Long‐term transportation,by road and air,of chub mackerel (Scomber japonicus) and atlantic bonito (Sarda sarda)

During the second semester of 2009, three trips were made from Olhão (Southern Portugal) to Stralsund (Northern Germany) carrying 2.122 animals, which included multiple teleosts, elasmobranchs and invertebrates. This group included scombrids, such as 1.869 Scomber japonicus and 9 Sarda sarda, which are notoriously difficult to transport. However, multiple adaptations to transport regimes adopted regularly have allowed the authors to successfully move these animals by road and air over a total of up to 25 hr. Such adaptations included maintaining oxygen saturation rates at approximately 200%, and also the constant addition of AmQuel^®, sodium bicarbonate, and sodium carbonate. Different formulations were used during the three trips, with the best results corresponding to 20/30/30 ppm of the three aforementioned chemicals, respectively. The authors suggest, however, that a modified formula of 20/40/40 ppm will allow for an even more stable pH on future trips. Zoo Biol 30:459–472, 2010. © 2010 Wiley‐Liss, Inc.     

Long‐term transportation of ratfish,Hydrolagus colliei,and tiger rockfish,Sebastes nigrocinctus

On December 10, 2000, five tiger rockfish, Sebastes nigrocinctus, and 10 ratfish, Hydrolagus colliei, were transported from Vancouver, Canada, to Lisbon, Portugal, for 51 and 44 hours, respectively. The transport vessels consisted of large circular tanks (1.0‐m diameter × 0.6 m high) and filtration was added to the rockfish tank only. Filtration consisted of a combination of cartridge and activated carbon, powered by a 12‐V bilge pump placed on the middle of the transport vessel. Water quality parameters (ammonia, temperature, and oxygen saturation) were tested during the transport, and an ammonia detoxifier was added to some of the transport vessels when ammonia concentration reached 0.25 mg/L. Sodium bicarbonate was also added for pH buffering. All animals survived the trip and the subsequent 4 weeks. The water quality monitoring and treatment regimen therefore seemed appropriate for this type of long‐term transportation. Zoo Biol 20:435–441, 2001. © 2001 Wiley‐Liss, Inc.     

Predation by White Sharks Carcharodon carcharias (Chondrichthyes: Lamnidae) Upon Chelonians, with New Records from the Mediterranean Sea and a First Record of the Ocean Sunfish Mola mola (Osteichthyes: Molidae) as Stomach Contents

The occurrence of marine turtles in the diet of white sharks, Carcharodon carcharias, is reviewed worldwide. Four records of chelonians eaten by white sharks in the Mediterranean Sea are described, which on the basis of carapace remnants confirmed both the loggerhead Caretta caretta and green turtle Chelonia mydas to be preyed upon in those waters. The condition of these remains indicates that large white sharks can ingest turtles essentially intact. As well as falling prey to white sharks, we suspect that some interactions involve turtles being grab-released in a non-predatory mannner and their survivability from such low-intensity bites or other mouthings may be quite high. The white shark may be the chief marine predator of adult chelonians in the Mediterranean Sea, albeit the impact of this predation upon turtle populations is nominal compared to other sources of mortality. Further, we give an account describing an adult ocean sunfish, Mola mola, in the stomach of a white shark taken in Italian waters.     

Comparison of growth and nutritional status of juvenile Komodo monitors (Varanus komodoensis) maintained on rodent or poultry‐based diets

Nutrition and growth data were collected on six juvenile Komodo monitors (Varanus komodoensis) over 20 months. The animals were captive‐bred from two separate clutches, laid 6 months apart. Three animals were fed a rodent diet, and the other three were fed a poultry‐based diet. The animals were all weighed and measured weekly. Blood samples were collected monthly. Blood samples were analyzed for sodium (Na), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), 25‐OH‐D, and cholesterol. Significant differences (P<0.05) were observed in dry matter intake (DMI) (1.12 vs. 0.90 %BW) and gross energy intake (GEI) (182.3 vs. 143.0 kcal GE/d), between rodent and poultry‐based diets, respectively. However, there were no differences in growth (average daily gain=6.13 vs. 6.33 g BW/d) between the two dietary treatments. Blood Na, K, Ca, P, Mg, and 25‐OH‐D concentrations were similar across treatments, while cholesterol levels were higher in the animals maintained on the rodent‐based diet. Zoo Biol 23:239–252, 2004. © 2004 Wiley‐Liss, Inc.     

