Tooth replacement in the red-backed salamander, Plethodon cinereus

The developmental cycle of the teeth in Plethodon cinereus is analyzed on morphological grounds using alizarin preparations. All the stages in development do not occupy the same proportion of the life cycle time. Functional teeth and germs at an early stage in development occupy a large proportion of the life cycle time, whereas the processes of tooth shedding and ankylosis occur very quickly. The time during which any locus does not bear a functional tooth, and is therefore a non-functional locus, is reduced to a minimum. P. cinereus has a basic pattern of tooth replacement which is consistent with Zahnreihen which are 2.0 tooth spaces apart. Variations in the replacement pattern are common and these are produced by relatively small fluctuations in the spacing of the Zahnreihen around the ?mean? of 2.0. Localized disturbances which produce breaks in the replacement pattern and cause waves to cross also occur. These may be due to the failure of tooth germs to develop, the fusion of tooth germs, or may be the result of the inherent variability in a complex biological system. This variability causes individual tooth germs to develop too slowly or too quickly and hence assume an ?abnormal”? position thus causing breaks in the replacement pattern. Tooth replacement may be controlled by an intra-local mechanism(s) rather than by stimuli which travel along the jaw.     

Retinal photoreceptor fine structure in the red-backed salamander (Plethodon cinereus).

The retinal photoreceptors of the red-backed salamander (Plethodon cinerus) have been studied by light and electron microscopy. Rods and single cones are present in this duplex retina in a ratio of about 25:1. The photoreceptors in this amphibian species are much larger than is reported for most vertebrates. In the light-adapted state, rods reach deep into the retinal epithelial (RPE) layer. The rod outer segment is composed of discs of uniform diameter displaying several very deep incisors. The rod inner segment displays a distal elliposid of mitochondria and a short stout myoid region. Rod nuclei are electron dense and often protrude through the external limiting membrane. Rod synaptic spherules are large and display several invaginated synaptic sites as well as superficial synapses. It is felt that the rods do not undergo retinomotor movements. The cone photoreceptors are much smaller than the rods and display a tapering outer segment, an unusual modified ellipsoid and a large parabolid of glycogen in the inner segment. Cone nuclei are less electron dense than rods and are located at all levels within the outer nuclear layer. The synaptic pedicle of the cones is larger, more electron lucent and display more synaptic sites (both invaginated and superficial) than that of rods. It is felt that cone photomechanical responses are minimal.     

Retinal pigment epithelial fine structure in the red-backed salamander (Plethodon cinereus).

The retinal pigment epithelium (RPE) of the red-backed salamander (Plethodon cinerus) consists of a single layer of large squamous shaped cells. The RPE cells are but minimally infolded basally (sclerally) but show many large apical (vitreal) processes interdigitating with the rod outer segments. These epithelial cells are joined laterally by prominent tight junctions located in the mid region of the cells. Internally smooth endoplasmic reticulum is very plentiful while rough endoplasmic reticulum is not. Polysomes, small dense mitochondria and small round to oval melanosomes are plentiful. Golgi zones and lysosome-like bodies are also present as are phagosomes of outer segment material and myeloid bodies. The RPE cell nucleus is large and vesicular. It is felt that the melanosomes undergo retinomotor movements but as only light-adapted specimens were examined it is not known how extensive are these movements. Bruch's membrane or complexus basalis shows the typical pentalaminate structure noted for most vertebrates. The choriocapillaris is a single layer of large anastomosing capillaries which are minimally fenestrated facing Bruch's membrane.     

A fresh look at meiosis and centromeric heterochromatin in the red-backed salamander,Plethodon cinereus cinereus (Green)

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a diffuse stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.     

Description of the postcloacal glands of Plethodon cinereus, the red-backed salamander, during bouts of scent marking

Plethodon cinereus, the red-backed salamander, is a small territorial vertebrate that defends refugia located on the forest floor. As a component of territorial defense, these animals use scent marks to advertise their refugia. Behavioral evidence indicates that scent marks are produced by the postcloacal glands located on the ventral surface of the tail just posterior to the cloaca. We placed animals on unmarked substrates and recorded changes in serous acini from the postcloacal and shoulder region over a 48-h period. Within the first hour there was an increase in the number of acini filled with secretory product in the postcloacal region. At 12 h the number of full acini decreased and the number of empty acini increased. By 24 h the number of empty acini had decreased and the number of renewing acini containing secretory cells producing product had increased. By 48 h the ratio of full to empty to renewing acini was similar to those observed at the start of the study. In the shoulder region, no significant changes in the ratio of full to empty to renewing acini were observed. Observations of the serous acini within the postcloacal region and the shoulder region indicate that the mode of secretory production is holocrine. These findings are additional evidence that the postcloacal glands are the site of scent mark production.     

