PREVALENCE OF SKELETAL AND EYE MALFORMATIONS IN FROGS FROM NORTH-CENTRAL UNITED STATES: ESTIMATIONS...

510

Journal of Wildlife Diseases, 39(3), 2003, pp. 510–521q Wildlife Disease Association 2003

PREVALENCE OF SKELETAL AND EYE MALFORMATIONS INFROGS FROM NORTH-CENTRAL UNITED STATES: ESTIMATIONSBASED ON COLLECTIONS FROM RANDOMLY SELECTED SITES

Patrick K. Schoff,1,4 Catherine M. Johnson,1 Anna M. Schotthoefer,2 Joseph E. Murphy,2

Camilla Lieske,2 Rebecca A. Cole,3 Lucinda B. Johnson,1 and Val R. Beasley2

1 Center for Water and the Environment, Natural Resources Research Institute, University of Minnesota—Duluth,5013 Miller Trunk Hwy, Duluth, Minnesota 55811-1442, USA2 Departments of Veterinary Biosciences and Veterinary Pathobiology, University of Illinois at Urbana–Champaign,2001 South Lincoln Ave., Urbana, Illinois 61802, USA3 U.S. Geological Survey, National Wildlife Health Center, 6006 Schroeder Rd., Madison, Wisconsin 53711, USA4 Corresponding author (email: [email protected])

ABSTRACT: Skeletal malformation rates for several frog species were determined in a set ofrandomly selected wetlands in the north-central USA over three consecutive years. In 1998, 62sites yielded 389 metamorphic frogs, nine (2.3%) of which had skeletal or eye malformations. Asubset of the original sites was surveyed in the following 2 yr. In 1999, 1,085 metamorphic frogswere collected from 36 sites and 17 (1.6%) had skeletal or eye malformations, while in 2000,examination of 1,131 metamorphs yielded 16 (1.4%) with skeletal or eye malformations. Hindlimbmalformations predominated in all three years, but other abnormalities, involving forelimb, eye,and pelvis were also found. Northern leopard frogs (Rana pipiens) constituted the majority ofcollected metamorphs as well as most of the malformed specimens. However, malformations werealso noted in mink frogs (R. septentrionalis), wood frogs (R. sylvatica), and gray tree frogs (Hylaspp.). The malformed specimens were found in clustered sites in all three years but the clusterlocations were not the same in any year. The malformation rates reported here are higher thanthe 0.3% rate determined for metamorphic frogs collected from similar sites in Minnesota in the1960s, and thus, appear to represent an elevation of an earlier baseline malformation rate.

Key words: Amphibian, anuran, broadleaf forest ecoregion, malformation, survey.

INTRODUCTION

Over the past several years, a number ofmalformed frogs and toads have been dis-covered in the USA and Canada; malfor-mations have been reported in specimensof 54 amphibian species in 44 USA statesand four Canadian provinces since 1995(Johnson et al., 2000; and see also NorthAmerican Reporting Center for Amphibi-an Malformations, 2002). Normal amphib-ian populations, like those of other verte-brates (Gilbert, 2000), appear to accom-modate a low number of malformed indi-viduals. Reports in the literature supportthe concept of a baseline malformationrate, which has been documented in sev-eral large-scale population studies (Mer-rell, 1969; Meyer-Rochow and Asashima,1988; Gray, 2000), as well as in numerousreports detailing spectacular anomaliessuch as ectopic or supernumerary legs (re-viewed by Van Valen, 1974 and Ouellet,2000). Occasionally, acute malformation

outbreaks occur in which an isolated pop-ulation has a very high malformation rate(Reynolds and Stephens, 1984; Volpe andRosenbaum, 2000). These usually involvea single species and last for just one gen-eration (Van Valen, 1974; Ouellet, 2000).Many of the current reports fall within theacute category. However, in contrast tohistorical reports, the current observationsare characterized by malformed animalsamong multiple species at a single site,multiple sites with high malformationprevalence spread over wide areas, and re-currence of malformed animals in thesame site in successive years (Ouellet etal., 1997; Gardiner and Hoppe, 1999;Johnson et al., 1999; Converse et al., 2000;Helgen et al., 2000; Hoppe, 2000). Mal-formation rates at some sites can be veryhigh. Johnson et al. (1999) reported an av-erage of 12.3% malformed Hyla regillafrom 13 sites and Helgen et al. (2000)sampled several sites that yielded 40–65%malformed Rana pipiens, while matching

SCHOFF ET AL.—PREVALENCE OF MALFORMED FROGS 511

reference sites yielded no greater than 1%malformed specimens. Ouellet et al.(1997) matched 14 agricultural sites, inwhich an average of 12% of the frogs hadmalformations, with 12 non-agriculturalsites, in which they found only 0.7% mal-formed specimens. Unfortunately, little isknown about the causes of the malforma-tions or whether acute malformation out-breaks represent transient elevations ofthe baseline rate or whether the two havedifferent etiologies.

