INTRODUCTION Information on the Gaping Behaviors

INTRODUCTION

Information on the gaping behaviors exhibited by bivalves is a potentially important tool for biological monitoring of water quality. Bivalves can filter tremendous quantities of water daily and can, therefore, accumulate pollutants in the tissues to a concentration of

1000 to 10,000 times that of surrounding waters (Pynn”nen, 1990). Several studies have used the concentrations of pollutants accumulated in bivalve tissue as indicators of water quality and pollutant levels (Green et al., 1989; Kramer et al., 1989, Muncaster et al., 1990;

M„kel„ and Oikari, 1990; M„kel„ et al., 1991). Since the degree of valve gaping has a linear relationship to the pumping rate (Jírgensen et al. 1988), gaping behavior directly affects both the efficiency of water filtration and the accumulation rate of pollutants and, hence, the sensitivity of bivalves as environmental monitors. In addition, the valve-closure response has been used as an early warning indicator of poor water quality (Borcherding and

Kramer et. al., 1989; Volpers, 1994), so the daily rhythmic gaping pattern can have diagnostic value.

Rhythms of gaping behavior in bivalves have been reported in several species from both marine (Morton, 1970a; Brand and Taylor, 1974) and freshwater environments

(Morton, 1970b). In marine species, these rhythms can be correlated to environmental factors such as diurnal (Sal nki, 1966), tidal (Morton, 1970a) or monthly rhythms (Brown et al., 1956). Much less research had been completed with freshwater mussels. Species living in an eutrophic lake or pond, such as Anodonta anatina and Unio tumidus (Englund & 1 Heino, 1994), and Ligumia subrostrata (McCorkle et al., 1979), have the greatest gaping

activity during the night. The physiological mechanisms for controlling diurnal behaviors are

still unclear. The purpose of this study was to examine gaping behavior of seven unionid

species and determine if this behavior is correlated with their habitats and environmental

factors. As heart rate and gaping behavior of bivalves have also been applied in

environmental monitoring, we also studied the relationship between these two variables in

Pyganodon grandis, which exhibits a diurnal pattern of gaping behavior.

MATERIALS AND METHODS

Mussels and Acclimation

Seven species of adult mussels of moderate size were collected in Virginia and

Tennessee from June to August, 1993 -1996. In Virginia, Pyganodon grandis (n=22) were collected from the profundal zone of Claytor Lake; Elliptio complanata (n=17) were collected from pools in the Nottoway River; and Villosa iris (n= 10) were obtained in riffles from the North Fork Holston River. Amblema p. plicata (n=19), Quadrula pustulosa

(n=21), Pleurobema cordatum (n=14) and Fusconaia ebena (n=21) were collected from

Kentucky Lake, in the lower Tennessee River, Tennessee. All of the mussels except V. iris were acclimated in 30 L aquaria with sand substratum and a flow-through system for 3 to 7 days before observations began. Male V. iris were transferred from another holding facility, and observations began 2 wk after acclimation in the laboratory. The animals were fed a

2 commercial algal diet (SUN Chlorella "A" granules by YSK international Corp., Japan) daily, in the morning at a concentration of approximately 60,000 cells/30L tank. The water temperature was controlled at 24.5 °C.

Valve gaping observations

After acclimation of the mussels in the laboratory, the presence of a possible diurnal cycle of activity was measured by examining a group of mussels partially burrowed in the sand bottom of the aquarium. The gaping behavior was observed for 96 hr (except A. p. plicata, which was observed for 48 hr). The photoperiod was controlled (12 light : 12 dark) by a timer, and the light period started at 0730. From 10 to 28 mussels of each species were observed. The number of specimens with shells gaped was counted every 1.5 hr during the day and every 3 hr at night. Observations during the night were made with the aid of a dim light. The percent gaped of the observed mussels during the dark and light periods were compared by Mann-Whitney Rank Sum Test (Siegel, 1956).

