J. Thermal Bh)h,qv. I.'ol. 3. pp 129 Io 131) 0306-4565 78 0"r01-0129502.00 0 © Pergamon Pres~ Ltd 1978. Printed it) Great Britain

FEBRILE RESPONSES OF BLUEGILL (LEPOMIS MACROCHIRUS) TO BACTERIAL PYROGENS

WILLIAM W. REYNOLDS*, MARTHA E. CASTERLIN* AND JERRY S. COVERTt *Biology Department, The Pennsylvania State University, Wilkes-Barre, PA 18708 U.S.A.

"['Biology Department, The Pennsylvania State University, Hazleton, PA 18201 U.S.A.

(Receiced 8 September 1977; accepted 23 November 1977)

Abstract--Bluegill exhibited a mean increase in preferred temperature of 2.5=C following intraperitoneal injection of endotoxin from the gram-negative bacterium Escherichia coil

2. lntraperitoneal injection of killed gram-positive Staphylococcus aureus caused a mean increase of 2.6°C.

3. These febrile responses are similar to the 2.7°C increase in response to injection of killed gram- negative Aeromonas hydrophila.

INTRODUCTION

FISHES (Reynolds et al., 1976a, Reynolds, 1977a: Reynolds & Covert, 1977) and other ectotherms (Vaughn et al., 1974; Satinoff et al., 1976; Kluger, 1977; Casterlin & Reynolds, 1977) exhibit a behav- iourai fever (increase in preferred temperature) when injected with live or killed bacteria. This response has survival value where infection with live pathogenic bacteria is involved (Covert & Reynolds, 1977), pre- sumably due to some thermal enhancement of host defence mechanisms.

Many pyrogenic (fever-inducing) agents have now been identified in both gram-negative and gram-posit- ive bacteria (Snell, 1971). The major pyrogen associ- ated with gram-negative bacteria is thought to be a lipopolysaccharide constituent of the cell wall, gener- ally referred to as endotoxin (Snell, 1971). Gram- positive bacteria contain other pyrogenic substances which are less well defined at present.

The bluegill sunfish Lepomis macrochirus--an excel- lent test animal for thermoregulatory studies because of its precise thermoregulatory behaviour and lack of a circadian rhythm of preferred temperature (Reynolds & Casterlin, 1976)--as well as the large- mouth bass Micropterus salmoides (Reynolds et al., 1976a) and the goldfish Carassius auratus (Reynolds & Covert, 1977; Covert & Reynolds, 1977) have been shown to exhibit a febrile response to injection with the gram-negative bacterium Aeromonas hydrophila. The present investigation was undertaken to deter- mine the thermoregulatory effects on bluegill of intra- peritoneal injection with purified endotoxin from gram-negative Escherichia coli, or with killed gram- positive Staphylococcus aureus, for comparison with febrile responses to killed A. hydrophila.

M A T E R I A L S A N D M E T H O D S

Thirty-six adult bluegills (60-100g; ~ = 8 5 g body weight) were tested individually in an electronic thermore- gulatory shuttlebox (Ichthyotron) described previously (Reynolds et al., 1976b; Reynolds, 1977b), which permitted them to control water temperatures, and thereby their own

body temperatures, by swimming between chambers moni- tored by photocells and associated circuitry which con- trolled heating and coolin~ units. Following an initial 24 h adjustment period (Richards et al., 1977), baseline data (normal preferred temperature or final preferendum) were collected for 24 h. A fish was then injected intraperitoneally with either sterile pyrogen-free saline (N = 8). or 4 x 109 killed A. hydrophila cells suspended in 0.75ml saline (N = 8), or 5 pg of purified E. coil endotoxin (Difco Labs., strain 055:B5; N = 10), or 3 x 10 a S. aureus cells sus- pended in 0.5 ml saline (N = 10), and mean preferred tem- perature was determined for the next 24 h.

RESULTS

The baseline preferred temperature (~ _ s.e.m.) was 30.5 _+ 0.6°C (Fig. IA). Saline injection produced no significant increase in the preferendum (Fig. IB). Killed A. hydrophila caused a significant mean in- crease of 2.7°C (Fig. IC). E. coil endotoxin produced a similar increase of 2.5°C (Fig. ID), while killed S. aureus stimulated a 2.6°C increase (Fig. IE).