Structural heterogeneity in the basal regions of the teeth of the red-backed salamander,Plethodon cinereus (Amphibia,Plethodontidae)

Ultrastructural and histochemical features of marginal (monostichous) teeth associated with the jawbones are compared with those of palatal (polystichous) teeth that compose two patches in the roof of the mouth. The apices and uncalcified regions are similar in both kinds of teeth, but the basal regions display distinctive differences. While bases (pedestals) of marginal teeth are essentially hollow cylinders that attach to the jawbones by their labial faces, bases of teeth in palatal patches are fused to form two horizontal plates which lack direct attachment to underlying bone. The plates are separated from each other by a pulp-filled space containing fibroblasts, blood vessels, and vertically oriented elements resembling bony spicules. Cylindrical pedestals like those of marginal teeth project from the ventral plate. While the identity of the material composing the basal regions remains controversial, the following evidence suggests that it is similar to “bone of attachment” (Tomes, '23): most of it, unlike dentin, does not develop in direct association with an enamel organ; alcian blue stains the bases of developing teeth but stains dentin, developing dentin, enamel, or mature bone very weakly (if at all); bases of teeth in palatal patches develop in isolation from the parasphenoid bone and thus cannot be considered extensions of it; and marginal teeth attach directly to the jawbones, but the material composing their bases does not blend with the bone. Structural heterogeneity of the basal regions appears to be linked to functional differences exhibited by these two types of teeth.     

Structure of the radially asymmetrical uncalcified region of the teeth of the red-backed salamander,Plethodon cinereus (Amphibia,Plethodontidae)

The teeth of the adult plethodontid salamander, Plethodon cinereus, were examined by light and electron microscopy with emphasis on the ringlike zone of uncalcified dentin that divides the calcified portion of each tooth into a proximal pedestal and a distal apex. The uncalcified region displays radial asymmetry, forming an integral part of the posterior wall of the tooth but bulging into the pulp cavity anteriorly, thus forming a hingelike structure. All portions of the dentin, including the uncalcified region, are composed predominantly of collagenous fibers but lack elastin. In scanning electron micrographs of teeth from which the oral mucosa has been removed, the location of the anterior uncalcified hinge is marked externally by a notch-like articulation of the apex and pedestal. Sites of transition between calcified and uncalcified areas of the dentin show no special modifications in transmission electron micrographs, but collagenous fibers in calcified portions are associated with more electron-dense amorphous material than are those in the uncalcified region. Odontoblasts associated with the uncalcified region possess ultrastructural features closely resembling those of odontoblasts found in calcified areas. The uncalcified region seems to afford the teeth a certain degree of flexibility, and the asymmetry of the region appears to allow the teeth to flex only in a posterior direction, thus facilitating the entry of living prey but hindering its escape. The uncalcified region also seems to permit the apex of a tooth to break away from its pedestal without damage to underlying bone.     

Female philopatry and male-biased dispersal in a direct-developing salamander, Plethodon cinereus

The local resource competition hypothesis and the local mate competition hypothesis were developed based on avian and mammalian systems to explain sex-biased dispersal. Most avian species show a female bias in dispersal, ostensibly due to resource defence, and most mammals show a male bias, ostensibly due to male-male competition. These findings confound phylogeny with mating strategy; little is known about sex-biased dispersal in other taxa. Resource defence and male-male competition are both intense in Plethodon cinereus, a direct-developing salamander, so we tested whether sex-biased dispersal in this amphibian is consistent with the local resource competition hypothesis (female-biased) or the local mate competition hypothesis (male-biased). Using fine-scale genetic spatial autocorrelation analyses, we found that females were philopatric, showing significant positive genetic structure in the shortest distance classes, with stronger patterns apparent when only territorial females were tested. Males showed no spatial genetic structure over the shortest distances. Mark-recapture observations of P. cinereus over 5 years were consistent with the genetic data: males dispersed farther than females during natal dispersal and 44% of females were recaptured within 1 m of their juvenile locations. We conclude that, in this population of a direct-developing amphibian, females are philopatric and dispersal is male-biased, consistent with the local mate competition hypothesis.     

