Many Benthic Marine Invertebrate Species Have Planktonic Larvae in Their Life Histories (Thorson, 1950)

Home , Larva, Marine invertebrates, Phytoplankton, Plankton

J. exp. mar. Biol. Ecol., 1980, Vol. 44, pp. 1-28 © Elsevier/North-Holland Biomedical Press

GROWTH AND ENERGY BALANCE DURING THE LARVAL LIVES OF THREE PROSOBRANCH GASTROPODS

JAN A. PECHENIK ! Graduate School of Oceanography, UniversiO, of Rhode Island, Kingston, RI02881, U.S.A.

Abstract: Larvae of most marine invertebrates postpone metamorphosis in the absence of a suitable environment for the adult stage. Larvae of three marine gastropod species, Ih,anassa obsoleta (Say), CrepidulaJbrnicata (L.), and Bittium alternatunl (Say), differed in the potential durations of their delay periods and in their ultimate fates in the absence of preferred substratum for metamorphosis in the laboratory. Experiments were conducted to test the hypothesis that differences in the delaying capabilities of the three species result from differences in the magnitude of metabolic adjustments made during the delay period. Carbon and caloric balances were calculated for larvae of llyanassa obsoleta before and after their attainment of competence, using data on feeding rates, respiration rates, and retention efficiencies. An excess of carbon and energy intake over expenditure was calculated in all experiments. As predicted from those calculations, but not from measurements of shell length increase, tissue biomass of I. obsoleta increases at a constant rate throughout larval life. Similarly, growth rate of larvae of Crepidulafornicata does not decline with the onset of metamorphic competence. Larvae of Bittiunl alter- nature also show substantial growth during delay of metamorphosis. Morphological differentiation continues during the delay periods of Ilyanassa obsoleta and Crepidula fornicata. Therefore, there is no indication that changes in energy balance directly limit the potential duration of the delay period in these Sl;~,cies. However, these data suggest a relationship between growth rate prior to the onset of competence and the potential duration of the delay period. The hypothesis is presented that there may be a program for larval development with a genetically fixed endpoint and that potential duration of the delay period may be determined by the rate at which larvae progress through this developmental program. Ecological factors potentially operating to select for differences in delay capabilities among species are discussed.

INTRODUCTION

Many benthic marine invertebrate species have planktonic larvae in their life histories (Thorson, 1950). Ev~,'ntually, the larvae reach a point in development at which metamorphosis to the benthos becomes possible, and then they begin asses- sing the suitability of substrata for settlement and metamorphosis (Wilson, 1958; Crisp, 1974). Termination of larval life involves two steps~, settlement, which is reversible, and metamorphosis, which is an irreversible morphological alteration to adult form (Crisp, 1974). For gastropod veligers, metamorphosis is defined as the loss of the larval swimming organ, the velum. Loss of the velar lobes is the first gross morphological indication that the transition from larval to adult form is taking place (Bonar & Hadfield, 1974).

I Present address: Biology Department, Tufts University, Medford, MA 02155, U.S.A. 1 2 JAN A. PECHENIK

If gastropod larvae fail to encounter a suitable substratum, they may enter a '~delay period" in which metamorphosis is postponed and additional substrata may be examined (Scheltema, 1961; Kriegstein et al., 1974; Hadfield, 1978; Switzer- Dunlap, 1978). The ability of larvae to delay metamorphosis is not restricted to gastropods but has been reported in many other groups of marine invertebrates (e.g., Wilson, 1948; Ryland, 1959; Bayne, 1965; Hinegardner, 1969; Chia & Spaul- ding, 1972; Bakke, 1974; Lewis, 1978). Larvae that metamorphose in marginally tolerable environments may produce short-lived or non-reproductive populations (Thorson, 1966; Mileikovsky, 1971). The ability of larvae to discriminate among different substrata and to prolong their planktonic existence until an appropriate habitat is encountered has substantial ecological significance, as it influences both future juvenile survival and larval dispersal potential (Thorson, 1950, 1966; Scheltema, 1971 ; Crisp, 1974). Despite the widespread occurrence and obvious ecological importance of the ability of larvae to delay metamorphosis, actual delaying capabilitiesare unknown for most species, and the factors controlling the potential duration of the delay period are poorly understood. It has been suggested that the potential duration of the delay period is determined by the ability of larvae to enter into a no growth, energetic steady state in which net energy intake balances basic metabolic demand. When such a state exists, no excess energy is available for further growth and differentiation (Scheltema, 1966, 1971; Vernberg, 1972; Culliney & Turner, 1976). Establishment of such a steady state would be adaptive for teleplanic (loag-distance) larvae, in particular, by limiting any further weight gain (Scheltema~ 1966). Theoretically, larvae which remain in energy balance can postpone metamorphosis indefinitely. The assumption that an energetic steady state occurs during the delay of metamorphosis of marine gastropods is based primarily upon measurements of shell length during development. Scheltema (1962, 1965, 1967) reported a decline in the rate of larval shell growth during the delay of metamorphosis of the mud snail, llyanassa obsoleta ( = Nassarius obsoletus) (Abbott, 1974). Similar declines in shell growth rate during the delay period have been reported for other gastropods raised in the laboratory (Werner, 1955; Kriegstein etal., 1974: Hadfield, 1978: Switzer- Dunlap, 1978). Teleplanic gastropod larvae also are thought to cease further growth after being carried out to sea (Scheltema, 1966, 1971). However, energy budgets for competent larvae have not been reported. Components of growth and energy balance were monitored during the development of three gastropod species, I. obsoleta (Say), Crepidula .fornicata (L.), and Bittium alternatum (Say), and the findings were related to observed differences in delaying capabilities among the three species. LARVAL ENERGY BALANCE 3

MATERIALS AND METHODS

GENERAL The basic reproductive biology, including larval development, has been described for llyanassa obsoleta (Scheltema, 1962) and for Crepidulafornicata (Conklin, 1897; Werner, 1955; Calabrese & Rhodes, 1974). A description of larval development for Bittium alternatum is given by Pechenik (1978). Evidence that larvae of Crepidula fornicata and Bittium ahernatum delay metamorphosis in the field is presented elsewhere (Pechenik, 1978). Larvae of B. alternatum used in experiments were picked from plankton samples taken in Buzzard's Bay, Massachusetts in July and transferred in groups of 200--300 individuals into 1-gal glass jars containing approximately 3.5 I of sea water passed through a Millipore filter (1 #m pore size). The sea water was well aerated by shaking before larvae were introduced but was not further aerated. Isochrvsis galbana cells from cultures in the exponential growth phase were added to each jar to attain an initial cell density of 1.2 x l0 s cells/ml, and additional L galbana was added the following day to restore this concentration if the water was not changed. Individuals were collected every one or two days by draining cultures through 150-,urn mesh Nitex screening which was suspended in sea water to minimize trauma to the animals. After examination larvae were transferred to freshly-filtered sea water and fed L gaibana. The jars were cleaned thoroughly at each water change. Larvae of Crepidula .]brnicata were spawned by adults collected from Quissett Harbor, Massachusetts and Bissell Cove, Rhode Island. After their release into the water, the larvae were collected by draining the cultures through 150-#m mesh Nitex screening and reared in I-gal glass jars as described above. Larvae of Ilyanassa obsoleta were obtained from egg capsules collected at Barn- stable Harbor, Massachusetts or from egg capsules produced in the laboratory by adults collected from Bissell Cove, Rhode Island. Larvae were collected and reared after hatching as described above. All larvae were reared at 19-20 °C under constant light from a General Electric cool, white fluorescent lamp. Although larval growth rates are suboptimal in this temperature range for L obsoleta (Scheltema, 1967) and Crepidula.[brnicata (Cala- brese & Rhodes, 1974), the average summer water temperature recorded at the Environmental Research Laboratory, Narragansett, Rhode Island for the period during which all three species are reproducing (June-September) was approximately 20 °C. This temperature also is favorable to the growth of the Isochrvsis galbana supplied as food for the larvae (Ukeles, 1961).