Simultaneous observation of heart rate and gaping of P. grandis

Four P. grandis were used to record heart rate. Heart activity was detected by using an impedance converter according to Trueman et al (1973). A small hole was drilled in one valve in the vicinity of the heart. Small insulated wires (with the ends bared) were inserted through the holes and sealed with glue. The resulting record, produced with a UFI model

2991 impedance converter, was displayed on a Grass polygraph recorder. The gaping observations and heart rate measurements were made simultaneously for 96 hr. As the pilot

3 studies showed that surgery was lethal to V. iris and E. complanata, consequently, the heart rate observations of these species were not conducted.

RESULTS

The pattern of valve movement is expressed as the percent (%) of animals with valves gaped at a particular time during the 24 hr period. For a continuous 96 hr of observation under photoperiod 12:12, P. grandis from Claytor Lake had the highest percent of gaping during the dark period (median = 96.4%), and the lowest percent of gaping during the light period (median = 82.1%) (Fig.1A). There was a statistically significant difference between the % gaping in the dark and light period (P < 0.0001). Conversely, the riverine species Q. pustulosa had the lowest percent of gaping at night (Fig. 1B). The median value during the day (61.9%) was significantly (P < 0.0001) higher than that at night (42.9%).

There was no obvious diurnal rhythm of shell gaping for the other five riverine species; P. cordatum (P = 0.42), F. ebena (P = 0.92), A. plicata (P = 0.58), E. complanata (P = 0.38), and V. iris (P = 0.68)(Fig. 2 A to E).

In the four P. grandis, heart rate varied with the closing and opening of the valves

(Fig. 3 A & B). Mussels had the highest heart rate of approximate 18 beats/min when they were open, and the lowest heart rate of approximate 3 beats/min when they were closed.

4 A Pyganodon grandis 100 N = 28 90

80

70

60

B 80 N = 21 Quadrula pustulosa 70 Percent gaped (%) 60

50

40

30

20 12 18 0 6 12 18 0 6 12 18 0 6 12 18 0 6 12

Time (hours)

Fig. 1 The 96-hr observations of the percent of specimens of P. grandis and Q. pustulosa

that were gaped, based on sample size (N). The median value of the dark period

(96.4%) was significantly higher than the median value of the light period (82.1%)

for P. grandis (P < 0.0001). The median value of the light period (61.9%) was

significantly higher than the median value of the dark period (42.9%) for Q.

pustulosa (P < 0.0001).

5 80 A N = 14 Pleurobema cordatum 70 60 50 40 B N = 21 Fusconaia ebena 100 90 80 70 C N = 19 80 Amblema plicata 70 60 50

Percent gaped (%) 100 D N = 17 Elliptio complanata 90 80 70 60 50 100 E N = 10 Villosa iris 80 60 40

12 18 0 6 12 18 0 6 12 18 0 6 12 18 0 6 12

Time (hours)

Fig. 2 The 96-hr-observations of the gaping of 5 species collected from riverine habitats.

Each point on the line represents the percent gaped, which was calculated based on

the sample size (N) indicated on the graph. There was no significant difference

between the median values of the light period versus dark period for these 5 species.

6 Pyganodon grandis A

Opened 3 2

Closed 1 0 9 12 0 15 3 18 6 21 9 12 0 15 3 18 6 21 9 12 0 15 3 18 6 21 9 12 0 15 3 18 6 21 9 12 Dark period Time ( hr )

B Pyganodon grandis

20

15

10

5 Heart beat / min 0 9 12 0 15 3 18 6 21 9 0 12 3 15 6 18 9 21 12 0 15 3 18 6 21 9 12 0 15 3 18 6 21 9 Dark period Time ( hr ) 12

Fig. 3 A. The 96-hour observations of opened vs. closed specimens of Pyganodon grandis,

where 1 is closed, 2 is opened and closed during the first half-hour observation, and

3 is opened. B. The 96-hour observations of heart beat of Pyganodon grandis under

12 light: 12 dark photoperiod.

7 DISCUSSION

The diurnal rhythm of gaping by Pyganodon grandis is similar to that recorded for

Anodonta cygnea L. (Barnes, 1955, 1962), Anodonta anatina and Unio tumidus (Englund and Heino, 1994), and Unio pictorum L. (Morton, 1970b). The habitats of these species are similar, as they all live in lentic habitats of ponds or lakes. My results for E. complanata differ from those of Imlay (1968), who reported that E. complanata has a rhythmic response to daily light changes, although the mussels in my study had a slight trend of greater opening during the night and greater closure during the day (Fig. 2D). Because mussels were fed in morning, food availability may have influenced the gaping behavior observed. A possible explanation for the missing rhythmic pattern for E. complanata is that the mussels were collected from habitats which had higher currents (shallow bank area of the Nottoway

River) than that of Imlay's studies (from Carolina Biological Supply Co., NC). This topic will be further discussed below.

