Pacific Northwest Conservation

The Year of the Frog
by David Shepherdson and Blair Csuti

Field Work
Captive Rearing at the Oregon Zoo
Dietary Requirements of Larval Oregon Spotted Frogs

Field Work at Conboy Lake

Conboy Lake National Wildlife Refuge sits next to the
Yakima Indian Reservation beneath the snowy southeast slopes
of Mt Adams, about 10 miles east of Troutlake. Controlled by a system of dykes and dams, the lake all but dries out in the summer when the exposed meadows are
mowed to produce a crop of hay. At this time of year, though, it is a huge shallow lake covering several square miles.

Aside from being an important migratory bird waypoint, Conboy Lake is also home to a large (probably the largest) population of Oregon spotted frogs (Rana pretiosia). Zoo Research Associate Dr. Marc Hayes, in conjunction with the U.S. Fish and Wildlife Service, has been studying this population intensively for several years now.

The spotted frog population at Conboy is particularly interesting because it co-exists with a healthy bullfrog population. Bullfrog predation is currently thought to be a major factor in the disappearance of spotted frogs from most of the rest of their range in California, Oregon, Washington, and British Columbia. Marc Hayes suspects that the healthy co-existence of these two species has something to do with the unique
microhabitat characteristics of Conboy Lake. Perhaps the spring water in the dykes, for example, allows the cold water-loving spotted frogs to grow too big for bullfrog predation before they disperse into the warmer water that bullfrogs prefer. If this could be confirmed it might be possible to use this information to manipulate some of the spotted frog's former habitats to allow co-existence with bullfrogs.

Each March for the last three years, Oregon Zoo employees, together with Zoo volunteers, have made a trip out to the lake to hunt for egg clusters. The work involves wading up and down the submerged meadows hunting for egg clusters. When found, the cluster is marked with a flag and then data on location, size,
water temperature, etc., are recorded. It may not sound like too much fun, but on a sunny day with Mt. Adams gleaming under a fresh coat of snow and Sandhill Cranes honking in the distance,
life could be worse!

Captive Rearing at the Oregon Zoo

The zoo hopes to raise some frogs to adulthood for exhibit, where we will tell zoo visitors the sad story of the vanishing Oregon spotted frog. As the zoo perfects its larval husbandry techniques, it will be in a position to step forward if fish and wildlife agencies launch a captive propagation/reintroduction program as part of the recovery plan for this species. The Oregon spotted frog is expected to be listed as Federally Threatened or Endangered within the next two years.

Dietary Requirements of Larval Oregon Spotted Frogs:

Final Report to the U.S. Fish and Wildlife Service

by Blair Csuti, Ph.D., Conservation Program Coordinator and Bret Seller, Senior Keeper

September 1, 2000

BACKGROUND

Growing alarm exists among conservationists that amphibian species are declining throughout the world (Blaustein and Wake 1990, Phillips 1990). While the factors or combinations of factors contributing to this decline have not been conclusively identified, the survival of many amphibian species is questionable. For example, the golden toad (Bufo periglenes) has apparently disappeared from the Monte Verde Cloud Forest Reserve in Costa Rica. Likewise, frogs native to the Pacific Northwest have been declining in recent decades (Marshall et al. 1996, Kiesecker and Blaustein 1998). The Oregon spotted frog (Rana pretiosa) is among the region's most endangered amphibians. It has disappeared from well over 70% of its relatively small range
in the Pacific Northwest and is completely extirpated from low elevation areas south of the Columbia River (Leonard et al. 1993; Hayes 1994, 1997; Marshall et al. 1996; McAllister and Leonard 1997). Currently,
about 35 known extant populations occur from the Lower Fraser Valley of southern British Columbia south to southern Oregon, most of which number less than 1,000 individuals. The species may be extinct in northeastern California, the southernmost extent of its historic range (Hayes 1997).

Systematic studies have resolved some questions about geographic variation in spotted frogs of western North America, including recognition of the genetically cohesive western complex of populations as a distinct unit, the Oregon spotted frog (Rana pretiosa)(Green et al. 1996, 1997). Populations from the remainder of spotted frog's geographic range are now assigned to a different species, R. luteiventris, the Columbia
spotted frog, which will require further taxonomic work to identify the several cryptic species (thought to be concealed) within this genetically discontinuous unit.

