Fabio Maria Guarino

Localization of FMRFamide-Like Immunoreactivity in the Brain of the Viviparous Skink (Chalcides chalcides)

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.

The blue lizard spandrel and the island syndrome

BackgroundMany small vertebrates on islands grow larger, mature later, lay smaller clutches/litters, and are less sexually dimorphic and aggressive than their mainland relatives. This set of observations is referred to as the Island Syndrome. The syndrome is linked to high population density on islands. We predicted that when population density is low and/or fluctuating insular vertebrates may evolve correlated trait shifts running opposite to the Island Syndrome, which we collectively refer to as the reversed island syndrome (RIS) hypothesis. On the proximate level, we hypothesized that RIS is caused by increased activity levels in melanocortin receptors. Melanocortins are postranslational products of the proopiomelanocortin gene, which controls pleiotropically pigmentation, aggressiveness, sexual activity, and food intake in vertebrates.ResultsWe tested the RIS hypothesis performing a number of behavioral, genetic, and ontogenetic tests on a blue colored insular variant of the Italian Wall lizard Podarcis sicula, living on a small island off the Southern Italian coast. The population density of this blue-colored variant was generally low and highly fluctuating from one year to the next.In keeping with our predictions, insular lizards were more aggressive and sexually dimorphic than their mainland relatives. Insular males had wide, peramorphic heads. The growth rate of insular females was slower than growth rates of mainland individuals of both sexes, and of insular males. Consequently, size and shape dimorphism are higher on the Island. As predicted, melanocortin receptors were much more active in individuals of the insular population. Insular lizards have a higher food intake rate than mainland individuals, which is consistent with the increased activity of melanocortin receptors. This may be adaptive in an unpredictable environment such as Licosa Island. Insular lizards of both sexes spent less time basking than their mainland relatives. We suspect this is a by-product (spandrel) of the positive selection for increased activity of melanocortins receptors.ConclusionsWe contend that when population density is either low or fluctuating annually as a result of environmental unpredictability, it may be advantageous to individuals to behave more aggressively, to raise their rate of food intake, and allocate more energy into reproduction.

Cytokines in the viviparous reproduction of squamate reptiles: Interleukin-1α (IL-1α) and IL-1β in placental structures of a skink

Placental viviparity is known in many species of squamate reptiles. Among these, some scincids have developed an epithelio-chorial chorio-allantoic placenta which in the structure of its central ridged zone is similar to those of certain therian mammalian species. A broad range of immunoregulatory peptides, cytokines, has been identified at the maternofetal interface of several species of mammals, either with invasive or non-invasive types of placenta. Thus we began to study whether interleukin-1, which is considered to play a crucial role in mammalian pregnancy, might also be involved in the viviparity of reptilian species. Placentae of Chalcides chalcides L. were processed by immunohistochemistry and incubated in a culture medium for different times. A very strong immunoreactivity for interleukin-1 alpha (IL-1 alpha) and for interleukin-1 beta (IL-1 beta) was present in the chorial epiblast and in uterine epithelial cells, with varying degree and localization in different periods of pregnancy. IL-1 beta was also released into the medium at different amounts during incubation. In light of the mammalian data, our results suggest that the role of cytokines in pregnancy may represent a significant event in the evolution of placental viviparity.

Natural history and larval morphology of Boophis occidentalis (Anura: Mantellidae: Boophinae) provide new insights into the phylogeny and adaptive radiation of endemic Malagasy frogs

During a zoological survey in north-west Madagascar (Sahamalaza Peninsula) we documented aspects of the natural history of Boophis occidentalis (formerly a subspecies of B. albilabris and here raised to species level). Individual age assessed by skeletochronology ranged from 4 to 11 years. Breeding behaviour was observed at a seasonal stream after heavy rainfalls: five choruses of eight to 90 males aggregated in shallow brook sections. The males emitted low frequency calls and engaged in scramble battles. Only two females were found. A couple laid a large number of eggs, attached as a single layer on submerged stones. Tadpoles reared from these eggs had the typical morphology of brook-breeding Boophis species with a 1:5+5/3 keratodont formula and a relatively flattened body. Adult males had an anteroventrally oriented cloaca, a morphological trait shared with B. albilabris. Advertisement calls were similar in general structure to those of B. albilabris recorded at Anjanaharibe-Sud. They were unharmonious and consisted of pulsed notes emitted at irregular intervals. Much lower pulse rates in B. albilabris (30‐38 pulses/s) than in B. occidentalis (90‐109 pulses/s) support the specific distinctness of these taxa. The combination of characters related to reproductive biology in B. occidentalis and B. albilabris is unique in Boophis. The acontinuous breeding behaviour and large number of eggs per clutch are traits found in the pond-breeding species of the B. tephraeomystax group, while larval morphology and habitat are characteristic of brook-breeding species. Available morphological and molecular data place B. albilabris and B. occidentalis into the brookbreeding Boophis radiation. The acontinuous timing of reproduction and large egg number may be secondary adaptations that allowed the species to colonize arid regions of western Madagascar.

