Woundfin, Ich, and Temperature / Charalito, Ich, y temperatura

KEYWORDS: woundfin; Plagopterus argentissimus; Cyprinidae; Ichthyophthirius multifilis; Dexter NFH; Utah; Arizona; Nevada

ABSTRACT

The woundfin, Plagopterus argentissimus, is an endangered cyprinid from the Virgin River system in Utah, Arizona, and Nevada. It was first brought into captivity at Dexter National Fish Hatchery in 1979 to maintain a population for reintroduction purposes. Problems in culture have occurred, ranging from loss in ponds due to excess aquatic vegetation, Lernaea infestation, and most notably Ichthyophthirius multifilis, or "Ich." Earlier this year the FDA banned the use of some chemotherapeutants traditionally used for Ich. The captive woundfin population is especially prone to acute Ich outbreaks in the spring, so alternative methods of control were investigated. All methods are basically water treatments and act on the free- swimming stage of the parasite. Non- chemical treatments include water ozonation, radiation with UV light, sub- micron filtration, increasing flow, and temperature extremes. This paper will describe the methods and results from an experiment using temperature control, and discuss the disease and reproductive status of woundfin currently at the station.

RESUMEN

El charalito, Plagopterus argentissimus, es una especie de ciprínido en peligro de desaparición en el sistema Virgin River, Utah. Esta especie fue mantenida por primera vez en cautiverio en el año de 1979 en la piscifactoría Dexter National Fish Hatchery, para sostener una población viable para propósitos de reintroducción. Diversos problemas concernientes con su cultivo han ocurrido, los cuales van desde la pérdida en los estanques debido a exceso de macrófitas acuáticas, infestación por el copépodo ancla Lernaea y más notablemente por el patógeno Ichthyophthirius multifilis o "Ich". A principios de 1992 la FDA (Federal Drug Administration) prohibió el uso de algunos quimioterapeúticos tradicionalmente utilizados para el tratamiento de Ich. La población en cautiverio de charalito es especialmente sensible a la fuerte proliferación de Ich en la primavera; por tal motivo, métodos alternativos de control fueron aquí investigados. Todos los métodos son básicamente tratamientos de agua, los cuales actúan en las etapas de nado libre de parásitos. El tratamiento no químico incluye la ozonización del agua, la radiación con luz ultravioleta, filtración sub- micron, incremento del flujo, y temperaturas extremas. Este trabajo, describe los métodos y resultados de un experimento utilizando control de temperatura, y discute el estatus reproductivo y patológico actual de los peces charalito cultivados en la piscifactoría.

CONTRIBUTED PAPER

Woundfin background

The endangered woundfin Plagopterus argentissimus is a small cyprinid, first described by Cope in 1874. Its dorsal fin rays are modified into a spine, which is presumably the source of the common name. It is the most highly modified of 3 species in the tribe Plagopterini, and the only species in its genus (Miller and Hubbs, 1960). In the 1800's it was found sporadically throughout the lower Colorado River basin, but is currently limited to a short reach of the Virgin River system, in Utah, Arizona, and Nevada (see Deacon, 1988 for distribution map). At times this unique species has comprised over 90% of the fish fauna in the Virgin River system (Minckley, 1973), and at other times is exceedingly rare. Upstream from Mesquite the populations have not changed much since the 1930's, but below Mesquite populations have decreased significantly (Cross, 1978). In a 1988 paper, Deacon found that since 1983 the population was approximately unchanged only in the 10 km section between Washington Fields Diversion and Quail Creek, and had declined dramatically in more than 60% of its remaining range. The species' decline during this century has been attributed to decreased water flow, introduction of exotic species (especially the red shiner, Cyprinella lutrensis), and other forms of habitat destruction (Cross, 1978). The woundfin was placed on the Endangered Species list in 1970 (USFWS, 1984), and the recovery plan for this species includes captive propagation. For this purpose, it was first brought to Dexter National Fish Hatchery in southeastern New Mexico in 1979 (DNFH files).

