Interaction of an insecticide with larval density in pond-breeding salamanders (Ambystoma)

1. Amphibian populations residing in or near agricultural areas are often susceptible to pesticide contamination. Recent evidence suggests that the effects of pesticides on amphibians often exceed those estimated in laboratory toxicity tests because other environmental factors (e.g. predators, resource abundance) can influence pesticide toxicity. 2. To examine the effects of an insecticide (carbaryl) on two species of Ambystoma salamanders experiencing the natural stress of competition, we manipulated chemical concentration (control, 3.5 and 7.0 mg L^?1) and larval density (low and high). We determined the effect of treatments on snout‐vent length (SVL), growth rate, lipid reserves, time to metamorphosis, per cent survival and per cent metamorphosis. 3. Carbaryl negatively affected all response variables of Ambystoma maculatum significantly, and significantly reduced survival and metamorphosis of A. opacum. Increased density significantly influenced SVL, lipid reserves, growth rate and metamorphosis of A. maculatum. 4. The effects of carbaryl and increased density on per cent metamorphosis were nearly additive, but were generally less than additive on other variables. 5. The negative effects of chemical contamination on salamanders were likely because of pesticide‐induced reductions of food resources, as zooplankton abundance decreased by as much as 97% following carbaryl application. 6. Our study demonstrates the importance of the interactive effects that chemical contamination and natural environmental factors have on salamanders.     

Stages of forelimb regeneration in Ambystoma maculatum.

A series of normal stages describing the regeneration of larval A. maculatum limbs after amputation through the upper arm or wrist is described. Nine discrete stages were recognized, based on external morphological and associated histological features. These stages are Initial Dedifferentiation (ID), Early Bud (EB), Medium Bud (MB), Late Bud (LB), Early Redifferentiation (ER), Notch (N), 2-Fingerbud (2-FB), 3-Fingerbud (3-FB) and 4-Fingerbud (4-FB). Similarities and differences between this and other staging systems for urodele limb regeneration are discussed. The absence of osteoclasts was a striking feature during dedifferentiation of the wrist, in contrast to their presence in large numbers during dedifferentiation of the upper arm.     

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.     

The effects of retinoic acid on mitosis during tail and limb regeneration in the axolotl larva,Ambystoma mexicanum

Summary Regenerating tails and limbs of axolotl larvae (A. mexicanum) were studied for overall growth and for mitosis after the animals received intraperitoneal injections of all-trans retinoic acid. Both processes were depressed to approximately the same extent (60–70%). Some mitosis always survived, even when the treatment was in effect during the entire history of the regenerate. The treatment duration was a major variable in the severity of the effect, whereas the post-amputation age of the regenerate was not. In limb regenerates the epithelial cap and the mesenchymal blastema were affected to roughly the same degree.Supported by PHS Grant 507RR7031H of the BMRG, Indiana University     

Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs

Hyaluronate-mediated expansion of the extracellular matrix has been suggested as an important element of growth and morphogenesis in several developing systems. In vitro, various growth factors have been shown to stimulate hyaluronate synthesis as well as cell proliferation. A similar link between proliferation and hyaluronate production during in vivo growth is difficult to demonstrate, because in most systems the source of growth-promoting factors is either not known or not amenable to experimental manipulation. During amphibian limb regeneration, cell proliferation depends upon paracrine release of factors from axons in the limb stump, and the nerve supply can be eliminated or augmented experimentally for study of growth in this system. Denervated and amputated limbs of larval salamanders do not begin to regenerate until distal areas of the limb stumps are reinnervated. We have used such limbs to examine the effect exerted by the reappearance of nerves on the amount of hyaluronate in the tissue undergoing the growth response. Hyaluronate was demonstrated by the metachromatic dye Ethyl Stains-all, which stains hyaluronate blue while sulfated glycosaminoglycans (GAGs) and proteins in the extracellular matrix stain various shades of violet, and by microspectrophotometry of alcian-blue-stained GAGs in serial sections pretreated with buffer or with Streptomyces hyaluronidase (SH) to remove hyaluronate specifically. Both methods showed little hyaluronate in the distal region of limb stumps prior to reinnervation, while reinnervated stumps had amounts of hyaluronate similar to those of control blastemas. Autoradiography of ³H-glucosamine-labeled limbs indicated that hyaluronate in the blastemas of reinnervated limb stumps included material newly synthesized by cells throughout the growing tissue. The microspectrophotometric study revealed that the relative concentration of hyaluronate increased during the time distal limb areas were undergoing reinnervation, which was monitored by staining of nerve fibers. The increase in hyaluronate concentration was followed immediately by an increase in mitotic activity and a decrease in mesenchymal cell density, two changes leading to blastema formation that others have shown to be associated with reinnervation in this system. These observations indicate that the growth-promoting influence of nerves includes stimulation of hyaluronate production, an effect similar to that of serum or purified mitogens on many cultured cells. Hyaluronate synthesis appears to promote expansion of the limb stump, which occurs when denervated-amputated larval limbs are reinnervated.     