The diurnal rhythm of freshwater mussels can be driven by an endogenous

"biological clock" or exogenous factors (i.e., photoperiod, availability of food, pheromones from other individuals). The rhythmic patterns producing an endogenous rhythm are generally not influenced by changes in environmental factors. For example, the common lake clam Ligumia subrostrata (Say) had a rhythmic oxygen consumption pattern under constant light for 14 days (McCorkle et. al., 1979). On the other hand, if the rhythm results from exogenous factors, changes in environmental factors should alter the diurnal pattern. There

8 seems to be more evidence that diurnal rhythms in bivalves are driven by exogenous factors

(Sal nki, 1966; McCorkle et. al, 1979; Graves and Dietz, 1980; McCorkle-Shirley, 1982).

Photoperiod is a major exogenous factor regulating diurnal rhythms. McCorkle et. al

(1979) found that Ligumia subrostrata had the highest valve gaping activity 1~2 hr after

onset of darkness, but the rhythmic valve gaping behavior was lost in constant light. Sal nki

(1966) also found that the diurnal rhythm in two Mediterranean lamellibranchs (Pecten

jacobaeus and Lithophaga lithophaga) was regulated by the light-dark period. In addition

to the gaping behavior, it was also noted that the active uptake of Na+ reached a maximum

during the dark period for Corbicula fluminea (McCorkle-Shirley, 1982) and in a unionid species, Carunculina (=Toxolasma) texasensis (Graves and Dietz, 1980). Both rhythmic

patterns were lost under constant light.

Several causes of rhythmic activity, other than photoperiod, have been suggested by

previous studies. Englund and Heino (1994) found that a high percentage of gaped

specimens of Anodonta anatina and Unio tumidus was correlated with a lower temperature,

which was also correlated with photoperiod. Sal nki and V‚r¢ (1969) proposed that the increased openness at night is related to decreasing oxygen levels. However, this seems doubtful because higher activities and oxygen demand were found in those species during the night, which would be maladaptive for hypoxia. Morton (1970b) suggested that gaping rhythm is endogenous and related to feeding and digestion. Englund and Heino (1994) stated that feeding may also be controlled by external factors such as availability of food 9 organisms. McMahon (1991) suggested that activity rhythms of Unionidae may be correlated with diurnal feeding and vertical migration cycles, because individuals can come to the substrate surface to feed at night and retreat below it during the day, hence avoiding visual predators. However, further research is required to confirm this. For mussels living in the profundal area of eutrophic lakes, where light intensity during the day is low, visual predators are probably not an important consideration.

The rhythmic gaping in many species would appear to depend on whether the habitat is lotic or lentic, and the associated environmental factors such as vertical migration of algae and plankton. Most of the species reported in earlier studies to have higher activities during the night were sampled from a relatively static water habitat. In such waters, several species of zooplankton, phytoplankton and algae have been found to vertically migrate upward during the day and downward at night (Hutchinson, 1967). For example, the alga Eudorina elegans was maximum at the surface at noon and later descended into deeper water.

Gonyostomum semen moved upward in the early morning, with the maximum concentration in the top meter of the pond. As phytoplankton is the principal live food of most marine and freshwater bivalves (Ukeles, 1971; Webb and Chu, 1983), freshwater mussels living in standing water may feed on the organisms that migrate vertically and thus would be gaped during the night. On the other hand, the diurnal pattern of vertical migration of algae or zooplankton has rarely been found in a stream, and thus there is no need for the diurnal pattern of gaping. In my study, the diurnal pattern of gaping at night was only found in P. grandis, which typically lives in lentic waters. The remaining 6 species collected from rivers

10 did not show such a diurnal pattern. I also found that mussels were siphoning during the day

in the river when we collected them in the field (personal observations). This pattern was

also observed by Englund and Heino (1994) and Sal nki and V‚r¢ (1969). Further support

for the above suggestion is seen in the work of Englund and Heino (1996), who found that

Anodonta anatina showed a diurnal rhythm of valve movement when the mussels were observed in a lake. However, when the same species was observed in the river, the diurnal rhythms were almost nonexistent.