While malformation rates at a single sitemight be estimated using a well designedsampling study, valid baseline malforma-tion rates for populations on local or re-gional scales can be difficult to glean fromstudies not specifically designed for thatpurpose. For instance, abnormalities gen-erated through injury or disease must bedistinguished from true developmental er-rors. Also, since relatively few malformedadults are seen (Helgen et al., 2000), stud-ies that do not distinguish between adultsand metamorphs may underestimate thetrue malformation rate. Despite these lim-itations, some information on historic mal-formation rates is available. By close ex-amination of preserved specimens, Hoppe(2000) determined a 0.3% malformationrate for 2,166 northern leopard frog (Ranapipiens) metamorphs collected fromknown sites in central Minnesota, USA inthe 1960s. Malformations in this group in-cluded ectrodactyly (missing digits), syn-dactyly (fused digits), polydactyly (multipledigits), and ectromely (missing limbs orlimb segments) all of which are commonin the current phenomenon. The study re-ported here addresses the issue of the cur-rent background malformation rates usingrandom site selection and a systematic col-lection protocol in wetlands in the north-central region of the US.

MATERIALS AND METHODS

Study area and site selection

The study area is encompassed by the broad-leaf forest ecoregion (Bailey, 1983) which spansnortheastern Illinois, southern Wisconsin, and

southern Minnesota (USA; Fig. 1). Currentland cover within this region ranges from forestto intensive agricultural or urban uses. Site se-lection was conducted using a stratified randomdesign. We randomly selected fourth order wa-tersheds from the broadleaf forest ecoregion.Wetlands within the selected watersheds weremapped using National Wetland Inventorydata. To capture the available gradient of con-ditions available within each watershed, we se-lected individual wetlands from regions repre-senting a spectrum of land use intensity (e.g.,less disturbed to most disturbed). Field recon-naissance was conducted to select four to eightwetlands in each watershed. Individual samplesites were also subject to the following criteria:1) classification as a palustrine aquatic bed oremergent wetland (Cowardin et al., 1979), 2)landowner permission, and 3) evidence that thesite would remain under a consistent manage-ment regime for the duration of the study. Werefer to groups of sites within watersheds aswetland clusters (WC), which are identified bywetland cluster and site numbers (e.g., WC 1,site 151.1; see Fig. 1 and Table 1). A total of62 sites in 13 wetland clusters were selectedand sampled in 1998. In 1999 and 2000 theobjectives of the larger project that encom-passed this malformation assessment requiredthe selection of 36 sites within a smaller region.This set was constructed using 18 of the exist-ing sites and 18 additional sites, 10 of whichwere associated with the existing wetland clus-ters (WC 9–13), and eight of which were ad-ditional sites adjacent to, but not contiguouswith the original watersheds. This set of eightadditional sites was designated as WC 14.

Field methods

Each site was visited three times during thespring and summer (March–April, May–June,and late July–August) to gather physical, chem-ical, and biological data. During the July–Au-gust visit, attempts were made to capture meta-morphosing frogs at all sites. A minimum effortof two person-hr was expended searching forfrogs at each site that was not completely dry.If no frogs were captured during that time, thesite was abandoned. At each site where frogswere found, an effort of approximately 16 per-son-hr was expended for collection and analy-sis. During 1998, the animals were measuredand examined in the field, and representativespecimens, including malformed individualsand randomly selected reference specimens,were collected for laboratory analyses. Frogswere euthanized by immersion in 1% MS 222(3-amino benzoic acid ethyl ester) and storedin 10% neutral buffered formalin. Only meta-

512 JOURNAL OF WILDLIFE DISEASES, VOL. 39, NO. 3, JULY 2003

FIGURE 1. Amphibian malformation study area. The study area included 78 wetland sites contained in 14spatial clusters distributed through southern Minnesota, Wisconsin, and northern Illinois.

morphic animals with emerged forelimbs (Gos-ner stage 42–46 and beyond; Gosner, 1960)were included in the malformation survey andcollection. Adults were not included because ofthe possibility that they could have migratedfrom other wetlands.

During the 1999 and 2000 collections, thefield researchers endeavored to capture 100metamorphic frogs; the first 40 frogs collectedfrom each site were distributed between the Na-tional Wildlife Health Center (Madison, Wis-consin, USA) and the University of Illinois Col-

lege of Veterinary Medicine (Urbana, Illinois,USA), for malformation assessment, as well asgross and histologic pathology and parasitologystudies (to be reported elsewhere). Any addi-tional frogs captured were examined in the field.Malformed frogs were kept for laboratory anal-ysis and the remaining specimens were released.

Staining

Specimens were fixed in neutral bufferedformalin or 70% ethanol. Cartilage and bone


were stained with alcian blue and alizarin red(Kelly and Bryden, 1983) with enzymatic tissueclearing (Dingerkus and Uhler, 1977). Clearedspecimens were stored in glycerin.

RESULTS

1998 collections

A total of 389 metamorphic anuransfrom seven species were examined in thefield (Table 1). The majority were R. pi-piens and R. clamitans, with R. septen-trionalis, R. sylvatica, R. catesbeiana, Hylaspp. (the gray tree frog, H. versicolor, andCope’s gray tree frog, H. chrysoscelis aredistinguishable in the field only by theircall), and Bufo americanus represented inmuch lower numbers (Table 2). Fifty-fourfrogs, including malformed and referencespecimens, were euthanized and returnedto the lab for skeletal analysis. After clear-ing and staining, nine (2.3%) frogs wereidentified with malformations (Table 2),which included anophthalmia, ectromely,ectrodactyly, taumely (a bony triangle, inwhich a long bone, such as the femur ortibiafibula, is bent in the middle, such thatthe heads of the bone are apposed), po-lymely, and polydactyly (Table 2). Twofrogs that were considered normal in thefield (i.e., reference specimens) werefound by bone staining to have asymmetricpelvises, in which the left ilial shaft waslonger than the right, resulting in torsionof the lower trunk. Both of these animalscame from the same wetland cluster (WC4; Table 1), although not from the samesite. This subtle malformation was notseen in cleared and necropsied specimensin the 1999 or 2000 collections (Tables 3and 4).

