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Polymorphic nature of cranial fluorescence in the fox squirrel (Sciurus niger) from Texas and Oklahoma.

Abstract. -- Congenital erythropoietic porphyria (CEP) is a rare pathological condition in man and some domestic animals. Reported diagnostic features include skin lesions, shortened life span, darkened bones and fluorescent skeletal tissues. Literature sources report the condition is characteristic of fox squirrels (Sciurus niger), and that the species suffers no ill effect of the disease. Examination under an ultraviolet light of 157 skulls and associated mandibles of S. niger from Texas and Oklahoma revealed that skeletal fluorescence is a polymorphic feature that affects only about 70 percent of examined specimens. Sexes did not vary in frequency of occurrence of this phenomenon, although adult specimens were significantly more likely to fluoresce than those of younger age classes. Among other findings are the purple fluorescence of nestling and juvenile specimens, in contrast to the orange coloration of older age classes. These results suggest several potential avenues for future study relative to the biogeography and genetics of this phenomenon in S. niger.

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Congenital erythropoietic porphyria (CEP) is a rare pathological condition that has been documented in humans and some domestic species of mammals. Onset of the diagnostic skin lesions and hemolytic anemia occurs in humans shortly after birth or early in childhood, and life expectancy is limited to a few years. The disease results from a defect in heme biosynthesis and the resulting porphyrins are distributed in the urine, feces, and tissues such as spleen, blood, bones and teeth. Deposition of these porphyrins imparts both a dark color to bone and a fluorescent quality is reported for the skeletal and other affected tissues of diseased individuals when exposed to long-wave ultraviolet light (Schmid et al. 1954; 1955; Bloomer et al. 1993).

The unusual dark pink to dull reddish hue of the skull and post-cranial skeleton of specimens of the fox squirrel (Sciurus niger) is a distinctive feature that has long been noted by naturalists and has even been used by some biologists (Jones et al. 1983; Lowery 1974) as a means to distinguish the skull of this species from that of the morphologically similar skull of the gray squirrel (S. carolinensis). Turner's (1937) documentation of porphyria as the causative factor of this phenomenon in S. niger was followed up in more detail by Levin & Flyger (1973) and Flyger & Levin (1977). Laboratory studies by these workers of the urine, blood and bone marrow demonstrated the presence of CEP in their series of fox squirrels, as well as an absence of the condition in a comparable series of gray squirrels.

Flyger & Levin (1977) noted that S. niger is asymptomatic for pathological aspects of CEP, which prompted them to suggest the species as a laboratory animal model for future studies of this physiological condition, but perhaps because of the rarity of the disease in humans, there has been no answer to their call for follow-up investigations of the species in this role. However, this study has been cited as the basis for a series of statements in the literature (Caire et al. 1989; Fitzgerald et al. 1994; Flyger 1999; Koprowski 1994) proclaiming CEP and the resulting fluorescent qualities of bones and teeth as characteristic of the fox squirrel.

The impetus for the present study was provided by a recent mammalogy laboratory exercise conducted by the third author, which unexpectedly demonstrated that a large proportion of fox squirrel skulls did not fluoresce when exposed to ultraviolet light. This study details the regional polymorphic aspect of this unusual feature in S. niger, and describes the frequency of fluorescence between sexes and relative age groups.

METHODS AND MATERIALS

Skulls of Sciurus niger deposited in the Collection of Recent Mammals of Midwestern State University comprised the basis for this investigation. This study was restricted to those specimens from Oklahoma and Texas because sample sizes from other states were judged insufficient to characterize any regional polymorphism for porphyria. Individual specimens were recorded as to gender (when known), specific locality (state and county), date of collection and relative age classes. Cranial dimensions and degree of ossification were useful age class indicators, as were the following dental criteria: nestlings (no visible formation of tooth crowns in alveoli); juveniles (incomplete battery of erupted molars--at least M3 not yet erupted); subadults (erupting or newly erupted P4, and all molars erupted); adults (full dental complement exhibiting wear on occlusal surface of all cheek teeth). Statistical analyses of the association of these parameters with the incidence of fluorescence were performed using the NCSS 97 statistical package (Hintze 1997).

Specimens were examined under a portable, battery-operated, long-wave ultraviolet lamp Model ML-49 (manufactured by UVP Incorporated, P.O. Box 1501, San Gabriel, California 91778). Fluorescence was treated as a qualitative character, although intensity and cranial distribution of the phenomenon varied considerably, and these features were recorded for each specimen.

An initial concern was that sometimes-harsh curatorial treatment or length-of-storage of specimens might have had a negative effect on the fluorescent property of some skulls. To address the issue of possible postmortem change, a skull of known fluorescent properties was placed in a solution of water and sodium carbonate ("soda ash"; sometimes used to soften persistent connective tissue for ease of removal from skeletal preparations) and boiled for 5 min, followed by 8 h in a drying oven at 80[degrees]C. The dried skull fluoresced as before. Similarly, length-of-storage of specimens appeared to have no effect, for some of the fluorescent specimens date back to the founding of MWSU collection in the early 1950s.

