Journal of Aquatic Animal Health 17:338±344, 2005 q Copyright by the American Fisheries Society 2005 [Article] DOI: 10.1577/H04-061.1 Effects of Temperature, Photoperiod, and Myxobolus cerebralis Infection on Growth, Reproduction, and Survival of Tubifex tubifex Lineages ROBERT DUBEY* Department of Fishery and Wildlife Sciences, New Mexico State University, Box 30003, MSC 4901, Las Cruces, New Mexico 88003, USA COLLEEN CALDWELL U.S. Geological Survey, New Mexico Cooperative Fish and Wildlife Research Unit, New Mexico State University, Box 30003, MSC 4901, Las Cruces, New Mexico 88003, USA WILLIAM R. GOULD University Statistics Center, New Mexico State University, MSC 3QC, Las Cruces, New Mexico 88003, USA Abstract.ÐTubifex tubifex is the de®nitive host for Myxobolus cerebralis, the causative agent of whirling disease in salmonid ®sh. Several mitochondrial lineages of T. tubifex exhibit resistance to M. cerebralis infection. Release of the triactinomyxon form of the parasite from T. tubifex varies with water temperature; however, little is known about the interactive effects of temperature and photoperiod on the susceptibility of T. tubifex lineages to M. cerebralis infection. In addition, the environmental effects on the growth, reproduction, and survival of T. tubifex lineages are unknown. Monocultures of lineages III and VI were subjected to infection (0 and 500 myxospores per worm), a range of temperatures (5, 17, and 278C), and various diurnal photoperiods (12:12, 14:10, and 16:8 dark : light) over a 70-d period by using a split±split plot experimental design. Lineage VI resisted infection by M. cerebralis at all temperatures, whereas lineage III exhibited infection levels of 4.3% at 58C, 3.3% at 178C, and 0% at 278C. Lineage VI exhibited signi®cantly higher adult survival, weighed more initially, gained more weight, and had higher natality (production of immature tubi®cids) than did lineage III regardless of temperature, photoperiod, or infection treat- ment. There was no detectable effect of lineage, infection, or photoperiod on cysting. Both lineages III and VI cysted at 58C but not at 178Cor278C. Competition between lineage VI and other lineages for resources may serve to decrease the overall infection levels among T. tubifex popu- lations, thereby reducing both triactinomyxon production and the occurrence of whirling disease among susceptible salmonids. Whirling disease is a parasitic infestation of car- de®nitive host for M. cerebralis (Wolf and Markiw tilage in salmonids by the myxosporean Myxobolus 1984; El-Matbouli and Hoffmann 1989). The myx- cerebralis. Evidence of recent introduction in osporean-type spores released by infected salmo- North America is supported by genetic sequence nids are ingested by T. tubifex and are transformed data from M. cerebralis isolates from Europe and into the actinosporean triactinomyxon (TAM) in the United States (Andree et al. 1999; Whipps et the gut epithelium of the oligochaete hosts (El- al. 2004). The devastating effects of whirling dis- Matbouli and Hoffmann 1989). Salmonids con- ease on wild salmonid populations were not fully tract whirling disease by brief contact with wa- realized until its discovery in the intermountain terborne TAMs (El-Matbouli et al. 1995). west in association with the rapid decline of sal- Tubifex tubifex is a cosmopolitan freshwater spe- monid populations in Montana (Vincent 1996) and cies with morphological characteristics exhibiting Colorado (Nehring and Walker 1996). phenotypic plasticity, such as pectinate chaetae, The aquatic oligochaete Tubifex tubifex is the that make it dif®cult to distinguish from closely related species (Chapman and Brinkhurst 1987; Kathman and Brinkhurst 1999). Crossbreeding * Corresponding author: [email protected] studies between sympatric morphological forms of Received December 3, 2004; accepted April 18, 2005 T. tubifex (tubifex and blanchardi) indicated that Published online November 3, 2005 they did not interbreed with suf®cient genetic dif- 338 RESISTANCE OF TUBIFEX TUBIFEX TO MYXOBOLUS CEREBRALIS 339 ferences to be considered distinct species (Paoletti Methods 1989). Using allozymes, Anlauf (1994, 1997) de- Tubifex tubifex are parthogenic (Poddubnaya scribed lineages of T. tubifex that differed between 1984). Thus, progeny from this form of reproduc- pond and lake habitats. Phylogenetic analysis of tion were used to establish laboratory monocul- mitochondrial 16S ribosomal DNA from geo- tures of lineages III and VI. Original stocks of T. graphically distinct populations of T. tubifex pro- tubifex lineages were obtained from the San Juan vided evidence that cryptic lineages within both River, New Mexico. The lineage of each mono- North American and European populations were culture was veri®ed by screening individuals with similar (Sturmbauer et al. 1999; Beauchamp et al. a polymerase chain reaction (PCR) ampli®cation 2001). Sturmbauer et al. (1999) described Euro- of 16S mRNA, developed by Beauchamp et al. pean lineages that exhibited differential tolerance (2002). The PCR methodology for lineage deter- to cadmium exposure. Beauchamp et