Dexter and Ich

Since that initial collection, there have been many more wild fish taken from the Virgin River (see Table 1). The profusion of fish brought to Dexter during 1988 was collected prior to the fateful red shiner eradication project, and consisted mainly of juveniles. The total population size at the hatchery has fluctuated greatly. As well as natural attrition, individuals have been lost in pond vegetation, overwhelmed with Lernaea infestations, killed during episodes of intense nitrogen supersaturation, and the population has been repeatedly decimated by Ichthyophthirius multifilis, or Ich. The woundfin population is also infected with Asian tapeworms Bothriocephalus acheilognathi (R. Major, pers. comm.). This parasite was introduced into the country with grass carp Ctenopharyngodon idella, and most likely introduced into the Virgin River drainage with red shiner Cyprinella lutrensis (Heckman, et al., 1986). Another species of tapeworm may also be present in the captive population; its identification is pending. Although fish may not die as a direct result of tapeworm infection, the added stress can affect feeding, reproduction, and immune responses. At Dexter, however, Ich is the main factor limiting woundfin production (see Figure 1). During the past 5 years, Ich outbreaks have occurred at least nine times (1988-2 occurrences, 1989-3, 1990-2, 1991-2).

Figure 1

	Year		Number		Location
	----		------		--------
	1979		240		Virgin River, AZ
	1981		395		Virgin River, AZ
	1983		460		Beaver Dam Wash area, AZ
	1987		235		Beaver Dam Wash area, AZ
	1988		28		St. George area, AZ
	1988		415		St. George area, UT
	1988		1600+		St. George area, UT
	1989		1060		St. George area, UT

Table 1 Numbers of wild woundfin Plagopterus argentissimus collected in the Virgin River, and brought to Dexter National Fish Hatchery and Technology Center.

Ich

Ichthyophthirius multifilis Fouquet is a protozoan with worldwide distribution. It is more commonly referred to as the tell-tale symptom which it causes in infected fish: "white spot." Host death occurs as a result of osmoregulatory imbalance, secondary infections, and finally malnutrition. Small-scaled or scaleless fish are more susceptible to Ich than those with large scales (Post, 1987). In fact, most Ich research is conducted on catfish. The woundfin, too is essentially scaleless, and therefore particularly at risk. It is the only species of 16 presently at Dexter which is chronically affected.

The protozoan life cycle lasts from 4 to 40 days, and is temperature dependent: at 18°C/64°F it is completed in 10-12 days. The embedded stage is called the trophozoite, which burrows into the epithelium of the host, ingesting tissue fluids and epidermal cells (Amlacher, 1970). In response the host produces extra epithelial tissue, which is the source of the white spots. Only recently has there been evidence of reproduction at this stage (Ewing et al, 1988). In general, a mature adult leaves the host. At this free-swimming stage, it is called a trophont, and is positively identified by the horseshoe-shaped nucleus (Meyer and Bullock, 1990). It then becomes submergent and secretes a cyst wall within 2-6 hours. While encysted, the tomont divides mitotically forming hundreds or thousands of new motile, infective units, called tomites. The tomite stage of this obligate parasite must encounter a fish host within 48 hours or die (Post, 1987). All ich treatments are directed at this free-living life stage, as both the encysted and the embedded stages are much more resistant.

Woundfin at Dexter

The hatchery water is supplied by a number of shallow wells, with pH of 8.5, 3500 mg/l of total dissolved solids, and constant temperature of 18°C/64°F. Woundfin at Dexter NFH are typically maintained in 12 m raceways, and do not spawn until the water reaches above 21°C/70°F. To achieve that temperature during the spawning season (usually April-May), the raceway water is usually recirculated via pumps, and the inflow of cool well water is decreased. Prior to this year, when an Ich outbreak was noticed, the fish were treated with a Malachite Green/Formalin bath daily for 10-14 days (DNFH notes). Malachite has a very small margin of safety between host and parasite toxicity (Cross, 1972), and mortality does occur. Dosages were adjusted for woundfin, and each Ich outbreak was treated accordingly and eventually dissipated for a time. However, Ich seemed always to return. Either the fish continued to harbor the parasite through each successive treatment, or Ich reinfection occurs every year.