In recognition of the threats to the continued survival of the Oregon spotted frog, the species was recently listed as endangered in Washington and British Columbia and is a Candidate for listing as Threatened or Endangered by the U.S. Fish & Wildlife Service. In Oregon, the Oregon spotted frog is Sensitive SpeciesCritical (Marshall et al. 1996), the highest of four categories of Sensitive Species recognized by
the state, whereas in California, it is a Species of Special Concern (Jennings and Hayes 1994). Many possible reasons have been postulated for the decline of the Oregon spotted frog, including predation pressure from non-native species, habitat loss and alteration, increased larval mortality resulting from fertilizer runoff, climate change, and increased susceptibility to pathogens or parasites. Because both
Oregon spotted frogs and introduced bullfrogs use warm water habitats, bullfrog predation is one factor that may have led to the spotted frog's extirpation from the Willamette Valley (Marshall et al. 1996, Csuti et al. 1997). Lawler et al. (1999) present experimental evidence that predation by bullfrog larvae reduces survivorship closely-related red-legged frog larvae. At least one site is known where spotted frogs and bullfrogs coexist (Conboy Lake National Wildlife Refuge, Washington; M. P. Hayes, ongoing research), which may be due, in part, to habitat partitioning in a relatively complex aquatic system. This example may provide a model for habitat modification that might allow successful reintroduction of the Oregon spotted
frog to low elevation sites in the Puget Trough and Willamette Valley.

Potential of reintroduction as a recovery strategy for the Oregon spotted frog, however, remains hypothetical because successful husbandry techniques for raising larval Oregon spotted frogs have not yet been developed. The Draft Oregon Spotted Frog Conservation Plan (Hall et al. 1999) recommends research on
a small scale captive-breeding program to develop techniques for possible future use in an introduction or augmentation program. This report describes husbandry methods for successfully rearing Oregon spotted frog larvae at the Oregon Zoo during spring and summer of 2000.

COLLECTION OF SPECIMENS

Frog eggs were collected from one of the two largest known populations of Oregon spotted frogs, found at Conboy Lake National Wildlife Refuge, Klickitat Co., Washington. During the 1998-1999 breeding seasons, over 13,000 egg masses were monitored for ongoing research on oviposition patterns and survivorship
(M. P. Hayes, pers. comm.). Oregon spotted frogs lay masses containing 600 to 1,500 eggs. We collected 110 late-stage embryos, with 10 eggs taken from each of 11 egg masses. This minimized the impact on
the contribution to recruitment for any female from which eggs were taken. This also insured that the cohort of frogs reared from these eggs represents a range of genotypes. Late-stage Oregon spotted frog embryos were collected on 13 March 2000 by Mr. Joe Engler, Conboy Lake National Wildlife Refuge. Dr. Marc P. Hayes transported eggs to experimental facilities on zoo grounds in Portland, Oregon, on 14 March 2000.

EXPERIMENTAL FACILITIES

Aquaria for this project have been assembled in the off-exhibit areas of the Oregon Zoo's Cascades Stream exhibit. Five separate aquarium systems, including three 35 gallon culture troughs, one 120 gallon culture trough, and one round 135 gallon circular tank, were maintained for this research. Each system was equipped with a heating/cooling element and water filter. The water temperature in each aquarium system was regulated at19C +2C. Each tank was monitored for ammonia levels and pH. During the course of the research ammonia levels were generally too low to be detected and reached a maximum of 0.02 mg/L. Water in all tanks remained slightly alkaline, with pH levels varying between 7.2-8.2, with most samples registering a pH of 7.5.

EXPERIMENTAL DIETS

Diet and husbandry techniques were adapted from successful husbandry protocols developed for Oregon spotted frogs by Mr. Frank Slavens, Woodland Park Zoo, Seattle, Washington. Four different dietary regimens were investigated, one in each 35 gallon culture trough (Tanks 1-3) and one in the 135 gallon tank (Tank 4). The 120 gallon culture trough was reserved to house frogs once the larvae transformed. Thirty eggs were placed in each 35 gallon culture trough. Twenty eggs were put into the 135 gallon circular tank. Each of the 10-egg samples from 11 egg masses was divided into two sub-samples of 5 eggs for a total of 22 sub-samples. Six unrelated sub-samples (30 eggs) were allocated to each 35 gallon tank and four unrelated sub-samples (20 eggs) were placed in the 135 gallon tank.