1998 distribution

Metamorphic frogs were collected in 45of the 62 sites sampled in 1998 (Table 1).Six sites contained only adult or larvalforms and 11 sites yielded no frogs duringthe collection period. The nine malformedspecimens were distributed among sevensites, with six sites having one malformedspecimen and one site having three mal-

formed specimens. Four of the nine mal-formed specimens came from one wetlandcluster (WC 11), and another wetlandcluster contained two malformed speci-mens (WC 4; Table 1).

1999 collections

Metamorphic frogs were collected from30 of the 36 sites sampled in 1999 (Fig. 1,Table 1). A total of 1,085 metamorphic ra-nid frogs were examined in the field or labfor skeletal malformations (Table 3). Ofthese, 267 were examined only in the field,306 were cleared and stained, and 512were subjected to necropsy, which in-volved histopathologic examination of softtissue (to be reported elsewhere). Skeletalmalformations were found in 17 (1.6%)frogs, including abnormalities in the limbs,mandible, pelvis, and spine (Table 3).Bone staining revealed several instances ofunique or rarely reported skeletal malfor-mations. One R. pipiens had a severelymalformed coccyx, which appeared to bein several separate pieces at the articula-tion with the spine. Several (13) other R.pipiens had less severe variations in nor-mal coccygeal form and position, but,since these did not appear to produce tor-sion or mispositioning of the pelvis or legs,they were considered to be variations innormal growth and not malformations.Several specimens also had asymmetricvertebrae and ribs, which, in one case, wassevere enough to produce scoliosis.

1999 distribution

Malformed frogs were found at 11 sites,with rates ranging from 0.8–8.8% by site(Table 1). Seven sites produced a singlemalformed specimen; two sites producedtwo malformed specimens; and two sitesproduced three malformed specimens.Malformed frogs tended to be grouped inparticular sites and wetland clusters. Spe-cifically, sites 64–68, which are all withinWC 13, produced 53% of the specimenswith skeletal malformations. Only one oth-er site (site 44, WC 10) yielded multiplemalformed specimens. These two geo-


TABLE 1. Amphibian metamorph collections and prevalence of malformations by site and year; 1998–2000.

Sitea

number Identification

1998

Prevb (Malfc/totald)

1999


2000


1234

1.11.21.31.4

00——

(0/2)(0/3)(0*)(0*)

————

————

————

————

5678

1.52.12.22.3

0—0—

(0/8)(0*)(0/2)(0*)

————

————

————

————

910111213

2.43.13.33.43.5

0—000

(0/1)(0**)(0/3)(0/1)(0/4)

—————

—————

—————

—————

141516171819

4.14.24.34.44.54.6

250

13000

(1/4)(0/1)(1/8)(0/6)(0/27)(0/16)

——————

——————

——————

——————

2021222324

4.74.85.15.25.3

00

170—

(0/12)(0/16)(1/6)(0/39)(0*)

—————

—————

—————

—————

252627282930

5.46.16.26.36.47.1

0—00—0

(0/14)(0*)(0/15)(0/7)(0**)(0/4)

——————

——————

——————

——————

3132333435

7.27.37.48.18.2

——009

(0*)(0*)(0/2)(0/1)(1/11)

—————

—————

—————

—————

363738394041

8.38.48.59.19.29.3

00——00

(0/8)(0/1)(0**)(0*)(0/5)(0/5)

————0—

———

(0*)(0/5)—

————1.7—

———

(0*)(2/117)

—4243444546

9.49.5

10.110.210.3

—0—00

(0*)(0/6)(0**)(0/3)(0/6)

046—0

(0/9)(1/23)(3/47)

—(0/8)

—00——

(0*)(0/7)(0/104)

—(0*)

474849505152

10.411.111.211.311.411.5

0—

250

270

(0/13)(0*)(1/4)(0/1)(3/11)(0/16)

0—0—0—

(0/4)—

(0/1)(0*)(0/13)(0*)

0—00—0

(0/1)—

(0/10)(0/2)(0*)(0/33)


TABLE 1. Continued.

Sitea

number Identification

1998


1999


2000


5354555657

11.611.711.811.912.2

—0——0

(0**)(0/16)

——

(0/11)

—000—

(0*)(0/2)(0/9)(0/41)

—

—0—2—

(0*)(0/4)(0*)(2/92)

—585960616263

12.312.412.512.612.712.8

0—————

(0/27)(0**)

————

00000—

(0/51)(0/1)(0/54)(0/48)(0/45)(0*)

0—1.83.30—

(0/142)(0*(2/113)(7/210)(0/39)(0*)

6465666768

13.113.213.313.413.5

00005

(0/1)(0/6)(0/4)(0/12)(1/20)

721.70.89

(1/14)(2/86)(2/118)(1/123)(3/34)

10000—2

(1/1)(0/1)(0/1)(0*)(1/42)

697971727374

13.613.713.814.114.214.4

——————

——————

—00202

(0*)(0/2)(0/29)(1/65)(0/18)(1/42)

——000—

(0*)(0*)(0/2)(0/1)(0/14)(0*)

75767778

14.514.614.714.8

————

————

2100

(1/47)(1/86)(0/48)(0/12)

0.9000

(1/111)(0/15)(0/60)(0/9)

Totals 2.3 (9/389) 1.6 (17/1,085) 1.4 (16/1,131)

a Site identification refers to wetland cluster (WC 1–14), followed by the individual site within that cluster. A total of 78 sitesin 14 wetland clusters were sampled during the 3 yr of the study. 1998: 62 sites (1.1–13.5); 1999 and 2000: 36 sites (9.1–14.8).

b Prevalence; percent of collected individuals with skeletal malformations.c Number of malformed metamorphic frogs collected/total number collected; 0* 5 collection attempted but no frogs captured;

0** 5 only larvae and adults collected; — 5 no collection attempted.d Skeletal and eye malformations only.

graphically separated clusters accountedfor 70% of the malformed frogs.

