Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Deltocyathiidae, an early-diverging family of Robust corals (Anthozoa, Scleractinia)

Over the last decade, molecular phylogenetics has called into question some fundamental aspects of coral systematics. Within the Scleractinia, most families composed exclusively by zooxanthellate species are polyphyletic on the basis of molecular data, and the second most speciose coral family, the Caryophylliidae (most members of which are azooxanthellate), is an unnatural grouping. As part of the process of resolving taxonomic affinities of caryophylliids', here a new Robust' scleractinian family (Deltocyathiidae fam. n.) is proposed on the basis of combined molecular (CO1 and 28S rDNA) and morphological data, accommodating the early-diverging clade of traditional caryophylliids (represented today by the genus Deltocyathus). Whereas this family captures the full morphological diversity of the genus Deltocyathus, one species, Deltocyathus magnificus, is an outlier in terms of molecular data, and groups with the Complex coral family Turbinoliidae. Ultrastructural data, however, place D.magnificus within Deltocyathiidae fam. nov. Unfortunately, limited ultrastructural data are as yet available for turbinoliids, but D.magnificus may represent the first documented case of morphological convergence at the microstructural level among scleractinian corals. Marcelo V.Kitahara, Centro de Biologia Marinha, Universidade de SAo Paulo, SAo SebastiAo, S.P. 11600-000, Brazil. E-mail:kitahara@usp.br

Combined levels of carotenoids and vitamin E in diets for gilthead seabream broodstocks (Sparus aurata L.): effect on the quality of egg spawning and composition

Watanabe et al. (1991 a,b) state that, vitamin E and carotenoids perform an essential role on the quality of egg spawning. Vitamin E is one of the main nutrients for the reproduction of fish (Izquierdo et al., 2001), and it has been proved that its inclusion in diets for broodstocks favors the quality of egg spawning in several species of fish (Watanabe and Takashima,1977; Takeuchi et al., 1981; Watanabe et al., 1985, 1991 a,b; Sutjaritvongsanon, 1987; Watanabe, 1990; Schimittou, 1993; Mushiake et al., 1993; Dube, 1996; Shiranee and Natarajan, 1996; Izquierdo et al., 2001; Morehead et al., 2001; Fernández- Palacios et al., 2005). On the other hand, the carotenoids which also perform an antioxidizing function (including the protection of lipids from oxidation), have been involved in the reproductive processes of marine organisms: crustaceans (Liñan-Cabello et al., 2002), marine fish (Watanabe y Kiron, 1995; Verakunpiriya et al., 1997 a,b; Vassallo-Agius et al., 2001 a,b,c, 2002; Watanabe and Vassallo-Agius 2003) and fresh water fish (Ahmadi et al., 2006). The results of this study suggest that the recommended levels of n-3 HUFA in diets for gilthead sea bream broodstocks could be increased up to 3,5 % when supplemented jointly with carotenoids from paprika oleoresin and vitamin E, thus favoring the quality of spawning.

Effects of diets, with different levels of n-3 HUFA, designed specifically for gilthead seabream (Sparus auratus L.) broodstock on spawning quality

The effects of dietary lipid levels in the spawning quality has been studied in several cultured fish species. Works like those of Watanabe et al. (1984 a); Mourente et al. (1989); Dhert et al. (1991); Bruce et al. (1993); Navas et al. (1997); Rodriguez et al. (1998); Lavens et al. (1999); Furuita et al. (2002, 2003 b); Mazorra et al. (2003); Fernandez- Palacios (2005) and Aijun et al. (2005) show that lipids and fatty acids are the dietetic components that have more influence in the spawning quality, specially in those species with continuous spawning which display short vitellogenesis periods and are able to incorporate these dietetic components in eggs during the spawning period. Diets for gilthead sea bream (Sparus aurata) broodstock with dietary levels of 2.84% n-3 HUFA, combined with levels of 250 mg/kg vitamine E rasure good spawning quality. Putting so indicative the importance for an effective utilization of essential fatty acids the use of adequate levels of antioxidants.

