Concentrations of lactic acid in neotenic and transformed tiger salamanders (Ambystoma tigrinum) before and after activity

Summary Concentrations of lactic acid in bodies of inactive neotenic tiger salamanders were similar to those recorded for inactive transformed animals. Transformed salamanders swimming against a water current for 5 min accumulated significantly more lactate in their body fluids than did neotenic salamanders, but subsequently metabolized the lactate at higher rates than characterized neotenic animals. Total body lactate in both morphs decreased rapidly in the first 1–1/2 h after activity, and within 6 h had returned to the level (common to both morphs) observed prior to activity. Whereas neotenic animals apparently swim with greater efficiency than transformed salamanders (4.26 mole ATP/gvs. 6.06 mole ATP/g expended by neotenic and transformed animals, respectively, during 5 min of continuous swimming), the transformed animals exhibit a metabolic adaptation to aquatic existence enabling them rapidly to recover from a lactacid debt.     

Interaction of endogenous and exogenous factors controlling locomotor activity rhythms in Carcinus exposed to tidal salinity cycles

The shore crab Carcinus maenas (L.) reported hitherto not to express endogenous circatidal rhythmicity in winter, is shown not to lose the ability to express such rhythmicity. Crabs maintained in constant reduced salinity in winter exhibit circatidal and circadian rhythms similar to the normal endogenous rhythms of summer caught crabs.In sinusoidal changes of salinity of tidal periodicity, reductions of salinity and increases to ambient sea water induced increased locomotor activity. The former were purely exogenous responses but the latter were also observed to entrain the underlying endogenously controlled circatidal pattern of behaviour.The occurrence of separate exogenous and endogenous responses to different phases of imposed salinity cycles has implications when seeking to understand rhythmic locomotor activity of crabs on the shore and in the search for components of the underlying physiological clock mechanism.     

Atherosclerosis induced by endogenous and exogenous toll-like receptor (TLR)1 or TLR6 agonists

Atherosclerosis is a chronic inflammatory vascular disease. Toll-like receptors (TLRs) are major initiators of inflammation. TLR2 promotes atherosclerosis in LDL receptor (LDLr)-deficient mice fed a high-fat diet (HFD). TLR2 forms heterodimers with TLR1 or TLR6 to enable inflammatory responses in the presence of distinct ligands. Here we asked whether TLR1 and/or TLR6 are required. We studied atherosclerotic disease using either TLR1- or TLR6-deficient mice. Deficiency of TLR1 or TLR6 did not diminish HFD-driven disease. When HFD-fed LDLr-deficient mice were challenged with Pam3 or MALP2, specific exogenous ligands of TLR2/1 or TLR2/6, respectively, atherosclerotic lesions developed with remarkable intensity in the abdominal segment of the descending aorta. In contrast to atherosclerosis induced by the endogenous agonists, these lesions were diminished by deficiency of either TLR1 or TLR6. The endogenous ligand(s) that arise from consumption of a HFD and promote disease via TLR2 are unknown. Either TLR1 or TLR6 are redundant for this endogenous ligand detection, or they are both irrelevant to endogenous ligand detection. However, the exogenous ligands Pam3 and MALP2 promote severe abdominal atherosclerosis in the descending aorta that is dependent on TLR1 and TLR6, respectively.     

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH.     

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum)

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a “critical-size defect” (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.     

Evaluation of two methods for measuring nonspecific immunity in tiger salamanders (Ambystoma tigrinum)

Study of amphibian immunotoxicology is a growing area of research, but very little information is available on how environmental contaminants affect disease resistance in urodele amphibians. Urodele amphibians lack the more highly evolved aspects of the specific immune system that are present in anurans, birds, and mammals. Instead, these animals rely more heavily on innate defense mechanisms than do anurans to provide rapid, nonspecific protection from pathogens. Thus, it is prudent that immunotoxicologic research with urodele amphibians includes an evaluation of effects of contaminant exposure on nonspecific immunity. The objectives of this study were to measure the phagocytic and oxidative-burst activity of peritoneal neutrophils collected from a urodele, the tiger salamander (Ambystoma tigrinum), and to evaluate the use of these assays in immunotoxicologic research using urodele amphibians. Using tiger salamanders collected in August 2000, phagocytosis and oxidative-burst assays modified from mammalian protocols were conducted through October 2001. Results indicated that large numbers of peritoneal neutrophils for use in immunotoxicologic tests can be collected from salamanders injected with thioglycollate. Moreover, these neutrophils readily engulfed foreign material (phagocytic activity) and produced measurable amounts of hydrogen peroxide (oxidative-burst activity). Phagocytosis was effectively inhibited by incubating cells with sodium azide (P<0.001), and quantification of phagocytosis using flow cytometry was well correlated with manual counts (r=0.84, P<0.001). Dexamethasone treatment reduced phagocytic activity as measured by manual counts (P<0.02), suggesting that this test is useful for detecting alteration by immunosuppressive agents. In contrast, oxidative function was unaffected by dexamethasone treatment, and results from the oxidative-burst assay were generally less consistent than those from the phagocytosis assay. Based on these results, phagocytic activity of peritoneal neutrophils may be a useful endpoint in immunotoxicologic studies to evaluate the impact of environmental contaminants on innate defense mechanisms in urodele amphibians.     