Effects of clove oil,essential oil of Lippia alba and 2‐phe anaesthesia on juvenile meagre,Argyrosomus regius (Asso, 1801)

The objectives of this experiment were to (i) determine the efficacy of essential oils of clove (CO) and Lippia alba (EOLA) to induce deep anaesthesia in juvenile specimens (49.0 ± 6.2 g body mass, 16.6 ± 0.8 cm; n = 8 per treatment) of meagre (Argyrosomus regius); and (ii) study the feasibility of these substances, together with 2‐phenoxyethanol (2‐PHE), as potential sedatives [low concentration: (i) EOLA: 12 mg L^?1; (ii) CO: 1 mg L^?1; and (iii) 2‐PHE: 33 mg·L ^?1; n = 8 per treatment] for live fish transport of this species. All test were performed at a constant temperature (18°C). Thus, the main primary stress indicator (plasma cortisol) and secondary factors (plasma metabolites) were evaluated. In addition, growth hormone (GH) mRNA expression was also evaluated in the pituitary gland. The results indicated that EOLA is considered to be effective for deep anaesthesia when the concentration is close to 160 mg L^?1, while CO produces the same effect when lower concentrations are added (40–50 mg L^?1). Regarding sedative concentrations, a significant ~3‐fold increase in plasma cortisol levels was detected in the EOLA group when compared to control specimens. In addition, glucose levels were not reduced and significantly increased (~1.6‐fold) for 2‐PHE in relation to the control fish. None of the anaesthetics promoted a significant difference for GH expression with respect to the control group, but a significant ~2‐fold increase for 2‐PHE treatment with respect to the EOLA exposition was found in this gene expression. Results show that none of the anaesthetics analysed, at least in the ranges of concentrations used in this study (EOLA 12 mg L^?1, CO 1 mg L^?1, 2‐PHE 33 mg L^?1), are recommended for live fish transport, as shown by the absence of inhibition on the stress parameters assessed.     

A combined RT‐PCR and dot‐blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin‐labelled probes resulted in the identification of both genotypes separately. The application of the RT‐PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT‐PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.     

Long‐term metapopulation study of the Glanville fritillary butterfly (Melitaea cinxia): survey methods,data management,and long‐term population trends

Long‐term observational studies conducted at large (regional) spatial scales contribute to better understanding of landscape effects on population and evolutionary dynamics, including the conditions that affect long‐term viability of species, but large‐scale studies are expensive and logistically challenging to keep running for a long time. Here, we describe the long‐term metapopulation study of the Glanville fritillary butterfly (Melitaea cinxia) that has been conducted since 1991 in a large network of 4000 habitat patches (dry meadows) within a study area of 50 by 70 km in the Åland Islands in Finland. We explain how the landscape structure has been described, including definition, delimitation, and mapping of the habitat patches; methods of field survey, including the logistics, cost, and reliability of the survey; and data management using the EarthCape biodiversity platform. We describe the long‐term metapopulation dynamics of the Glanville fritillary based on the survey. There has been no long‐term change in the overall size of the metapopulation, but the level of spatial synchrony and hence the amplitude of fluctuations in year‐to‐year metapopulation dynamics have increased over the years, possibly due to increasing frequency of exceptional weather conditions. We discuss the added value of large‐scale and long‐term population studies, but also emphasize the need to integrate more targeted experimental studies in the context of long‐term observational studies. For instance, in the case of the Glanville fritillary project, the long‐term study has produced an opportunity to sample individuals for experiments from local populations with a known demographic history. These studies have demonstrated striking differences in dispersal rate and other life‐history traits of individuals from newly established local populations (the offspring of colonizers) versus individuals from old, established local populations. The long‐term observational study has stimulated the development of metapopulation models and provided an opportunity to test model predictions. This combination of empirical studies and modeling has facilitated the study of key phenomena in spatial dynamics, such as extinction threshold and extinction debt.     

Metabarcoding for stomach‐content analyses of Pygmy devil ray (Mobula kuhlii cf. eregoodootenkee): Comparing tissue and ethanol preservative‐derived DNA