A staging system for forelimb regeneration in the axolotl,Ambystoma mexicanum

A staging system has been devised for normal regeneration from the upper arm in the mature axolotl. It consists of seven externally definable stages: (1) Wound healing (WH); (2) Dedifferentiation (DD); (3) Early bud (EB); (4) Medium bud (MB); (5) Late bud (LB); (6) Palette (Pal), and (7) Digital outgrowth (DO). Serial histological sections of 38 regenerating limbs were used to correlate gross stages with microscopic events in the regenerative process.     

Isolation of microsatellite loci in spotted salamanders (Ambystoma maculatum)

To assess the level and spatial pattern of genetic diversity in the spotted salamander, Ambystoma maculatum, we characterized hypervariable molecular markers by screening genomic libraries enriched for microsatellite motifs. We designed primers that reliably amplify twelve polymorphic loci and checked for variability in individuals from populations in the vicinity of Ithaca, New York. Loci show high variability in the number of alleles and heterozygosities, suggesting they will be useful for determining local population differentiation and mating systems in this pond‐breeding amphibian.     

Effect of microhabitat on fitness components of larval tiger salamanders,Ambystoma tigrinum nebulosum

Summary Survivorship and growth of larval tiger salamanders, Ambystoma tigrinum nebulosum, in the White Mountains of east-central Arizona were compared in six microhabitats using field enclosures during summers, 1983–85. Microhabitats were vegetated and nonvegetated shallows, surface, middle, and bottom horizontal layers of limnetic areas, and the vertical limnetic water column. Initial enclosure densities (0.025 larvae per 1) were identical among microhabitats. Three enclosures were placed in each microhabitat in two ponds. Larval survivorship and growth were usually greatest in vegetated shallows in lowest in middle and bottom limnetic enclosures, despite several population dieoffs. Lower fitness, as reflected in survivorship and growth, in these latter enclosures was correlated with lower food levels, temperatures, and oxygen concentrations in deeper limnetic areas. Relative fitness varied greatly between years while food levels, temperatures, and oxygen concentrations within microhabitats remained relatively constant indicating additional factors affected fitness. Disparities in fitness between microhabitats apparently affect habitat use patterns of larvae.     

Rescue of blocked cells by reinnervation in denervated forelimb stumps of larval Ambystoma

Cells of amputated, denervated larval Ambystoma forelimbs dedifferentiate and enter the cell cycle but do not subsequently proliferate sufficiently to form a blastema. The denervated limb stump resorbs slowly until reinnervation stimulates regeneration. We used this system to investigate the fate of cells in denervated limbs which undergo early but limited cycling in response to amputation. In Experiment 1, cells were labeled with [3H]thymidine (3H-T) on Day 4 postamputation (PA)/Day 3 postdenervation (PD). Labeled cells were still present on Day 7 PA, but were less frequently observed on Day 13 PA when the limbs were reinnervated and beginning to regenerate. In Experiment 2 we denervated 1 day preamputation to obtain earlier reinnervation and prevent loss of Day 4 PA labeled cells. Cells labeled with 3H-T on Day 4 PA/Day 5 PD were present throughout the denervation period and most were still present on Day 13 PA. Little or no mitotic activity was found among the labeled cells after the initial round of cycling. The apparent cell cycle block was released upon reinnervation on Days 12 and 13 PA when cycling resumed. Labeled mitotic figures were present on Day 13 PA, and the mitotic index of the labeled population increased as a result of reinnervation. These results demonstrate that blocked cells are rescued by nerves, re-enter the cell cycle, and thus contribute to the reinnervation blastema.     

Metamorphosis of the feeding mechanism in tiger salamanders (Ambystoma tigrinum): the ontogeny of cranial muscle mass

Most previous research on metamorphosis of the musculoskeletal system in vertebrates has focused on the transformation of the skeleton. In this paper we focus on the transformation of the muscles of the head during metamorphosis in tiger salamanders ( Ambystoma tigrinum ) in order (1) to provide new data on changes in myology during ontogeny, and (2) to aid in interpreting previous data on the metamorphosis of function in the head of salamanders.
The physiological cross-sectional area of nine head muscles was calculated by measuring fibre angles, fibre lengths, and muscle mass in two samples of tiger salamanders obtained just before and just after metamorphosis. The major mouth-opening muscles (rectus cervicis and depressor mandibulae) exhibit a significant decrease in estimated maximum tetanic tension (MTT) across metamorphosis of about 36%. The jaw-closing muscles (adductor mandibulae internus and externus) and the head-lifting muscles (epaxials) also decrease in MTT but not significantly. The muscles associated with tongue projection during feeding on land (the subarcualis rectus I, geniohyoideus, interhyoideus and intermandibularis) all show a slight increase in MTT at metamorphosis.
Metamorphic transformation of feeding behaviour in Ambystoma tigrinum involves changes in performance, the design of skeletal elements, changes in muscle force-generating capability, and changes in hydrodynamic design from unidirectional flow in larvae to bidirectional flow during aquatic feeding after metamorphosis. Although muscle activity patterns during aquatic feeding do not change across metamorphosis, tongue-based terrestrial feeding involves a suite of novel muscle activity patterns, morphological characters acquired at metamorphosis, and a metamorphic increase in the masses of muscles important in tongue projection.     