Diet 1 (Tank 1). Algae on Suspended Plastic Strips (Algae Diet). Oregon spotted frog tadpoles are grazers (Licht 1974, McAllister and Leonard 1997). They scrape plant tissue, algae, bacteria, and other detritus off of plant surfaces and, as with selected other larval western ranids studied, the grazing of diatoms growing on these surfaces may be favored (Kupferberg 1996). Oregon spotted frog tadpoles have been successfully raised to metamorphosis by Mr. Frank Slavens, Woodland Park Zoo, on a diet of algae grown on the surface of leaves and other plant debris in an outdoor tank. At Oregon Zoo, we established a 220 gallon outdoor tank for production of algae and associated organisms on plastic 3"x9"x1/16 plastic strips suspended in the upper water column. Subsets of these strips were suspended in one 35-gallon trough on 24-hour rotating basis to supply a diet of natural algae.

Diet 2 (Tank 2). Algae Encrusted Leaves (Leaf Diet). Leaves of trees and shrubs native to the Pacific Northwest (black cottonwood, red alder, Oregon ash) were placed in 30 two gallon shallow trays approximately one month prior to arrival of larvae. Each tray was inoculated with leafy debris collected at Conboy Lake NWR and placed outdoors in a sunny portion of zoo grounds not accessible to the public. Leaf-litter with encrusted algal growth was offered to larvae hatched in a second 35 gallon culture trough. A fresh supply of algae-encrusted leaves was offered daily.

Diet 3 (Tank 3). Cooked Spinach/Bloodworm Cubes (Spinach Diet). The third diet was developed by Mr. Mike Demlong, formerly Curator of Ectotherms at the Phoenix Zoo, for rearing Ramsey Canyon leopard frog larvae (Rana subaquavocalis). The diet consists of spinach leaves supplemented with a protein source. Organic spinach leaves were blended and frozen into cubes and thawed as needed prior to feeding. In this case, frozen bloodworm cubes, a common protein supplement in zoo animal husbandry, were the protein source of choice. This diet differs from the first two diets in that it is artificial. However, it also repeatable and reduces the possibility of introducing diseases or parasites found in natural ecosystems to the experimental environment.

Diet 4 (Tank 4). Mixed Vegetable/Mixed Protein Supplement (Mixed Diet). The fourth diet, fed to 20 larvae in the 135 gallon tank, was intended to provide tadpoles with a variety of vegetable materials and protein supplements. This diet explored the potential of ad libitum mixed artificial food resources to substitute for the natural diet of this species. Frozen mixed vegetable diet cubes were prepared by combining 150 g blended kale, 300 g blended yellow squash, and 300 g blended zucchini squash with 600 ml water. Organically grown vegetables were used in every case. Cubes were thawed as needed prior to feeding. A protein supplement of frozen bloodworm cubes or boiled egg white was offered, changing on alternate days. These foods have been used successfully to raise Rana subaquavocalis larvae at the Phoenix Zoo and represent a less intensive alternative to raising natural foods for Oregon spotted frog larvae. The increased volume of water per larva may also affect larval growth and survival. This variable will not be investigated in 2000, but may be the subject of future research.

RESULTS

At 19C, Oregon spotted frog eggs developed rapidly. By 26 March, 12 days after arriving at the Oregon Zoo, 95 of 110 eggs had hatched. Three eggs did not hatch and another 12 larvae died within two days of hatching, reducing the total number of tadpoles to 95. Larvae in all diet trials began feeding by the third day following hatching. Larval growth, expressed as total length in millimeters, for each diet is displayed in Figure 1. Larvae feeding on algae-covered strips grew slower than those on other diets, but all other diets displayed similar growth for the first 35 days after hatching. Once larvae reached approximately 25 mm in length, their growth accelerated on all diets. The two samples fed artificial diets grew faster than the larvae feeding on algae and other natural foods.

The number of larvae remaining in each diet trial is depicted in Figues 2-5. These figures also plot the appearance and survivorship of metamorphosed frogs. The number of larvae declines with time due to mortality or metamorphosis. In most cases there was relatively low larval mortality beyond the first few days after hatching. The one exception is the sharp drop in larval numbers feeding on the leaf surface diet between 16 May and 30 May. Five slow-growing larvae that displayed reduced vigor were lost when they were trapped on the tank's outflow screen on 18 May. On 27 May, a failure on the outflow screen fastener resulted in the loss of 10 larvae when they were drawn into the recirculating filter system. Table 1 charts the onset and completion of metamorphosis for each diet trial. The duration of metamorphosis was much shorter for larvae fed artificial diets. The group fed on algae covered plastic strips not only had a protracted period of metamorphosis, but onset of metamorphosis was much later than other diet groups.