RESULTS

Of the 157 skulls and associated mandibles examined in this study, 108 exhibited some degree of fluorescence. Sites most commonly found to be fluorescent were the palatal complex, frontals and parietals of the skull, and the masseteric fossa of the mandible. Among the upper and lower dentition, the incisors and premolars were most commonly observed to react under UV light. Incidence of fluorescent skulls per examined specimens (arranged by state and county) are as follows: OKLAHOMA (6/6): Bryan Co., 1/1; Commanche Co., 2/2; Cotton Co., 1/1; Pontoco Co., 1/1; Rogers Co., 1/1. TEXAS (97/151): Archer Co., 1/3; Baylor Co., 0/3; Bowie Co., 1/1; Brown Co., 1/1; Clay Co., 2/5; Collin Co., 0/1; Cooke Co., 1/1; Ellis Co., 1/1; Fannin Co., 1/2; Grayson Co., 1/1; Haskell Co., 0/1; Jack Co., 1/1; Kimble Co., 9/12; Knox Co., 2/3; Milam Co., 1/1; Montague Co., 17/22; Stephens Co., 1/1; Sutton Co., 1/1; Tarrant Co., 2/2; Wichita Co., 50/77; Wilbarger Co. 1/8; Young Co., 3/3.

Cranial and mandibular materials of subadult and adult specimens reflected an orange to reddish color under the UV lamp. Fluorescence among the two younger age categories was a distinctive dull purple color, which was restricted to the frontal-parietal complex of the skull and upper incisors, although the palate of one nestling also fluoresced.

Frequency of fluorescence between age categories was significant, with adult-aged specimens significantly more likely to exhibit fluorescence than specimens of younger age (Table 1). Juveniles represented the smallest sample (one of three specimens of this age category exhibited fluorescence), and slightly more than half of the subadult specimens fluoresced. Adults, comprising nearly two-thirds of the total sample, exhibited a significantly greater frequency of fluorescence than the pooled number of individuals representing younger age categories.

DISCUSSION

Fluorescent coloration and bone pigmentation. -- Porphyrin compounds and heme pigments deposited in the calcified tissues produce the dark coloration reported of skeletal materials in Sciurus niger. However, bone coloration was found to be a poor indicator of fluorescence, for some paler materials fluoresced brightly, whereas some darker materials failed to evince fluorescence. The observed lack of fluorescent characteristics may be the result of deposition of intermediates of porphyrin biosynthesis, such as the partially oxidized "porphomethenes" characterized by Mauzerall & Granick (1958). These intermediates, which would contribute to bone-darkening features, are purported to exhibit no fluorescence (Schwartz et al. 1980).

Color and regional distribution of fluorescent pigments in materials used in this study contrast somewhat with the findings of Levin & Flyger (1973), who reported that their freshly dissected skeletal materials of S. niger fluoresced a "brilliant crimson" over the entire surface of the skull. Perhaps areas of lesser deposition of porphyrins lose fluorescent properties soon after death, while regions of heavier deposition remain fluorescent for at least decades.

Color changes from the crimson of fresh material (Levin & Flyger 1973) to the orange of MWSU museum specimens of the two older age categories may be attributed to pH changes incurred in the museum preparatory, preservation and storage processes, for Schwartz et al. (1980) observed that porphyrins fluoresce orange in acidic solutions and appear red under alkaline conditions. However, no mention exists in earlier studies of this purple coloration unique to specimens of the two younger age categories, which leads one to believe that the specimens reported by Flyger & Levin (1977) were adults. This distinctive purple fluorescence of nestling and juvenile fox squirrels likely represents the differential fluorescent qualities of a precursor product to porpyrin I, although laboratory verification is warranted.

Geographic aspects. -- The study by Levin & Flyger (1973) is responsible for subsequent inferences in the literature that all members of Sciurus niger are characterized by congenital erythropoietic porphyria, and that skeletal tissues of the species fluoresce diagnostically under ultraviolet light. The bases for these assumptions presumably were the widely separated (but not specifically detailed) geographic localities from which their samples were procured: Maryland and a commercial squirrel farm in Palestine, Texas. However, results reported in this study suggest that these conclusions were premature. Levin & Flyger (1973) never stated their total sample size, although from the varying number of specimens subjected to their hematological and biochemical assays, it is deduced that no more that 15 animals of unknown age were at their disposal. Their remarks on the fluorescent nature of skeletal tissues suggest that the phenomenon occurs in all animals, although it is not clear whether all specimens were tested for fluorescence.

This study clearly demonstrates the polymorphic nature of cranial and dental fluorescence in the fox squirrel in Texas and Oklahoma. Nine of the 12 counties represented by two or more specimens produced both fluorescent and non-fluorescent specimens, and no discernable pattern of distribution of the phenomenon was evident. However, the species is usually abundantly represented in systematic mammal collections, and the examination of holdings from across a wide range of the species' distribution will aid in determination of the geographic scope of this polymorphism.

