Molecular Genetics Unveiled Unknown Family Relationships and Hybrids in an Ex-Situ Colony of African Penguins (Spheniscus demersus).

Genealogical relationships among colony members, inbreeding status, and presence of hybrids are crucial data that can assist zoo curators in captive colony management and decision-making on relocation for reproduction. This study employed molecular markers to study a large colony (n = 56) of African Penguin hosted in an Italian biopark. A panel of 15 STRs (single tandem repeats) was selected, and genotype data were analyzed using COLONY software to determine parentage relationships and compare the existing studbook information to a pedigree built from genetic analyses. The existence of extra-pair mating and the presence of hybrids were investigated: discrepancies in kinship relationships emerged following molecular parentage analysis and 10 unknown genetic relationships were revealed. Infidelity of one member of the pair was observed in 6 cases and extra-pair copulation was assessed by genetic analysis in 2 episodes. One member of the colony was found to be a hybrid (S. demersus × S. humboldti); his progeny, derived by extra-pair copulation, was traced. Three other hidden hybrids were discovered and assessed using the identified candidate private alleles. Overall, our results demonstrate that molecular methods to confirm parentage and analyze relatedness among colony members are a valuable tool to complement studbook-based genetic management of African penguin captive populations. Because a variety of behavioral dynamics (e.g., extra-pair mating) can make observations ineffective in some species and because molecular markers outperform studbook in identifying the presence of hybrids, reliance on studbook information alone is not recommended.

Evidence suggests vocal production learning in a cross-fostered Risso's dolphin (Grampus griseus).

Vocal learning is a rare skill in mammals, and we have limited information about the contexts in which they use it. Previous studies suggested that cetaceans in general are skilled at imitating sounds, but only few species have been studied to date. To expand this investigation to another species and to investigate the possible influence of the social environment on vocal learning, we studied the whistle repertoire of a female Risso's dolphin (Grampus griseus) that was stranded at an early age and was subsequently raised in a group of bottlenose dolphins (Tursiops truncatus). We show that this cross-fostered animal produced vocal signals more akin to those of its Tursiops poolmates than those of Risso's dolphins in the wild. This is one of very few systematic cross-fostering studies in cetaceans and the first to suggest vocal production learning in the Risso's dolphin. Our findings also suggest that social experience is a major factor in the development of the vocal repertoire in this species.

Sex- and age-related variation in metal content of penguin feathers.

The presence of xenobiotics, such as metals, in ecosystems is concerning due to their durability and they pose a threat to the health and life of organisms. Moreover, mercury can biomagnify in many marine food chains and, therefore, organisms at higher trophic levels can be adversely impacted. Although feathers have been used extensively as a bio-monitoring tool, only a few studies have addressed the effect of both age and sex on metal accumulation. In this study, the concentrations of trace elements were determined in the feathers of all members of a captive colony of African Penguins (Spheniscus demersus) housed in a zoological facility in Italy. Tests were performed by inductively coupled plasma-mass spectrometry to detect aluminum, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium, tin, vanadium, and zinc. Mercury was detected by a direct mercury analyzer. Sexing was performed by a molecular approach based on analyzing the chromo-helicase-DNA-binding1 gene, located on the sex chromosomes. Sex- and age-related differences were studied in order to investigate the different patterns of metal bioaccumulation between male and female individuals and between adults and juveniles. Juvenile females had significantly higher arsenic levels than males, while selenium levels increased significantly with age in both sexes. Penguins kept in controlled environments-given that diet and habitat are under strict control-represent a unique opportunity to determine if and how metal bioaccumulation is related to sex and age.

Postnatal development of echolocation abilities in a bottlenose dolphin (Tursiops truncatus): temporal organization.

In spite of all the information available on adult bottlenose dolphin (Tursiops truncatus) biosonar, the ontogeny of its echolocation abilities has been investigated very little. Earlier studies have reported that neonatal dolphins can produce both whistles and burst-pulsed sounds just after birth and that early-pulsed sounds are probably a precursor of echolocation click trains. The aim of this research is to investigate the development of echolocation signals in a captive calf, born in the facilities of the Acquario di Genova. A set of 81 impulsive sounds were collected from birth to the seventh postnatal week and six additional echolocation click trains were recorded when the dolphin was 1 year old. Moreover, behavioral observations, concurring with sound production, were carried out by means of a video camera. For each sound we measured five acoustic parameters: click train duration (CTD), number of clicks per train, minimum, maximum, and mean click repetition rate (CRR). CTD and number of clicks per train were found to increase with age. Maximum and mean CRR followed a decreasing trend with dolphin growth starting from the second postnatal week. The calf's first head scanning movement was recorded 21 days after birth. Our data suggest that in the bottlenose dolphin the early postnatal weeks are essential for the development of echolocation abilities and that the temporal features of the echolocation click trains remain relatively stable from the seventh postnatal week up to the first year of life.

