Evaluation of fixatives and autofluorescence reduction treatments for marine bivalve larvae

Improved understanding of the occurrence and spatio-temporal distribution of bivalve larvae holds significant benefits for ecological studies, shellfisheries management and aquaculture. Morphological methods for identification have proved difficult to develop because of the small size of these larvae and similarities in their shape and colour. Molecular methods based on DNA extraction can confirm the presence of a species in a plankton sample, but without sample sorting and individual larval analysis, provide no estimate of larval abundance and are incapable of providing an estimate of larval growth rate. Fluorescencein situhybridization (FISH) using species-specific DNA probes has the potential to resolve these issues. However, utilization of this technique is constrained by the strong autofluorescence, common in marine larvae. Here we evaluate the effect of eight different fixatives on the autofluorescence intensity of bivalve larvae using fluorescein isothiocyanate (FITC) and Cy3 filters. In addition, fifteen autofluorescence reduction treatments were evaluated and their compatibility with FISH assessed. Relative to fresh larvae, chemically fixed larvae had significantly higher autofluorescence in both filter sets. Larvae preserved by freezing at –80°C exhibited no significant increase in autofluorescence over a 3-year period. Autofluorescence levels were generally lower with the FITC filter set than the Cy3 filter set. For archived larvae preserved in modified saline ethanol and exhibiting fixative-induced autofluorescence, the autofluorescence intensity could be reduced to 20–30% with saturated Sudan Black B and to 30–40% with Chemicon™. Both of these autofluorescence reduction treatments were compatible with subsequent FISH protocols using a FITC-labelled probe.

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Short and long term consequences of larval stage exposure to constantly and ephemerally elevated carbon dioxide for marine bivalve populations

Biogeosciences Discussions ◽

10.5194/bgd-9-15901-2012 ◽

2012 ◽

Vol 9 (11) ◽

pp. 15901-15936

Author(s):

C. J. Gobler ◽

S. C. Talmage

Keyword(s):

Larval Development ◽

Larval Stage ◽

Growth Rates ◽

Larval Growth ◽

Marine Bivalve ◽

High Co2 ◽

Legacy Effect ◽

Bivalve Larvae ◽

Co2 Concentrations

Abstract. While larval bivalves are highly sensitive to ocean acidification, the basis for this sensitivity and the longer term implications of this sensitivity are unclear. Experiments were performed to assess the short term (days) and long term (months) consequences of larval stage exposure to varying CO2 concentrations for calcifying bivalves. Higher CO2 concentrations depressed both calcification rates assessed using 45Ca uptake and RNA:DNA ratios in Mercenaria mercenaria and Argopecten irradians larvae with RNA:DNA ratios being highly correlated with larval growth rates r2 > 0.9). These findings suggested that high CO2 has a cascading negative physiological impact on bivalve larvae stemming in part from lower calcification rates. Exposure to elevated CO2 during the first four days of larval development significantly depressed A. irradians larval survival rates, while a 10 day exposure later in larval development did not, demonstrating the extreme CO2-sensitivity of bivalve larvae during first days of development. Short- (weeks) and long-term (10 month) experiments revealed that individuals surviving exposure to high CO2 during larval development grew faster when exposed to normal CO2 as juveniles compared to individuals reared under ambient CO2 as larvae. These increased growth rates could not, however, overcome size differences established during larval development, as size deficits of individuals exposed to even moderate levels of CO2 as larvae were evident even after 10 months of growth under normal CO2 concentrations. This `legacy effect' emphasizes the central role larval stage CO2 exposure can play in shaping the success of modern day bivalve populations.

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Species identification of bivalve larvae using random amplified polymorphic DNA (RAPD): differentiation between Cerastoderma edule and C. lamarcki

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315498000691 ◽

1999 ◽

Vol 79 (3) ◽

pp. 563-565 ◽

Cited By ~ 24

Author(s):

C. André ◽

M. Lindegarth ◽

P.R. Jonsson ◽

P. Sundberg

Keyword(s):

Random Amplified Polymorphic Dna ◽

Macoma Balthica ◽

Morphological Characters ◽

Marine Bivalve ◽

Species Identity ◽

Quick Method ◽

Cerastoderma Edule ◽

Bivalve Larvae ◽

Scrobicularia Plana ◽

Species Specific

The polymerase chain reaction (PCR) was used to produce species-specific DNA markers (RAPDs) from two sibling cockle species and five other co-occurring intertidal bivalves. Amplification reactions with one single primer readily distinguished larvae and adults of Cerastoderma edule from larvae and adults of C. lamarcki, and from adults of Mya arenaria, Macoma balthica, Scrobicularia plana, Venerupis pulastra and Mytilus edulis. Random amplified polymorphic DNA (RAPD) is suggested as a simple and quick method to determine species identity in taxa that are difficult to identify on the basis of morphological characters alone, such as marine bivalve larvae.

