Influence of Overwintering Stresses on Respiration

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Influence of Overwintering Stresses on Respiration Throughout the Life History of the Freshwater Leech, Nephelopsis obscura Ronald W. Davies' and V. Kalarani D i v i s i ~ nof E c o l s g ~Aquatic Ecology Research Croup, Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N 1 N4

Davies, R.W., and V. Kalarani. 1993. Influence of overwintering stresses on respiration throughout the life history of the freshwater leech, Nephelopsis obscura, Can. 1. Fish. Aquat. Sci. 50: 841-845. The effects of overwintering stresses (low temperature, low oxygen concentration) on the life history patterns of activity-specific oxygen consumption by Nephelopsis obscura were determined using a flow-through respirometer system and compared with leeches hatched and maintained under summer conditions. While resting and active oxygen consumption increased with body dry weight, weight-specific resting (Rm) and active (Ra) oxygen consumption and aerobic scope (AS) decreased with increase in body weight in both winter and summer N. obscura. Rm in winter leeches was higher than in summer leeches, probably reflecting the higher ~metaboliccosts of tissue repair and maintenance after winter stresses. Although Ra and AS in winter leeches were initially lower than in summer leeches, by stage 4, compensation in winter leeches was complete and by stage 6 , over-compensation occurred. The effects of overwintering sn oxygen consumption and AS persisted throughout the life history and help explain some of the differences in allocation of energy storage observed in winter and summer leeches. Les effets des stress de I'hiver (faible temperature, faible concentration d'oxygene) sur t'6vslution de la consommation d1oxygPne spkcifique de I'activite au cours du cycle vital de Nephelopsis obscura ont kt6 determines 3 Itaide d k n e systeme respirom4trique A renouvellement de I'eau et les r6sultats ont 6t6 compares aux valeurs sbtenues chem des sangsues ecloses et gardees dans des conditions caracteristiques de I'ete. Bien que la consommation droxyg+ne au repos et en activit6 ait augment6 avec le poids corporel sec, la conssmmation dloxyg+ne au repos (Cr) et en activite (Ca) specifiques du poids et Ilktendue aerobic (EA) snt diminue avec ['augmentation du poids corporel chem N. obscura aussi bien I'hiver que I'ete. La Cr etait plus elevee chez les sangsues I'hiver que I'ete, ce qui reflgte probablement les coots metaboliques plus eleves li6s a la reparation et a J'entretien des tissus aprks les stress de I'hiver. Bien que la Ca et I'EA aient 4t6 initialement plus faibles chem les sangsues d'hiver que chez les sangsues d'ete, la compensation chez les sangsues d'hiver etait complgte au stade 4, et au stade 6 , il y avait surcompensation. Les effets de I'hiver sur la consommation dloxyg+ne et sur I'EA ont persist6 pendant tsute la dur6e du cycle vital et aident 3 expliquer certaines diffkrences dans l'affectatiow de I'6nergie 3 la croissance somatique et au stockage d'energie observees chez les sangsues d'hiver et d'ete. Received duly 14, 7 992 Accepted October 15, 7992 (JB552)

T

he influence of body size on oxygen consumption has been well documented for numerous freshwater poikilothems and leeches (Mann B 956; Wamamurthi 1955; Mulkarni 1978). The effects of temperature, oxygen saturation, osmotic stress, and food deprivation on oxygen consumption have also been studied in a diversity of freshwater invertebrates (e.g., Wockwood et al. 1990; Urabe and Watanabe 1998; Glazier 1991; Mornbach 1991), including leeches (Eindeman 1932, 1935; Mann 1956, 1958, 1951 ; Madanrnohanrao 1968; Wamamurthi 1965, 1968; Ca%eswand Riley 1982; Wrona and Davies 1984; Milne and Calow 1998; Davies et al. 1992). Closed respirometers have been used most frequently in these studies despite their many associated problems (Eampert 1984; Wrona and Davies 1984). Flow-through systems overcome these problems and allow accurate quantification of the activity patterns of an organism concurrent with the measurement of oxygen consumption (Wrona and Davies 1984; Davies et a%. 1 992). Nephelopsis obsekcra Venill (Erpobdellidae) is a predatory 'Author to whom correspondence should be addressed. Can. J8 Fish. aqua^. Sci., Vol. 50, 1993

R e p le 74 juillet 1992 Accept4 le 15 octobre 1992

leech widely distributed in lentic freshwater ecosystems of Canada and the northern parts of the United States (Davies 1991). In southern Alberta, two generations of N . obseura are produced annually, one in early summer and the second in late summer, both generations overwintering prior to reaching maturity the following summer. Each generation produces young after either 2 2 and 15 or 12 and 19 mooBefore entering winter, N . obseura!of the early summer generation are >98 mg and those from the late summer generation are between 9 and 19 mg (Davies and Everett 1977). Thus, 3- to 5-mg individuals collected at the end of spring before reproduction commences belong to the Bate summer generation which overwinters when the lakes are anoxic or severely hypxic for 98-120 d (Baird et al. 1987) and when, like the majority of freshwater invertebrates in temperate zones, it also experiences low water temperatures and restricted food availability. Dratnal and Davies (1998) and Reddy et al. (1992) showed that small overwintered N . obscura and similar sized individuals hatched in the summer differed not only in age beat also in other ecophysiological processes (although respiration was not measured) resulting in differences in life history and success. The physiological differences between winter and summer N . obscura! persisted 84 B