LARVAL GROWTH RATES AND SIZE AT COMPETENCE Groups of larvae of Crepidula .fornicata and llyanassa obsoleta were killed in buffered formalin and preserved in buffered 80~', ETOH for shel!length determina- 4 JAN A. PECHENIK tions, which were made at 50 x using an ocular micrometer. Samples were taken until all larvae in the cultures metamorphosed or died. Attempts to induce settlement and metamorphosis of Crepidula fornicata larvae were made by pipetting small groups of advanced larvae (having a well-developed propodium on the foot) from the cultures and transferring them to glass finger bowls to which adult C. fornicata, shell fragments of Mercenaria mercenaria, or small algal-filmed stones were added. To serve as a control one group of larvae was transferred to a clean finger bowl to which no substrata were added. Larvae were examined after 15-24 h, and the shell lengths of metamorphosed individuals determined. These experiments provided estimates of the size at which metamorphosis first becomes possible in this species. Similar experiments were undertaken with larvae of Bittium alternatum hatched from eggs and reared in the laboratory. Filaments of red algae, Neoagardiella and Ceramium, were used as the inducing substrata.

RELATIVE DELAYING CAPABILITIES OF THE THREE SPECIES

Bittium alternatum larvae were obtained from plankton samples and held in the laboratory as described above. One group of 20 larvae was transferred from each of two cultures to finger bowls to which filaments of red algae were added. The number of individuals metamorphosing (i.e., losing the velum) was determined after 8-12 h. Every 2-3 days the experiment was repeated on two other groups of 20 larvae until 100% of the tested individuals metamorphosed in response to thealgal substratum. Then the larvae remaining in the cultures were presumed competent to metamorphose and were examined for the presence of a velum at 1- to 2-day intervals thereafter. Isochrysisgalbanawas provided at an initial cell density of 1 x 105 ceUs/ml, and this density was re-established daily. Observations continued until all larvae either had metamorphosed or died. This methodology determines a minimum delay capability since many individuals may have become competent well before the beginning of the experiment. Larvae of llyanassa obsoh,ta become competent to metamorphose at a shell length of 550-600/~m (Scheltema, 1967). Experiments using larvae of I. obsoleta were initiated by selecting individuals of ~600 /~m shell length from four batch cultures and rearing them in two groups of approximately 20 individuals each. Larvae were fed Isochrysis galbana daily at an initial cell density of about 5 x 104 cells/ml; shells became badly fouled with debris if highel cell densities were used. Animals were examined for the presence of a velum t intervals of.l to 2 days and transferred to fresh sea water after each observation. The number of larvae metamorphosing or dying between observations was determined at each observation, and shell lengths were determined. Larvae of Crepidulafornicata were reared from hatching in the laboratory and transferred to clean vessels and freshly filtered sea water at intervals of 1 to 2 days. Isochrysis galbana was provided daily at an initial cell density of 1.5 x 105 cells/ml. LARVAL ENERGY BALANCE 5

Larvae that metamorphosed between observations were killed in buffered formalin and preserved for shell length measurements. Shell measurements of larvae which died between observations also were made. The length of the delay period was estimated by assuming that larvae first become competent to metamorphose at 700 /~m (Werner, 1955, pers. obs.) and then, using the growth rate equations presented below, to calculate the number of days required to grow from 700/~m to the size at which metamorphosed individuals were taken in the cultures. Glassware was cleaned thoroughly at each larval transfer to new food and freshly filtered sea water.

DETERMINATION OF LARVAL BIOMASS

Due to differences in sh.ell geometry among species comparisons of growth rates between species are not possible on the basis of shell length measurements. There- fore, the relationship between shell length and actual tissue biomass was determined for larvae of Ilyanassa obsoleta and CrepMula/brnicata. In one set of determinations, 5-day-old veligers of each species were divided into two groups of 50 individuals each. One group of larvae was transferred to small, pre-weighed platinum foil containers, and the containers were re-weighed after drying for 24 h at 70 °C. Then the larvae were ashed at 500 °C for 20 h, and the weight loss was computed. Weighings were made on a Cahn electrobalance in the presence of CaSO4 desiccant. Tissue weight of the second 50 snail larvae of each species was determined directly. The larvae were pipetted into 0.25 M HCI for ~l min and removed to sea water as soon as the larval shells appeared to have dissolved. Then the de-shelled larvae were pipetted onto a piece of absorbent paper towel, rinsed with two drops of distilled water to remove adhering salt, and transferred to pre-weighed aluminium foil pans. These individuals then were dried overnight at 60 °C, and the final weight was determined in the presence of desiccant. The two methods gave essentially identical results. The decalcification method was used for all subsequent determinations of larval biomass. The relationship between larval shell length and larval tissue biomass was obtained for Ilyanassa obsoleta and Crepidula.lbrnicata by weighing groups of individuals similar in shell size. Larval weights were determined using either a Cahn electrobalance or a Perkin-Elmer electrobalance. Freshly killed, non-preserved individuals were used for all measurements.

ENERGY BALANCE The growth rate of an animal is determined largely by the rate at which energy is accumulated in excess of basic metabolic demands, assuming that the food supply contains all nutrients needed for larval growth (Pechenik & Fisher, 1979). Thus, G = A-(R + E), where G = growth or production, A =assimilation, R- respiration~ and E = excretion. A decline in growth during delay of metamorphosis 6 JAN A. PECHENIK could be caused by decreased rate of ingestion, decreased efficiency of assimilation of ingested food, increased rates of energy expenditure relative to rates of energy accumulation, or by some combination of these factors. Rate of assimilation by larvae of different sizes or ages was determined by measuring the rate of ingestion and estimating the efficiency of assimilation as "retention efficiency" (see definition below). It has been shown elsewhere that rates of carbon excretion by larvae of llyanassa obsoleta are negligible with respect to rates of carbon respiration (Pechenik, 1979). Therefore, the complete equation of carbon balance is G = I x RE- R, where I = ingestion rate and RE = retention efficiency. All components of the budget were independently determined. The smzll, naked flagellate, Isochrysis galbana, was used as a food in most feeding rate and assimilation experiments. A few additional experiments were conducted to test for changes in particle size preference with increasing larval age of llyanassa obsoleta. Feeding rate experiments were conducted simultaneously using cells of Isochpysis galbana and cells of a larger naked flagellate, Dunaliella tertiolecta. Physical characteristics of the two algal species are reported elsewhere (Pechenik & Fisher, 1979). Details of the methodology used for grazing rate and assimilation experiments are given by Pechenik and Fisher (1979). The average number of algal cells ingested per h by individual larvae was determined from the equation: Y = (C0 - Ct)" (ml of algal suspension). (number of larvae) -~. (duration of experiment) -~, where Y - cells eaten, h -~ • larva- ~and C0and C~ are initial and final algal cell densities, respectively. All experiments were initiated at a cell density of about 2 x 105 cells, ml -~ and run for only 3 h so that final cell density after grazing never fell below the range of cell densities over which ingestion rates appear to be maximal for larvae of llyanassa obsoleta (Pechenik & Fisher, 1979) and Crepidula fornicata (Pechenik, 1978). Algal cell densities were determined using a Hauser Ultra-Plane hemacyto- meter in early experiments and a Model TAll multichannel particle counter (Coulter Electronics, Inc.) in later experiments. The efficiencies with which veliger larvae of different ages retained ingested carbon from cells of IsochtTsis galbana were determined using ~4C-labelled phytoplankton cells, following procedures given by Pechenik & Fisher (1979). The volume of water in tile grazing vessels and the length of time the animals were allowed to graze on radioactive algae were selected so that neither the algal cell density nor the radioactivity of each cell would change substantially during the grazing period. Care also was taken to minimize the amount of soluble 14C present in the grazing vessel. Uptake of soluble ~4C from the medium by the larvae is negligible at the levels found in my experiments, based upon results for llyanassa ohsoleta (Pechenik & Fisher, 1979). The duration of each experiment was kept short to minimize recycling of ~4C in the system. Veligers were allowed to graze on radio-labelled Isochrvsis galbana under identical conditions of temperature (19-20 °C) and low light for exactly 3 h in all experiments. LARVAL ENERGY BALANCE 7