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Short- and long-term consequences of larval stage exposure to constantly and ephemerally elevated carbon dioxide for marine bivalve populations

Biogeosciences ◽

10.5194/bg-10-2241-2013 ◽

2013 ◽

Vol 10 (4) ◽

pp. 2241-2253 ◽

Cited By ~ 50

Author(s):

C. J. Gobler ◽

S. C. Talmage

Keyword(s):

Larval Development ◽

Larval Stage ◽

Growth Rates ◽

Larval Growth ◽

Marine Bivalve ◽

High Co2 ◽

Legacy Effect ◽

Bivalve Larvae ◽

Co2 Concentrations

Abstract. While larval bivalves are highly sensitive to ocean acidification, the basis for this sensitivity and the longer-term implications of this sensitivity are unclear. Experiments were performed to assess the short-term (days) and long-term (months) consequences of larval stage exposure to varying CO2 concentrations for calcifying bivalves. Higher CO2 concentrations depressed both calcification rates assessed using 45Ca uptake and RNA : DNA ratios in Mercenaria mercenaria and Argopecten irradians larvae with RNA : DNA ratios being highly correlated with larval growth rates (r2>0.9). These findings suggested that high CO2 has a cascading negative physiological impact on bivalve larvae stemming in part from lower calcification rates. Exposure to elevated CO2 during the first four days of larval development significantly depressed A. irradians larval survival rates, while a 10-day exposure later in larval development did not, demonstrating the extreme CO2 sensitivity of bivalve larvae during first days of development. Short- (weeks) and long-term (10 month) experiments revealed that individuals surviving exposure to high CO2 during larval development grew faster when exposed to normal CO2 as juveniles compared to individuals reared under ambient CO2 as larvae. These increased growth rates could not, however, overcome size differences established during larval development, as size deficits of individuals exposed to even moderate levels of CO2 as larvae were evident even after 10 months of growth under normal CO2 concentrations. This "legacy effect" emphasizes the central role larval stage CO2 exposure can play in shaping the success of modern-day bivalve populations.

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Analysis of spatio-temporal distribution of mortality due to gastric and colon cancer in Hungary and in Japan between 1986–2002

Zeitschrift für Gastroenterologie ◽

10.1055/s-2008-1079715 ◽

2008 ◽

Vol 46 (05) ◽

Cited By ~ 1

Author(s):

K Tamássy ◽

A Páldy ◽

G Nádor ◽

A Laborczi ◽

L Pásztor ◽

...

Keyword(s):

Colon Cancer ◽

Temporal Distribution ◽

Spatio Temporal

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Spatial ecology and growth in early life stages of bay anchovy Anchoa mitchilli in Chesapeake Bay (USA)

Marine Ecology Progress Series ◽

10.3354/meps13431 ◽

2020 ◽

Vol 651 ◽

pp. 125-143

Author(s):

TD Auth ◽

T Arula ◽

ED Houde ◽

RJ Woodland

Keyword(s):

Chesapeake Bay ◽

Spatial Ecology ◽

Environmental Variability ◽

Larval Growth ◽

Larval Abundance ◽

Spatial And Temporal Variability ◽

Estuarine Ecosystem ◽

Anchoa Mitchilli ◽

Larval Production ◽

Bay Anchovy

The bay anchovy Anchoa mitchilli is the most abundant fish in Chesapeake Bay (USA) and is a vital link between plankton and piscivores within the trophic structure of this large estuarine ecosystem. Baywide distributions and abundances of bay anchovy eggs and larvae, and larval growth, were analyzed in a 5 yr program to evaluate temporal and spatial variability based on research surveys in the 1995-1999 spawning seasons. Effects of environmental variability and abundance of zooplankton that serve as prey for larval bay anchovy were analyzed. In the years of these surveys, 97.6% of eggs and 98.8% of larvae occurred in the polyhaline lower bay. Median egg and larval abundances differed more than 10-fold for surveys conducted in the 5 yr and were highest in the lower bay. Within years, median larval abundance (ind. m-2) in the lower bay was generally 1-2 orders of magnitude higher than upper-bay abundance. Salinity, temperature, and dissolved oxygen explained 12% of the spatial and temporal variability in egg abundances and accounted for 27% of the variability in larval abundances. The mean, baywide growth rate for larvae over the 5 yr period was 0.75 ± 0.01 mm d-1, and was best explained by zooplankton concentration and feeding incidence. Among years, mean growth rates ranged from 0.68 (in 1999) to 0.81 (in 1998) mm d-1 and were fastest in the upper bay. We identified environmental factors, especially salinity, that contributed to broadscale variability in egg and larval production.

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