DISCUSSION AND CONCLUSIONS

Adult bluegill exhibit a febrile response of similar magnitude (2.5-2.7°C) to i.p. injection with purified endotoxin from gram-negative E. coil or with killed gram-positive S. aureus, as with killed gram-negative A. hydrophila. Previous work (Reynolds, 1977a) indi- cated that smaller juvenile bluegill (10--30g) show a sfightly higher fever (3.2°C) in response to killed A. hydrophila, possibly either an ontogenetic or a size- related dosage effect. The antipyretic acetaminophen (Tylenoi) has been shown previously to counteract the pyrogenic effect of A. hydrophila in juvenile bluegill (Reynolds, 1977a), possibly by inhibiting prosta- glandin production or release.

Previous demonstrations of fever in fishes have util- ized live or killed whole gram-negative A. hydrophila cells as the pyrogenic agent (Reynolds et al., 1976a; Reynolds, 1977a; Reynolds & Covert. 1977; Covert & Reynolds, 1977). We have now extended these results to show a similar pyrogenic effect of extracted

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W. W. REYNOLDS. MARTHA E. CASTERLIN AND J. B. COVERT

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A B C D E

Fig. I. Changes in preferred temperature (mean f S.E.M. for 24 h) of bluegill relative to a baseline prefebrile prefer- endum (final preferendum) of 30.5 + 0.6”C (A), after i.p. injection of (B) sterile saline; (C) killed Aeromonas hydro-

phi/u ceils;(D) E. co/i endotoxin; or (E) killed Staphylococ-

cus aureus cells.

E. co/i lipopolysaccharide endotoxin (exogenous pyro- gen), and also of killed gram-positive S. aureus, which presumably contains some other pyrogenic sub- stance(s). The pyrogenic action of these exogenous pyrogenic agents is thought to be indirect, via stimu- lation of endogenous pyrogen release from leukocytes (Snell, 1971).

Ac~rlo,~/edyeme,lr-This study was aided by PSU Grant No. SAF-77-113.

REFERENCES

CASTERLIN M. E. & REYNOLDS W. W. (1977) Behavioral fever in anuran amphibian larvae. Li/r Sci. 20, 593-596.

COVERT J. B. & REYNOLDS W. W. (1977) Survival value of fever in fish. Nature 267. 4345.

KLUGER M. J. (1977) Fever in the frog Hyla cinrrra. J. Thermal Biol. 2. 79-82.

REYNOLDS W. W. (1977~) Fever and antip)-resis in the blue- gill sunfish Lepomis macrochirus. Comp. Biochem. Phy-

siol. 5X, 165-167. REYNOLDS W. W. (1977b) Fish orientation behavior: an

electronic device for studying simultaneous responses to two variables. J. Fish. Res. Bd Can. 34. 3OG304.

REYNOLDS W. W. & CASTERLIN M. E. (1976) Thermal pre- ferenda and behavioral thermoregulation in three cen- trarchid fishes. In: Thermal Ecology II (Edited by ESCH G. W. & MCFARLANE R. W.) USERDA Symp. Ser. CONF-750425, U.S. Natl. Tech. Info. Serv.. Springfield. Va., pp. 185-190.

REYNOLDS W. W., CASTERLIN M. E. & COVERT J. B. (1976a) Behavioural fever in teleost fishes. .&attire 259, 4142.

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(Eds) (1977) Temperature preference studies in environ- mental impact assessments: an overview with procedural recommendations. J. Fish. Res. Bd Can. 34. 728-761.

SATINOFF E.. MCEWEN G. N. JR. & WILLI.~~S B. A. (1976) Behavioral fever in newborn rabbits. Science 193.

1139-I 140. SNELL E. S. (1971) Endotoxin and the pathogenesis of fever.

In: Microbial Toxins, Vol. 5, Bacterial Endoroxins pp. 277-340, Academic Press, New York.

VAUGHN L. K.. BERNHEIM H. A. & KLLGER M. J. (1974) Fever in the lizard Dipsosaurus dorsulis. Nature 252,

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Key Word Index-Fever; Thermoregulation; Bluegill; Lepomis macrochirus; Endoto.rin; Aeronwnas hydrophila; Escherichia coli; Staphylococcus aureus; Bacteria.