Because the radioactive algae were grown for many generations in the presence of [~4C]-bicarbonate in airtight vessels, the cells were probably uniformly labelled with radioactive carbon when harvested for use in experiments. Uptake of the label by the veligers therefore should reflect total carbon uptake from the algal cells. Larval ingestion rates were determined by monitoring changes in cell density of non-radioactive phytoplankton. Simultaneously, larvae from the same cultures grazed on ~4C-labelled I. galbana. Determinations of ingestion rates and radioactivity per algal cell allow calculation of total radioactivity ingested during the 3 h period. "Assimilation" is defined as the movement of food material across the gut wall and into the animal (Conover, 1968). A major difficulty in determining assimilation is in measuring the amount of assimilated material that is lost by the animal during the grazing period (Conover & Francis, 1973). Therefore, the amount of radioactivity retained after a 3-h grazing period followed by a l- to 3-h period of gut evacuation in non-radioactive sea water was measured. During this 4- to 6-h period an unknown amount of assimilated radioactivity is lost, primarily through regpiration of ~4CO, (Pechenik, 1979). Dividing the amount of radioactivity retained at the end of the gut evacuation period by the amount of radioactivity ingested during the 3-h grazing period therefore gives a minimum estimate of assimilation efficiency which I call "retention efficiency." Retention efficiency was calculated as: (dpm retained, larva -~). (number of algal cells ingested, dpm per cell) - ~. Short-term assimilation experiments were conducted in an attempt to minimize the magnitude of the discrepancy between assimilation and retention efficiencies. Respiration rates were determined as/~l 02 consumed, h - i. larva -~ using all-glass differential microrespirometry (Grunbaum et al., 1955). Determinations were made at 20 °C and readings were taken at 15- to 20-min intervals for at least 1.5 h. At the end of each experiment, larvae were counted and shell lengths determined. Larvae were allowed to empty their guts in filtered sea water for approximately l h prior to experimentation. Rates of net carbon and energy accumulation during the development of Ilyanassa obsoleta were calculated using estimates of assimilation efficiency, rates of ihges- tion and respiration, and appropriate conversion factors. A carbon content of 11.63 pg carbon .cell -I was assumed for Isochrysis galbana (Strathmann, 1967). Since the caloric value of a phytoplankton sample may be predicted with high precision from its carbon content (Platt & Irwin, 1973), I assumed 1.33 x 10- 7 calories per cell for I. galbana based upon a caloric equivalence of I 1.4 calories per mg carbon (Platt & Irwin, 1973). Respiration rates were converted from #l O, consumed, h-~. larva -j to #g carbon respired, h-~. larva-~ by assuming a respiratory quotient (RQ) of 0.8 (Brody., 1945). Respiration rates also were converted to calories burned, h-~- larva -~ by assuming an oxycaloric equivalence of 4.8 x l0 - calories per #l 02 consumed (Brody, 1945; Anderson, 1977). Larval growth rates were expressed as/~g carbon accumulated, day ~by assuming that 50% of dry tissue weight is carbon (Nichols, 1975). 8 JAN A. PECHENIK

RESULTS

COMPETENCE AND METAMORPHOSIS Larvae of Bittium alternatum were reared from hatching in the laboratory with 0~o mortality and became competent to metamorphose within 2.5 wk of hatching at room temperature. Shells were ~, 300 #m in length and had 2.8-3.0 shell whorls at the onset of competep "~ Competent larvae of B. alternatum were induced to metamorphose with 100~ success by providing small pieces of a variety of filamentous algal species (Ce~ra- mium, Polysiphonia, or Neoagardiella). The velum generally was lost within 2 h of introducing the substratum. The total dry weight of individuals metamorphosing soon after becoming competent to do so was 4.2 #g larva -~, including the shell (SE = 0.04 #g; n = 8 determinations using 1-3 individuals each). The total dry weight of individuals induced to metamorphose after a delay period of ~,30 days was 5.35 #g. larva -t (SV = 0.09 #g; n = 4 determinations using 1-3 larvae each). Thus, growth continued during delay of metamorphosis. A detailed description of larval development of Ilyanassa obsoleta was given by Scheltema (1962). The only addition I would make to his description is that developing gill rudiments appear in creeping-swimming, competent larvae by the time veligers have reached a shell length of about 635/am. Veligers of Crepidulafbrnicata metamorphosed with the addition of Mercenaria mercenaria shell fragments and juvenile C. fornicata (about 4-6 mm shell length). Juveniles of C../brnicata were the most successful inducers of larval metamorphosis (Table I), as predicted by Walne (1956). The three smallest juveniles recovered after

TABLI- I Occurrence of metamorphosed Crepidula ./brnicata at the end of 3 days in two experiments: seven "'brimmed" veligers were used in each replicate experiment.

Treatment No. metamorohosed (A) No. metamorphosed (B)

Control 0 0 Shell fragments 2 2 Juvenile C..lbrnicata 9 11 metamorphosis were 676-716.4 #m. No larvae metamorphosed spontaneously ia glass vessels at shell lengths of <1 mm. Veligers attained shell lengths of up to 1.5 mm with velar lobes approximately as long as the shell before the larvae metamorphosed spontaneously. Conklin (1897), Werner (1955), and Fretter (1972) reported a dramatic change in shell geometry following metamorphosis in C..lbrnicata; shell growth became linear rather than spiral. This same shift in the pattern of shell growth occurred in LARVAL ENERGY BALANCE 9 competent larvae of C. fornicata in my cultures. Such larvae resemble brimmed hats (see Fretter, 1972, Fig. 43 of the related species Crucibulum spinosum). The smallest "brimmed" veliger of Crepidula Jbrnicata observed in my cultures was 696.5 ,um long. Many larvae were brimmed by the time they reached =750 ,um in shell length, and all ~,00/~m veligers had brimmed shells. Competent larvae developed brown adult shell pigmentation during the delay period, although pronounced shell calcification did not occur before metamorphosis. Gill differentiation proceeded during the delay of metamorphosis of C../brnicata. Veligers of shell length 686-736 ,um possessed gill rudiments; most larvae > 720 ~um had large, apparently functioning gills. Coordinated beating of gill cilia was easily visible through the larval shell.

INGESTION RATE AS A FUNCTION OF SHELL LENGTH Data obtained on the feeding rates of larvae as a function of larval shell length are summarized in Figs. l and 2 for larvae of llyanassa obsoleta and Crepidula/brnicata, respectively. The complete data are given elsewhere (Pechenik, 1978, Ap- pendices I and If). Initially, ingestion rate increases linearly with increased shell length for both species. This is followed by a plateau or decline in feeding rate later in development with the leveling off of ingestion rate apparently correlating with the onset of the delay period. To examine whether the observed decline in ingestion rate resulted from a gradual decrease in the ability of larvae to ingest the small Isochn,sis galbana cells, six

14000

12000

~) i0000 -r 8000 f, o • 8 Kit o o <1:6000 o o _J

Or) 4000 J _I iii~~0 = Y =22.64 X - 3920.48 bd :I!00 0 (r =0.78, F =54 3) 0 2000

0 I I°° I I ! I 300 400 500 600 700 SHELL LEN~3TH, ,,urn

Fig. 1. Ingestion of L~ochrysis galhana by larvae of llyanassa obsoleta as a function of shell length" different symbols represent data taken in different years; arrow indicates approximate size at which larvae become competent to metamorphose. 10 JAN A. PECHENIK

I0000 Y = ?.4.23 X - 5966.78 o - (r = 0.78, F = 82.4) - o .ooo_. o

,~ o o o ..1 gOl~- o

N eoool"° ,*

'-' • o • Vi 300o 1"° P I I I I I , _..I 400 500 600 700 BOO 900 I000 I100 SHELL LENGTH (/~m)

Fig, 2. Ingestion of isochrvsis galhana by larvae of Crephhda ,lbrnicata as a function of shell length: different symbols represent data taken in different years; arrow indicates approximate size at which larvae attain competence. experiments were conducted in which larvae of Ilyanassa obsoh, ta were allowed to graze for 3 h on either lsochrysis galbana or Dunaliella tertiolecta. No discernible changes in particle size preference during development were revealed (Table II).