Dose-dependent effect of a single GnRHa injection on the spawning of meagre ("Argyrosomus regius") broodstock reared in captivity

[EN]The present study aimed to determine the spawning efficacy, egg quality and quantity of captive breed meagre induced with a single gonadotrophin-releasing hormone agonist (GnRHa) injection of 0, 1, 5, 10, 15, 20, 25, 30, 40 or 50 μg kg–1 to determine a recommended optimum dose to induce spawning. The doses 10, 15 and 20 μg kg–1 gave eggs with the highest quality (measured as: percentage of viability, floating, fertilisation and hatch) and quantity (measured as: total number of eggs, number of viable eggs, number of floating eggs, number of hatched larvae and number of larvae that reabsorbed the yolk sac). All egg quantity parameters were described by Gaussian regression analysis with R2 = 0.89 or R2 = 0.88. The Gaussian regression analysis identified that the optimal dose used was 15 μg kg–1. The regression analysis highlighted that this comprehensive study examined doses that ranged from low doses insufficient to stimulate a high spawning response (significantly lower egg quantities, p < 0.05) compared to 15 μg kg–1 through to high doses that stimulated the spawning of significantly lower egg quantities and eggs with significantly lower quality (egg viability). In addition, the latency period (time from hormone application to spawning) decreased with increasing doses to give a regression (R2 = 0.93), which suggests that higher doses accelerated oocyte development that in turn reduced egg quality and quantity. The identification of an optimal dose for the spawning of meagre, which has high aquaculture potential, represents an important advance for the Mediterranean aquaculture industry.

Climate signatures in corals of the tropical-temperate transition zone (Late Miocene, Crete/Greece)

The present study describes a Late Miocene (early Tortonian - early Messinian) transitional carbonate system that combines elements of tropical and cool-water carbonate systems (Irakleion Basin, island of Crete, Greece). As documented by stratal geometries, the submarine topography of the basin was controlled by tilting blocks. Coral reefs formed by Porites and Tarbellastrea occurred in a narrow clastic coastal belt along a „central Cretan landmass“, and steep escarpments formed by faulting. Extensive covers of level-bottom communities existed in a low-energy environment on the gentle dip-slope ramps of the blocks that show the widest geographical distribution within the basin. Consistent patterns of landward and basinward shift of coastal onlap in all outcrop studies reveal an overriding control of 3rd and 4th order sea level changes on sediment dynamics and facies distributions over block movements. An increasingly dry climate and the complex submarine topography of the fault block mosaic kept sediment and nutrient discharge at a minimum. The skeletal limestone facies therefore reflects oligotrophic conditions and a sea surface temperature (SST) near the lower threshold temperature of coral reefs in a climatic position transitional between the tropical coral reef belt and the temperate zone. Stable isotope records (δ18O, δ13C) from massiv, exceptionally preserved Late Miocene aragonite coral skeletons reflect seasonal changes in sea surface temperature and symbiont autotrophy. Spectral analysis of a 69 years coral δ18O record reveals significant variance at interannual time scales (5-6 years) that matches the present-day eastern Mediterranean climate variability controlled by the Arctic Oscillation/North Atlantic Oscillation (AO/NAO), the Northern Hemisphere’s dominant mode of atmospheric variability. Supported by simulations with a complex atmospheric general circulation model coupled to a mixed-layer ocean model, it is suggested, that climate dynamics in the eastern Mediterranean and central Europe reflect atmospheric variability related to the Icelandic Low 10 million years ago. Usually, Miocene corals are transformed in calcite spar in geological time and isotope values are reset by diagenetic alteration. It is demonstrated that the relicts of growth bands represent an intriguing source of information for the growth conditions of fossil corals. Recrystallized growth bands were measured systematically in massive Porites from Crete. The Late Miocene corals were growing slowly with 2-4 mm/yr, compatible with present-day Porites from high latitude reefs, a relationship that fits the position of Crete at the margin of the Miocene tropical reef belt. Over Late Miocene time (Tortonian - early Messinian) growth rates remained remarkably constant, and if the modern growth temperature relationship for massive Porites applies to the Neogene, minimum (winter) SST did not exceed 19-21°C.