Morphology of planktonic zoeal stages of Palicus caronii (Decapoda, Brachyura), identified by DNA barcoding, provides novelties to Palicoidea larval systematics.

The zoeal development of the brachyuran crab, Palicus caronii, comprises two zoeal stages and the morphology is described and illustrated in detail. The zoeae were collected in plankton samples from the Southern Ligurian Sea (Western Mediterranean). Although the morphology of the larval stages of this species was unknown, a combination of characters allowed the zoeae to initially be assigned to the Palicidae, based on the previous unique known first zoeal description of one species of this family. Later, the identification of the larvae as Palicus caronii was confirmed through molecular analysis. The morphological features of the zoeae that characterize the Palicidae and separate them from the Crossotonotidae are confirmed. Also, the larval development comprising only two zoeal stages observed in Palicus caronii, the peculiar and uncommon carapace surface setation, and the presence of anterodorsal and posterodorsal sensory dorsal organs suggest that these characters could be common to the Palicoidea.

Description of the zoeal stages of Periclimenes aegylios Grippa d'Udekem d'Acoz, 1996 (Crustacea: Decapoda: Palaemonidae) reared in the laboratory.

The eight zoeal stages of the Mediterranean shrimp Periclimenes aegylios are described and illustrated in detail, using laboratory-reared specimens. This study improved the partial and unpublished descriptions of the zoeae of this species. The complete and accurate definition of the morphology of all the stages now allows comparison of the zoeae of P. aegylios with those of other Mediterranean Periclimenes species, as well as the allopatric Atlantic P. sagittifer, from which P. aegylios was separated in 1996.

Trace elements, rare earth elements and inorganic arsenic in seaweeds from Giglio Island (Thyrrenian Sea) after the Costa Concordia shipwreck and removal.

The occurrence of trace elements, REE and iAs was investigated in macroalgae collected from Giglio Island (Grosseto, Italy), 3 years after the Costa Concordia shipwreck recovery operations. There was a high variability of metals and REE between species, even those belonging to the same phylum. Arsenic level was found within the range of the Tuscany marine environment; the inorganic fraction was from 9% to 31%. Al, Be, Pb and Zn levels in seaweeds from Giglio Island were found to be significantly higher than in macroalgae from other islands of the Tuscany archipelago. REE were double the values found in the Tuscan islands of Elba and Capraia. We suggest that concentrations of metals and REE were influenced by the 3-year-long naval operations carried out for the Costa Concordia shipwreck rescue, and that macroalgae could be suitable bioindicators of perturbations in metal concentrations in the marine environment. CAPSULE: Inorganic contaminants in seaweeds for Giglio Island.

Trace metal occurrence in Mediterranean seaweeds.

Seaweeds have been used as animal feed since a long time and are consumed as food in several cultures. In fact, macroalgae are a source of protein, fiber, polyunsaturated fat, and minerals. The concentration of trace elements was determined in dominant macroalga species from three sites of the northwestern Mediterranean Sea. A high interspecies variability was observed, with higher metal levels in brown and green than those in red seaweeds. The maximum values set by European regulations for arsenic, mercury, and cadmium in food and feed were never exceeded, but a few samples were very close to limits set for mercury. Conversely, the maximum limit for lead in feed was exceeded in all species from one of the considered sites. Analogously, lead in seaweeds could constitute a potential risk for human health, due to the exceeding of the maximum value set for food supplements.

Acoustic correlates of body size and individual identity in banded penguins.

Animal vocalisations play a role in individual recognition and mate choice. In nesting penguins, acoustic variation in vocalisations originates from distinctiveness in the morphology of the vocal apparatus. Using the source-filter theory approach, we investigated vocal individuality cues and correlates of body size and mass in the ecstatic display songs the Humboldt and Magellanic penguins. We demonstrate that both fundamental frequency (f0) and formants (F1-F4) are essential vocal features to discriminate among individuals. However, we show that only duration and f0 are honest indicators of the body size and mass, respectively. We did not find any effect of body dimension on formants, formant dispersion nor estimated vocal tract length of the emitters. Overall, our findings provide the first evidence that the resonant frequencies of the vocal tract do not correlate with body size in penguins. Our results add important information to a growing body of literature on the role of the different vocal parameters in conveying biologically meaningful information in bird vocalisations.

A first report of rare earth elements in northwestern Mediterranean seaweeds.