The application of high‐throughput sequencing to retrieve multi‐taxon DNA from different substrates such as water, soil, and stomach contents has enabled species identification without prior knowledge of taxon compositions. Here we used three minibarcodes designed to target mitochondrial COI in plankton, 16S in fish, and 16S in crustaceans, to compare ethanol‐ and tissue‐derived DNA extraction methodologies for metabarcoding. The stomach contents of pygmy devilrays (Mobula kuhlii cf. eregoodootenkee) were used to test whether ethanol‐derived DNA would provide a suitable substrate for metabarcoding. The DNA barcoding assays indicated that tissue‐derived operational taxonomic units (OTUs) were greater compared to those from extractions performed directly on the ethanol preservative. Tissue‐derived DNA extraction is therefore recommended for broader taxonomic coverage. Metabarcoding applications should consider including the following: (i) multiple barcodes, both taxon specific (e.g., 12S or 16S) and more universal (e.g., COI or 18S) to overcome bias and taxon misidentification and (ii) PCR inhibitor removal steps that will likely enhance amplification yields. However, where tissue is limited or no longer available, but the ethanol‐preservative medium is still available, metabarcoding directly from ethanol does recover the majority of common OTUs, suggesting the ethanol‐retrieval method could be applicable for dietary studies. Metabarcoding directly from preservative ethanol may also be useful where tissue samples are limited or highly valued; bulk samples are collected, such as for rapid species inventories; or mixed‐voucher sampling is conducted (e.g., for plankton, insects, and crustaceans).     

Long‐term persistence,density dependence and effects of climate change on rosyside dace (Cyprinidae)

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Changes in hormonal profile, gonads and sperm quality of Argyrosomus regius (Pisces, Scianidae) during the first sexual differentiation and maturation

In the present study, sexual gonadal differentiation and first sexual maturation of Meagre (Argyrosomus regius) was studied, based upon the annual changes in gonadosomatic index (GSI), gonadal histology, and the plasma steroid hormones, testosterone (T), 11-ketotestosterone (11-KT), and estradiol (E2). In addition, spermatozoa characteristics were evaluated by measuring sperm motility and morphology. Results demonstrated that Meagre completes sex differentiation at 10 to 12 mo of age, and are group-synchronous spawners, which reach puberty at 2 (mean length 26.8 ± 0.7 cm, mean weight 920 ± 75 g; N = 10) and 3 (mean length 35.8 ± 0.8 cm, mean weight 1610 ± 89 g; N = 10) years of age for males and females, respectively. In males, during the sex differentiation period, T levels were significantly higher with respect to those of 11-KT; this suggests that T has a key role in the early phases of the sex differentiation. During the spawning season an increase in plasma concentrations of all hormones was observed with 11-KT levels being significantly higher that those of T. In females, during the sex differentiation period, there was an increase in E2 plasma levels, while during the first spawning season, a significant increase of T and E2 levels were measured. Regarding sperm characteristics, the measured curvilinear velocity (VCL) and straight-linear velocity (VSL), resulted in the same order of magnitude with respect to those measured in other marine fish, while the average path velocity (VAP) was similar to that measured in the European Eel. The head of Meagre spermatozoa presents as oval shaped with a surface area of approximately 3.66 μm² and a perimeter of approximately 6.65 μm. All these findings represent an important basis for further investigation on the reproductive biology of this specie and may assist the farmers to improve seed production in aquaculture.     

Long‐term monitoring of B‐chromosome invasion and neutralization in a population of Prospero autumnale (Asparagaceae)

B chromosomes have been reported in about 15% of eukaryotes, but long‐term dynamics of B chromosomes in a single natural population has rarely been analyzed. Prospero autumnale plants collected in 1981 and 1983 at Cuesta de La Palma population had shown the presence of B chromosomes. We analyze here seven additional samples collected between 1987 and 2015, and show that B frequency increased significantly during the 1980s and showed minor fluctuations between 2005 and 2015. A mother–offspring analysis of B chromosome transmission, at population level, showed significant drive on the male side (k_B = 0.65) and significant drag on the female side (k_B = 0.33), with average B transmission rate being very close to the Mendelian rate (0.5). No significant effects of B chromosomes were observed on a number of vigor and fertility‐related traits. Within a parasite/host framework, these results suggest that B chromosomes’ drive on the male side is the main pathway for B chromosome invasion, whereas B chromosome drag on the female side might be the main manifestation of host genome resistance in this species. Prospero autumnale thus illuminates a novel evolutionary pathway for B chromosome neutralization by means of a decrease in B transmission through the nondriving sex.     

Corticosterone responses of hand‐reared and parent‐reared grey‐faced petrel chicks (Pterodroma macroptera gouldi)

Hand‐reared grey‐faced petrel (Pterodroma macroptera gouldi) chicks (Order Procellariformes) that were subjected to a standardised blood sampling protocol immediately before they fledged showed a reduced corticosterone response compared to parent‐raised chicks. Serum corticosterone concentrations were lower in hand‐reared than parent‐reared birds 30 and 60 min after handling was initiated (21.5±6.7 vs. 105.4±7.4 ng/ml at 30 min, and 11.3±3.6 vs. 93.8±11.4 ng/ml at 60 min, respectively). The total integrated corticosterone response and the response corrected for initial corticosterone concentrations were similarly lower in hand‐reared than parent‐reared birds (927±489 vs. 4639±1924 ng/ml.min, and 529±352 vs. 4110±1896 ng/ml.min, respectively). Habituation to handling associated with hand‐rearing, which is expressed as a subdued corticostrone response, is likely to reduce the physiological stress associated with the translocation and reintroduction of these birds. However, the longer‐term consequences of this attenuated response remain unclear. Zoo Biol 0:1–8, 2005. © 2005 Wiley‐Liss, Inc.     