TABLEI. Mean ( + SD) WWT and DWT (mg) of winter and summer M. obscura ((n = 5) at each of the six life history stages. Stage


Leech Winter WWT DWT Summer WWT DWT

9.9 1.8

+ k

3.0 0.4

1 1 . 1 2 2.4

2.8

+ 0.3

21.9 4.1

+ 3.1 + 0.5

23.1 + 2.0 4.5 2 1.1

165.4 30.8

+ +

17.9 1.8

163.1 + 9.4 31,5 2 1.8

throughout the life history. Winter N. obscum started the summer season with significantly lower total lipid, total protein, and glycogen concentrations than similar sized summer leeches. Summer leeches allocated energy to storage approximately isometrically with an increase in body size. In contrast, winter leeches compensated for winter stress by enhanced energy allocation to total lipid, total protein, and glycogen during the early life history stages. This is the first study to determine the effects of a stress (in this case, overwintering) on activity-related oxygen consumption (a measure sf respiration) throughout the life history of a species (N. obscura). Specifically, we determined if exposure to winter stresses results in changes in resting oxygen consumption, active oxygen consumption, and aerobic scope compared with summer (control) leeches and whether compensation occurs in winter leeches.

Materials and Methods Small (8-12 mg wet weight (WVVT)) N. obscura that had overwintered in the field (termed winter N. obscura) were collected from Stephenson's Pond near Calgary, Alberta, during May 1990 when the water temperature was 4°C. They were gradually acclimated to 20°C (1'-d-') with 100% oxygen-saturated filtered pond water and a 12 h light: 12 h dark regime and provided ad libitum with Tubifex tubifex Miiller for 2 h three times a week until they reached maturity. In July 1990, N. obscura cocoons were collected form the pond immediately after deposition (water temperature 20°C) md maintained in the laboratory in filtered aerated pond water at 20°C with 100% oxygen saturation far approximately 4 wk until they hatched (termed summer N. obscum). After hatching, they were maintained under identical conditions to the winter N. cabscura until they reached maturity. Both winter and summer N. obscura showed sigmoid growth, and six representative life history stages were selected (Table I), each characterized by differences in growth rates and stages of sexual maturity: (1) post-hatchling size, (2) initially accelerating, relatively flat growth, (3) late accelerating growth, (4) inflection point of growth curve, (5) appearance of clitellum indicating sexual maturity, and (6) decelerating growth in fully mature animals (Reddy et al. 1992). At each stage, oxygen consumption measurements were made for leeches starved for 48 h. The WWT of individual leeches was taken prior to the animal being placed in a computer-monitored, Wow-through respirometer system (Davies et al. 1992) with the flow rate adjusted to maintain at least a 5-Tom difference (1 Ton = 133.322 Pa) between the control and the three experimental chambers. To avoid reading biased by stress of handling (Davies et al. 1992)-oxygen consumption

279.1 + 29.1 56.7 2 2.7

344.3 77.2

28.6 3.4

515.3 2 74.5 107.5 + 4.6

297.4 2 12.2 60.8 + 5.4

478.7 2 31.5 86.4 k 6.3

7694.2 + 58.7 145.6 2 I 1.5

+ +

measurements were taken 1 h after introducing the animals to the system. Two activity states were defined using the impedance monitoring system: (1) random movement associated with crawling, short bursts of swimming and randona probing by the leeches' anterior (termed active) and (2) no measurable activity (termed resting) (Wrona and Davies 1984). Readings from the control and each experimental chamber were taken for 20 and 40 min, respectively, and then repeated until each experimental animal provided resting and active rate measurements for at least 20 min (i .e., about 1000 replicate readings for resting and active oxygen consumption). At the end of each run, animals were freeze-dried and the dry weight (DWT) of individuais determined (Table 1). Volumetric oxygen uptake by whole animal (microlitres of oxygen per total individual DWT per hour) and weight-specific oxygen uptake (microlitres of oxygen per milligram DWT per hour) were determined following Bavies et al. (1992). Aerobic scope (AS), which indicates the amount of oxygen available for aerobic respiration above maintenance, was calculated as the difference between active (Ra) and resting (Rm) weight-specific oxygen consumption (Fry 1947). a m , Ra, and AS may be converted to energy units using an oxycaloric equivalent of 0.0202 JepL 9,- ' (Elliott and Davison 1975). At each life history stage, five summer and five winter N. obscura were tested and the double log-transformed data used in regression analysis (re = 30 for each group). Covariance analysis was used to test for differences between slopes. The mean Rm and Ra were calculated separately at each stage for winter and summer N. cshscura using the appropriate regression equation. Tn all statistical analyses, significance is assessed at the p < 0.85 value.