TABLE I1 Ingestion rate (cells ingested • h - !. larva - 1) of Isochrysis gaihana and Dunaliella tertiolecta as a function of larval shell length for Ilyanassa obsoleta" data are means _+ l SE (n).

Mean shell length (#m) I. galhana (a) D. tertiolecta (b) Ratio a/b

285.5 __ 1.8 (58) 2845.0 __+ 481.6 (3) 1730.0 +__ 176.8 (2) 1.6 312.4 +__ 3.0 (15) 3451.7 +__ 441.9 (3) 3110.8 __+ 383.3 (3) l.l 366.0 +_ 5.1 (14) 3034.3 _+ 828.3 (3) 2065.0 +_ 286.4 (2) !.5 386.5 +_ I !.3 (18) 5250.2 +_ 73.2 (3) 3320.0 +_ 190.0 (2) !.6 656.2 __ 10.4 (! l) 6789.5 _+ 195.5 (3) 3884.7 +_ 479.1 (2) !.7 68(!.3 _+ 1 !.6 (6) 7877.0 (i) 3697.9 (I) 2.1

CARBON RETENTION

The most detailed examination of carbon assimilation during development was made for larvae of llyanassa obsoleta since growth of this species was previously reported to decline in the delay period (Scheltema, 1965, 1967). Data used in calculation of retention efficiencies for the eight experiments conductedwith larvae of this species are given in Table Ill. There is no evidence of a decline in assimilation efficiency during the development and delay of metamorphosis of larval I. obsoleta. Indeed, since the degree to which retention efficiency underestimates assimilation efficiency probably varies with the magnitude of excretion and respiration rates, LARVAL ENERGY BALANCE 11

TABLE 1II Summary of 14C retention experiments conducted with larval gastropods: data are means _+_SE (n); R.E., retention efficiency.

Shell length (#m) dpm/cell Cells eaten • h - l. larva - l dpm retained/larva R.E. (o,,) llyanassa obsoleta 297.3 + 1.6 (15) 0.0330 3641.4 + 235.3 (3) 89.0 + 1.3 (24) 24.7 313.0.+_ i.8(12) 0.0434 3548.0+211.4(3) 79.1 + 4.0(6) 17.1 329.7 + 4.2 (57) 0.0410 4030.0 + 318.9 (3) 259.4 + 7.9 (9) 52.3 404.4 + 4.2 (51) 0.0257 7120.3 + 137.6 (3) 204.2 + 10.5 (9) 37.2 478.3 + 7.7 (18) 0.0374 4480.4 + 320.6 (3) 270.9 + 18.3 (6) 53.9 520.9 + 12.3 (15) 0.0410 14406.0 + 618.1 (3) 503.7 + 26.8 (4) 28.4 563.6 + il.8 (21) 0.0433 5643.2 + 376.8 (3) 318.9 + 21.4 (4) 43.5 603.2 + II.0 (25) 0.0481 7168.3 (I) 514.1 + 19.5 (3) 49.7 667.1 + 10.1 (15) 0.0418 7380.1 (1) 537.7 + 31.2 (6) 58.1 Bitthtm alternatum 07;; in delay 0.0416 889.7 + 113.2 (3) 49.6 + 3.1 (3) 44.7 5Y'.i, in delay 0.0349 1435.2 + 218.4 (3) 53. I + 4.2 (3) 35.4 100,~i; in delay 0.0530 1230.2 + 193.4 (3) 82.7 + 8.7 (3) 42.3 Crepidula.lbrnicata 540.5 +_ 5.0 (25) 0.0382 8381.8 +_ 351.5 670.8 +_ 20.5 (6) 69.8 and since rate of oxygen consumption increases with increasing body size, the data suggest that actual assimilation efficiency may increase during the delay period. Only three experiments were conducted using larvae of Bittium alternatum, but again there seems to be no substantial change in the efficiency with which the larvae of this species assimilate phytoplankton as they enter the delay period (Table III). Evidence based on larval growth, feeding, and respiration rates of larvae of Crepidula.lbrnicata suggests that no decline in assimilation efficiency occurs during the delay of metamorphosis for this species (see below). The single retention efficiency recorded for C.fornicata exceeded values found for larvae of tlyanassa obsoleta and Bittium alternatum (Table III). lARVAL BIOMASS A nonlinear relationship relating shell length to dry tissue weight was found for llyanassa obsoleta and is given by the equation:

Y = (2.428 x 10-'r)X 345'~ (r = 0.992), (1) where X = length in/~m and Y = dry tissue weight in/~g. (See Appendices III and IV in Pechenik's paper (1978) for raw data.) Thus, a given increase in the shell length of older individuals corresponds to a disproportionately greater increase in tissue biomass than does the same length increment in smaller individuals. This is probably because the larval shell does not grow linearly but rather in spiral fashion, with ever increasing width of each succeeding whorl (Fretter~ 1972). A linear rela- 12 JAN A. PECHENIK tionship is obtained between log shell length and log dry tissue weight (Fig. 3). This linear relationship was used to convert measurements of shell length to actual tissue biomass.

m 1.2 I.-- .1- (,,9 0.8 m IJJ

W :~0.4

oo o

)" - 0,4 B log Y = 5.458 IogX - 8.611

B (r = 0.996)

/ -0,8 - I I I ~I I I 2.4 2.6 2.8 3.0 log /zm SHELL LENGTH

Fig. 3. Relationship between log shell length and log dry tissue weight for nonpreserved veligers of llyanassa ohsoh, ta: arrow iladicates size at which larvae are competent to metamorphose.

In contrast to results obtained for larvae of I. obsoleta, the relationship between shell length and tissue biomass is linear for larvae of Crepidula fornicata. This probably is explained by the gradual change in shell geometry which occurs as larvae of this species approach competence, i.e., as the pattern o? C. fornicata shell growth changes from spiral to linear as discussed above. The relationship between shell length (X) and tissue dry weight (Y) for larvae of C..[brnicata is: Y = 0.0306X- 10,77 (r = 0.979). (2)

RESPIRATION MEASUREMENTS

Oxygen consumption rates, Y(/~I 02 consumed, h -t. larva-'), for larvae of llyanassa obsoleta increased as a power function of shell length in vm (X) until larvae became competent to metamorphose (raw data are in Pechenik, 1978; Appendices V and VI). The relationship between rate of oxygen consumption (Y), as/d O., consumed, h-' .larva-' and shell length (X) obtained for larvae <550/~m shell length was:

log Y -= 4.7833 log X- 14.2501 (r= 0.989). (3) The data were converted from #m to/~g dry tissue weight using the regression equation shown in Fig. 3 and a linear relationship between log respiration rate and log dry tissue weight was obtained for larvae of L obsoleta" log Y = 1.35 log X - 2.32 (r -- 0.991). (4) LARVAL ENERGY BALANCE 13

Larvae of L obsoleta continued to show increased respiration rates with increased shell sizes in the delay period, but the rates increased more slowly than prior to the onset of competence. The change in the pattern of larval respiration is seen most clearly when oxygen consumption is expressed per unit of dry tissue weight (Fig. 4). I0 '_o]R 4)in 8

6 "o

0,m,, o__t. = I I i I ' ' I , i I i , I , * l 0 3 6 9 12 15 16 Fg DRY TISSUE/ LARVA Fig. 4. The relationship between weight-specific respiration rate and larval dry tissue weight tbr larvae of llyanassa ohsoh, ta: the curve for precompetent larvae was generated using the linear relationship between log 02 consumption and log dry weight given in Equation 4.

