyi: Role of physiological and environmental variables. Aquatic Toxicology 12:245-271 (1988).

Landrum, P.F., M.D. Reinhold, S.R. Nihart, and B.J. Eadie. Predicting the bioavailabilty of organic

xenobiotics to Pontoporeia hoyi in the presence of humic and fulvic materials and natural dissolved

organic matter. Environmental Toxicologyand Chemistry 4:459-467 (1985).

Lehninger, A.L. Biochemistry. New York: Worth Publishers Inc. (1975).

Mayzaud, P., and R.J. Conover, R.J. O:N atomic ratio as a tool to describe zooplankton metabolism.

Marine Ecology Progress Series 45:289-302 (1988).

Nalepa, T.F. Estimates of macroinvertebrate biomass in Lake Michigan. Journal of Great Lakes Research 15:437-443 (1989).

Nalepa, T.F., M.A. Quigley, K. Childs, J.M. Gauvin, T.S. Heatlie, M. Parker, and L. Vanover. The

macrobenthos of southern Lake Michigan. NOAA Data Report ERL GLERL-28, Great Lakes Environmental Research Laboratory, Ann Arbor, MI (1975).

Orzechowski, B. The correlation between the respiration metabolism of Gammarus fossarum (Koch)

and the presence of some ammonium salts in water. Acta Universitatis Nicolai Copernici, Nauki

Matematyczno-Pryzrodnicze, Zeszyt. 34:1-16 (1974).

Quigley, M.A. Gut fullness of the deposit-feeding amphipod, Pontoporeia hoyi, in southeastern Lake

Michigan. Journal of Great Lakes Research 14:178-187 (1988).

Quigley, M.A., J.F. Cavaletto, and W.S. Gardner. Lipid composition related to and size and maturity of

the amphipod Pontoporeia hoyi. Journal of Great Lakes Research 15:601-610 (1989).

Segerstrale, S.G. The distribution and morphology of Pontoporeia affinis Lindstrom f. brevicornis

(Crustacea: Amphipoda) inhabiting North American lakes, with a description of a new aberrant form

from the area. Comment. Biol. Soc. Sci. Fennica 32:3-19 (1971).

Sutcliffe, D.W. 1984. Quantitative aspects of oxygen uptake by Gammarus (Crustacea, Amphipoda): a

critical review. Freshwater Biology 14:443-489 (1984).

Tedengren, M., M. Arner, and N. Kautsky. Ecophysiology and stress response of marine and brackish

water Gammarus species (Crustacea, Amphipoda) to changes in salinity and exposure to cadmium and

diesel-oil. Marine Ecology Progress Series 47:107-116 (1988).

Vainola, R., and S. Varvio. Molecular divergence and evolutionary relationships in Pontoporeia (Crustacea: Amphipoda). Canadian Journal of Fisheries and Aquatic Sciences 46:1705-1713 (1989).
16

Van Oosten, J., and H.J. Deason. The food of the lake trout (Cristivomer namaycush namaycush) and of

the lawyer (Lota maculosa) of Lake Michigan. Transaction of the American Fisheries Society 67:155-

177 (1938).

Vernberg, W.B., B.C. Coull, and D.D. Jorgensen. Reliability of laboratory metabolic measurements of

meiofauna. Journal of Fisheries Research Board of Canada 34:164-167 (1977).

Wells, L. Food of alewives, yellow perch, spottail shiners, trout-perch, and slimy and four-horn sculpins

in southeastern Lake Michigan. U.S. Fish. and Wild. Serv. Tech. Pap. 98 (1980).

Well, L., and A.M. Beeton. Food of the bloater, Coregonus hoyi in Lake Michigan. Transactions of the

American Fisheries Society 92:245-255 (1963).

Wright, E.A., A.A. and Wright. The respiratory quotient of Gammarus pulex L. Comp. Biochem.

Physiol. 53A:45-46 (1976).

Zar, J.H. 1984. Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall Inc. (1984).