Experiment: Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Impact of ocean acidification in the metabolism and swimming behavior of the dolphinfish (Coryphaena hippurus) early larvae

Since the industrial revolution, [CO2]atm has increased from 280 µatm to levels now exceeding 380 µatm and is expected to rise to 730-1,020 µatm by the end of this century. The consequent changes in the ocean's chemistry (e.g., lower pH and availability of the carbonate ions) are expected to pose particular problems for marine organisms, especially in the more vulnerable early life stages. The aim of this study was to investigate how the future predictions of ocean acidification may compromise the metabolism and swimming capabilities of the recently hatched larvae of the tropical dolphinfish (Coryphaena hippurus). Here, we show that the future environmental hypercapnia (delta pH 0.5; 0.16 % CO2, ~1,600 µatm) significantly (p < 0.05) reduced oxygen consumption rate up to 17 %. Moreover, the swimming duration and orientation frequency also decreased with increasing pCO2 (50 and 62.5 %, respectively). We argue that these hypercapnia-driven metabolic and locomotory challenges may potentially influence recruitment, dispersal success, and the population dynamics of this circumtropical oceanic top predator.

The larvae of congeneric gastropods showed differential responses to the combined effects of ocean acidification, temperature and salinity

The tolerance and physiological responses of the larvae of two congeneric gastropods, the intertidal Nassarius festivus and subtidal Nassarius conoidalis, to the combined effects of ocean acidification (PCO2 at 380, 950, 1250 ppm), temperature (15, 30 degrees C) and salinity (10, 30 psu) were compared. Results of three-way ANOVA on cumulative mortality after 72-h exposure showed significant interactive effects in which mortality increased with pCO(2) and temperature, but reduced at higher salinity for both species, with higher mortality being obtained for N. conoidalis. Similarly, respiration rate of the larvae increased with temperature and pCO(2) level for both species, with a larger percentage increase for N. conoidalis. Larval swimming speed increased with temperature and salinity for both species whereas higher pCO(2) reduced swimming speed in N. conoidalis but not N. festivus. The present findings indicated that subtidal congeneric species are more sensitive than their intertidal counterparts to the combined effects of these stressors. (c) 2014 Elsevier Ltd. All rights reserved.

Seawater carbonate chemistry and shell length of Mediterranean pteropod Cavolinia inflexa larvae during experiments

Larvae of the Mediterranean pteropod Cavolinia inflexa were maintained at controlled pHT values of 8.1, 7.82 and 7.51, equivalent respectively to pCO2 levels of 380, 857 and 1713 µatm. At pHT 7.82 larvae exhibited malformations and lower shell growth, compared to the control condition. At pHT 7.51 the larvae did not make shells but were viable and showed a normal development. However, smaller shells or no shells will have both ecological (food web) and biogeochemical (export of carbon and carbonate) consequences. These results confirm that 1pteropods, as well as the species dependent upon them as a food resource, will be severely impacted by ocean acidification.

Seawater carbonate chemistry and severe tissue damage in Atlantic cod larvae under increasing ocean acidification, 2012

Ocean acidification, caused by increasing atmospheric concentrations of CO2, is one of the most critical anthropogenicthreats to marine life. Changes in seawater carbonate chemistry have the potential to disturb calcification, acid-base regulation, blood circulation and respiration, as well as the nervous system of marine organisms, leading to long-term effects such as reduced growth rates and reproduction. In teleost fishes, early life-history stages are particularly vulnerable as they lack specialized internal pH regulatory mechanisms. So far, impacts of relevant CO2concentrations on larval fish have been found in behaviour and otolith size, mainly in tropical, non-commercial species. Here we show detrimental effects of ocean acidification on the development of a mass-spawning fish species of high commercial importance. We reared Atlantic cod larvae at three levels of CO2, (1) present day, (2) end of next century and (3) an extreme, coastal upwelling scenario, in a long-term ( 2.5 1/2 months) mesocosm experiment. Exposure to CO2 resulted in severe to lethal tissue damage in many internal organs, with the degree of damage increasing with CO2 concentration. As larval survival is the bottleneck to recruitment, ocean acidification has the potential to act as an additional source of natural mortality, affecting populations of already exploited fish stocks.