The concentrations of rare earth elements (REE) were determined by ICP-MS in dominant seaweed species, collected from three locations of the northwestern Mediterranean Sea. This is the first study to define levels and patterns of REE in macro algae from these coastal areas. Rare elements are becoming emerging inorganic contaminants in marine ecosystems, due to their worldwide increasing applications in industry, technology, medicine and agriculture. Significant inter-site and interspecies differences were registered, with higher levels of REE in brown and green macro algae than in red seaweeds. Levels of light REE were also observed to be greater compared to heavy REE in all samples. One of the investigated locations (Bergeggi, SV) had higher REE and ΣREE concentrations, probably due to its proximity to an important commercial and touristic harbor, while the other two sites were less affected by anthropogenic contaminations, and showed comparable REE patterns and lower concentrations. CAPSULE: Rare earth elements in seaweeds.

Vocal individuality and species divergence in the contact calls of banded penguins.

Penguins produce contact calls to maintain social relationships and group cohesion. Such vocalisations have recently been demonstrated to encode individual identity information in the African penguin. Using a source-filter theory approach, we investigated whether acoustic cues of individuality can also be found in other Spheniscus penguins and the acoustic features of contact calls have diverged within this genus. We recorded vocalisations from two ex-situ colonies of Humboldt penguin and Magellanic penguin (sympatric and potentially interbreeding in the wild) and one ex-situ group of African penguins (allopatric although capable of interbreeding with the other two species in captivity). We measured 14 acoustic parameters from each vocalisation. These included temporal (duration), source-related (fundamental frequency, f0), and filter-related (formants) parameters. They were then used to carry out a series of stepwise discriminant function analyses (with cross-validation) and General Linear Model comparisons. We showed that contact calls allow individual discrimination in two additional species of the genus Spheniscus. We also found that calls can be classified according to species in a manner far greater than that attributable by chance, even though there is limited genetic distance among African, Humboldt, and Magellanic penguins. Our results provide further evidence that the source-filter theory is a valuable framework for investigating the biologically meaningful information contained in bird vocalisations. Our findings also provide novel insights into penguin vocal communication and suggest that contact calls of the penguin family are affected by selection for individuality.

Pond use by captive African penguins (Spheniscus demersus) in an immersive exhibit adjacent to human bathers.

Nonhuman animals in zoos are exposed to a continuous human presence, which affects their behaviors and welfare. However, little is known about what role the "visitor effect" has on penguins in captivity. The African penguin (Spheniscus demersus) is an endangered species commonly housed in zoos worldwide. The aim of this study was to evaluate whether the abundance of human bathers could reduce the average time spent in the water of a colony of African penguins housed in an exhibit where their pond habitat was adjacent to a swimming pool. Observations were carried out on 7 penguins in the summer of 2009. Data were collected during 3 time periods (Time 1 [T1] = opening of the swimming season, Time 2 [T2] = core of the season, Time 3 [T3] = late season) of 14 days each. The human disturbance caused by bathers strongly reduced the pond use by penguins at T1 and T2, especially when there were large numbers of visitors. However, at T3, we observed that the overall use of the pond by penguins increased, and the average duration of their diving was no longer dependent on the number of visitors.

Hydrophilic extract from Posidonia oceanica inhibits activity and expression of gelatinases and prevents HT1080 human fibrosarcoma cell line invasion.

Posidonia oceanica (L.) Delile is an endemic Mediterranean sea-grass distributed in the infralittoral zones, where it forms meadows playing a recognized ecological role in the coastal marine habitat. Although its use as a traditional herbal remedy is poorly documented, recent literature reports interesting pharmacological activities as antidiabetic, antioxidant and vasoprotective. Differently from previous literature, this study presents a hydrophilic extraction method that recovers metabolites that may be tested in biological buffers. We showed for the first time in the highly invasive HT1080 human fibrosarcoma cell line that our hydrophilic extract from P. oceanica was able to strongly decrease gene and protein expression of gelatinases MMP-2 and MMP-9 and to directly inhibit in a dose-dependent manner gelatinolytic activity in vitro. Moreover, we have revealed that our extract strongly inhibited HT1080 cell migration and invasion. Biochemical analysis of the hydrophilic extract showed that catechins were the major constituents with minor contribution of gallic acid, ferulic acid and chlorogenic plus a fraction of uncharacterized phenols. However, if each individual compound was tested independently, none by itself was able to induce a direct inhibition of gelatinases as strong as that observed in total extract, opening up new routes to the identification of novel compounds. These results indicate that our hydrophilic extract from P. oceanica might be a source of new pharmacological natural products for treatment or prevention of several diseases related to an altered MMP-2 and MMP-9 expression.

Vocal individuality cues in the African penguin (Spheniscus demersus): a source-filter theory approach.