Uptake and metabolism of exogenous and endogenous thyroxine in the brain of young rats.

The uptake of labelled exogenous thyroxine by the brain was determined in 10- and 30-day-old rats. It was three times higher and thyroxine was more deiodinated on day 10 than on day 30. On the other hand, the endogenous hormonal iodine content was estimated in both the brain and the isolated cortical neurones in the young rats of the same ages equilibrated with 125I. On day 10, brain and neurones contained five times more thyroid hormones than on day 30. These results are discussed in the context of the relations previously discovered between development of thyroid function and brain maturation.     

Stimulation of maize invertase activity following infection by Ustilago maydis

The effect of U. maydis infection on the invertase activity of maize leaves has been studied. Infection causes a specific stimulation of an acid invertase in soluble and pellet fractions of homogenized tissue. Invertase activity is stimulated within one day after infection, and is maintained at a high level until 10 days after infection, in contrast to the progressive decline in activity with age, in healthy leaves. Analyses of soluble extracts by gel electrophoresis suggest that the invertase showing this increase is derived from the host and not the pathogen.     

The relationship between locomotor activity and sexual behaviour in ebony strains of Drosophila melanogaster

Male flies homozygous for the ebony¹¹ mutant allele suffer a severe visual impairment and consequently show poor mating success. However, ebony males show improved mating in darkness because they initiate courtship more quickly than in the light. They also exhibit a competitive superiority over wild-type males under darkened conditoons that correlates with the observed increase in the mutant's locomotor activity. Locomotor activity levels were subsequently shown to correlate with several indices of mating success under both dark and illuminated conditions. The evolutionary implications of this relationship between the two characters are discussed.     

The effect of dusk and dawn on the locomotor activity rhythm of Talitrus saltator (Montagu) (Crustacea: Amphipoda)

Entrainment of the nocturnal, endogenous locomotor activity rhythm of Talitrus saltator (Montagu) by the natural light-dark cycle is non-parametric, the phase of the rhythm shifting only in response to changes in the time of an experimentally simulated ‘dawn’ transition. The difference in response to light at ‘dusk’ and ‘dawn’ illustrates a phase-dependent responsiveness common to endogenous rhythms. Activity begins after complete darkness, with cessation always occurring during the ‘dawn’ transition and never continuing past the onset of total experimental illumination. The point of activity cessation is taken to be the position of a synchronizing cue controlling entrainment. The ‘dawn’ cue appears to be an absolute irradiance level of approximately 1.5 × 10^?4 W/m² (1.5 lux). The implications of such a cue are discussed in relation to field behaviour.     

Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins

Immature rat ovaries increase their secretion of estradiol (E₂) when stimulated by gonadotropins but only after a lag period of several hours. Once established, estrogen secretion can be maintained, or increased, by the continued presence of gonadotropin. A combination of ovine FSH+LH given at 2 hr intervals stimulated the estrogen synthesizing system ($\text{ESS}$) of the ovary and serum E₂ showed a pronounced rise between 16 and 20 hrs after the initial injection. When given every 2 hrs for 5 doses (0–8 hrs) serum E₂ was undetectable. However, it was increased if 20 IU PMS was injected at the time of the last dose of FSH+ LH. Endogenous FSH&LH, increased by hourly injections of LH-releasing hormone for a period of 8 hrs, stimulated the $\text{ESS}$; serum E₂ increased at the expected time when this treatment was followed by an injection of PMS.Anti-PMS antiserum given 12 hrs after PMS, prevented the expected rise in serum E₂ at 24 hrs. However, FSH, LH or a combination of the two given every 2 hrs beginning at the time of the anti-PMS produced an increase in E₂ secretion; the combination was more effective than either hormone alone.These results are consistent with the interpretation that a combined FSH-LH action is responsible for induction of the $\text{ESS}$ in the immature rat ovary. The combination of hormones is also very effective in maintaining estrogen secretion but some function appears possible with FSH or LH alone.     

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum)

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, transspecies evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relict of their unique historical demography, not their phylogenetic legacy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Dopamine enhances locomotor activity for mating in male honeybees (Apis mellifera L.)

