Skip to main content
SpringerLink
Log in

Examining bivalve fecundity: oocyte viability revealed by Neutral Red vital staining

  • Published:
Aquaculture International Aims and scope Submit manuscript

Abstract

Estimation of realized fecundity (Freal, number of viable oocytes produced) is an essential, yet seldom-achieved element in the understanding of marine animal production and population dynamics. We used the Neutral Red (NR) vital stain to determine oocyte viability in spawns and gonad strippings of four species of commercially-important bivalves: Cerastoderma edule (L), Crassostrea gigas Thunberg, Mytilus edulis L, and Tapes philippinarum (Adams and Reeve). The utility of Trypan Blue as a complementary mortal stain was also assessed, and found to be unnecessary. Normal, live oocytes stained with NR, and were either spherical (mature oocytes) or pedunculated (immature oocytes); atresic/abnormal oocytes also stained with NR, allowing their ready recognition due to their abnormal shapes and cytoplasmic retraction. Dead oocytes did not stain with NR. Across the species studied, a considerable and highly variable proportion of spawned or stripped oocytes was either dead or non-viable; quantitative counts were performed for Cerastoderma edule, the only species for which > 5 spawns occurred. The high level and variability (34–85%) of dead or non-viable oocytes is consistent with a reproductive Red Queen dilemma, in which greater oocyte numbers do not translate to commensurately greater real fecundities, and also with a Sweepstakes Reproductive Success strategy, in which a large range of Freal confronts the considerable variability of intertidal environmental conditions. Neutral Red vital staining is a promising tool for the elucidation and optimization of crucial yet previously intractable aspects of bivalve hatchery production, genetic improvement, restocking, stock management, and conservation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

All files from this study are available from the authors upon reasonable request.

References

  • Adiyodi K, Adiyodi R (1983) Reproductive biology of invertebrates. Vol. I: Oogenesis, oviposition, and oosorption. Wiley, New York

  • Aguirre-Martínez GV, Buratti S, Fabbri E, DelValls AT, Martín-Díaz ML (2013) Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin. J Environ Sci 25:1408–1418

    Google Scholar 

  • Angel-Dapa MA, Rodríguez-Jaramillo C, Cáceres-Martínez CJ, Saucedo PE (2010) Changes in lipid content of oocytes of the penshell Atrina maura as a criterion of gamete development and quality: a study of histochemistry and digital image analysis. J Shellfish Res 29:407–413

    Google Scholar 

  • Aoyama S (1989) The Mutu Bay scallop fisheries: scallop culture, stock enhancement, and resource management. In: Caddy JF (ed) Marine invertebrate fisheries: their assessment and management. Wiley, New York, pp 525–539

    Google Scholar 

  • Arnold WS (2008) Application of larval release for restocking and stock enhancement of coastal marine bivalve populations. Rev Fish Sci 16:65–71

    Google Scholar 

  • Babich B, Borenfreund E (1993) Applications of the neutral red cytotoxicity assay to risk assessment of aquatic contaminants: an overview. In: Environmental toxicology and risk assessment, W. Landis, J. Hughes and M. Lewis. ASTM International, West Conshohocken, PA, pp 215–219

  • Barros J, Velasco L, Winkler F (2018) Heritability, genetic correlations and genotype by environment interactions in productive traits of the Caribbean scallop, Argopecten nucleus(Mollusca: Bivalvia). Aquaculture 488:39–48

    Google Scholar 

  • Beninger PG (2017) Caveat observator: the many faces of pre-spawning atresia in marine bivalve reproductive cycles. Mar Biol:164–163. https://doi.org/10.1007/s00227-017-3194-x

  • Beninger PG, Boldina I (2018) Quantitative considerations in mudflat ecology. In: Mudflat ecology. P.G. Beninger. Springer, Cham, Switzerland, pp 389–419

    Google Scholar 

  • Beninger PG, Boldina I, Katsanevakis S (2012) Strengthening statistical usage in marine ecology. J Exp Mar Biol Ecol 426:97–108

    Google Scholar 

  • Borenfreund E, Puerner JA (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Cult Meth 9:7–9

    Google Scholar 

  • Borenfreund E, Puerner JA (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett 24:119–124

    CAS  PubMed  Google Scholar 

  • Borenfreund E, Babich H, Martin-Alguacil N (1988) Comparisons of two in vitro cytotoxicity assays—the neutral red (NR) and tetrazolium MTT tests. Toxicol in vitro 2:1–6

    CAS  PubMed  Google Scholar 

  • Boudry P (2009) Genetic variation and selective breeding in hatchery-propagated molluscan shellfish. In: New technologies in aquaculture. G. Burnell and G. Allan. CRC Press-Woodhead Publishing Ltd, Boca Raton FL, pp 87–108