Age class variation. -- The high frequency of occurrence of fluorescence among nestling-aged specimens deserves some clarification. This sample is comprised of three litters (n = 1, 2, 5), and each of the five fluorescent specimens were siblings of the largest litter. With such a limited series, sampling error cannot be discounted, although a genetic basis sufficiently simple to be readily demonstrated by breeding studies seems plausible.

The authors are reluctant to offer an explanation for the significantly greater occurrence of fluorescence among adult-aged specimens, especially given the limited samples of the two younger age categories (Table 1). The younger age classes for most species are commonly underrepresented in systematic collections (Stangl & Jones 1987), and concerted efforts to address this age bias is warranted before addressing any possible differential fitness topics.

Medical implications. -- The observed polymorphism for bone fluorescence in S. niger is based on museum specimens subjected to a series of environmental variables during the course of procurement and preparation, and not on living tissues. Nevertheless, findings from this study suggest the need to address the potential utility of S. niger as an animal model for porphyria research, as first proposed by Flyger & Levin (1977). If CEP is indeed the normal physiological condition in the fox squirrel, then fluorescence may not be an accurate indicator of the condition in this species (porphyrins deposited in tissues other than bone in some animals). If skeletal fluorescence proves to be a reliable indicator of CEP, then the fox squirrel could prove useful in determining the genetic basis for this condition.
Table 1. Statistical analyses for frequency of occurrence by sex and
relative age category of cranial and mandibular fluorescence among 157
specimens of Sciurus niger from Oklahoma and Texas.

Parameters (n) No. normal skulls Frequency
 (No. fluorescent skulls) of fluorescence

Specimens of known sex (146)
 Males (79) 25 (54) 0.68
 Females (67) 18 (49) 0.73
Specimens of unknown sex (11) 6 (5) 0.45
Age classes *
 Nestlings (8) 3 (5) 0.63
 Juveniles (3) 2 (1) 0.33
 Subadults (37) 17 (20) 0.54
 Adults (109) 27 (82) 0.75
Pooled age classes **
 Adults (109) 27 (82) 0.75
 Subadults and younger (48) 22 (26) 0.54

* ANOVA; P < 0.05
** Two-tailed t-test; P < 0.01.


ACKNOWLEDGMENTS

We thank the administration of Midwestern State University for their continued support and encouragement of our research efforts. Jim R. Goetze, John V. Grimes and Clyde Jones kindly reviewed an earlier draft of this manuscript, and Rodney Cate provided critical references on porphyrin biochemistry. The Oklahoma Department of Wildlife and Texas Department of Parks and Wildlife have long cooperated through the issuance of collecting permits that allowed the gradual accumulation of specimens used in this study.

LITERATURE CITED

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Koprowski, J. L. 1994. Sciurus niger. Mamm. Species, 479:1-9.

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Lowery, G. H., Jr. 1974. The Mammals of Louisiana and Its Adjacent Waters. Louisiana State University Press, Baton Rouge, xxiii + 565 pp.

Mauzerall, D. & S. Granick. 1958. Porphyrin biosynthesis in erythrocytes. III. Uroporphyrinogen and its decarboxylase. J. Biol. Chem., 232(4):1141-1162.

Schmid, R., S. Schwartz & C. J. Watson. 1954. Porphyrin content in bone marrow and liver in the various forms of porphyria. Arch. Internal Med., 93(2):167-190.

Schmid, R., S. Schwatrz & R. D. Sundberg. 1955. Erythropoietic (Congenital) Porphyria: A Rare Abnormality of the Normoblasts. Blood, 10(3):416-428.

Schwartz, S., M. H. Berg, I. Bossenmaier & H. Dinsmore. 1980. Determination of Porphyrins in Biological Materials. Pp. 221-293, in Methods of Biochemical Analysis (D. Glick, ed.). Interscience Publishers, New York, ix + 400 pp.

Stangl, F. B., Jr. & E. M. Jones. 1987. An assessment of geographic and seasonal biases in systematic mammal collections from two Texas universities. Tex. J. Sci., 39(2):129-137.

Turner, W. J. 1937. Studies on porphyria. I. Observations on the fox squirrel, Sciurus niger. J. Biol. Chem., 118(3):519-531.

Kimberly D. Spradling, Bonnie L. Blossman-Myer and Frederick B. Stangl, Jr.

Department of Biology, Midwestern State University

Wichita Falls, Texas 76308

FBS at: stanglf@nexus.mwsu.edu
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Author:Spradling, Kimberly D.; Blossman-Myer, Bonnie L.; Stangl, Frederick B., Jr.
Publication:The Texas Journal of Science
Geographic Code:1U7OK
Date:Nov 1, 2000
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