Regulation of proximal-distal intercalation during limb regeneration in the axolotl (Ambystoma mexicanum)

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.     

Intergenomic translocations in unisexual salamanders of the genus Ambystoma (Amphibia, Caudata)

Intergenomic interactions that include homoeologous recombinations and intergenomic translocations are commonly observed in plant allopolyploids. Homoeologous recombinations have recently been documented in unisexual salamanders in the genus Ambystoma and revealed exchanged chromosomal segments between A. laterale and A.jeffersonianum genomes in individual unisexuals. We discovered intergenomic translocations in two widespread unisexual triploids A.laterale--2 jeffersonianum (or LJJ) and its tetraploid derivative A.laterale--3 jeffersonianum (or LJJJ) by genomic in situ hybridization (GISH). Two different types of intergenomic translocations were observed in two unisexual populations and one contained novel chromosomes generated by an intergenomic reciprocal translocation. We also observed chromosome deletions in several individuals and these chromosome fragmentations were all derived from the A. jeffersonianum genome. These observed intergenomic reciprocal translocations are believed to be caused by non-homologous pairing during meiosis followed by breakage-rejoining events. Genomes of unisexual Ambystoma undergo complicated structural changes that include various intergenomic exchanges that offer unisexuals genetic and phenotypic complexity to escape their evolutionary demise. Unisexual Ambystoma have persisted as natural nuclear genomic hybrids for about four million years. These unisexuals provide a vertebrate model system to examine the interaction of distinct genomes and to evaluate the corresponding genetic, developmental and evolutionary implications of intergenomic exchanges. Intergenomic translocations and homoeologous recombinations appear to be frequent chromosome reconstruction events among unisexual Ambystoma.     

Group size structure affects patterns of aggression in larval salamanders

The potential importance of intrapopulation phenotypic variabilityto population-level ecology has been demonstrated in both theoreticaland field studies. One way to connect individuals to the dynamicsof populations they compose is to study behavioral response(an individual-level characteristic) to variability in conspecificphenotypes (a population-level characteristic). We examinedeffects of variation in size of individuals on patterns of aggressionin larval tiger salamanders (Ambystoma tigrinum nebulosum) byobserving aggressive behavior in groups of three larvae in alaboratory experment. We assessed effects of variability insize of conspecifics independently of mean larval size and larvaldensity Overall levels of aggression were generally higher ingroups in which all individuals were similprly sized than ingroups of variably sized individuals. Medium-sized individualsexhibited significantly higher levels of aggression and wereattacked significantly more often when in groups consistingonly of similarly sized larvae as compared to groups composedof larvae representing a wider range of body sizes. Activitylevels of larvae were also generally lower when all individualswere the same size, resulting in a negative correlation betweenactivity and levels of iggressititi. These results suggest thatgioups of similarly sized individuals experience a more aggressivesocial environment than groups of variably sized individuals,and they suggest a promising avenue of research for connectingindividual behavioral and physiological responses to size structure(phenotypic variability) with population dynamics.     

Population structure of spotted salamanders (Ambystoma maculatum) in a fragmented landscape

Understanding the impacts of landscape-level processes on the population biology of amphibians is critical, especially for species inhabiting anthropogenically modified landscapes. Many pond-breeding amphibians are presumed to exist as metapopulations, but few studies demonstrate the extent and consequences of this metapopulation structure. Gene flow measures may facilitate the construction of more realistic models of population structure than direct measures of migration. This is especially true for species that are cryptic, such as many amphibians. We used eight polymorphic microsatellite loci to determine the genetic population structure of spotted salamanders ( Ambystoma maculatum ) breeding at 17 ponds in northeastern Ohio, a landscape fragmented by roads, agriculture, urban areas and the Cuyahoga River. Using a variety of analyses (Bayesian clustering, F -statistics, AMOVA) we generated a model of salamander population genetic structure. Our data revealed patterns of genetic connectivity that were not predicted by geographical distances between ponds (no isolation by distance). We also tested for a relationship between population structure and several indices of landscape resistance, but found no effect of potential barriers to dispersal on genetic connectivity. Strong overall connectivity among ponds, despite the hostile habitat matrix, may be facilitated by a network of riparian corridors associated with the Cuyahoga River; however, high gene flow in this system may indicate a general ability to disperse and colonize beyond particular corridors.