Acetazolamide does not disrupt limb regenerate morphogenesis in the salamander, Plethodon cinereus

Acetazolamide, a potent and highly specific inhibitor of carbonic anhydrase, is teratogenic in mammalian embryos and when administered during early limb development causes unique limb defects in a time- and dose-dependent manner. The regenerating urodele limb is often considered to be a good experimental analog of limb development and, if it employs the same mechanisms of tissue interactions during pattern formation, should be susceptible to teratogens which selectively disrupt developmental limb patterning. This study demonstrates that while carbonic anhydrase inhibition is toxic to the red-backed salamander, Plethodon cinereus, it does not have the same teratogenic effect on limb regeneration as seen in mammalian limb development. Several points are considered as to why the regenerating limb, at least in this salamander species, may not be suitable for studying this class of teratogen.     

Antibacterial and hemolytic activity of the skin of the terrestrial salamander, Plethodon cinereus

As resistance increases against fungal antibiotics, antimicrobial peptides are receiving attention as possible replacements. The dermal glands of frogs secrete, among other things, antimicrobial peptides. As part of the innate immune system, stressors may affect the production of antimicrobial peptides by dermal glands. The dermal secretions of some salamanders have been examined for their toxic secretions, but little attention has been given to salamander antimicrobial peptides. This study examines the skin from the tail region for the production of antimicrobial peptides in the terrestrial salamander, Plethodon cinereus. Fractions of tail extracts were isolated using cation-exchange chromatography and reverse-phase HPLC. An HPLC fraction eluting at 15.75 min (HPLC run: 30 min, 30-80% acetonitrile/water gradient, Aquapore RP-300 C18 column) showed activity against Staphylococcus aureus but not against Escherichia coli. The antibacterial activity gradually increased over a 4-hr incubation time up to about 85% inhibition of bacterial growth. Lysis of guinea pig red blood cells also increased gradually over a 1-hr time period. J. Exp. Zool. 287:340-345, 2000.     

Sperm aggregations in the spermatheca of the red back salamander (Plethodon cinereus)

The spermatheca of Plethodon cinereus is a compound tubular gland that stores sperm from mating in early spring (March–April) to oviposition in summer (June–July). The seasonal variation of sperm storage in this species has previously been studied by light and transmission electron microscopy. In this paper, sperm aggregations, interaction of sperm with the spermathecal epithelium, and spermathecal secretions are studied using scanning electron microscopy. Within spermathecal tubules, relatively small groups of sperm are aligned along their entire lengths in parallel arrays. This pattern is similar to other plethdontids with complex spermathecae. Lumina of spermathecal tubules are filled with secretory material in April prior to the arrival of sperm, and after sperm appear, a coating of secretory material persists on the apices of the spermathecal epithelium. Sperm peripheral to the central luminal mass can become embedded in the secretory matrix or pushed deeper into the spermathecal epithelium. The spermathecal secretions may serve to attract and prolong the viability of sperm, but sperm that become enmeshed in the secretions or epithelium are phagocytized. Sperm and spermathecal secretions are largely absent after ovulation and in summer months, and new secretory vacuoles are formed in fall, although mating does not occur until spring.     

Structural changes of drosopterinosomes (red pigment granules) during the erythrophore differentiation of the frog,Rana japonica,with reference to other pigment-containing organelles

Summary Structural changes in drosopterinosomes (red pigment granules) of Rana japonica in the process of erythrophore differentiation were studied by light and electron microscopy. On the basis of the degree of pterinosome differentiation, three types can be recognized: Typ-I drosopterinosomes appear first during metamorphosis and have clear limiting membranes and amorphous materials within. Those of type-II are found in abundance shortly after metamorphosis and have inner structures, consisting of fibrillae and/or small lamellae in dense concentric arrangement. Type-III is found abundantly in adults and acquires an almost homogeneously electron-dense mature morphology, probably from the deposition of electron-dense materials. On the basis of counts of pterinosomes, a successive transformation from type I to III is suggested. The differences among red drosopterinosomes, yellow sepiapterinosomes in xanthophore and melanosomes are not always distinguishable electron microscopically. Discrimination is possible by careful examination of lamellar patterns characteristic of the respective granules and by a simultaneous application of light and electron microscopy. From this viewpoint, a re-evaluation of the identification of granules previously reported was effected.This work was supported by a grant in aid to T. H. from the Ministry of Education (No. 92112, 1971).     