Reproduction of Mediterranean zooxanthellate and azooxanthellate scleractinian corals along environmental gradients

Despite extensive studies focus mainly on sexual reproductive characteristics in tropical scleractinian species, there is limited knowledge on temperate regions. The Mediterranean is a biodiversity hotspot under intense pressure from anthropogenic impacts. Climatic models further predict that the Mediterranean basin will be one of the most impacted regions by the ongoing warming trend. This makes it a potential model of more global patterns to occur in the world’s marine biota, and a natural focus of interest for research on climate. The present research contributed to increase data on reproductive modes and sexuality of temperate scleractinian corals, highlighting their developmental plasticity, showing different forms of propagation and different responses to environmental change. For the first time, sexuality and reproductive mode in Caryophyllia inornata were determined. An unusual embryogenesis without a clear seasonal pattern was observed, suggesting the possibility of an asexual origin. Sexual reproduction of Astroides calycularis was governed by annual changes in seawater temperature, as observed for other Mediterranean dendrophylliids. Defining the reproductive biology of these species is the starting point for studying their potential response to variations of environmental parameters, on a global climate change context. The results on the influence of temperature on reproductive output of the zooxanthellate (symbiosis with unicellular algae) Balanophyllia europaea and the non-zooxanthellate Leptopsammia pruvoti suggest that the latter may be quite tolerant to temperature increase, since the zooxanthellate species resulted less efficient at warm temperatures. A possible explanation could be related to their different trophic system. In B. europaea thermal tolerance is primarily governed by the symbiotic algae, making it more sensitive to temperature changes. On the contrary, the absence of symbionts in L. pruvoti might make it more resistant to temperature. In a progressively warming Mediterranean, the efficiency on scleractinian reproduction could be influenced in different ways, reflecting their extraordinary adaptability.

Controlled reproduction in Anguilla anguilla (L.): advanced studies on broodstock management, spawning techniques and system design for artificial seed production.

The thesis aims to expose the advances achieved in the practices of captive breeding of the European eel (Anguilla anguilla). Aspects investigated concern both approaches livestock (breeding selection, response to hormonal stimulation, reproductive performance, incubation of eggs) and physiological aspects (endocrine plasma profiles of players), as well as engineering aspects. Studies conducted on various populations of wild eel have shown that the main determining factor in the selection of wild females destined to captive breeding must be the Silver Index which may determine the stage of pubertal development. The hormonal induction protocol adopted, with increasing doses of carp pituitary extract, it has proven useful to ovarian development, with a synchronization effect that is positively reflected on egg production. The studies on the effects of photoperiod show how the condition of total darkness can positively influence practices of reproductions in captivity. The effects of photoperiod were also investigated at the physiological level, observing the plasma levels of steroids ( E2, T) and thyroid hormones (T3 and T4) and the expression in the liver of vitellogenin (vtg1 and vtg2) and estradiol membrane receptor (ESR1). From the comparison between spontaneous deposition and insemination techniques through the stripping is inferred as the first ports to a better qualitative and quantitative yield in the production of eggs capable of being fertilized, also the presence of a percentage of oocytes completely transparent can be used to obtain eggs at a good rate of fertility. Finally, the design and implementation of a system for recirculating aquaculture suited to meet the needs of species-specific eel showed how to improve the reproductive results, it would be preferable to adopt low-flow and low density incubation.

Daily modulation of the Heat shock proteins (Hsps) in three different species of scleractinian corals

Temperature and light intensity is the most important environmental parameters that influence circadian cycle of scleractinian corals. In this context, modulation of the biomarkers Hsp60 and Hsp70 in situ was investigated by three different healthy coral species (Acropora tenuis, Echinopora lamellosa and Porites lobata) not stress induced during time course of 24h. Significance species-specific modulation under natural conditions is displayed by all corals under study. A strong fluctuation in Hsps expression is shown by the most susceptible, branched coral A. tenuis, instead of fine and low modulation is shown by the massive coral P. lobata. From the results match between morphology difference and physiological difference response its suggest and similarity pattern between Hsps with different cellular compartments location is suggested too. Starting from this study health of coral reefs could be able to be investigated in the future with a set of biomarkers composed also by Hsps which will be set up.