Larval Crepidula.lbrnicata also displayed increased individual respiration rates with increased shell size prior to the attainment of competence for metamorphosis. The relationship between #1 O, consumed, h -~. larva -~ (Y) and shell length in /~m (X) for larvae < 700 ,um shell length is given by the power function Y = (4.89 x 10- ")X 284') (r= 0.90). (5)

DRY WEIGHT (/u. g ) 5 I0 15 20 25 I~ 15 II I = I I I I t I" I I I I I I i t = 1 r i I I I

b_ ,a

e- "~.~::1..6~'->'~9 ~

0(%1 3 • • • J • • :t. °

380 580 780 980 i leo SHELL LENGTH (/.zm ) Fig. 5. The relationship between weight-specific respiration rate and larval shell length and weight-specific respiration rate and larval dry tissue weight for Crepidula.lbrnicata: the curve for precompetent larvae t was generated using Equations 2 and 5. TABLE IV

Carbon budget for larvae of llyanassa obsoleta as a function of larval shell length" respiration rates for larvae < 550/~m shell length were calculated from Equation 3; respiration rates listed for larger veligers represent actual measurements.

! ngestion Retention Respiration Excess _! Shell length Cells • h - I. larva - I /ag C- h - i. larva - I Efficiency ~ug C h - I. larva - ! ~ul 0 2 - h - I. larva - i /ag C • h - ! • larva - ! #g C. h - !. larva (t~m~ C°,,)

313.0 3548 4.13 x i0-- 18.9 0.78 x 10-- 4.86 x 10 -3 0.208 × 10 -2 0.57 × I0 " 329.7 4030 4.69 x 10 -2 48.9 2.29 x 10 -2 6.23 x 10 -3 0.267 × 10 -2 2.02 x 10 -2 404.4 7120 8.28 × !0 .....- 37.2 3.08 × !0-- 16.6 x !0 3 0.711 × 10 -'~ 2.37 x 10 -2 478.3 4480 5.21 x !0- 2 53.9 2.81 x i0 --"~ 37.0 × I0 - 3 i.59 x !0-" "- !.22 x 10 -2 520.9 14406 16.75 x 10 -2 28.4 4.76 x 10 -2 55.6 x 10 -3 2.38 x 10 -2 2.38 x 10 -2 563.6 5643 6.56 × 10 -2 43.0 2.82 × 10 -2 58.9 x 10 -3 2.52 x 10 -2 0.30 x 10-2 603.2 7168 8.34 x 10 -2 49.7 4.14 x 10 -2 72.4 × I0 ~ 3.10 x 10 -2 !.04 x 10 -2 667.1 7380 8.58 x 10 -2 58.1 4.99 x 10 -2 87.1 × 10 -3 3.73 × 10 -2 !.26 x!0 -2 Z

¢3

t'rl TABLE V Z 7~N Energy budget for larvae of llyanassa obsoleta as a function of larval shell length: respiration rates for larvae < 550 #m shell length were calculated from Equation 3; respiration rates listed for larger veligers represent actual measurements; Cal. = calories.

Ingestion Retention Respiration Excess

Shell length Cells - h - I. larva - I Cal • h - i. larva - I Efficiency Cal • h - I. larva - I ~1 0 2 - h - I. larva - ! Cal - h - !. larva - I Cai • h - I. larva - I ~/~m) t0,,)

313.0 3548 4.72 × 10 -4 18.9 8.99.?, × 10 -5 4.86 x 10 -3 2.33 x 10 -5 6.59 x 10 -5 329.7 4030 5.36 x 10-4 48.9 2.62 × 10 -4 6.23 × 10 -3 2.99 × 10 -5 2.32 x 10 -4 404.4 7120 9.47 × 10 -4 37.2 3.52 x 10 -4 16.6 × 10 -3 7.95 × 10 -5 2.72 × 10 --4 478.3 4480 5.96 x 10 -4 53.9 3.21 x 10 -4 37.0 × 10 -3 !.78 x 10 -4 !.43 × 10 -4 520.9 14406 1.92 x i0 3 28.4 5.44 × 10 -4 55.6 x 10 -3 2.67 x 10 -4 2.77 x 10 -4 563.6 5643 7.51 × IO -4 43.0 3.23 × 10 -4 58.9 x 10 -3 2.82 x 10 -4 4.07 × 10 -5 603.2 7168 9.53 x i0-4 49.7 4.74 x 10 -4 72.4 x 10 -3 3.48 x 10 -4 !.26 × 10 -4 667.1 7380 9.82 x 10 -4 58.1 5.70 x 10 -4 87.1 × !0-3 4.18 × 10 -4 1.52 x 10 -4 LARVAL ENERGY BALANCE 15

No further increase in respiration rates was observed as development continued. When respiration is expressed as ,ul 02 consumed, h-~ .,ug dry tissue weight -l, a pronounced change in the relationship between respiration rate and shell length is seen to occur at about the time the larvae enter the delay period (Fig. 5).

GROWTH AND ENERGY BALANCE

The equation used to calculate rates of net carbon and energy accumulation at different times during the larval life of Ilyanassa obsoleta was G = I x RE - R. Since retention efficiency underestimates true assimilation, this calculation yields a minimum estimate of excess carbon available for growth. The calculations have been made for L obsoleta based upon the eight assimilation experiments using larvae of this species (Tables IV and V). In terms of both carbon and calories, there is no indication that the larvae ever enter into an energetic steady state during the delay of metamorphosis, despite the apparent decrease in rate of shell growth seen in competent veligers. Using the linear regression tuation relating log shell length to log dry tissue weight for L obsoleta (Fig. 3), the original data of Scheltema (1967) on the growth rate of larval I. obsoleta have been replotted (Fig. 6). As predicted from energy

m 800 24

I E :L 600 z l- (.9 Z l.d .J -I >, 400 .J ttl ',r t/1

4 200 o I,l,lllllll,I- o ,, 8 ,2 ,6 z0 DAY

Fig. 6. Growth of larval llyanassa ohsoh, ta in terms of shell length and dry tissue weight: arrows in- dicate approximate size at onset of competence for metamorphosis; shell length data are those of Scheitema (1967); data were converted from/Jm to/~g dry tissue weight using the equation shown in Fig. 3. balance considerations, veligers continue to accumulate biomass throughout larval life. Larval growth continues at a constant rate, even while larvae are delaying metamorphosis. The same is true for larvae of Crepidula Jbrnicata. The relationships between dry tissue weight in vg (Y) and number of days in culture (X) for the two cultures of C. fornica,a that were monitored are: 16 JAN A. PECHENIK

Y --- 0.7205X + 2.2070 (r -- 0.995) (6) Y = 0.7547X + 2.6506 (r --- 0.997). (7) Average growth rates (#g dry tissue accumulated, day-~) were compared for precompetent larvae of llyanassa obsoleta and Crepidula fornicata using shell length measurements made on eight cultures of llyanassa obsoleta and two cultures of Crepidula fornicata (Table VI). First, the data were converted from shell length to gg dry weight of tissue using the appropriate regression equation for each species. Then the relationship between days of culture and ,ug dry weight was determined for cultures N1, N4, N5, CI, and C2 (see Table VI) by linear regression analysis.

TABLE V!

Laboratory growth of larval llyanassa obsoleta and CrepidulaJbmicata: data are mean shell lengths in ~am; at least 20 shells were measured for each determination.

Age of culture (days)

Culture 0 ! 2 4 6 8 ! 0 11 15 l. obso&ta NI 285 302 - 351 - 420 - - - N4 255 - 315 400 - - 529 - 605 N5 255 - 310 338 - 422 - 478 - N7 261 - 305 - - 414 475 - - NI0 265 - - - 388 .... PFNA 295 - - - 416 .... PFNB 295 - - - 397 .... PFNC 295 - - - 389 ....