al. (2002) mination used the DNA template from DNeasy reported four lineages of T. tubifex (I, III, V, and (Qiagen, Valencia, California) mouse-tail protocol VI) at two sites on the Colorado River, Colorado. extractions and three rounds of ampli®cation using Those authors also observed varied susceptibility primers. In this protocol, four North American lin- of the lineages to experimental infection of M. eages were distinguished by size-speci®c PCR cerebralis. Recently, three lineages (I, III, and VI) product. Of the two lineages tested the PCR prod- were identi®ed in sympatric populations from the uct is 147 base pairs (bp) for lineage III and 125 tailwater of the San Juan River, New Mexico bp for lineage VI. (DuBey and Caldwell 2004). No infection was de- Myxobolus cerebralis spores were isolated from tected in lineages I and VI, in contrast to lineage infected rainbow trout Oncorhynchus mykiss. The III. Furthermore, lineage III exhibited higher in- ®sh heads were de¯eshed by heating at 458C, and fection levels in pools (3.0%) than in rif¯e habitats ¯esh and noncartilaginous material were manually (0.5%), demonstrating varied infection responses removed. The cartilaginous material was pulver- presumably attributable to different environmental ized and centrifuged to concentrate spores into a conditions. pellet. The pellet was resuspended and spores were Anlauf (1994) observed differences in growth, quanti®ed by microscopic examination and count- reproduction, and survival among cultures of three ing within a known volume. T. tubifex lineages originating from different hab- Tubifex tubifex lineages III and VI were ran- itats at 5, 15, and 208C. A positive linear rela- domly assigned to each of 18 treatment combi- tionship between T. tubifex fecundity, temperature, nations within nine 38-L glass aquaria by using a split±split plot experimental design. Each aquari- and percentage of organic matter in sediments was um was divided in half with clear Plexiglas. Eight observed in Montana headwater streams (Kaster 350-mL plastic tubs were placed within each 1980). Myxobolus cerebralis development in in- aquarium half, four tubs containing lineage III and fected T. tubifex was also shown to be temperature- four tubs containing lineage VI. Before placement dependent, the spore loads being highest at 158C, within the aquaria, 25 immature tubi®cids were whereas nonviable degenerating M. cerebralis randomly assigned to each tub with a mixture of 8 spores were detected at 30 C (El-Matbouli et al. sterilized silt, sand, and water. Subsequently, T. 1999). Furthermore, allopatric geographic popu- tubifex in eight tubs were challenged individually lations of T. tubifex differed in biomass, repro- with an infection of 500 spores per worm, a rate duction, growth, and TAM production when sub- similar to Stevens et al. (2001) and Blazer et al. jected to controlled infection of M. cerebralis (Ste- (2003). Eight plastic tubs received no spores. The vens et al. 2001). However, little is known re- challenged T. tubifex tubs were then assigned to garding lineage-speci®c responses of M. cerebralis an aquarium half; the nonchallenged T. tubifex tubs infection to combinations of temperature and pho- were placed in the other half to ensure indepen- toperiod. Thus, the objective of our research was dence of the treatment and control groups. to characterize the responses of two lineages of T. A range of temperatures (5, 17, and 278C) were tubifex found in the San Juan River tailwater selected to represent the optimum temperature at (DuBey and Caldwell 2004) to a range of photo- which T. tubifex is commonly found. Each tem- period and thermal regimes and determine whether perature treatment was applied to three aquaria at infection by M. cerebralis would affect growth and three separate locations. Treatment at 5 6 18C was survival of lineages. conducted in a Living Stream (Frigid Units, To- 340 DUBEY ET AL. ledo, Ohio) in a laboratory with a temperature- cerebralis infection was determined for individual controlled refrigeration unit (model DI-500; worms in the infected treatment and for worms Aquarium Systems, Mentor, Ohio). Treatment at composited from noninfected treatments. The in- 17 6 18C was conducted within the same labo- fection level for each treatment combination was ratory on a bench top. Treatment at 27 6 18C was computed by dividing the total number of infected conducted in a walk-in Sheerer environmental worms by the total number of worms examined chamber having a UP 780 digital control system (lineage-speci®c) from each aquarium half. to maintain the desired temperature (Yokogama The presence or absence of M. cerebralis infec- Corporation of America, Newton, Georgia). tion was determined with a nested PCR ampli®- Chamber temperature was measured with a Na- cation of 18S rRNA. The PCR methodology for tional Institute of Standards and Testing±certi®ed M. cerebralis used DNA template from phenol ex- sensor (Model MRHT3±2±1-D; General Instru- tractions, two rounds of ampli®cation according to ments, Woburn, Massachusetts). protocol, and primers developed by Andree et al.