    Google Scholar 

  • Buttner JK, Weston S (2010) Softshell clam culture: hatchery phase, broodstock care through seed production. Northeastern Regional Aquaculture Center Publication No. 202-2010, 12 pp

  • Caers M, Coutteau P, Cure K, Morales V, Gajardo G, Sorgeloos P (1999) The Chilean scallop Argopecten purpuratus (Lamarck, 1819): II. Manipulation of the fatty acid composition and lipid content of the eggs via lipid supplementation of the broodstock diet. Comp Biochem Physiol B: Biochem Mol Biol 123:97–103

    Google Scholar 

  • Caers M, Utting S, Coutteau P et al (2002) Impact of the supplementation of a docosahexaenoic acid–rich emulsion on the reproductive output of oyster broodstock, Crassostrea gigas. Mar Biol 140:1157–1166

    CAS  Google Scholar 

  • Cannuel R, Beninger PG (2005) Is oyster broodstock feeding always necessary? A study using oocyte quality predictors and validators in Crassostrea gigas. Aquat Liv Res 18:35–43

    Google Scholar 

  • Chérel D, Beninger PG (2017) Oocyte atresia characteristics and effect on reproductive effort of Manila clam Tapes philippinarum (Adams and Reeve, 1850). J Shellfish Res 36:549–557

    Google Scholar 

  • Chérel D, Beninger PG (2019) Oocyte atresia and its effect on reproductive effort of the common cockle Cerastoderma edule (Linneaus, 1758). J Shellfish Res 38:603–609. https://doi.org/10.2983/035.038.0300

    Article  Google Scholar 

  • Chérel D, Beninger PG, Le Pennec G (2020) Two enigmas may solve each other: the oocyte coat and atresia in the common cockle, Cerastoderma edule (Linnaeus, 1758). Mar Biol 167:104. https://doi.org/10.1007/s00227-020-03718-6

    Article  CAS  Google Scholar 

  • Chiba K, Kawakami K, Tohyama K (1998) Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells. Toxicol in vitro 12:251–258

    CAS  PubMed  Google Scholar 

  • Corporeau C, Vanderplancke G, Boulais M, Suquet M, Quéré C, Boudry P, Huvet A, Madec S (2012) Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas. J Proteom 75:5554–5563

    CAS  Google Scholar 

  • Crippen RW, Perrier J (1974) The use of neutral red and Evans blue for live-dead determinations of marine plankton (with comments on the use of rotenone for inhibition of grazing). Stain Technol 49:97–104

    CAS  PubMed  Google Scholar 

  • Da Luz DS, Da Silva DG, Souza MM et al (2016) Efficiency of Neutral Red, Evans Blue and MTT to assess viability of the freshwater microalgae Desmodesmus communis and Ediastrum boryanum. Phycol Res 64:56–60

    Google Scholar 

  • Dressel DM, Heinle DR, Grote MC (1972) Vital staining to sort dead and live copepods 1, 2, 3. Chesapeake Sci 13:156–159

    Google Scholar 

  • Ehrlich P (1894) Ueber Neutralrot. Allg med Zentral-Zeit 20:

  • Elliott DT, Tang KW (2009) Simple staining method for differentiating live and dead marine zooplankton in field samples. Limnol Oceanogr: Methods 7:585–594

    Google Scholar 

  • Gallager SM, Mann R (1986) Growth and survival of larvae of Mercenaria mercenaria (L.) and Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs. Aquaculture 56:105–121

    CAS  Google Scholar 

  • Gallager SM, Mann R, Sasaki GC (1986) Lipid as an index of growth and viability in three species of bivalve larvae. Aquaculture 56:81–103

    CAS  Google Scholar 

  • García-Corona JL, Rodríguez-Jaramillo C, Saucedo PE, López-Carvallo JA, Arcos-Ortega GF, Mazón-Suástegui JM (2018) Internal energy management associated with seasonal gonad development and oocyte quality in the Horsemussel Modiolus capax (Bivalvia; Mytilidae). J Shellfish Res 37:475–484

    Google Scholar 

  • Gomez ED, Mingoa-Licuanan SS (2006) Achievements and lessons learned in restocking giant clams in the Philippines. Fish Res 80:46–52. https://doi.org/10.1016/j.fishres.2006.03.017

    Article  Google Scholar 

  • Gómez-Robles E, RodrIguez-Jaramillo C, Saucedo PE (2005) Digital image analysis of lipid and protein histochemical markers for easuring oocyte development and quality in pearl oyster Pinctada mazatlanica (Hanley, 1856). Journal of Shellfish Research 24:1197–1202. https://doi.org/10.2983/0730-8000(2005)24[1197:DIAOLA]2.0.CO;2