0 2 4 7 8 9 10 !1 14 18 20 30

C.fornicata C! 406 - 545 599 - 623 - 660 720 865 - il09 C2 - 481 - - 620 - 698 - - - 960 !160

The resulting equations are given in Table VII. From these equations, the time required for larvae to reach a given weight was calculated~ and growth rate thereby was determined as llg dry tissue added per day (Table VII). Growth rates of larvae in cultures N10--N13 (Table VI) were calculated by converting from shell length to biomass using the appropriate regression equation and then dividing the weight difference between Day 0 and Day 6 by the number of days in culture. The results also are presented in Table VII. The average growth rate for larvae of llyanassa obsole was 0.33/~g dry iissue- day-I (SE = 0.04, n = 8). while the average growth of larval Crepidula fornicata was 0.75 /~g tissue .day -! (Table VII). One-way analysis of variance affirms a statistically significant difference in the growth rate of the two species (F- 22.4, P < 0.01). LARVAL ENERGY BALANCE 17

TABLE VII

Larval growth rates based upon shell length measurements given in Table VI" Y, `ag glry tissue weight; X, days after escape from egg capsule.

Correlation coefficient Growth rate Culture Regression equation (r) `ag dry tissue/day llyanassa obsoleta NI Y = 0.2669X + 0.6584 0.992 0.28 N4 Y = 0.6604X + 0.0249 0.996 0.60 N5 Y = 0.361 IX + 0.2585 0.986 0.34 N7 Y = 0.3624X + 0.3541 0.973 0.35 NI0 - - 0.26 PFNA - - 0.32 PFNB - - 0.26 PFNC - - 0.23 Crepidula.l'ornicata CI Y = 22.9563X + 424.0085 0.995 0.70 C2 Y = 24.6639X + 438.5049 0.997 0.80

Comparisons of ingestion rates, respiration rates, and rates of potential carbon accumulation (assuming an assimilation efficiency of 100%) were made for larvae of llyanassa obsoleta and Crepidula jbrnicata (Table VIII). Ingestion rates (cells eaten, h -~.larva -l) were calculated for larvae of 500 /~m shell length using the appropriate regression equations, and the results then were converted to carbon equivalents.

TABLi-VIII Energy relationships for 500 ,am shell length larvae of Crepidula.lbrnicata and ilyanassa ohsoleta" an assimilation efficiency of l_vOfloJ ,, was assumed for the calculation of potential carbon accumulation rates.

1. obsoleta C. fornicata

àg dry wt 5.267 4.532 àg carbon ingested/day 2.065 !.716 àg carbon respired/day 0.4114 0.2159 àg carbon respired • day - I .àg dry wt 7.812 x 10 -2 4.761 x 10 -2 àg carbon accumulated/day !.65 ! .50

Larvae of equivalent size have similar ingestion rates, but the respiration rate oflar- val llyanassa obsoleta is approximately double that of Crepidulafornicata on a per individual basis and 1.6 times greater on a dry weight basis. In the absence of differences in assimilation efficiency, llyanassa obsoleta larvae would be expected to accumulate carbon at a slightly faster rate than larvae of Crepidula fornicata. However, larvae of C. fornicata grew much more rapidly than larvae of llyanassa obsoleta under the conditions of the present experiments. Larvae of Crepidula Jbr- nicata therefore must assimilate ingested food with greater elficiency than do veligers 18 JAN A. PECHENIK of I. obsoleta, and thus the general results of the ~4C experiments are confirmed (Table III). A summary of larval encrgetics for llyanassa obsoleta and Crepidula :[brnicata is given in Table IX. The average time required from hatching to reach the approximate dry tissue weight at which metamorphosis first becomes possible (days of growth) was calculated for each species from average growth rates (Table VII). Retention efficiency given for llyanassa obsoleta is the mean of nine determinations (Table III).

Tam_~ IX Summary of larval energetics for l/yanassa obsoleta and Crepithda./brnicata from hatching (initial shell length) until the attainment of competency (final shell length).

!. ohsoh, ta C..[brnicata

Initial shell length (#m) 285 400 Final shell length (#m) 55() 700 Growth (.ug of tissue carbon) 3.29 4.59 Days of growth 19.88 12.24 Growth rate (#g tissue carbon/day) 0.165 0.375 Retention eMciency 40.5". 69.8". Gross growth efficiency (G/I x 100,'...) !0.72", 18.26"0

Gross growth efficiencies (K~) (Conover, 1968) for development prior to attainment of competency for metamorphosis were estimated for larvae of I. ohsoleta and Crepidulalornicata using the regression equations determined previously. Gross growth efficiency was calculated as: t'change in carbon content)x (carbon ingested) -~ x 100'~,,. Larvae of C..[brnicata were found to grow nearly twice as ef- ficiently as larvae of llyam:ssa obsoleta. Note that shell growth is not considered in these calculations.

RELATIVE DELAYING CAPABILITIES One hundred percent of the field-collected larvae of Bittium alternatum were assumed to be in the delay period by the l lth day following their capture from the plankton (Table X). These individuals delayed their metamorphosis for up to 60-73 additional days, with slightly more than half of the larvae metamorphosing spontaneously by the end of this time (Fig. 7; Table XI). The remaining individuals died during the experiment. Shells of dead or metamorphosed individuals all had between 3 and 3.25 whorls. In contrast to the results for larvae of B. alternatum, most larvae of llvanassa obsoleta died during the experiment. Only 177,0 of the individuals metamorphosed spontaneously (Fig. 7; Table XI). Larvae delayed metamorphosis for up to 60 days after the initiation of the experiment. Mean shell length of individuals dying during LARVAL ENERGY BALANCE 19

TABLE X Percentage of larval Bittium alternaturn tested which metamorphosed in response to strands of red algae as a function of time since capture from the plankton: twenty larvae were generally tested from each of two cultures, a and b, on each day.

Percent metamorphosing

Days after capture a b

1 35 40 3 45 45 6 75 85 9 95 100 !1" 100 100

* Ten larvae/experiment on this day.

28 W 24 > 8. ol/ernotum n,* 20 _I 16- 0 12

W 8

4 Z I I I I t I I I I I I I I I i I I I i I I i I I 0 "3 9 15 21 2T 33 39 45 51 57 63 69 "r5 DAYS OF DELAY

> ft," 24 <1 --120

0 16

~J 12 ¢n ::De Z 4 iw v v vvv J j••j• i I I I I I li I l I I I i I I I t I I I 15 20 25 30 35 40 45 50 55 60 DAYS OF DELAY

Fig. 7. Fate of competent larvae of Bittium alternatum and ll.vanassa ohsoh'ta in the absence of algal and mud st, bstrates, respectively: e, spontaneous metamorphosis on the glass of the culture vessels; O, larval mortality. 20 JAN A. PECHENIK

TABLE Xl Delaying capabilities of the three species examined and the fates of the larvae in the absence of the preferred stimulus.