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Table 1. Time and Duration of Metamorphosis by Diet

3/15 3/31 4/15 4/30 5/15 5/31 6/15 6/30 7/15 7/31

Algae Diet XXXXXXXXXX

Leaf Diet XXXXXXXXXX

Spinach Diet XXXXX

Mixed Diet XXXXXX

________________________________________________________________________

The survivorship of larvae in different diet groups also differed (Table 2), although results for the group fed algae-covered leaves are not comparable to other groups because of mortality from the equipment failure described above. The percentage of successful metamorphosis is nearly identical for both artificial diets and substantially higher than either larval group offered natural algae and associated microflora and microfauna.

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Table 2. Total Metamorphosing and Surviving by Diet

Individuals Total Length at Total Percent

at Start Metamorphs Metamorphosis Surviving Surviving

to 8/31/00

Algae Diet 30 20 1/2" 7 23%

Leaf Diet 30 4 1/2" 0 0%

Spinach Diet 30 22 3/4" 19 63%

Mixed Diet 20 16 3/4" 12 60%

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DISCUSSION

Diet 1 (Algae Diet). Based on slow growth rates, delayed initiation of metamorphosis, and small size at metamorphosis, we believe that larave feeding on algae-covered plastic strips were food-limited. Algae growth did not keep up with consumption as larvae matured. Twenty of 30 larvae on this diet completed metamorphosis, but survival of metamorphs was below 50%. We do not recommend this method of growing food for Oregon spotted frog larvae.

Diet 2 (Leaf Diet). Larvae fed on algae and other organisms growing on leaf litter initiated metamorphosis at approximately the same time as those fed artificial diets. The size of metamorphs (1/2" total length) was smaller than individuals from either artificial diet (3/4" total length). Due to accidental mortality, we are unable to assess the ultimate survivorship of frogs fed this larval diet. This diet introduced practical difficulties to obtaining accurate daily counts of larvae because larvae would take refuge among leaves of irregular color, shape, and size. Although we did not record any simultaneous mortality that could have been attributed to disease, we also had no control over which species of plants or animals present in the outdoor environment colonized the leaf litter culture trays. The possibility of introducing pathogens in amphibian reintroduction programs represents a major risk factor to wild amphibian populations and, given acceptable artificial diets as an alternative, we would not recommend using cultured leaf litter to feed larval Oregon spotted frogs intended to be released into the wild.

Diet 3 (Spinach Diet). Larvae fed a blended spinach diet with a protein supplement grew rapidly, underwent metamorphosis rapidly, and had the highest survivorship as frogs. There were some adverse consequences of this diet, including formation of gas bubbles in the digestive tract, which caused the larvae to float to the surface following feeding. There were no apparent long-lasting consequences of this condition. Tail deformities were also noticed in spinach-fed larvae, although no residual effects were evident in metamorphs. Although metamorphs from this diet trial appear vigorous and have grown rapidly (some approximately 3" long as of 31 August 2000), tail deformities and gas accumulation displayed by spinach-fed tadpoles suggest that this would not be the diet of choice for raising Oregon spotted frog larvae in the future.

Diet 4 (Mixed Diet). Larvae fed a mixed vegetable diet (with bloodworm or egg white protein supplement) grew nearly as fast as those fed spinach and displayed fewer tail deformities prior to metamorphosis. Survivorship of metamorphs was similar to spinach-fed larvae, as was overall survivorship from the egg stage. Larvae fed this diet did not present any symptoms of gas accumulation in the digestive tract. Given the survival rate, ease of diet preparation and feeding, and reduced chance of exposure to pathogens, we conclude that a mixed vegetable diet, such as the formula used in this trial, is preferable for rearing Oregon spotted frog larvae in captivity.

Following metamorphosis, young frogs were provided a moss-covered floating platform on which to feed. Frogs also took refuge in the moss matrix, increasing the difficulty of making accurate daily counts of individuals. As a result, data sheets reflected the number of frogs observed out of a possible total, rather than the absolute number of frogs present. Metamorphs were fed small crickets raised on a calcium-rich diet. Growth in metamorphs raised on spinach or mixed-vegetable diet was rapid. Within 6-8 weeks of metamorphosis, surviving frogs reached two to three inches in snout-vent length.