In the fall of 1991, the Federal Drug Administration (FDA) banned the use of Malachite Green at federal hatcheries, citing its oncogenic, teratogenic, and mutagenic properties. The FDA also banned the use of formalin for all but a few species, for only a few specific uses. Since our chemical arsenal against Ich was now emptied, we had to find alternative treatment methods.

Many non-chemical treatments have potential for treating Ich. These include filtration (with mesh size that must effectively screen out particles the size of Ich tomites, < 1 mm); radiation with ultraviolet light (at 100,000 microwatts/second/cm2); ozonation; pasteurization; and treatment with temperature extremes. The critical thermal maxima of woundfin is very high, even for a desert adapted fish (Deacon et al., 1987). As the temperature was quickly raised, it did not lose equilibrium (which would lead to death in the wild) until the water reached 39.5°C/103°F. Ich dies at 32°C/89.5°F (Meyer, 1984), stops reproducing at 30°C/86°F (Dr. Nick St. Erne, DVM, pers. comm.), and does not infect new fish at 29.4°/85°F (Johnson, 1976). Based on these findings, it was decided to try heat as the non-chemical alternative with which to treat Ich.

Methods

Early this spring, in an effort to avoid Ich in the first place, none of the aforementioned techniques were used to warm the water. Instead of recirculating between raceways and decreasing the inflow, the inflow of cool water was left open. With flow-through well water, the temperature in the raceways does not get above 20°C/68°F even in the summer. Ich was not observed by May, but neither was any spawning behavior. Lockhart (1980) suggested that woundfin live » 2.5 years in the wild. Since the majority of adults at Dexter are 3-4 years old, and this spring was possibly their last spawning season, it was decided then to increase the temperature, and the water inflow was cut down. This would allow spawning to occur, and enable us to try heat treatments as a prophylactic to the inevitable (Hamman, pers. comm.) spring Ich outbreak.

From the raceways, most fish were brought into the holding house and held in 70-gallon aquaria; the remainder were left outside as a primary control for this stage of handling. The holding aquaria were maintained at 23°C/73°F. The treatment tanks consisted of 10, 10-gallon aquaria, each with an airstone and a heater. Ten fish were placed in each tank and allowed to acclimate for 3 days. Temperature was then raised and lowered in each of the treatment tanks daily for 5 days (van Duijn, 1967; Amlacher, 1970). The temperature was gradually brought up from 23°C/73°F to at least 31°C/88°F during the course of a day, using 300-watt, thermostatically controlled Visitherm_at_* heaters, and monitored at two hour intervals. The water temperature was then lowered to the acclimation temperature during the course of 30-60 minutes by making a series of partial water changes. Cross (1972) suggested that changing water may in itself be a treatment (separating the host from the infective stage of the parasite). To test this possibility, two tanks in each trial were held at constant temperature and water changes were made daily. As an additional control, two tanks in each trial were not manipulated at all. Mortalities were recorded, and the maximum temperature tolerated by survivors was compared to that of those that died, using ANOVA. After the 5-day treatment, fish were transferred to 70-gallon aquaria. They were held there for observation before being released into a disinfected raceway. Eventually, all adults (except the primary raceway controls) and 10 juveniles were subjected to the treatments.

Observations and Results

Woundfin behavior changed when subjected to temperature increases. Dissolved oxygen also varied with temperature, from 83 to 75 % saturation at temperatures 23-31.5°C/73-89°F, respectively. When the water first reached 31°C/87F°, fish would become very active, gilling faster, and taking forays to the surface where they would skim their bodies along the surface. Perhaps this splashing cooled the water. After about 20 minutes at this temperature the fish would calm down, but gilling rate would remain elevated.

First, I conducted a preliminary run by heating just one tank of 10 fish (see Table 2). Two fish died in control tanks during this first week in aquaria. The average daily maximum temperature was 33°C/92°F. A total of three fish died when the temperature reached its peak of 34°C/93°F, on the second and 4th days. Lockhart reported similar results in 1980, when death occurred at 33.8°C/93F° for fish acclimated at 21.5°C/71°F.