2000 collections

The 36 sites that were sampled in 1999were sampled again in 2000. A total of1,131 metamorphic frogs were collectedfrom 24 sites (Table 1). Of these, 478 wereexamined for malformations in the fieldand returned and 653 were subjected tonecropsy. Sixteen (1.4%) were determinedto be malformed. As observed in previousyears, the majority of malformations oc-curred in the hindlimbs (Table 4), how-ever, five specimens with eye malforma-tions were also collected.

2000 distribution

Seven of the 24 sites that yielded frogshad at least one malformed frog (Table 1).As observed in previous years, the malfor-mations were clustered, with two sites (60and 61, WC 12) contributing nine of the16 malformed specimens.

Annual variation

Frog captures were highly variableamong sites within the same year. Of the62 sites sampled in 1998, 11 yielded nofrogs and 35 yielded fewer than 10 meta-morphic frogs during the collection peri-od. In 1999, 15 of the 36 sites yielded zeroto nine metamorphic frogs, and in 2000,


TABLE 2. Summary of malformations in anurans collected in 1998.

Species na Malfb Prevc (%) Malformation typed and (number)e

Rana pipiens 232 6 2.6 pelvic asymmetry (2), polydactyly (2), anophthalmia (1)ectromely (1), polymely (1), taumely (1)

R. clamitans 95 1 1 brachydactyly and brachymelyR. septentrionalisR. sylvaticaR. catesbeiana

2210

1

000

000

Hyla spp. 13 2 10 bilateral amely (1), brachydactyly and brachymely (1)Bufo americanusunknown

412

00

00

Total 389 9 2.3

a Number of metamorphic frogs collected.b Skeletal and eye malformations.c Prevalence; percentage of specimens malformed in sample.d All limb malformations occurred in the hindlimbs, unless otherwise noted; pelvic asymmetry-ilial shaft longer on one side;

polydactyly-multiple digit; anophthalmia-missing eye; ectromely-missing limb or segment; polymely-multiple limbs; taumely-bony triangle, where long bones are bent such that the proximal and distal ends are apposed and the middle forms the apexof a triangle; brachydactyly-reduced metatarsal or phalanges; brachymely-reduced limb segment; amely-absence of hindlimband acetabulum.

e Some specimens had multiple anomalies.


Species na Malfb Prevc (%) Malformation typed and (number)e

Rana pipiens 931 13 1.4 brachydactyly (3), forelimb brachydactyly (1), brach-ygnathia (2), brachymely (1), craniofacial (1), ectro-dactyly (1), ectromely (1), polymely (1) pelvic (1),polydactyly (1)

R. septentrionalis 119 3 2.5 brachydactyly (1), ectromely (1), spinal (1)R. clamitans 23 1 4 brachymely with cutaneous fusionR. sylvatica 12 0 0Total 1,085 17 1.6

a Number of metamorphic frogs collected.b Skeletal and eye malformations.c Prevalence; percentage of specimens malformed in sample.d All limb malformations occurred in the hindlimbs, unless otherwise noted; brachydactyly-reduced metatarsal or phalanges;

brachygnathia-reduced mandible; brachymely-shortened limb; craniofacial-anomalous zygomatic arch; ectrodactyly-missingdigit, including metatarsal; ectromely-missing limb or segment; polymely-multiple limbs; pelvic anomalous pelvic develop-ment; polydactyly-multiple toes; spinal-abnormality causing scoliosis; cutaneous fusion-skin joined over two limb segments.


22 of 36 sites yielded zero to nine frogs(Table 1). Also, there appeared to be littleconsistency in the number of metamorphicfrogs captured in the same site from yearto year, even though the collecting teamwas similar in all years. Although somesites produced relatively high numbers offrogs in 1999 and 2000 (e.g., WC 12), mostothers showed no pattern. In addition, thedistribution of malformed frogs was notconsistent in the three collection years. Ofthe 36 sites that were surveyed in both

1999 and 2000, 15 contained malformedspecimens (Table 1). However, only threesites contained malformed frogs in bothyears, and of these, only one had multiplemalformed frogs in a single year (site 13.5,1999). Twenty sites were sampled in all 3yr. Two of these sites yielded no frogs andthree sites yielded fewer than 10 totalfrogs across all 3 yr. Even though the 22malformations from these sites represent-ed over half of the total malformations ob-served during the 3 yr, only one site (13.5)



Species na Malfb Prevc (%) Typed and (number)e

Rana pipiens 1,107 15 1.4 anophthalmia (5), brachymely (2), ectromely (2), fore-limb ectromely (1), bony projection (1), brachydac-tyly (1), ectrodactyly (1), lateral asymmetry (1), pel-vic (1), polymely (1)

R. clamitans 21 0 0R. septentrionalis 3 1 33 brachydactyly (1)Total 1,131 16 1.4

a Number of metamorphic frogs collected.b Skeletal and eye malformations.c Prevalence; percentage of specimens malformed in sample.d All limb malformations occurred in the hindlimbs, unless otherwise noted; anophthalmia-missing eye; brachymely-reduced

limb; ectromely-missing limb or segment; bony projection-anomalous appendage, in this case originating in the pelvic area;brachydactyly-reduced metatarsal or phalanges; ectrodactyly-missing digit, including metatarsal; lateral asymmetry-one halfof body smaller than the other; pelvic-anomalous pelvic development; polymely, multiple limbs.