PLANS FOR CONTINUING RESEARCH

We suspect that maintaining water temperature continuously at 19C contributed to the rapid growth of larvae fed either spinach or mixed vegetable diets. While we have not observed any adverse consequences of this rapid development, we would like to raise larvae and metamorphs at lower temperatures, and study the relationship between growth and survivorship at different temperatures when the diet is standardized. While surface water temperatures in the field may exceed 19C during parts of the day, temperatures are usually lower at night. When eggs were collected on 13 March 2000, the daytime water temperature at Conboy Lake NWR was 12C. We propose raising Oregon spotted frog larvae at 12C, 14C, 16C, and 18C to explore additional husbandry options that might result in survivorship above the 60% rate we experienced in 2000.

DISPOSITION OF SPEICMENS

All surviving larvae were maintained at the Oregon Zoo through metamorphosis. The Oregon Zoo will raise 10 frogs to adulthood, with the possibility of maintaining a small breeding population. Interpretive materials, as specified in our Cooperative Agreement with USFWS, will be developed in cooperation with USFWS. Frank Slavens, Woodland Park Zoo, has expressed interest in placing some adult Oregon spotted frogs at Woodland Park or other zoos for exhibit and environmental education about declining amphibian populations in the Pacific Northwest.

LITERATURE CITED

Blaustein, A. R., and D. B. Wake. 1990. Declining amphibian populations: A global phenomenon. Trends in Ecology and Evolution 5:203-204.

Csuti, B., A. J. Kimerling, T. A. O'Neil, M. M. Shaughnessy, E. P. Gaines, and M. M. P. Huso. 1997. Atlas of Oregon Wildlife. Oregon State University Press, Corvallis.

Green, D. M., H. Kaiser, T. F. Sharbel, J. Kearsley, and K. R. McAllister. 1997. Cryptic species of spotted frogs, Rana pretiosa complex, in western North America. Copeia 1997:1-8.

Green, D. M., T. F. Sharbel, J. Kearsley, and H. Kaiser. 1996. Postglacial range fluctuation, genetic subdivision and speciation in the western North American spotted frog complex, Rana pretiosa. Evolution 50:374-390.

Hall, S., T. Bloxton, E. Goldstein, A. Jennings, S. Jensen, K. Kaczorowski, J. Klavitter, and J. Luginbuhl. 1999. Draft Oregon Spotted Frog Conservation Plan. Unpublished report, College of Forest Resources, University of Washington, Seattle, March 1, 1999.

Hayes, M. P. 1994. The spotted frog in western Oregon. Technical Report #94-1-01, Wildlife Diversity Program, Oregon Department of Fish and Wildlife, Portland.

Hayes, M.P. 1997. Status of the Oregon spotted frog (Rana pretiosa sensu stricto) in the Deschutes Basin and selected other systems in Oregon and northeastern California with a rangewide synopsis of the species' status. Final report prepared for The Nature Conservancy under contract to the US Fish and Wildlife Service.

Jennings, M. R., and M. P. Hayes. 1994. Amphibian and reptile species of special concern in California. California Department of Fish and Game, Sacramento. 255 pp.

Kiesecker, J. M., and A. R. Blaustein. 1998. Effects of introduced bullfrogs and smallmouth bass on microhabitat use, growth, and survival of native red-legged frogs (Rana aurora). Conservation Biology 12:776-787.

Kupferberg, S. J. 1996. The ecology of native tadpoles (Rana boylii and Hyla regilla) and the impact of invading bullfrogs (Rana catesbeiana) in a northern California river. Unpublished Ph.D. Dissertation, University of California, Berkeley.

Lawler, S. P., D. Dritz, T. Strange, and M. Holyoak. 1999. Effects of introduced mosquitofish and bullfrogs on the threatened California red-legged frog. Conservation Biology 13:613-622.

Leonard, W. P., H. A. Brown, L. L. C. Jones, K. R. McAllister, and R. M. Storm. 1993. Amphibians of Washington and Oregon. Seattle Audubon Society, Seattle, Washington.

Licht, L. E. 1974. Survival of embryos, tadpoles, and adults of the frogs Rana aurora aurora and Rana pretiosa pretiosa sympatric in southwestern British Columbia. Canadian Journal of Zoology 52:613-627.

Marshall, D. B., M. W. Chilcote, and H. Weeks. 1996. Species at risk: sensitive, threatened and endangered vertebrates of Oregon, 2nd edition. Oregon Department of Fish and Wildlife, Portland, Oregon.

McAllister, K. R., and W. P. Leonard. 1997. Washington State status report for the Oregon Spotted Frog. Washington Department of Fish and Wildlife., Olympia. 38pp.

Phillips, K. 1990. Where have all the frogs and toads gone? BioScience 40:422-424.