Spawning coordination of mates in a shell brooding cichlid

Aim. External fertilisation requires synchronisation of gamete release between the two sexes. Adequate synchronisation is essential in aquatic media because sperm is very short-lived in water. In the cichlid Lamprologus callipterus, fertilisation of the eggs takes place inside an empty snail shell, where females stay inside the shell and males have to ejaculate into the shell opening. This spawning pattern makes the coordination of gamete release difficult. Methods. This study examined the synchronisation of males and females during egg laying. Results. The results showed that the male initiates each spawning sequence and that sperm release and egg laying are very well synchronised. 68% of all sperm releases occurred at exactly the same time when the female laid an egg, and 99% of ejaculations occurred within ±5 seconds from egg deposition. On average 95 eggs are laid one by one with intervals of several minutes between subsequent eggs, leading to a total spawning duration in excess of six hours. Conclusions. We discuss this exceptional spawning pattern and how it might reflect a conflict between the sexes, with males attempting to induce egg laying and females extending the egg laying period to raise the chance for parasitic males to participate in spawning.

Estimation of vertical groundwater fluxes into a streambed through continuous temperature profile monitoring and the relationship of groundwater fluxes to coaster brook trout spawning habitat

We hypothesized that the spatial distribution of groundwater inflows through river bottom sediments is a critical factor associated with the selection of coaster brook trout (a life history variant of Salvelinus fontinalis,) spawning sites. An 80-m reach of the Salmon Trout River, in the Huron Mountains of the upper peninsula of Michigan, was selected to test the hypothesis based on long-term documentation of coaster brook trout spawning at this site. Throughout this site, the river is relatively similar along its length with regard to stream channel and substrate features. A monitoring well system consisting of an array of 27 wells was installed to measure subsurface temperatures underneath the riverbed over a 13-month period. The monitoring well locations were separated into areas where spawning has and has not been observed. Over 200,000 total temperature measurements were collected from 5 depths within each of the 27 monitoring wells. Temperatures within the substrate at the spawning area were generally cooler and less variable than river temperatures. Substrate temperatures in the non-spawning area were generally warmer, more variable, and closely tracked temporal variations in river temperatures. Temperature data were inverted to obtain subsurface groundwater velocities using a numerical approximation of the heat transfer equation. Approximately 45,000 estimates of groundwater velocities were obtained. Estimated velocities in the spawning and non-spawning areas confirmed that groundwater velocities in the spawning area were primarily in the upward direction, and were generally greater in magnitude than velocities in the non-spawning area. In the non-spawning area there was a greater occurrence of velocities in the downward direction, and velocity estimates were generally lesser in magnitude than in the spawning area. Both the temperature and velocity results confirm the hypothesis that spawning sites correspond to areas of significant groundwater influx to the river bed.

Rugose Corals of the Madison Group

One of the best types of index fossils, and one which occurs most plentifully in the Madison group of formations in Montana, and yet, one which has been little studied, is the group of rugose, or cup, corals. Perhaps this group of fossils has not been studied in detail because their distinguishing characteristics are mainly internal, and labo­ratory preparation in the form of thin section or polished surfaces is necessary to bring out these characteristics.

Reproductive parasitism: male and female responses to conspecific and heterospecific intrusions at spawning in a mouth-brooding cichlid Ophthalmotilapia ventralis

A rare form of alternative reproductive behaviour without simultaneous parasitic spawning was observed in Ophthalmotilapia ventralis, a lekking mouth-brooding cichlid from Lake Tanganyika. Floater males attempted to sneak opportunistically into the territory to actively court the female, while the owner (bourgeois male) defended the territory against other potential intruders. Floater males had more body fat than territory owners and generally higher condition factors. In field experiments, the response of bourgeois males and courted females was tested towards floaters and egg predators (a catfish Synodontis multipunctatus) present in the territories. Territory owners responded aggressively particularly to floaters, and female responsiveness to bourgeois male courtship tended to decline when floaters were present. The potential influence of reproductive parasitism on sexual selection in mouth-brooding cichlids is discussed.