TABLE 5. Summary of severe malformations foundin anurans collected in 1998–2000.

YearNumberof frogs

Malformations

Totalmalfa

Prev(%)b Severec

Prevb

(%)

199819992000

3891,0851,131

91716

2.31.61.1

61113

1.51.01.1

Total 2,605 42 1.6 30 1.2

a Skeletal and eye malformations.b Prevalence; percentage of specimens malformed in sample.c Severity was based on an estimation of the effect of the

malformation on fitness.

produced malformed specimens in each ofthe 3 yr.

Severity

Malformations were categorized as ‘‘se-vere’’ (Table 5) if they were estimated tocause a major reduction in fitness. On thisbasis, minor skeletal malformations, suchas missing or reduced phalanges, were notconsidered severe. Similarly, abnormal col-oration noted on the hindlimb skin wouldprobably not impair fitness, although it isrecognized that skin color pattern could,under certain circumstances, be relevantto mate selection and susceptibility to pre-dation.

DISCUSSION

Comparisons of current and historicalmalformation rates

Gray (2000) reported that in a set ofover 10,000 cricket frogs (Acris crepitans)collected in Illinois between 1968 and1971, 0.4% were abnormal. The majorityof these were missing limbs or limb seg-ments which could have resulted from ei-ther developmental malformations or trau-matic amputations. A survey of Illinoiscricket frogs in the early 1990s also iden-tified only one skeletal malformation in341 animals (Reeder et al., 1998). Thesestudies suggest that, either A. crepitans isresistant to etiologic agents causing mal-formations among other anurans or thatthe causative factor(s) were not widely dis-tributed during that time. Hoppe (2000)conducted a retrospective analysis of pre-served R. pipiens metamorphs collected incentral Minnesota between 1958 and 1963in which malformations were distinguishedfrom traumatic deformations on the basisof scar tissue formation. Six of 2,166 frogs(0.3%) had malformations, including hind-limb ectrodactyly, syndactyly, and ectro-mely, as well as three cases of forelimb ec-tromely, syndactyly, and polydactyly. No-tably absent from these studies were cu-taneous fusions and eye and jaw


malformations which were common mal-formations in more recent observations ofmetamorphic R. pipiens from the samearea (this study; Hoppe, 2000), as well asthe bony triangles and ectopic bonesfound in this and other recent studies(Gardiner and Hoppe, 1999). Thus, ifthese 1960s collections were unbiased, theavailable data suggest that baseline mal-formation rates for R. pipiens and A. cre-pitans in the north-central region of theUSA was 0.3 to 0.4% 30 yr ago and thatthere may be both qualitative and quanti-tative differences in the malformationtypes currently observed. Of 2,605 meta-morphic frogs collected over 3 yr in thecurrent study, 42 (1.6%) had skeletal oreye malformations, representing a four-tofive-fold increase over the historic back-ground prevalence reported for this re-gion.

Causes of malformation

Although no single cause has beenfound to explain all amphibian malforma-tions, three major hypotheses, each withlimited theoretical and empirical support,have emerged. These are: trematode par-asites, chemical mimics of retinoic acid,and ultraviolet-B radiation (UV-B). Ankleyet al. (1998, 2000) showed that R. pipiensembryos and larvae exposed to artificial ornatural UV-B developed extensive limbmalformations, consisting almost exclu-sively of hindlimb deletions or reductions.Militating against the UV hypothesis is thefact that only limb deletions and trunca-tions were produced which does not re-flect the current situation in the fieldwhere R. pipiens and other ranids arefound with a wide variety of malformationsin limb and other tissue.

A second hypothetical cause of malfor-mations is exposure to natural or anthro-pogenic chemicals that act as retinoidswhich are potent regulators of develop-ment in the vertebrate brain, eye, mandi-ble, and limbs. Although essential forproper development, retinoids can be ter-atogenic if applied at inappropriate times

or in abnormal concentrations. For exam-ple, retinoic acid produced duplicate limbswhen applied directly to developing froglimb buds (Bryant and Gardiner, 1992), orregenerating tails (Maden, 1993), and pro-duced deletions when applied to regener-ating axolotl limbs (Scadding and Maden,1986a). Few studies have been performedwith native frogs, but Degitz et al. (2000)established that retinoic acid caused hind-limb malformations in R. sylvatica thatwere similar to those seen in field speci-mens of R. septentrionalis (Gardiner andHoppe, 1999). Significantly, these malfor-mations included bony triangle malforma-tions of the long bones of the leg whichhave also been observed in retinoic acid-treated Xenopus (Scadding and Maden,1986b) and other vertebrates (Summer-bell, 1983; Tickle et al., 1985). AlthoughOuellet et al. (1997) observed a correlationbetween malformed amphibians, agricul-tural land use, and implied exposure tochemical pesticides, no direct evidence forthe presence of active retinoids has beenestablished in the current malformationphenomenon.