The second run included all ten tanks and 100 fish, with tank treatment randomly assigned. Of the heated tanks, the average daily maximum temperature was 32°C/90°F, and a total of 10 fish died. The highest overall temperature reached during this run was 35°C/95°F, after which 5 of the 10 deaths occurred.

The third run included 4 adult tanks and one juvenile (or 1 year old) tank. The average daily maximum temperature was 31°C/87.5°F, overall maximum for the run was 32/90°F, and no mortalities occurred during this treatment.

There appears to be a direct relationship between average maximum temperature and mortality. Of the 13 fish that died, the mean maximum temperature was 34°C/93°F, while that of survivors was 31.5°C/88.6°F, a significant difference (t = -6.577, p < 0.01, df 53).

Mean

Trial Max Temp n Mort Max Temp

    I        33.5°C        10       30%          34°C         
   II        32°C          60       16%          35°C         
  III        31°C          40        0%          32°C         

Table 2 Results from experimental high temperature treatment for control of Ichthyophthiriasis, at Dexter National Fish Hatchery and Technology Center during spring of 1992 (see text for further explanation).

Discussion

No treated fish got Ich this year, however no control fish got Ich this year, either. Therefore, at best the results of this experiment on the effectiveness of treating Ich-infected woundfin with heat are inconclusive. Some explanations come to mind: It may be that the last malachite/formalin treatment finally did get rid of the parasite. However, after so many unsuccessful trials, this is unlikely. Another possibility is that the usual procedure of recirculating the raceway water gives the tomites a better chance of encountering a host. Continuous flushing (as was done this year) may decrease the chance of such an encounter. Also, water quality would be enhanced by continuous flushing, and therefore minimize that stress on the fish.

Although inconclusive about Ich, conducting this experiment did provide some additional insight into the biology of these little-known fish. The woundfin's ability to withstand high temperature is surely an advantage in the Virgin River system. Woundfin have been collected there at 36°C/97°F (Deacon, 1987). The maximum temperature tolerated by woundfin in this experiment was 35°C/95°F, while in Deacon's CTM experiment (1987) it was 39.5°C/103°F. The difference in maximum temperatures obtained in these experiments may be explained by the rate at which temperatures were raised. In this experiment, temperature was raised at an average rate of 0.03°C per minute, while in Deacon's experiment it was raised 1°C per minute. As woundfin can withstand higher temperatures when raised quickly, this would imply a phsiological mechanism in place to buffer against the effects of variable water temperatures.

Even with this thermal treatment and intensive handling this year, the fish still spawned. On the first days of treatment I observed spawning behavior in the aquaria, and after all fish had been cycled through the primary holding aquarium, I found fry. Since the last adults were removed from that aquarium on June 7, the fry were spawned sometime prior to that. On July 7, fry were dipnetted from the raceway of treated fish. This continued until Sept 23, for a total of 257 fry from treated fish. It is possible that some fry could have escaped our nets, making this recruitment number a conservative one. Only 1 fry was observed in the raceway of untreated fish during the same period. At Dexter National Fish Hatchery, fry are usually collected between May 18 and June 20 (DNFHTC files), a period of 4 1/2 weeks. This year's experimentally delayed spawning season was at least 15 weeks long, and therefore the longest recorded season of production at Dexter. Although Lockhart (1980) found peak gonad maturation in May, he also suggested that under favorable conditions woundfin could spawn over a longer period of time. Perhaps favorable conditions include a delayed summer, as was simulated by these experiments.

I found that upon heating more tapeworms evacuated their hosts. Over the 3 week course of the experiment, I collected 13 independent adult tapeworms. Given that the tapeworm is exotic to the Virgin River system, and has probably been in Utah only since 1984 (Heckmann et al., 1986), it may not be well adapted to the temperature extremes common there and imitated in this experiment. Temperature extremes should be investigated as a non-chemical parasiticide for tapeworms.