    Article  Google Scholar 

  • González-Wangüemert M, Basso L, Balau A, Costa J, Renault L, Serrão EA, Duarte CM, Hendriks IE (2018) Gene pool and connectivity patterns of Pinna nobilis in the Balearic Islands (Spain, Western Mediterranean Sea): implications for its conservation through restocking. Aquat Conserv: Mar Freshw Ecosyst 29:175–188. https://doi.org/10.1002/aqc.2976

    Article  Google Scholar 

  • Hammond M, Goodwin J, Dvorak H (1980) Quantitative measurements of neutral red uptake and excretion by mammalian cells. J Reticuloendothel Soc 27:337

    CAS  PubMed  Google Scholar 

  • Hedgecock D, Pudovkin AI (2011) Sweepstakes Reproductive Success in highly fecund marine fish and shellfish: a review and commentary. Bull Mar Sci 87:971–1002. https://doi.org/10.5343/bms.2010.1051

    Article  Google Scholar 

  • His E, Beiras R, Seaman M (1999) The assessment of marine pollution-bioassays with bivalve embryos and larvae. Adv Mar Biol 37:1–178

    Google Scholar 

  • Horvath TG, Lamberti GA (1999) Mortality of zebra mussel, Dreissena polymorpha, veligers during downstream transport. Freshw biol 42:69–76

    Google Scholar 

  • Hu W, Culloty S, Darmody G, Lynch S, Davenport J, Ramirez-Garcia S, Dawson K, Lynch I, Doyle H, Sheehan D (2015) Neutral red retention time assay in determination of toxicity of nanoparticles. Mar Environ Res 111:158–161. https://doi.org/10.1016/j.marenvres.2015.05.007

    Article  CAS  PubMed  Google Scholar 

  • Jacques PJ (1969) Endocytosis. Lysosomes in biology and pathology 2:395–420

    CAS  Google Scholar 

  • Jennings S, Kaiser MJ, Reynolds JD (2001) Marine fisheries ecology. Blackwell Scientific Publication

  • Joaquim S, Gaspar MB, Matias D, Ben-Hamadou R, Arnold WS (2008) Rebuilding viable spawner patches of the overfished Spisula solida (Mollusca: Bivalvia): a preliminary contribution to fishery sustainability. ICES J Mar Sci 65:60–64

    Google Scholar 

  • Koslow JA (1992) Fecundity and the stock–recruitment relationship. Can J of Fish Aquat Sci 49:210–217

    Google Scholar 

  • Kreeger DA, Gatenby CM, Bergstrom PW (2018) Restoration potential of several native species of Bivalve Molluscs for water quality improvement in Mid-Atlantic Watersheds. Journal Shellfish Res 37:1121. https://doi.org/10.2983/035.037.0524

    Article  Google Scholar 

  • Kwok AKH, Lai TYY, Li WWY, Yew DTW, Wong VWY (2004) Trypan blue- and indocyanine green-assisted epiretinal membrane surgery: clinical and histopathological studies. Eye 18:882–888. https://doi.org/10.1038/sj.eye.6701359

    Article  CAS  PubMed  Google Scholar 

  • Liu X, Rodeheaver DP, White JC, Wright AM, Walker LM, Zhang F, Shannon S (2018) A comparison of in vitro cytotoxicity assays in medical device regulatory studies. Regul Toxicol Pharmacol 97:24–32. https://doi.org/10.1016/j.yrtph.2018.06.003

    Article  CAS  PubMed  Google Scholar 

  • Lowe D, Moore M, Evans B (1992) Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from dab Limanda limanda. Mar Ecol Prog Ser 91:135–140. https://doi.org/10.3354/meps091135

    Article  CAS  Google Scholar 

  • Lowe D, Fossato V, Depledge M (1995a) Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar Ecol Prog Ser 129:189–196. https://doi.org/10.3354/meps129189

    Article  Google Scholar 

  • Lowe DM, Soverchia C, Moore MN (1995b) Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene. Aquat Toxicol 33:105–112. https://doi.org/10.1016/0166-445X(95)00015-V

    Article  CAS  Google Scholar 

  • Massapina C, Joaquim S, Matias D, Devauchelle N (1999) Oocyte and embryo quality in Crassostrea gigas (Portuguese strain) during a spawning period in Algarve, South Portugal. Aquat Living Resour 12:327–333

    Google Scholar 

  • Maunder MN, Thorson JT (2019) Modeling temporal variation in recruitment in fisheries stock assessment: a review of theory and practice. Fish Res 217:71–86

    Google Scholar 

  • Mejuto J (1984) Primeros datos sobre la dinámica de la población de Cerastoderma edule (L.) de la Ría do Pasaxe (NW de Galicia): estrategias de explotación. Actas IV Simp Iber Estud Bentos Mar 1:83–102

    Google Scholar 

  • Militz TA, Southgate PC (2021) Chapter 3: Culture of giant clams. In: Shumway, S (ed) Molluscan shellfish aquaculture: a practical guide. 5M Books Ltd, Essex, pp 61-85