Repellent effects of Melaleuca alternifolia (tea tree) oil against cattle tick larvae (Rhipicephalus australis) when formulated as emulsions and in β-cyclodextrin inclusion complexes

Rhipicephalus australis (formerly Boophilus microplus) is a one host tick responsible for major economic loss in tropical and subtropical cattle production enterprises. Control is largely dependent on the application of acaricides but resistance has developed to most currently registered chemical groups. Repellent compounds that prevent initial attachment of tick larvae offer a potential alternative to control with chemical toxicants. The repellent effects of Melaleuca alternifolia oil (TTO) emulsions and two β-cyclodextrin complex formulations, a slow release form (SR) and a modified faster release form (FR), were examined in a series of laboratory studies. Emulsions containing 4% and 5% TTO applied to cattle hair in laboratory studies completely repelled ascending tick larvae for 24 h whereas 2% and 3% formulations provided 80% protection. At 48 h, 5% TTO provided 78% repellency but lower concentrations repelled less than 60% of larvae. In a study conducted over 15 days, 3% TTO emulsion applied to cattle hair provided close to 100% repellency for 2 days, but then protection fell to 23% by day 15. The FR formulation gave significantly greater repellency than the emulsion and the SR formulation from day 3 until the end of the study (P < 0.05), providing almost complete repellency at day 3 (99.5%), then decreasing over the period of the study to 49% repellency at day 15. Proof of concept is established for the use of appropriately designed controlled-release formulations to extend the period of repellency provided by TTO against R. australis larvae.

Biofloc contribution to antioxidant defence status, lipid nutrition and reproductive performance of broodstock of the shrimp Litopenaeus stylirostris: Consequences for the quality of eggs and larvae

The aim of this study was to determine biofloc contributions to the antioxidant status and lipid nutrition of broodstock of Litopenaeus stylirostris in relationship with their reproductive performance and the health of larvae produced. Shrimp broodstock reared with Biofloc technology (BFT) compared to Clear water (CW) exhibited a higher health status with (i) a better final survival rate during the reproduction period (52.6% in CW against 79.8% in BFT); (ii) higher glutathione level (GSH) and total antioxidant status (TAS), reduced oxidized/reduced glutathione ratio and a higher spawning rate and frequency as well as higher gonado-somatic index and number of spawned eggs. Finally, larvae from broodstock from BFT exhibited higher survival rates at the Zoe 2 (+ 37%) and Post Larvae 1 (+ 51%) stages when compared with those from females from CW treatment. The improved reproductive performance of the broodstock and higher larvae survival rate resulting from BFT treatment may be linked to the dietary supplement obtained by the shrimp from natural productivity during BFT rearing. Indeed, our study confirms that biofloc particulates represent a potential source of dietary glutathione and a significant source of lipids, particularly essential phospholipids and n-3 highly unsaturated fatty acids (HUFA) for shrimps. Thus, broodstock from BFT treatment accumulated phospholipids, n-3 HUFA and arachidonic acid, which are necessary for vitellogenesis, embryogenesis and pre-feeding larval development. The predominant essential fatty acids, arachidonic acid (ARA), eicopentaeonic acid (EPA) and docosahexaenoic acid (DHA), had levels in the eggs that were, respectively, 2.5, 2.8 and 3 fold higher for BFT compared to the CW treatment. Statement of Relevance Today, the influence of biofloc technology on shrimp broodstock is not enough described and no information was available on the larvae quality. Moreover, two key pieces of new information emerge from the present study. Firstly, biofloc is a source of further dietary lipids that can act as energetic substrates, but also as a source of phospholipids and essential fatty acids necessary to sustain reproduction, embryonic and larval development. Second, improving the reproduction of the broodstock also leads to an improvement in the quality of the larvae. We think that our research is new and important to increase knowledge on biofloc topic. We believe the paper will contribute to the development of more efficient and therefore more sustainable systems.

Evaluation of the impact of polyethylene microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae

Microplastics are present in marine habitats worldwide and may be ingested by low trophic organisms such as fish larvae, with uncertain physiological consequences. The present study aims at assessing the impact of polyethylene (PE 10-45µM) microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Fish were fed an inert diet including 0, 104 and 105 fluorescent microbeads per gram from 7 until 43 days post-hatching (dph). Microbeads were detected in the gastrointestinal tract in all fish fed diet incorporating PE. Our data revealed an efficient elimination of PE beads from the gut since no fluorescent was observed in the larvae after 48h depuration. While the mortality rate increased significantly with the amount of microbeads scored per larvae at 14 and 20 dph, only ingestion of the highest concentration slightly impacted mortality rates. Larval growth and inflammatory response through Interleukine-1-beta (IL-1) gene expression were not found to be affected while cytochrome-P450-1A1 (cyp1a1) expression level was significantly positively correlated with the number of microbeads scored per larva at 20 dph. Overall, these results suggest that ingestion of PE microbeads had limited impact on sea bass larvae possibly due to their high potential of egestion

Identification, diversity and distribution patterns of fish larvae in Khark & Kharko Coralline Islands ecosystem – Persian Gulf, by GIS method