Percent eventually Length of delay Species period (days) Metamorphosing Dying

llyanassaobsoleta >I 60 17 83 Bittiumalternatum >t 60 54 46 Crepidulafornicata < 30 94 6 the experiment was 715.6/am (SE = 16.9 #m, n --- 35). Mean shell length of metamorphosed individuals was 761.9/am (SE --- 16.8/am, n = 7). Larvae of Crepidula.]brnicata delayed metamorphosis for less than 40 days after the initiation of observations. About 95'~ of all veligers metamorphosed during the delay period (Fig. 8; Table XI). All metamorphosed individuals had prominent adult shell morphology and pigmentation when recovered from experimental containers. The mean size of metamorphosed C..[brnicata recovered from the experiment was 1.42 mm (SE = 14.5/am, n = 124). Active veliger larvae with shells about 1.5 mm in length occasionally were observed during the experiments, although the smallest recovered juvenile had a shell length of only 1.09 mm. Extra- polatin~ from the regression equations relating mean shell length to age of culture for larva~ of C. fornicata (Table VII), an average of 41.75 days from hatching is r,:quired to reach a shell length of 1.42 mm. This estimate seems reasonable since most of the larvae metamorphosed between 35 and 40 days after hatching (Fig. 8). Since an average of 12.24 days is required to reach 700/am (Table VII), these larvae are presumed to have delayed metamorphosis for about 30 days (41.75 - 12.24 = 29.51 days).

r~ I,.d 14o CO 0 -r" ~2o I~, n- I C. fornicata O ~oo

~:~ 8o I-- LU ~o

n- 40 IM rn 20

Z 0 o ~: DAYS OF CULTURE z Fig. 8. Fate of larvae of Crepuhda.lbrnicatain the absence of preferred substratum for metamorphosis" symbol definitions as in Fig. 7. LARVAL ENERGY BALANCE 21

Thirty days is a maximum estimate of the delay period since the computation for C. fornicata assumes that metamorphosed individuals were recovered immediately after the velum had been lost. If it is assumed that the smallest juveniles measured (mean shell length = 1.12 mm, SE = 23.3 Ilm, n = 3) were collected nearest the time of actual metamorphosis, then a delay period of about 17 days is calculated to have occurred (29.16 - 12.24 = 16.92 days). Therefore, the actual period observed lies between 17 and 30 days for larvae of C.]brnicata. Results for all three species studied are summarized in Table XI.

DISCUSSION

LARVAL ENERGETICS

There are few reports of feeding rates for molluscan veligers. Published information on the feeding biology of gastropod veligers is limited primarily to descriptions of the mechanisms of food collection (Werner, 1955; Fretter, 1967; Fretter & Mont- gomery, 1968; Strathmann et al., 1972) and the mechanics of digestion (Fretter & Montgomery, 1968). Advanced larvae of the bivalve Mytilus edulis (240 #m shell length) have been reported to filter Isochrysis galbana at an average rate of 0.45 ml • larva-J, day- ~at 18 °C (Bayne, 1965). When the results shown in Fig. 2 are converted for small larvae of Ilyanassa obsoleta (300 Ilm shell length) from ingestion rates to filtration rates by dividing ingestion rate by mean algal cell density during an experiment, the larvae are found to filter approximately 0.48 ml .larva -~ .day -j. In experiments of Walne (1965) larvae of the oyster, Ostrea edulis (219 #m shell length), ingested Isochrysis galbana cells at a rate of approximately 1400 cells, h i. larva ~at 23-24 °C. This is about 40'~o of the feeding rate exhibited by the smallest larval llyanassa obsoh, ta in the present experiments, which is in keeping with the size differences between larvae of the two species. Respiration rates of veliger larvae have been determined infrequently. Respiration rates of about 1.5 Ill 02 consumed per mg total dry weight were determined for recently hatched larvae of I. obsoleta using Cartesian divers at 20 °C and 30%0 salinity (Vernberg, 1972; Vernberg & Vernberg, 1975). Neither larval dry weight nor shell length were reported. To enable comparisons with the present data, two determinations were made of total dry weight for recently hatched L obsoleta (mean shell length +_l SE = 270.0 +_ 1.5 Ilm, n = 25), using 16-17 veligers per measurement. Dry weights of 1.79 and 1.86 #g larva -~ were obtained. Assuming an average of 1.825 x l0 -3 mg. larva -~, 1.5 Ill O2 consumed • h -z. mg ~is equivalent to 2.7 x 10- 3 #l 02 consumed, h-~. larva- I. This is comparable to the present determinations of ~, 5 x 10 - 3 Ill 02" h i. larva -~. Zeuthen (1947) used Cartesian divers to measure larval respiration rates of 400-430 Ilm larvae of Nassarius reticulatus at 16°C. Ra~es of approximately 22 JAN A. PECHENIK

12 x 10-3 ,ul 02 consumed, h-~. larva J were measured when all three individuals were swimming actively. An active 480 #m individual respired about 18.5 x 10-3 #1 02' h-~. These results are substantially lower than the values which were recorded for comparably sized larvae of llyanassa obsoleta. At least part of the discrepancy may be due to differences in experimental temperatures. However, respiration rates of larval Nassarius reticulatus at 16 °C (Zeuthen, 1947) were similar to the respiration rates of comparably sized larvae of Crepidula fornicata at 20°C in the present experiments. Larval respiration and feeding rates of llyanassa obsoleta and Crepidula.[brnicata were found to plateau or decline during the delay period. Feeding rates are reported to decline in the delay period of Mytilus edulis as well, but this decline is due to a gradual resorption of the velum and of the cilia of the food groove (Bayne, 1965). Degradation of larval structure was not observed in the present experiments. More- over, the decline in ingestion rate which was observed is probably not attributable to a decreasing ability of the larvae to handle the small cells of lsoch~Tsis galbana since no shell-size-dependent changes in relative ingestion rates were recorded for llyanassa obsoleta larvae feeding on food particles of two different sizes. Additional experiments using a wider range of particle sizes are needed, however. The change in pattern of feeding and oxygen consumption observed in the present experiments is probably due to behavioral changes on the part of the larvae as they shift from continuous swimming to alternating periods of swimming and crawling (Vernberg, 1972). Larval respiration rates are strongly affected by changes in larval activity. Zeuthen (1947) found that oxygen consumption rate dropped by up to 500.i, when larvae of Nassarius reticulatus shifted from swimming to nonswimming. Respiration rates of the isolated velum of M).tihts e&dis and Mya arenaria accounted for up to 50'I~; and 251'.o, respectively, of the respiration rates of whole, swimming animals (Zeuthen, 1947).

FACTORS INFLUENCING THE DELAY OF METAMORPHOSIS

Relative delaying capability and the ultimate fate of larvae in the absence of suitable substratum ~aries greatly among species. For example, larvae of the echir~o- derm, Lvtechhnts picta, can delay metamorphosis for up to 60 days (Hinegardner, 1969). larvae of the bivalve, Lvro&~s pedicellatus, can delay only 7 days (Turner & Johnson, 1971). larvae of the bivalve. Mvtilus edulis, can delay metamorphosis for 2-5 days (16-21 °C) (Bayne, 1965), and larvae of the polychaete, Spirorbis borealis. can delay metamorphosis for only ~12 h (Williams, 1964). Reported mortality rates during delay of metamorphosis are very high for the above species. However, larvae of the hydrozoan, Tealia crassicornis (Chia & Spaulding, 1972), and those of the gastropod, CrepMula .lbrnicata (this study), can delay metamorphosis for ,~2 wk to 1 month, with most larvae metamorphosing spontaneously during this time. LARVAL ENERGY BALANCE 23