The African penguin is a nesting seabird endemic to southern Africa. In penguins of the genus Spheniscus vocalisations are important for social recognition. However, it is not clear which acoustic features of calls can encode individual identity information. We recorded contact calls and ecstatic display songs of 12 adult birds from a captive colony. For each vocalisation, we measured 31 spectral and temporal acoustic parameters related to both source and filter components of calls. For each parameter, we calculated the Potential of Individual Coding (PIC). The acoustic parameters showing PIC ≥ 1.1 were used to perform a stepwise cross-validated discriminant function analysis (DFA). The DFA correctly classified 66.1% of the contact calls and 62.5% of display songs to the correct individual. The DFA also resulted in the further selection of 10 acoustic features for contact calls and 9 for display songs that were important for vocal individuality. Our results suggest that studying the anatomical constraints that influence nesting penguin vocalisations from a source-filter perspective, can lead to a much better understanding of the acoustic cues of individuality contained in their calls. This approach could be further extended to study and understand vocal communication in other bird species.

The vocal repertoire of the African Penguin (Spheniscus demersus): structure and function of calls.

The African Penguin (Spheniscus demersus) is a highly social and vocal seabird. However, currently available descriptions of the vocal repertoire of African Penguin are mostly limited to basic descriptions of calls. Here we provide, for the first time, a detailed description of the vocal behaviour of this species by collecting audio and video recordings from a large captive colony. We combine visual examinations of spectrograms with spectral and temporal acoustic analyses to determine vocal categories. Moreover, we used a principal component analysis, followed by signal classification with a discriminant function analysis, for statistical validation of the vocalisation types. In addition, we identified the behavioural contexts in which calls were uttered. The results show that four basic vocalisations can be found in the vocal repertoire of adult African Penguin, namely a contact call emitted by isolated birds, an agonistic call used in aggressive interactions, an ecstatic display song uttered by single birds, and a mutual display song vocalised by pairs, at their nests. Moreover, we identified two distinct vocalisations interpreted as begging calls by nesting chicks (begging peep) and unweaned juveniles (begging moan). Finally, we discussed the importance of specific acoustic parameters in classifying calls and the possible use of the source-filter theory of vocal production to study penguin vocalisations.

Support vector machines to model presence/absence of Alburnus alburnus alborella (Teleostea, Cyprinidae) in North-Western Italy: comparison with other machine learning techniques.

Alburnus alburnus alborella is a fish species native to northern Italy. It has suffered a very sharp decrease in population over the last 20 years due to human impact. Therefore, it was selected for reintroduction projects. In this research project, support vector machines (SVM) were tested as possible tools for building reliable models of presence/absence of the species. A system of 198 sites located along the rivers of Piedmont in North-Western Italy was investigated. At each site, 19 physical-chemical and environmental variables were measured. We verified that performances did not improve after feature selection but, instead, they slightly decreased (from Correctly Classified Instances [CCI]=84.34 and Cohen's k [k]=0.69 to CCI=82.81 and k=0.66). However, feature selection is crucial in identifying the relevant features for the presence/absence of the species. We then compared SVMs performances with decision trees (DTs) and artificial neural networks (ANNs) built using the same dataset. SVMs outperformed DTs (CCI=81.39 and k=0.63) but not ANNs (CCI=83.03 and k=0.66), showing that SVMs and ANNs are the best performing models, proving that their application in freshwater management is more promising than traditional and other machine-learning techniques.

Performance comparison among multivariate and data mining approaches to model presence/absence of Austropotamobius pallipes complex in Piedmont (North Western Italy).

Freshwater inhabitants in Piedmont (Italy) have been deeply disadvantaged by environmental changes caused by human disturbance. Hence there are engendered species that need human intervention of an entirely different kind - better management through the development of innovative practical tools. The most ecologically important of the river-dwelling invertebrates is a threatened species, the native white-clawed crayfish Austropotamobius pallipes. This is the species that we focused on in our effort to contribute to species conservation. Specifically we contrasted three different techniques of managing data relating to the presence/absence of this species: logistic regression, decision-tree models and artificial neural networks (ANN). Logistic regression and decision tree models (unpruned and pruned) performed worse than ANN. In this case, tree-pruning techniques did not make these models significantly more reliable, but did make the trees less complex and therefore did make the models clearer. ANN performed the best. Therefore we have judged them to be the most effective techniques.

The role of water chemistry in the distribution of Austropotamobius pallipes (Crustacea Decapoda Astacidae) in Piedmont (Italy).

Over the last decades, the populations of Austropotamobius pallipes have decreased markedly all over Europe. If we evaluate the ecological factors that determine its presence, we will have information that could guide conservation decisions. This study aims to investigate the chemical-physical demands of A. pallipes in NW Italy. To this end, we investigated 98 sites. We performed Principal Component Analysis using chemical-physical parameters, collected in both presence and absence sites. We then used principal components with eigenvalue > 1 to run Discriminant Function Analysis and Logistic Regression. The statistics on the concentration of Ca(2+), water hardness, pH and BOD(5) were significantly different in the presence and in the absence sites. pH and BOD(5) played the most important role in separating the presence from the absence locations. These findings are further evidence that we should reduce dissolved organic matter and fine particles in order to contribute to species management and conservation.