Khark & Kharko Islands are the last Northern point for fringing coral reefs in Iranian side of the Persian Gulf. These Coralline habitats are the Protected Area and Wildlife Refugees with the total area of 2400 ha which located in the territory of Bushehr Province. This research carried out during 2006-2007 with monthly sampling from 12 stations, which selected around Islands and inshore waters with maximum depth of 20 meter. Sampling was conducted using by Bongo-Net plankton sampler with 500μ of mesh size. Totally, 1808 specimen from 45 family fish larvae was identified in studied area, including: 21 coralline fish larva families and 24 shore fish larvae such as pelagic and demersal fishes which some of them known as indicator, sentinel or endemic species for coral reef ecosystems. The results was shown that coral reef diversity in coral reefs (Khark & Kharko Islands) is more than other habitats such as estuary and river mouth, creeks, mangrove forest sites, and off shore water of the Persian Gulf and Oman Sea Iranian side. Among Identified families, Clupeidae, Blenniidae, Sillaginidae, Atherinidae and Tripterygiidae; with more abundance were dominant families in studied area. The pick of fish larvae abundance family were estimated in spring. There were significant differences between seasonally abundance and sub areas, but there were not significant differences in diversity indexes between Khark and Kharko stations with coastal stations (p< 0.05). The mean abundance of fish larvae were estimated 18.7083 larvae under 10m² of sea surface, and the mean diversity indexes and evenness were estimated 0.7135 and 0.565342 consequently, that was showed the area is under ecological stress for fish larvae, and wasn’t stable. Therefore, from the ecological point of view, only some of the fish larvae groups as like Clupeidae were dominant. Thus, they were the main cause of the fish larvae abundance change in studied area. Due to geographical location of Khark and Kharko Islands and among the environmental parameters, Its seems that the condition of sea current is the main cause for present or absent and distribution patterns of fish larvae in area. Abundance of fish larvae in west of Islands was higher than eastern parts in the spring. But this condition will be reversed in eastern part of Island and several coastal stations, so that the Islands surrounding clock wise current to cause fish larvae distribution patterns.

First insight into personality traits in Northern pike (Esox lucius) larvae: a basis for behavioural studies of early life stages

Personality traits have been studied for some decades in fish species. Yet, most often, studies focused on juveniles or adults. Thus, very few studies tried to demonstrate that traits could also be found in fish larvae. In this study, we aimed at identifying personality traits in Northern pike (Exos lucius) larvae. Twenty first-feeding larvae aged 21 days post hatch (16.1 +/− 0.4 mm in total length, mean +/− SD) were used to establish personality traits with two tests: a maze and a novel object. These tests are generally used for evaluating the activity and exploration of specimens as well as their activity and boldness, respectively. The same Northern pike twenty larvae were challenged in the two tests. Their performances were measured by their activity, their exploratory behaviour and the time spent in the different arms of the maze or near the novel object. Then, we used principal component analysis (PCA) and a hierarchical ascendant classification (HAC) for analysis of each data set separately. Finally, we used PCA reduction for the maze test data to analyse the relationship between a synthetic behavioural index (PCA1) and morphometric variables. Within each test, larvae could be divided in two sub groups, which exhibited different behavioural traits, qualified as bold (n = 7 for the maze test and n = 13 for the novel object test) or shy (n = 9 for the maze test and n = 11 for the novel object test). Nevertheless, in both tests, there was a continuum of boldness/shyness. Besides, some larvae were classified differently between the two tests but 40 % of the larvae showed cross context consistency and could be qualified as bold and/or proactive individuals. This study showed that it is possible to identify personality traits of very young fish larvae of a freshwater fish species.

Investigation on possibility of survival and growth enhancing by selected seaweeds from Persian Gulf in post larvae shrimp Litopenaeus vannamei