Dopamine plays multiple roles in the regulation of reproduction in female honeybees where it appears to act independently of juvenile hormone (JH). In males the role of dopamine and its relationship to JH control have not been elucidated. In the present study we determined hemolymph levels of dopamine and its metabolite (N-acetyldopamine) in males at post-emergence days 0-16. The development of locomotor and flight activities were recorded over the same period. Hemolymph levels of dopamine and N-acetyldopamine were found to increase at the time of onset of mating flight activity and those of dopamine decreased thereafter. Both locomotor and flight activities increased in parallel with hemolymph dopamine levels but the increased activity levels were maintained following decline of dopamine levels. Brain and meso-metathoracic ganglia levels of dopamine showed a similar developmental profile to hemolymph dopamine levels. Locomotor activities were temporarily inhibited by injection of a dopamine-receptor antagonist (cis(Z)-flupenthixol) into the thorax, and were enhanced by injection of a dopamine-receptor agonist (6,7-ADTN). These results suggest that dopamine regulates locomotor activities for mating and plays a role downstream of JH in premature males in honeybees.     

Habitat preferences of two estuarine burrowing crabs Helice crassa Dana (Grapsidae) and Macrophthalmus hirtipes (Jacquinot) (Ocypodidae)

The horizontal and vertical distributions of two species of endemic, burrowing mud crabs Helice crassa Dana 1851 (Grapsidae) and Macrophthalmus hirtipes (Jacquinot 1853) (Ocypodidae) are described for the Avon-Heathcote Estuary (43°33′S: 172°44′E), Christchurch, New Zealand. Substratum preference is shown to be the most important factor influencing mud crab distribution, but lack of tolerance to salinities below 4‰ is also a significant factor preventing M. hirtipes from occurring at points close to freshwater input. Both species had similar sediment organic content and particle size requirements. Helice crassa was concentrated in well-drained, compacted sediments above mid-tide level, whilst Macrophthalmus hirtipeswas found in waterlogged areas below mid-tide level. This vertical separation is shown not to be caused by differential desiccation tolerances, but by feeding and burrowing adaptations related to these different substrata.     

Developmental regulation of hepatic ceruloplasmin mRNA and serum activity by exogenous thyroxine and dexamethasone.

Ceruloplasmin (Cp) is a copper-dependent oxidase with roles that include the regulation of iron metabolism, participation in the acute-phase response to inflammation, and antioxidant systems. Although developmental increases in hepatic Cp gene expression and serum activity have been described, the molecular mechanisms that are responsible for this regulation are not fully understood. The studies described here explored the possible role of glucocorticoids and thyroxine (T4) in the early neonatal development of Cp by the administration of these hormones on postnatal Day 1 (24 hr after birth), and the measurement of both hepatic Cp mRNA and serum activity through postnatal Day 10. Administration of the synthetic glucocorticoid hormone, dexamethasone (2 micrograms/g body wt), resulted in an increase in Cp mRNA on Days 3-7 that was accompanied by an increase in serum Cp activity that reached statistical significance at Day 10. Exogenous T4 (2 micrograms/g body wt) significantly increased Cp mRNA 24 hr after administration. Serum Cp activity was also significantly elevated by the early neonatal administration of T4. Furthermore, gestational hypothyroidism resulted in a significant decrease in Cp activity after postnatal Day 3. These data suggest a role for thyroid hormone and possibly glucocorticoids in the normal developmental regulation of Cp.     

Identification of endogenous and exogenous activity in a molluscan neurone by spike train analysis

The statistical properties of background spike train activity recorded from a molluscan neurone are used to identify lengths of discharge which are produced by endogeneous pacemaker mechanisms. Such pacemaker discharge has an infinitely divisible interspike interval probability density function.     

Metamorphosis of cranial design in tiger salamanders (Ambystoma tigrinum): A morphometric analysis of ontogenetic change

While ontogenetic analyses of skull development have contributed to our understanding of phylogenetic patterns in vertebrates, there are few studies of taxa that undergo a relatively discrete and rapid change in morphology during development (metamorphosis). Morphological changes occurring in the head at metamorphosis in tiger salamanders (Ambystoma tigrinum) were quantified by a morphometric analysis of cranial osteology and myology to document patterns of change during metamorphosis. We employed a cross-sectional analysis using a sample of larvae just prior to metamorphosis and a sample of transformed individuals just after metamorphosis, as well as larvae undergoing metamorphosis. There were no differences in external size of the head among the larval and transformed samples. The hyobranchial apparatus showed many dramatic changes at metamorphosis, including shortening of ceratobranchial 1 and the basibranchial. The subarcualis rectus muscle increased greatly in length at metamorphosis, as did hypobranchial length and internasal distance. A truss analysis of dorsal skull shape showed that at metamorphosis the snout becomes wider, the maxillary and squamosal triangles rotate posteromedially, and the neurocranium shortens (while maintaining its width), resulting in an overall decrease in skull length at metamorphosis. These morphometric differences are interpreted in light of recent data on the functional morphology of feeding in salamanders. Morphological reorganization of the hyobranchial apparatus and shape changes in the skull are related to the acquisition of a novel terrestrial feeding mode (tongue projection) at metamorphosis. Metamorphic changes (both internal and external) that can be used to judge metamorphic condition are discussed.