Mothers influence offspring body size through post-oviposition maternal effects in the redbacked salamander, Plethodon cinereus

In the terrestrial salamander (Plethodon cinereus), previous work has shown that mothers body size is positively correlated to offspring size at the time of hatching even after controlling for the effects of egg size. This study was designed to determine whether maternal body size affects offspring size via pre-oviposition factors (e.g., yolk quality, jelly coat composition, or maternal genes) or post-oviposition factors (e.g., parental care behaviors, parental modification of environment). Gravid females were captured and induced to lay eggs in experimental chambers in which the environment was standardized. Fifteen clutches were exchanged, or cross-fostered, between female pairs differing in body size. Ten females whose eggs were taken away and then returned served as controls for the crossing treatment. Foster mothers did not significantly differ from control mothers in the time spent with eggs, body position, or number of egg movements during brooding. Average egg mass measured midway through development was not significantly correlated to the body size of either the genetic or foster mother, but was correlated to pre-oviposition oocyte size. At hatching, offspring body length was positively correlated to egg size and the foster mothers body size. This correlation suggests that in P. cinereus post-oviposition maternal effects have a greater impact on offspring size than other maternal factors incorporated into the egg prior to oviposition. While our study showed that larger mothers moved their eggs less often and tended to spend more time in contact with their eggs, further work needs to be done to identify the specific mechanisms through which larger mothers influence the body size of their offspring. This is the first experimental demonstration of post-oviposition maternal effects for any amphibian with parental care.     

Sperm storage in the spermatheca of the red-back salamander,Plethodon cinereus (Amphibia: Plethodontidae)

In northern Indiana, the mating season of Plethodon cinereus occurs after hibernation from March until June, when oviposition begins. During the mating season, a female stores sperm in its spermatheca, a compound tubular gland in the roof of the cloaca. The apical cytoplasm of the spermathecal epithelium is filled with large secretory vacuoles whose product is released while sperm are stored. Females induced to oviposit in June and July by injections of human chorionic gonadotropin (hCG) still retain much sperm 1 month after oviposition, but secretory vacuoles are absent in all specimens sacrificed in July and August. Instead, some sperm are embedded in the spermathecal epithelium with resultant spermiophagy involving lysosomes. A female sacrificed in September 2 months after oviposition possesses scant sperm, but spermiophagy alone does not seem extensive enough to account for the decrease in sperm numbers. Females sacrificed in October prior to hibernation lack sperm in their spermathecae; some secretory vacuoles are present, but they are not as numerous or as enlarged as in specimens collected in March and May. Inter- and intrafamilial differences in the cytology of sperm storage may not be phyletically informative at the family level but related to species-specific reproductive adaptations. J. Morphol. 234:131–146, 1997. © 1997 Wiley-Liss, Inc.     

Lead-phase and red-stripe color morphs of red-backed salamanders Plethodon cinereus differ in hematological stress indices:A consequence of differential predation pressure?

<正> Throughout the animal kingdom there are species that have two or more phenotypic forms or 'morphs', and many ofthese are amphibians. In North America, the red-backed salamander Plethodon cinereus can have either a red dorsal stripe or nodorsal stripe (lead-phase form), and evidence to date indicates the lead-phase form incurs a greater number of attacks from predators.In a recent collection of 51 P. cinereus, blood smears of both color morphs (35 red-stripe, 16 lead-phase) were examined toobtain numbers of circulating leukocytes (via light microscopy), which can be used to indirectly estimate levels of stress hormonesin vertebrates via a 'hematological stress index', which is the ratio between the number of two leukocyte types (neutrophilsand lymphocytes). Our results showed that lead-phase salamanders tended to have greater numbers of circulating neutrophils andlower numbers of circulating lymphocytes than red-stripe morphs, leading to higher average neutrophil-lymphocyte ratios inlead-phase individuals. Since the salamanders were held (refrigerated) for 7 days before sampling, we cannot be certain if this effectis a stress reaction to the captivity or the normal level for this morph. However comparison with two sets of related salamandersthat were captured and sampled immediately indicates the red-stripe salamanders were either not stressed from the captivityat all, or their white blood cell distributions had returned to normal after 7 days of captivity. Taken together, our results indicatethat lead-phase forms of P. cinereus have higher stress levels than the red-stripe forms, which may be a consequence of theirhigher exposure to, and/or attacks from, predators. They may also indicate that the lead-phase form is less-suited to captivity thanthe red-stripe form of this species     