The remaining hypothesis is that trem-atode parasites cause malformations whenthey physically or chemically disrupt thedeveloping limb, jaw, or eye fields (Ses-sions et al., 1999). Experimental infectionsof Hyla regilla (Johnson et al., 1999) andBufo boreas (Johnson et al., 2001) withcercariae from the trematode parasite Ri-beiroia sp. produced multiple types ofhindlimb malformations, including missingor shortened limbs as well as multiple dig-its and limbs. In addition, these infectionscaused the bony triangle formation inhindlimbs that has been noted as a reti-noid effect (Gardiner and Hoppe, 1999)suggesting that the parasites may produceretinoids or stimulate retinoid-regulatedpathways. Malformation induction was de-pendent on the parasite species becauseinfection with Alaria mustelae cercariaedid not result in abnormal limb develop-ment (Johnson et al., 1999). These exper-imental infections produced only hindlimb


and forelimb malformations and not theother forms that have been consistentlyfound in wild populations. Also, eventhough Ribeiroia sp. has been found insome of our Minnesota study sites, noneof the malformed frogs identified in ourstudy harbored these metacercariae(Schotthoefer, data not shown). The ap-parent lack of Ribeiroia sp. in malformedindividuals indicates that either trematodeparasites other than Ribeiroia sp. cancause malformations, that Ribeiroia cer-cariae are cleared from the frogs, or thatother mechanisms are at work.

Malformation and declining amphibianpopulations

A recent analysis documented the de-cline in amphibian populations over thelast several decades and also showed thatthe phenomenon exists on a global scale(Houlahan et al., 2000). Despite the factthat malformations appear to occur atscales that would not contribute signifi-cantly to population declines (Carey, 2000)the increased malformation prevalenceand declining populations are occurring inthe same time frame, which may be morethan coincidental. Many population reduc-tions can be associated with habitat de-struction, degradation, fragmentation, andcontamination (Reeder et al., 1998; Beas-ley et al., 2002) but others appear to beoccurring in areas less affected by humaninfluences (Berger et al., 1998). Infectiousdiseases have been implicated as the prox-imate cause of declines or extinctions insome areas (for review, see Dazsak et al.,1999). Diseases caused by a chytrid fungus(Berger et al., 1998) and by Saprolegniaferax (Kiesecker et al., 2001) have contrib-uted to widespread amphibian deaths inareas described as ecologically pristine.Other diseases implicated in recent deci-mation of amphibian populations includeAeromonas hydrophila, the bacterium re-sponsible for red leg disease (Cunninghamet al., 1996) and iridoviruses (Bollinger etal., 1999). Additional stressors include in-troduced predators or competitors (Corn,

2000), increased temperatures and otherdeleterious aspects of climate change(Pounds et al., 1999), and increased ex-posure to UV-B, which has been suggestedto reduce reproductive success and surviv-al of amphibians at high altitudes (Blau-stein et al., 1994).

Biotic stressors may cause amphibianmalformations and declining populationsdirectly (Blaustein et al., 1994; Ankley etal., 2000), or they may contribute indi-rectly by suppressing immune competence(Carey et al., 1999), or by increasing sus-ceptibility to pathogens. Thus, the appar-ent concurrence of amphibian populationdeclines and elevated malformation ratesin the preceding 40 yr may be coincidentalor the two phenomena may have respond-ed to changing environmental conditions.Anthropogenic factors, such as elevatedtemperature and pH, widespread exposureto chemical toxicants, and increased ex-posure to ambient UV radiation, may com-bine to reduce reproductive success, in-crease susceptibility to disease, and sup-press behavioral and immunologic defens-es against pathogens. Long-term studies ofamphibian population dynamics and theoccurrence and causes of malformationswill be necessary to determine whetherthe factors that contribute to high malfor-mations rates also contribute significantlyto health problems of amphibians that, inturn, influence population sustainability onlocal, regional, and global scales.

ACKNOWLEDGMENTS

The authors wish to acknowledge the privatelandowners, as well as the Departments of Nat-ural Resources of Wisconsin and Minnesota forgranting permission for this study to be con-ducted on their property. We also thank T.Treml, University of Minnesota—Duluth, J. As-hamallah, W. Baumgartner, M. Bhuthimethee,J. Creasey, A. Fronczke, L. Morgan, L.Schmoldt, J. Steele, Z. Steinberg, and S. Tuf-tedal, University of Illinois, Urbana-Cham-paign, for assistance in collections, A. Koehler,National Wildlife Health Center, for skeletalstaining, and T. Hollenhorst, Natural ResourcesResearch Institute, for assistance in site selec-tion, collections, and graphics. Although the re-


search described in this article has been fundedwholly or in part by the United States Environ-mental Protection Agency through grant GADR82558667 to VRB, it has not been subjectedto the Agency’s required peer and policy reviewand therefore does not necessarily reflect theviews of the Agency and no official endorse-ment should be inferred.

LITERATURE CITED

ANKLEY, G. T., J. E. TIETGE, D. L. DEFOE, K. M.JENSEN, G. W. HOLCOMBE, E. H. DURHAN, AND

S. A. DIAMOND. 1998. Effects of ultraviolet lightand methoprene on survival and development ofRana pipiens. Environmental Toxicology andChemistry 17: 2530–2542.

, , G. W. HOLCOMBE, D. L DEFOE, S.A. DIAMOND, K. M. JENSEN, AND S. J. DEGITZ.2000. Effects of laboratory ultraviolet radiationand natural sunlight on survival and developmentof Rana pipiens. Canadian Journal of Zoology 78:1092–1100.