In captivity, food availability can play a role in valve movements. Higgins (1980)

found that introduction of food induces the unfed quiescent bivalves to open and suggested

that bivalves can detect ambient suspended food. Sprung and Rose (1988) also found that

the pumping rates of mussels were increased when particle concentrations in the water were

increased. Perhaps the diurnal pattern is more important when food availability is low

because of the energetic cost of pumping. Several laboratory studies of the rhythmical valve

movement of freshwater mussels indicate that the animals have their valves closed for most

of the time, and that the rhythms are very regular (Barnes, 1955 & 1962; Morton, 1970b),

which may result from the low food availability and static water. Englund and Heino (1994)

also found that in situ, Unio tumidus in a eutrophic lake has less evident gaping behavior

than Anodonta anatina in an oligotrophic lake which has less food available. While food

availability may be an important factor in gaping behavior, rhythmic activity may persist

even in the absent of food in some species. McCorkle et. al. (1979) found that L. subrostrata

11 (Say) collected from ponds exhibited rhythmic activity even after being in the laboratory for

longer than one year without feeding.

I found that heart rate of P. grandis was highest when the animals were active and siphoning water, and lowest when the valves were closed. This is similar to L. subrostrata

(Dietz and Tomkins,1980) and Lampsilis radiata (Counts et. al., 1977). As the heart rate can increase from ~3 beats/min to ~18 beats/min when going from closed to gaping, the metabolic cost during gaping must be much higher than that for remaining closed. Famme

(1980) found that under starvation the "open" oxygen consumption of M. edulis is six times higher than the "closed" oxygen consumption; but for those in the fed condition, the "open" oxygen consumption was only 3 times higher than "closed" oxygen consumption. Hence, from the perspective of energy cost, the pattern of gaping could be more important when food availability is low.

For mussels living in running water, energy expenditure in pumping can be reduced by adjusting the body position to the current. Hence, the diurnal pattern will not be as beneficial as when living in a lake or pond. This may help explain why the mussels sampled from running water show a less evident diurnal pattern than those living in standing water.

Englund and Heino (1994) indicated that A. anatina (which has a more evident diurnal gaping) can be found in both oligotrophic and eutrophic lakes, but U. tumidus (in which diurnal gaping is less evident) can only be found in eutrophic lakes. This may be evidence that a diurnal pattern of gaping in static water has an ecological advantage.

12 The reason for the higher activity of Q. pustulosa during the day is unclear. As the algae solution was fed daily in the morning, the rhythmic pattern might be exogenously driven by food. To test this hypothesis, the feeding time was switched to the dark period after one week of observations of the diurnal pattern. The rhythmic pattern remained unchanged for the first two days, but then the rhythmic pattern disappeared and the percent of specimens open was reduced. As the mussels were closed during the night at the beginning of the observation, they may not have detected food for the first two days.

However, it is difficult to conclude whether the lost rhythmic patterns resulted from change of feeding time or to stresses caused by acclimation in the laboratory.

Reproductive activities can also affect valve gaping. For example, female V. iris have a diurnal pattern of gaping during the reproductive season, with the mantle extended to attract the host fish (Henley, 1997). In my study, only male V. iris were used and did not show evidence of diurnal gaping. Therefore for some species, it is important to consider the reproductive period and sex of mussels.

I suggest that before using the valve activity of freshwater mussels for environmental monitoring or early warning systems, it is important to establish the diurnal gaping behavior for the species to be used. For early warning systems in static water, mussels from standing waters may be good indicators for monitoring the water conditions at night but not during the day. In a running water system, maintaining the health condition and continuous opening

13 of the mussels needs to be watched, so that the shutting of the valves can reflect the actual deterioration of water quality instead of poor mussel health. From a practical perspective of holding mussels in an artificial environment, the best feeding time for mussels sampled from a standing water system seems to be during darkness, although for mussels removed from riverine systems, the feeding time may not be important.

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