Long‐term administration of α‐tocopherol in captive Asian elephants (Elephas maximus)

After the loss of an African elephant (Loxodonta africana) in February 1989 at the Denver Zoological Gardens (DZG) with very low circulating serum α‐tocopherol, a long‐term study was initiated with three Asian elephants (Elephas maximus) to evaluate the effect of an oral micellized, water‐soluble, natural source d‐α‐tocopherol supplement. Baseline α‐tocopherol levels were evaluated and found to be approximately 3.75‐fold less than those reported for semi‐free‐ranging Asian Nepalese work camp and free‐ranging African elephants. The DZG elephants were then administered a liquid d‐α‐tocopherol (Emcelle^®) at 2.2 IU/kg body weight orally once daily. Serum samples were obtained and analyzed at 1, 2, 8, and 12 months and then annually for 96 months. The oral vitamin E supplement significantly elevated serum levels above baseline and were found to be comparable with levels reported for semi–free‐ranging and free‐ranging elephants. Zoo Biol 20:245–250, 2001. © 2001 Wiley‐Liss, Inc.     

Development and ossification of the feeding apparatus in the larvae of two co‐occurring species of kob (Sciaenidae), Argyrosomus japonicus and Argyrosomus inodorus,in South Africa

The teeth of the oral jaws of two sympatric species of Argyrosomus, Argyrosomus japonicus and Argyrosomus inodorus, found along the South African coast developed first on the premaxilla and then on the dentary of the lower jaw. Teeth were observed on the premaxilla of A. inodorus [head length (L_H) = 1·0 mm; notochord length (L_N) = 2·7 mm] at a smaller size than in A. japonicus (L_H = 1·2 mm; L_N = 4·7 mm). The ventral elements of the gill arches (hypo‐ and basibranchials) were not ossified by the end of preflexion. The fifth ceratobranchial began ossifying and possessed pharyngeal teeth by 1·2 mm L_H (L_N = 4·7 mm) in A. japonicus and 1·1 mm L_H (L_N = 3·2 mm) in A. inodorus. To complement the osteological data, stomach contents were also analysed as a proxy for feeding apparatus functionality. Prey were first present in the stomach of A. japonicus at 1·2 mm L_H (L_N = 4·7 mm) and only 22% of the stomachs contained no prey suggesting that A. japonicus is already actively foraging by preflexion. In comparison, 83% of the stomachs of A. inodorus contained no prey and a single prey item was present in the largest examined specimen (L_H = 1·6 mm; L_N = 5·4 mm). Elements of the feeding apparatus begin to ossify early during ontogeny. While the overall pattern of ossification is similar between the two species, A. japonicus may be able to begin feeding at smaller head lengths relative to A. inodorus in their nursery habitats.     

Long‐term isolation of the coastal plant Calystegia soldanella (Convolvulaceae) in ancient freshwater Lake Biwa,Japan

Lake Biwa is an ancient freshwater lake that was formed approximately 4 Mya and harbours many coastal plants that commonly inhabit the seashore. We used chloroplast DNA haplotype analysis using two spacer sequences and simple sequence repeat (SSR) analysis using eight nuclear microsatellite markers to detect genomic signatures indicating long‐term isolation of inland populations of Calystegia soldanella in Lake Biwa from coastal populations. We used 348 samples from 63 populations for haplotype analysis and 478 samples from 27 populations for SSR analysis covering the inland and coastal distribution of the species. We detected seven haplotypes, and the distribution pattern of these haplotypes was geographically highly structured between Lake Biwa and the coast. Nuclear SSR analysis also supported genetic differentiation between Lake Biwa and coastal populations (analyses of molecular variance, 43%), and the grouping of Lake Biwa and coastal populations by a Neighbour‐joining tree. In addition, genetic diversity of the inland populations (mean H_E = 0.153) was significantly lower than that of coastal populations (mean H_E = 0.328). These results suggested that inland populations at Lake Biwa have been isolated from coastal populations for a very long time. The inland populations most likely experienced a bottleneck effect, resulting in sufficient in situ genetic divergence to clearly distinguish them from coastal populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 51–66.