The limited potential for delay of some species (e.g., Lyrodus pedicellatus, M),tilus edulis, and Spirorbis borealis) is probably related to a finite larval energy supply at hatching (Knight-Jones, 1953; Eggleston, 1972; Pechenik et al., 1979) or to resorption of feeding structures during the delay period (Bayne, 1965). Differences in the delaying capabilities reported for most other species are possibly due to differences in rearing conditions. Bayne (1965) showed that the potential length of the delay period in Mytilus edulis varies with temperature, salinity, and pH of the rearing medium. However, differences in delay potential and the ultimate fates of larvae in the absence of ideal conditions for metamorphosis were observed in this study even though all larvae were reared under the same conditions. The high mortality of competent larval llvanassa obsoh,ta observed in this study contrasts with results of Scheltema (1961) who observed at least 50')~0 spontaneous metamorphosis of larval 1. obsoleta deprived of mud substratum. This difference in results possibly reflects differences in rearing conditions. Scheltema ( 1961 ) reared his larvae on a diet of Phaeodactylum tricornutum at 22-24 °C. It should be noted, however, that although about 50~o of the larvae of Bittium alternatum eventually died in the experinaents during the delay period, these larvae were reared from hatching through the onset of competence with 0% mortality under the same conditions of food and temperature. Growth of bivalve and gastropod larvae is generally estimated in terms of shell length (e.g., Bayne, 1965; Scheltema, 1965, 1966, 1967, 1971; Walne, 1965; Pilking- ton & Fretter, 1970). Shell length accurately reflected growth of Crepidula.lbrnicat~. in my experiments, and no decline in growth rate was observed for competent larvae of this species in terms of shell length or tissue biomass. Lucas & Costlow (1979) report a decline in growth of this species with increasing larval age at 25 °C, but this may reflect a dietary insufficiency; the algal food used in their experiments is a poor food for larvae of llyanassa obsoleta (Pechenik & Fisher, 1979). Growth of I. obsoleta is not well represented by changes in shell length, particularly in larger larvae. Similarly, nonlinear relationships between shell length and N content or total dry weight have been reported for larvae of Nassarius reticulatus, Mvtilus ethdis, Littorina littorea, and Ostrea edulis (Zeuthen. 1947; Walne, 1965). Whereas changes in shell length proceed more slowly in competent larvae of llyanassa obsoleta (Scheltema, 1965, 1967; Vernberg, 1972), no decline in tissue growth rate actually occurs in the delay period of this species. Not only does growth continue at a constant rate in competent larvae of at least some species, but also differentiation may continue as well. Specifically, the gills, which generally develop after the metamorphosis of bivalve and gastropod larvae (Sastry, 1965; Hickman & Gruffydd, 1971; Bonar, 1976), were seen to develop in delaying larvae of Crepidulafornicata and Hyanassa obsoh, ta. Moreover, the dramatic changes in shell morphology which invariably follow metamorphosis of gastropod larvae (Conklin, 1897; Fretter & Manly, 1977) take place during larval life in competent larvae of Crepidula fornicata. Similarly. substratum-deprived larvae of 24 JAN A. PECHENIK the sea anemone, Tealia crassicornis, begin developing tentacles within about 3 days after the onset of competency. Apparently, "the development of tentacles is more or less fixed in a certain age and independent of settling" (Chia & Spaulding, 1972, p. 213). Thus there is no evidence of a developmental hiatus in competent larvae of these species. Good evidence that opisthobranch larvae do cease growth and differentiation in the delay period has been presented (Bonar & Hadfield, 1974; Kriegstein etal., 1974; Switzer-Dunlap, 1978). It is clear, however, that a prolonged delay period is not universally dependent upon entrance into such a steady-state situation. Metamorphosis of larvae of benthic marine invertebrates involves radical changes in the life style of the organism, including habitat and dietary shifts as well as changes in the manner of locomotion. Although adult and larval forms are ecologically distinct (Istock, 1967), they share the same genetic material. Consequently, a single genome codes for larval structures and enzymes, adult characteristics, and characteristics which persist throughout the lifespan of the organism (Grave, 1936). Metamorphosis clearly involves "shutting off' that portion of the genome coding for specifically larval characteristics and "turning on" that portion of the genome which functions only in the adult stage. An hypothesis consistent with the present results and published literature is that the portion of the genome concerned specifically with larval functioning may have a genetically fixed endpoint. It is commonly believed that senescence is regulated by a genetic clock (Hayflick, 1975; Holliday & Pugh, 1975) which can termirmte the activity of specific loci at the end of a predetermined number of cell divisions (Holliday & Pugh, 1975). Shifts in environmental factors which accelerate rate of larval development (Thorson, 1950; Bayne, 1965; Scheltema, 1967) also seem to foreshorten the potential length of the delay period (Bayne, 1965) in keeping with the concept of a genetically fixed larval endpoint. Similarly, the time of met~.,- morphosis in amphibians may be reliably predicted from the rate of differentiation during development (Smitb.-Gill & Berven, 1979), and 17-yr cicadas may emerge 4 yr ahead of schedule due to acceleration of developmental rate (Lloyd & White, 1976). Larvae of some invertebrate species exhibit morphological degeneration while they delay metamorphosis (Wilson, 1935; Bayne, 1965; Sastry, 1965; Hinegardner, 1969) and eventually may lose the ability to metamorphose even when presented with favorable conditions (Wilson, 1935; Grave, 1936; Knight-Jones, 1953; Hine- gardner, 1969). Hadfield (1978) has suggested that attainment of competency may involve the development of certain receptors or neural connections. If this is the case, then the loss of ability to respond to substrata may reflect degeneration of the larval neural system as the organism reaches the end of the program for larval development. The rate at which a gastropod larva runs through its developmental program. including the delay period, may be related to the rate at which net energy is ac- LARVAL ENERGY BALANCE 25 cumulated. In support of this hypothesis, larvae of Crepidulafornicata were found to have a higher retention efficiency than larvae of llyanassa obsoleta, a corres- pondingly more rapid growth rate, and a shorter delay period (Tables IX, XI). Further insight into the proximate controlling factors of the delay period will re- quire more data on larval growth rates and delay periods and a more complete under- standing of the regulation of gene action in developmental systems. Ultimately, the functional lifespan of the genes associated with larval characteristics must be determined by ecological factors. It has been suggested or implied that the length of larval life is related to the degree of difficulty which larvae will experience in locating the appropriate environment for the adult (Wilson, 1958; Bayne, 1965; Grassle, 1973; Crisp, 1974). Species with very general adult habitat requirements would be expected to produce larvae with only limited delaying capabilities while species with more patchily distributed and specialized adult niches would be expected to be able to prolong larval life to a greater extent. The present data on the relative delaying abilities of Crepidula/brnicata, Ilyanassa obsoleta, and Bittium alternatum appear consistent with this hypothesis. Crepidula.lbrnic'ata is a scdentary gastropod commonly found on a variety of firm substrates from the intertidal zone to a depth of at least 50 ft (Conklin, 1897; Abbott, 1974). Dense aggrega- tions of Bittium alternatum occur subtidally on a variety of macroalgal substrates, while llyanassa obsoleta are restricted to intertidal mud fiats (Scheltema, 1967). Based upon energy balance data obtained from the present experiments, one can hypothesize a mechanism through which natural selection could act to control the potential length of the delay period. The greater delaying capabilities of larval I. obsoleta and Bittium aiternatum relative to those of Crepidula fornicata could result from selection for energetic inefficiency. By having low assimilation efficiencies and high respiration r~tes, larvae of llyanassa obsoleta run through their developmental program less r,~_pidly than do larvae of Crepidulafqrnicata and consequently have greater delay capabilities. The speculative nature of this hypothesis must be emphasized, but others also have suggested that natural selection do~: not neces- sarily act to maximize the efficiency of energy utilization (Odum & Pinkerton, 1955). It is interesting to note that many tropical gastropod species have particularly long-lived larvae (Thorson, 1961 ; Crisp, 1974) and seem to have very great capacities for delay of metamorphosis (Scheltema, 1966, 1971). Such species have evolved a high degree of specialization on physical and biotic components of their environments (Kohn, 1971; Grassle, 1973) so that their larvae must experience difficulty in locating the proper substratum for adult existence. The great dispersal capabilities of many tropical species therefore may be a consequence of selection for long delay potential rather than selection for dispersal capability per se. 26 JAN A. PECHENIK

ACKNOWLEDGEMENTS

I wish to acknowledge the encouragement and helpful comments of colleagues and/or members of my thesis committee: Drs. D.B. Bonar, R. Bullock, H.P. Jef- fries, D.C. Miller, T. Napora, A.N. Sastry, and R.D. Turner. Comments of Drs R. Strathmann and L. Halderman improved the manuscript substantially. Portions of the research were supported by National Science Foundation Dissertation Im- provement Grant GA 38895 and the Environmental Research Laboratory of the United States Environmental Protection Agency.

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