BAILEY, R. G. 1983. Delineation of ecosystem re-gions. Environmental Management 7: 365–373.

BEASLEY, V. R., S. A. FAEH, B. WIKOFF, J. EISOLD,D. NICHOLS, R. COLE, A. M. SCHOTTHOEFER, C.STAEHLE, M. GREENWELL, AND L. E. BROWN.2003. Risk factors and the decline of the north-ern cricket frog, Acris crepitans: Evidence forthe involvement of herbicides, parasitism, andhabitat modifications. In The status and conser-vation of United States amphibians, M. Lannoo(ed.). University of Iowa Press, Iowa City, Iowa,in press.

BERGER, L., R. SPEARE, P. DASZAK, D. E. GREEN, A.A. CUNNINGHAM, C. L. GOGGIN, R. SLOCOMBE,M. A. RAGAN, A. D. HYATT, K. R. MCDONALD,H. B. HINES, K. R. LIPS, G. MARANTELLI, AND

H. PARKES. 1998. Chytridiomycosis causes am-phibian mortality associated with population de-clines in the rain forests of Australia and CentralAmerica. Proceedings of the National Academyof Sciences USA 95: 9031–9036.

BLAUSTEIN, A. R., P. D. HOFFMAN, D. G. HOKIT, J.M. KIESECKER, S. C. WALLS, AND J. B. HAYS.1994. UV repair and resistance to solar UV-B inamphibian eggs: A link to population declines.Proceedings of the National Academy of Scienc-es USA 91: 1791–1795.

BOLLINGER, T. K., J. MAO, D. SCHOCK, R. M. BRIG-

HAM, AND V. G. CHINCHAR. 1999. Pathology, iso-lation, and preliminary molecular characteriza-tion of a novel iridovirus from tiger salamandersin Saskatchewan. Journal of Wildlife Diseases 35:413–429.

BRYANT, S. V., AND D. M. GARDINER. 1992. Retinoicacid, local cell-cell interactions, and pattern for-mation in vertebrate limbs. Developmental Bi-ology 152: 1–25.

CAREY, C. 2000. Infectious disease and worldwidedeclines of amphibian populations, with com-ments on emerging diseases in coral reef organ-isms and in humans. Environmental Health Per-spectives 108 (Supplement 1): 143–150.

, N. COHEN, AND L. ROLLINS-SMITH. 1999.Amphibian declines: An immunological perspec-tive. Developmental and Comparative Immunol-ogy 23: 459–472.

CONVERSE, K. A., J. MATTSSON, AND L. EATON-POOLE.2000. Field surveys of midwestern and north-eastern Fish and Wildlife Service lands for thepresence of abnormal frogs and toads. Journal ofthe Iowa Academy of Science 107: 160–167.

CORN, P. S. 2000. Amphibian declines: Review ofsome current hypotheses. In Ecotoxicology ofamphibians and reptiles, D. W. Sparling, G. Lin-der and C. A. Bishop (eds.). SETAC Press, Pen-sacola, Florida, pp. 663–696

COWARDIN, L. M., V. CARTER, F. GOLET, AND E. LAR-

OE. 1979. Classification of wetlands and deep-water habitats of the United States. U.S. FishWildlife Service FWS/OBS-79/31, 131 pp.

CUNNINGHAM, A. A., T. E. LANGTON, P. M. BENNETT,J. F. LEWIN, S. E. DRURY, R. E. GOUGH, AND S.K. MACGREGOR. 1996. Pathological and micro-biological findings from incidents of unusualmortality of the common frog (Rana temporaria).Philosophical Transactions of the Royal Society:Biological Sciences 351: 1539–1557.

DASZAK, P., L. BERGER, A. A. CUNNINGHAM, A. D.HYATT, D. E. GREEN, AND R. SPEARE. 1999.Emerging infectious diseases and amphibianpopulation declines. Emerging Infectious Dis-eases 5: 735–748.

DEGITZ, S. J., P. A. KOSIAN, E. A. MAKYNEN, K. M.JENSEN, AND G. T. ANKLEY. 2000. Stage- andspecies-specific developmental toxicity of all-trans retinoic acid in four native North Americanranids and Xenopus laevis. Toxicological Sciences57: 264–274.

DINGERKUS, G., AND L. D. UHLER. 1977. Enzymeclearing of alcian blue-stained whole small ver-tebrates for demonstration of cartilage. StainTechnology 52: 229–232.

GARDINER, D. M., AND D. M. HOPPE. 1999. Envi-ronmentally induced limb malformations in minkfrogs (Rana septentrionalis). Journal of Experi-mental Zoology 248: 207–216.

GILBERT, S. F. 2000. Developmental biology. SinauerAssociates, Inc., Sunderland, Massachusetts, pp.3–24.

GOSNER, K. L. 1960. A simplified table for staginganuran embryos and larvae with notes on iden-tification. Herpetologica 16: 183–190.

GRAY, R. H. 2000. Morphological abnormalities inIllinois cricket frogs, Acris crepitans, 1968–71.Journal of the Iowa Academy of Science 107: 92–95.

HELGEN, J. C., M. C. GERNES, S. M. KERTEN, J. W.


CHIRHART, J. T. CANFIELD, D. BOWERS, J. HAF-

ERMAN, R. G MCKINNEL, AND D. M. HOPPE.2000. Field investigations of malformed frogs inMinnesota 1993–97. Journal of the Iowa Acade-my of Science 107: 96–112.