I also observed woundfin mouthing, picking at, and ingesting such free-living tapeworms. In the Virgin River system, tapeworms are newcomers to the community, and woundfin feeding behavior probably does not include discrimination of tapeworms from other worms. This may not be the typical mode of tapeworm transmission to a host, but it may be the case for native fish in the Virgin River.

Currently on hand at DNFH&TC, we have on hand 77 adults, 82 juveniles, and 173 y-o-y. The '91 and '92 year classes are being maintained overwinter in 4, »75 gallon fiberglass aquaria, and the adults are overwintering in an outdoor raceway. Fifty one mixed-age fish were sent to Dr. Steven Vives of Georgia Southern University on Oct 7. There he will conduct experiments on spawning and reproductive behavior.

In order to maintain viable numbers of the captive population, and eventually produce enough fish to begin a restocking program for woundfin, it is necessary to maintain the highest standards of water quality, be ever vigilant for changes in fish behavior, and continue development of alternative treatment methods.

* Use of tradenames does not imply U. S. Government endorsement of commercial products.

Literature Cited

Amlacher, E. 1970. Textbook of Fish Diseases, TFH Publications, Inc., Neptune City, New Jersey.

Cross, D.G. 1972. A review of methods to control Ichthyophthiriasis. Progressive Fish Culturist 34(3): 165-170.

Cross, J. N. 1978. Contributions to the biology of the woundfin, Plagopterus argentissimus (Pisces: Cyprinidae), an endangered species. Great Basin Naturalist 38 (4): 463-468.

Deacon, J.E., P.B. Schumann and E.L. Stuenkel. 1987. Thermal tolerances and preferences of fishes of the Virgin River system (Utah, Arizona, Nevada). Great Basin Naturalist 47(4):538-546.

Deacon, J. E. 1988. The Endangered Woundfin and Water Management in the Virgin River, Utah, Arizona, Nevada. Fisheries 13 (1):18-24.

Ewing, M. S., S. A. Ewing, and K. M. Kocan. 1988. Ichthyophthirius (Ciliophora): Population Studies Suggest Reproduction in Host Epithelium. Journal of Protozoology 35(4): 549-552.

Heckmann, R. A., J. E. Deacon, and P. D. Greger. 1986. Parasites of the woundfin minnow, Plagopterus argentissimus, and other endemic fishes from the Virgin River, Utah. Great Basin Naturalist 46 (4):662-676.

Johnson, S. K. 1976. Laboratory evaluation of several chemicals as preventatives of Ich disease. U. S. Fish and Wildlife Service. Fisheries Disease Leaflet 10.

Lockhart, J. N. 1980. Ecology of the Woundfin Minnow, Plagopterus argentissimus, Cope. Manuscript, Univ. Nevada, Las Vegas. 187 pp.

Meyer, F. P. and G. L. Bullock. 1990. Protozoan parasites of freshwater fishes. Fish Health Bulletin 8. Dept. of the Interior, U. S. Fish and Wildlife Service.

Meyer, F. P. 1984. Parasites of freshwater fishes; II, Protozoa Ichthyophthirius multifilis. U.S. Department of the Interior, Fisheries Disease Leaflet 2.

Miller, R. R. and C. L. Hubbs. 1960. The spiny-rayed cyprinid fishes (Plagopterini) of the Colorado River System. Misc. Publ. Mus. Zool., Univ. Mich. 115: 1-39, 3 pls.

Minckley, W. L. 1973. Fishes of Arizona. Arizona Game and Fish Department, Tucson, Arizona.

Post, G. 1987. Textbook of Fish Health, TFH Publications, Inc., Neptune City, New Jersey.

U. S. Fish and Wildlife Service. 1984. Recovery Plan for Woundfin, Plagopterus argentissimus Cope. U. S. Fish and Wildlife Service, Albuquerque, New Mexico.

van Duijn, C. 1967. Diseases of Fishes, Charles C. Thomas, Publisher, Springfield, Illinois.

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