The use of antibiotics in aquaculture has been limited. Scientifics seeking for natural substitutes to prevent of aquatic animals diseases. Considering seaweeds are rich of nutritions and bioactive compounds, the purpose of this study is: investigation the potential and use possibility of native seaweeds from Persian Gulf in shrimp aquculture industry to improve growth, survival of postlarvae and to resistance against pathogens such as vibriosis. For this propose 7 macroalgae species from Bushehr province coast, inclouding: green algae (C. iyengarii), brown algae (S. angutifolium and S. ilicifolium) and red algae (L. snyderiae, K. alvarezii and G. corticata) were collected and identified. Then seaweed extracts abtained by Water, Ethanol, Methanol and Chloroform solvents by soaking method. In vitro antibacterial activity of extracts against Gr+ bacteria (S. aureus and B. subtilis) and Gr- bacteria (V. harveyi, V. alginolyticus and E. coli) was conducted by Agar diffusion, MIC and MBC methods. Antioxidant activity also by DPPH and EC50 methods was investigated. According to results of these two tests four seaweeds species (S. angutifolium, L. snyderiae, K. alvarezii and G. corticata) were selected for use in shrimp postlarvae (PL22) diets by Bio-Encapsulation (Artemia enrichment). Before of enrichment, toxicity effect of extracts to Artemia nauplii were evaluated by determination of LC50 24 h method. From results of this section Ethanol extracts were selected to bioencapsulation. After encapsulation shrimp postlarvae divided to 12 groups in triplicate, namely: C-, C+, S (200), S (400), S (600), L(200), L(400), L(600), G(300), G(600), K(300) and K(600). During 30 days of reared period C- and C+ use of basal diet and unenriched Artemia, but the other groups use of basal diet and enriched Artemia. Except C-, the shrimps in first day of culture put in 107 cfu/ml v. harveyi suspension for 30 minutes, and after water exchange 10 ml of this dose was added to reared aquaria. After 30 days survival percentage, obtained weight and SGR% were investigated. To evaluate vibrio loading, every 10 days 5 postlarvae were sampled randomly for vibrio count. Results showed that vibrio count in C- was less than the others and in C+ was more than the others. In treatments vibrio count in L(200) was the most and L(600) was the less. Survival rate in C- was the most and after that G(600) with 79.4±6.6% and then S(300) and K(600) were 73.3±7.3% and 70.6±6.6% respectively that were significantly compare the other (P < 0.01). Also the C+ was the less with 33.3±6.6% that difference was significant (P< 0.01). In this study growth parameters of all groups that fed by enriched Artemia were better than C+ (P<0.05). After cultre period 10 shrimp of every aquarium disinfected and reared for 10 days like before treatment. After 10 days the shrimps were challenged by 3×108 cfu/ml V. harveyi and mortality was recorded for 7 days. The all of animals in C- were survive but more than 90% of C+ were dead. And survival in all of treatments were better the C+ (P<0.05). The study showed the ethanol extracts of selected seaweed from Persian Gulf is a good source for growth, Survival and disease control in shrimp larviculture.

Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos and larvae

Batrachoidids, which include midshipman and toadfish are less known among embryologists, but are common in other fields. They are characteristic for their acoustic communication, and develop hearing and sound production while young juveniles. They lay large benthic eggs (>5mm) with a thick chorion and adhesive disk and slow development, which are particularly challenging for studying embryology. Here we took advantage of a classical tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series of embryos and larvae from 3 to 30mm in length, which allowed detailed 3D anatomical reconstructions non-destructively. We documented some of the developmental stages (early and late in development) and the anatomy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other techniques accessible to developmental biology labs, OPenT provided advantages in terms of image quality, cost of operation and data throughput, allowing identification and quantitative morphometrics of organs in larvae, earlier and with higher accuracy than is possible with other imaging techniques.

Age estimation in common sole Solea solea larvae: validation of daily increments and evaluation of a pattern recognition technique

Close similarities have been found between the otoliths of sea-caught and laboratory-reared larvae of the common sole Solea solea (L.), given appropriate temperatures and nourishment of the latter. But from hatching to mouth formation. and during metamorphosis, sole otoliths have proven difficult to read because the increments may be less regular and low contrast. In this study, the growth increments in otoliths of larvae reared at 12 degrees C were counted by light microscopy to test the hypothesis of daily deposition, with some results verified using scanning electron microscopy (SEM), and by image analysis in order to compare the reliability of the 2 methods in age estimation. Age was first estimated (in days posthatch) from light micrographs of whole mounted otoliths. Counts were initiated from the increment formed at the time of month opening (Day 4). The average incremental deposition rate was consistent with the daily hypothesis. However, the light-micrograph readings tended to underestimate the mean ages of the larvae. Errors were probably associated with the low-contrast increments: those deposited after the mouth formation during the transition to first feeding, and those deposited from the onset of eye migration (about 20 d posthatch) during metamorphosis. SEM failed to resolve these low-contrast areas accurately because of poor etching. A method using image analysis was applied to a subsample of micrograph-counted otoliths. The image analysis was supported by an algorithm of pattern recognition (Growth Demodulation Algorithm, GDA). On each otolith, the GDA method integrated the growth pattern of these larval otoliths to averaged data from different radial profiles, in order to demodulate the exponential trend of the signal before spectral analysis (Fast Fourier Transformation, FFT). This second method both allowed more precise designation of increments, particularly for low-contrast areas, and more accurate readings but increased error in mean age estimation. The variability is probably due to a still rough perception of otolith increments by the GDA method, counting being achieved through a theoretical exponential pattern and mean estimates being given by FFT. Although this error variability was greater than expected, the method provides for improvement in both speed and accuracy in otolith readings.