HOPPE, D. M. 2000. History of Minnesota frog ab-normalities: Do recent findings represent a newphenomenon? Journal of the Iowa Academy ofScience 107: 86–89.

HOULAHAN, J. E., C. S. FINDLAY, B. R. SCHMIDT, H.MEYER, AND S. L. KUZMIN. 2000. Quantitativeevidence for global amphibian population de-clines. Nature 404: 752–755.

JOHNSON, D. H., S. C. FOWLE, AND J. A. JUNDT. 2000.The North American reporting center for am-phibian malformations. Journal of the IowaAcademy of Science 107: 123–127.

JOHNSON, P. T. J., K. B. LUNDE, E. G. RITCHIE, AND

A. E. LAUNER. 1999. The effect of trematodeinfection on amphibian limb development andsurvivorship. Science 284: 802–804.

, , R. W. HAIGHT, J. BOWERMAN, AND

A. R. BLAUSTEIN. 2001. Ribeiroia ondatrae(Trematoda: Digenea) infection induces severelimb malformations in western toads (Bufo bo-reas). Canadian Journal of Zoology 79: 370–379.

KELLY, W. L., AND M. M. BRYDEN. 1983. A modifieddifferential stain for cartilage and bone in wholemount preparations of mammalian fetuses andsmall vertebrates. Stain Technology 58: 131–134.

KIESECKER, J. M., A. R. BLAUSTEIN, AND L. K. BEL-

DEN. 2001. Complex causes of amphibian pop-ulation declines. Nature 410: 681–684.

MADEN, M. 1993. The homeotic transformation oftails into limbs in Rana temporaria by retinoids.Developmental Biology 159: 379–391.

MERRELL, D. J. 1969. Natural selection in a leopardfrog population. Journal of the Minnesota Acad-emy of Science 35: 86–89.

MEYER-ROCHOW, V. B., AND M. ASASHIMA. 1988.Naturally occurring morphological abnormalitiesin wild populations of the Japanese newt Cynopspyrrhogaster (Salamandridae; Urodela; Amphib-ia). Zoologischer Anzeiger 221: 70–80.

NORTH AMERICAN REPORTING CENTER FOR AM-PHIBIAN MALFORMATIONS. 2002. Jamestown,North Dakota, Northern Prairie Wildlife Re-search Center Home Page, http://www.npwrc.usgs.gov/narcam (Version 30 Sept., 2002).

OUELLET, M. 2000. Amphibian deformities: Currentstate of knowledge. In Ecotoxicology of amphib-ians and reptiles, D. W. Sparling, G. Linder and

C. A. Bishop (eds.). SETAC Press, Pensacola,Florida, pp. 617–661.

, J. BONIN, J. RODRIGUE, J.-L. DESGRANGES,AND S. LAIR. 1997. Hindlimb deformities (ectro-melia, ectrodactyly) in free-living anurans fromagricultural habitats. Journal of Wildlife Diseases33: 95–104.

POUNDS, J. A., M. P. L. FOGDEN, AND J. H. CAMP-

BELL. 1999. Biological response to climatechange on a tropical mountain. Nature 398: 611–615.

REEDER, A. L., G. L. FOLEY, D. K. NICHOLS, L. G.HANSEN, B. WIKOFF, S. FAEH, J. EISOLD, M. B.WHEELER, R. WARNER, J. E. MURPHY, AND V. R.BEASLEY. 1998. Forms and prevalence of inter-sexuality, and effects of environmental contami-nants on sexuality in cricket frogs (Acris crepi-tans). Environmental Health Perspectives 106:261–266.

REYNOLDS, T. D., AND T. D. STEPHENS. 1984. Mul-tiple ectopic limbs in a wild population of Hylaregilla. Great Basin Naturalist 44: 166–169.

SCADDING, S. R., AND M. MADEN. 1986a. Compari-son of the effects of vitamin A on limb devel-opment and regeneration in the axolotl, Ambys-toma mexicanum. Journal of Embryology and Ex-perimental Morphology 91: 19–34.

, AND . 1986b. Comparison of the ef-fects of vitamin A on limb development and re-generation in Xenopus laevis tadpoles. Journal ofEmbryology and Experimental Morphology 91:35–53.

SESSIONS, S. K., R. A. FRANSSEN, AND V. L. HORNER.1999. Morphological clues from multileggedfrogs: Are retinoids to blame? Science 284: 800–802.

SUMMERBELL, D. 1983. The effect of local applica-tion of retinoic acid to the anterior margin of thedeveloping chick limb. Journal of Embryologyand Experimental Morphology 78: 269–289.

TICKLE, C., J. LEE, AND G. EICHELE. 1985. A quan-titative analysis of the effect of all-trans-retinoicacid on the pattern of chick wing development.Developmental Biology 109: 82–95.

VAN VALEN, L. 1974. A natural model for the originof some higher taxa. Journal of Herpetology 8:109–121.

VOLPE, E. P., AND P. A. ROSENBAUM. 2000. Under-standing evolution, 6th Edition, McGraw Hill,Boston, Massachusetts, 281 pp.

Received for publication 14 March 2002.

PREVALENCE OF SKELETAL AND EYE MALFORMATIONS IN FROGS FROM NORTH-CENTRAL UNITED STATES: ESTIMATIONS...

Documents

Transcript of PREVALENCE OF SKELETAL AND EYE MALFORMATIONS IN FROGS FROM NORTH-CENTRAL UNITED STATES: ESTIMATIONS...