The influence of habitat provisioning: use of earthworm burrows by the terrestrial salamander, Plethodon cinereus

Studies examining the influence of habitat provisioning by one species on the behavior of other species can provide key insights regarding impacts of ecosystem engineers on the availability of resources to other species. More specifically, an organism’s use of additional habitat provided by ecosystem engineers may affect the interpretation of observational or demographic data. We chose to examine the possible influence of earthworms, as ecosystem engineers, on the behavior of terrestrial salamanders, common forest vertebrates in North America. We conducted two experiments in microcosms to examine whether the earthworm Lumbricus terrestris acted as an ecosystem engineer by providing additional habitat (burrows) for Plethodon cinereus, a common woodland salamander. We also examined whether the behavioral changes of the non-burrowing, fossorial P. cinereus differed across age classes. Adults and juveniles responded similarly, with both adults and juveniles found under cover objects more often when earthworms were absent and using earthworm burrows when available. Our field data supported our experimental data in that greater numbers of earthworms on field plots were associated with a lower proportion of cover objects being occupied by salamanders (i.e., salamanders were likely underground). We found no differences in the response of adults and juveniles to the presence of earthworms. By providing underground habitat for a common terrestrial amphibian, the presence of earthworms may impact demographic studies on salamanders and conclusions drawn from those studies regarding aboveground faunal communities in eastern North America.     

Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus)

Beneficial cutaneous bacteria on amphibians can protect against the lethal disease chytridiomycosis, which has devastated many amphibian species and is caused by the fungus Batrachochytrium dendrobatidis. We describe the diversity of bacteria on red-backed salamanders (Plethodon cinereus) in the wild and the stability of these communities through time in captivity using culture-independent Illumina 16S rRNA gene sequencing. After field sampling, salamanders were housed with soil from the field or sterile media. The captive conditions led to different trajectories of bacterial communities. Eight OTUs present on >90% of salamanders in the field, through time, and in both treatments were defined as the core community, suggesting that some bacteria are closely associated with the host and are independent of an environmental reservoir. One of these taxa, a Pseudomonas sp., was previously cultured from amphibians and found to be antifungal. As all host-associated bacteria were found in the soil reservoir, environmental microbes strongly influence host–microbial diversity and likely regulate the core community. Using PICRUSt, an exploratory bioinformatics tool to predict gene functions, we found that core skin bacteria provided similar gene functions to the entire community. We suggest that future experiments focus on testing whether core bacteria on salamander skin contribute to the observed resistance to chytridiomycosis in this species even under hygenic captive conditions. For disease-susceptible hosts, providing an environmental reservoir with defensive bacteria in captive-rearing programs may improve outcomes by increasing bacterial diversity on threatened amphibians or increasing the likelihood that defensive bacteria are available for colonization.     

Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.     

Red-backed salamander (Plethodon cinereus) as a bioindicator of mercury in terrestrial forests of the northeastern United States

Despite concerns over the widespread deposition of mercury (Hg) in remote forested areas of the northeastern United States (U.S.), little information is available on the bioaccumulation of Hg in this region's terrestrial fauna. There is a strong need to develop baseline data on appropriate bioindicator species for this area, and here we report Hg concentrations in one of the most widely distributed vertebrates in forested areas, the red-backed salamander (Plethodon cinereus). To inform the use of this species as a bioindicator of Hg accumulation, we assessed ratios of bioavailable methylmercury (MeHg) to total Hg, and techniques for non-destructive sampling along an elevational gradient of increasing forest floor Hg concentrations. Total Hg in body samples was 70% MeHg, and Hg concentrations in tail-clips, which can be collected non-lethally, were positively correlated with body concentrations. Mercury concentrations in salamanders increased 2.4-fold along an increasing elevational gradient of Hg in soils. We conclude that Hg concentrations in P. cinereus can act as a biomonitoring tool for broad areas of remote terrestrial forests, and may help identify regions and landscape characteristics of particular concern for Hg bioaccumulation.