Results While resting and active rates of oxygen consumption (microlitres of oxygen per total individual DWT per hour) in both groups of leeches increased with body DWT (Fig. I ) , the slopes for resting and active respiration were significantly different between winter and summer N. obscura. Conversely, weight-specific Rm and Ra (microlitres of oxygen per milligram DWT per hour) of both winter and summer N . obsc.uru decreased with increase in body DWT (Fig. 21, and the slopes for Rm and Ra were significantly different between winter and summer leeches. Although stage 1 winter leeches had 64.8% higher Rm than summer leeches, the difference reduced to 10.2% by stage 6 (Table 2). Stage 1 winter leeches had 5.6% lower Ra than summer leeches, but by stage 6 the Ra of winter leeches was 20.9% higher than that of summer leeches (Table 2). AS decreased with increase in body DWT in both winter and

TABLE2. Calculated mean (n = 5) of oxygen consumption rates and aerobic scope (AS) (p,L OI*rng DkfTT ' . h ' ) and percentage differences between winter (W) and summer (S) N o obscura. Rm = resting respiration rate; Ra = active respiration rate. Ra

Rm


Stage

S

W

%,

S

W

LOG DRY WEIGHT

.4S 57r

S

\V

.4SiR1n C;/c

S

W

LOG DRY WEIGHT

FIG. 1 . Resting (open circles) and active (solid circles) rates of oxygen consumption (pE 02.total individual DWT - 'eh ') in relation to DWT (mg) of (A) summer and (B) winter N. obsc.ul-ca.

summer leeches, with the slope for winter leeches significantly different from that of summer leeches (Fig. 3). While stage l winter N . obsrura had 24.9% lower AS than summer leeches, by stage 6 the AS of winter leeches was 20.9% higher than that of summer leeches. Reflecting the changes in Ran and Wa, the AS/Wm ratio increased from 1.7 at stage 1 to 3.0 at stage 6 in winter N . obscura but decreased from 3.7 at stage 1 to 2.6 at stage 6 in summer leeches (Table 2).

Discussion The observed differences in respiratory oxygen consumption between life history stages sf N. olascura reflect the influence sf size and/or physiological state, and the differences between winter and summer N. obscura reflect the influence of the winter stresses of the animals. Levels of resting and active whole-animal oxygen csnsumption in summer and winter IV. sbscura are proportional to the power function of the body weight with slopes similar to those observed by Ultsch (1973) and Calow (1975). The slopes recorded by Mann (1956) for five species of British leeches Cm. J. Fish. Aquat. Sci., V d . PO, 1993

were higher (0.78- 1 .O6) than those recorded for Ibr. obscurer. This could be attributed to Malmn9suse of WWTs rather than DWTs over a restricted body weight range, the inability to determine activity-specific respiration, or interspecific differences. The decrease in weight-specific resting oxygen consumption (Wm) was asymptotic in both winter and summer N . sbscura . The higher Rm in winter compared with summer N . obscsrra (Table 2) probably reflects the higher costs of maintaining, mobilizing, and transporting materials for tissue repair after experiencing winter stresses. Although winter N . cahscura maintained higher Rm than summer leeches throughout their life history, the initial decrease suggests a relatively fast metabolic recovery. Earlier studies on the effects of stress on respiration have not measured activity-specific respiration and have looked at only one life history stage, e.g., Baird et al. (1990) inferred that Rrn increased in Daphnia rlaagna Straus exposed to cadmium and 3,4-dichloroaniline (BCA), and Barber et al . ( 1 990) observed that such increases in oxygen consumption were possibly due to the costs associated with protein turnover. Maltby and Nay843


LOG DRY WEIGHT

LOG DRY WEIGHT

FIG.2. Relationship between resting (open circles) and active (solid circles) weight-specific rates of oxygen consumption (pL 0;mg and DWT (mg) of ( A )summer and (B) winter N . obscura.

lor (1990) and Maltby et al. (1998) found that zinc and DCA stresses did not result in changes in respiration by Garnrnarus pule-x (L.), although exposure to ammonia did. The significantly higher values of Ra than Rm at each life history stage of winter (2.7-4.0 times) and summer (3.6, 4.7 times) N. sbscurca fa11 within the range (1.4-10.0) quoted by Newell (1970) for a variety of invertebrates. Overwintering resulted in lower Ra values in winter N . obricura during the early stages of growth, but they show a rapid respiratory recovery with overcompensation at the later stages. However, the smaller percentage differences in Ra compared with Rm in stage B and 2 winter leeches suggests either that Ra is less influenced by winter stresses or that there is greater ability to csmpensate (Table 2). Like N . sbscura, a decrease in AS with increase in body weight has also been observed in shrimp (Heterosquikh tricarinata) by Hnnes (1985) and some smaH (