Growth and Longevity by Skeletochronological Analysis in Mantidactylus microtympanum, a Rain-Forest Anuran from Southern Madagascar

dant bone matrix and lines of arrested growth (LAGs). Conversely, amphibians and reptiles from tropical and subtropical areas, and particularly those inhabiting rain forests with relatively constant environments, are expected to have either ill-defined or no LAGs, since these species presumably have no or scarce interruptions and hence continuous growth. Alternatively, if these species undergo annual patterns of inactivity, then a seasonal pattern of LAGs should be detectable. However, despite the great number of skeletochronological papers recently published on amphibians (Castanet et al., 1993, 1996; Guarino et al., 1995), no one has ever investigated amphibians from tropical rain forests. The aim of this study was to examine skeletochronology in Mantidactylus microtympanum, an endemic anuran from the Malagasy rain forest, and to determine whether this technique was appropriate for estimating age structure of tropical herpetofauna. Blommers-Schlosser (1993) includes M. microtympanum in the subfamily Mantellinae (Ranidae), which is endemic to Madagascar. This large-sized species (snoutvent length up to 100 mm) inhabits rain-forest streams in the southeast of the GrandIle, and it is active and visible mainly at night. Males and females differ in some characters, such as belly convexity, biometric ratios, and peculiar structure of cloaca in the female. Both juveniles and adults have a brownish back, although it is mottled in the former. The large size of the species allowed us to perform skeletochronological analysis on phalanges alone and, therefore to mark and release the individuals on the site of

Lethal Thermal Impact at Periphery of Pyroclastic Surges: Evidences at Pompeii

Background The evaluation of mortality of pyroclastic surges and flows (PDCs) produced by explosive eruptions is a major goal in risk assessment and mitigation, particularly in distal reaches of flows that are often heavily urbanized. Pompeii and the nearby archaeological sites preserve the most complete set of evidence of the 79 AD catastrophic eruption recording its effects on structures and people. Methodology/Principal Findings Here we investigate the causes of mortality in PDCs at Pompeii and surroundings on the bases of a multidisciplinary volcanological and bio-anthropological study. Field and laboratory study of the eruption products and victims merged with numerical simulations and experiments indicate that heat was the main cause of death of people, heretofore supposed to have died by ash suffocation. Our results show that exposure to at least 250°C hot surges at a distance of 10 kilometres from the vent was sufficient to cause instant death, even if people were sheltered within buildings. Despite the fact that impact force and exposure time to dusty gas declined toward PDCs periphery up to the survival conditions, lethal temperatures were maintained up to the PDCs extreme depositional limits. Conclusions/Significance This evidence indicates that the risk in flow marginal zones could be underestimated by simply assuming that very thin distal deposits, resulting from PDCs with poor total particle load, correspond to negligible effects. Therefore our findings are essential for hazard plans development and for actions aimed to risk mitigation at Vesuvius and other explosive volcanoes.

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4′ -′ 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

Potential role of interleukin-1 at the peri-ovulation stage in a species of placental viviparous reptile, the three-toed skink, Chalcides chalcides (squamata: scincidae)

We recently showed that interleukin-1 (IL-1) is secreted by the placenta of a species of squamate reptile, the three-toed skink, Chalcides chalcides. In this study, we used immunohistochemical techniques to investigate the expression of IL-1 (in the two isoforms, IL-1α and IL-1β) and its specific membrane receptor IL-1 RtI in uterine oviduct during the peri-implantation period. We found that both IL-1 and its receptor were expressed in uterine tissues before and after ovulation (in the pre-ovulatory stage, even before the yolk had formed in the ovary). However, while IL-1α was mostly localized in the uterine mesenchyme tissue, IL-1β and IL-1RtI were present in the uterine epithelium. Our data provide a further comparison between the reproduction of mammals and squamate reptiles.

Giant and long-lived? Age structure in Macroscincus coctei, an extinct skink from Cape Verde

The age structure of the extinct giant Cape Verde skink Macroscincus coctei was studied using skeletochronology applied to 11 preserved males, 15 females, and one juvenile, collected at the end of the 19th century. Counts of lines of arrested growth (LAGs) indicated that in this species — which lived on islets with extreme natural conditions (e.g., hard aridity, lack of rainfall) — the maximum life span was estimated as 16 years for males and 12 years for females; the single juvenile was 4 years old. Although the sexes had similar mean ages, mean body length of males was significantly higher than that of females (283 versus 255 mm). There was no clear relationship between body length and estimated age, although the largest males were also the oldest. The analysis of LAGs revealed a sexual difference in growth rates, males reaching greater size earlier than females.

Reproductive activity of bombina pachypus from southern Italy

Abstract Seasonal changes in gonadal morphology, secondary sexual characters, and plasma sex steroids were studied in male and female Bombina pachypus from southern Italy. In this species, frogs are active and breed from May to September. During the long breeding season, sperm were always present in the testes. However, the testes did not always exhibit all stages of spermatogene‐sis. In July, spermatogenesis did not exceed the stage of sperma‐tocyte I, whereas in August it included all stages. It is possible that males use sperm originating from the preceding fall for the earliest spring matings. Throughout the breeding season, females showed many follicles in advanced or complete vitellogenesis and many atretic vitellogenic follicles. Postovulatory follicles, if any, were rare. Females laid few eggs at each oviposition. Concerning male sex steroids, testosterone was at comparable levels from May to September, and it markedly decreased in October. In females, wide variations in plasma estradiol and proge...