  • Moens T, Beninger PG (2018) Meiofauna: an inconspicuous but important player in mudflat ecology. In: Beninger PG (ed) Mudflat Ecology. Springer International Publishing, Cham, pp 91–147

    Google Scholar 

  • Nemes Z, Dietz R, Lüth JB, Gomba S, Hackenthal E, Gross F (1979) The pharmacological relevance of vital staining with neutral red. Experientia 35:1475–1476. https://doi.org/10.1007/BF01962793

    Article  CAS  PubMed  Google Scholar 

  • Nevejan N, Courtens V, Hauva M, Gajardo G, Sorgeloos P (2003) Effect of lipid emulsions on production and fatty acid composition of eggs of the scallop Argopecten purpuratus. Mar Biol 143:327–338

    CAS  Google Scholar 

  • Patetsini E, Dimitriadis VK, Kaloyianni M (2013) Biomarkers in marine mussels, Mytilus galloprovincialis, exposed to environmentally relevant levels of the pesticides, chlorpyrifos and penoxsulam. Aquat Toxicol 126:338–345. https://doi.org/10.1016/j.aquatox.2012.09.009

    Article  CAS  PubMed  Google Scholar 

  • Pérez Camacho A, Román G (1984) Crecimiento, reproducción, mortalidad y producción del berberecho Cerastoderma edule (L.), en la Ría de Arousa. Cuad Area Cienc Mariñ Sem Est Gal 499–507

  • Plough LV (2018) Fine-scale temporal analysis of genotype-dependent mortality at settlement in the Pacific oyster Crassostrea gigas. J Exp Mar Biol Ecol 501:90–98

    Google Scholar 

  • Plough L, Shin G, Hedgecock D (2016) Genetic inviability is a major driver of type III survivorship in experimental families of a highly fecund marine bivalve. Mol Ecol 25:895–910

    CAS  PubMed  Google Scholar 

  • Rajab GZE-A, Demer JL (2019) Long-term results of surgical excision of conjunctival retention cyst using trypan blue with methylcellulose. Am J Ophthalmol Case Rep 14:28–31. https://doi.org/10.1016/j.ajoc.2019.01.010

    Article  Google Scholar 

  • Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3:1125–1131. https://doi.org/10.1038/nprot.2008.75

    Article  CAS  PubMed  Google Scholar 

  • Shantharam AK, Padilla DK, Peterson BJ, Doall M, Lobue C, Webb A (2019) Macrofaunal community structure following the restocking of Northern Quahog (Mercenaria mercenaria) to Great South Bay, Long Island, NY. J Shelf Res 38:259–270. https://doi.org/10.2983/035.038.0206

    Article  Google Scholar 

  • Smolarz K, Hallmann A, Zabrzańska S, Pietrasik A (2017) Elevated gonadal atresia as biomarker of endocrine disruptors: field and experimental studies using Mytilus trossulus (L.) and 17-alpha ethinylestradiol (EE2). Mar Poll Bull 120:58–67. https://doi.org/10.1016/j.marpolbul.2017.04.007

    Article  CAS  Google Scholar 

  • Strober W (2015) Trypan Blue exclusion test of cell viability. Curr Protoc Immunol 111:A3.B.1-A3.B.3. doi:https://doi.org/10.1002/0471142735.ima03bs111

  • Svendsen C, Spurgeon DJ, Hankard PK, Weeks JM (2004) A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker? Ecotoxicol Environ Safety 57:20–29. https://doi.org/10.1016/j.ecoenv.2003.08.009

    Article  CAS  PubMed  Google Scholar 

  • Talbot P, Chacon RS (1981) A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool 215:201–208. https://doi.org/10.1002/jez.1402150210

    Article  CAS  PubMed  Google Scholar 

  • Tang KW, Freund CS, Schweitzer CL (2006) Occurrence of copepod carcasses in the lower Chesapeake Bay and their decomposition by ambient microbes. Estuar Coast Shelf Sci 68:499–508

    Google Scholar 

  • Triglia D, Braa SS, Yonan C, Naughton GK (1991) In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model. In Vitro Cell Devel Biol-Animal 27:239–244

    Google Scholar 

  • Uttig SD, Spencer BE (1991) The hatchery culture of bivalve mollusc larvae and juveniles. Laboratory Leaflet, Ministry of Agriculture, Fisheries and Food Fisheries Research, Lowestoft 68:31 pp

  • Utting SD, Millican P (1997) Techniques for the hatchery conditioning of bivalve broodstocks and the subsequent effect on egg quality and larval viability. Aquaculture 155:45–54

    Google Scholar 

  • Valdez-Ramirez ME, Donval A, Le Pennec M (2002) Ultrastructural and histochemical criteria for determining normality in mature oocytes of the Pacific oyster Crassostrea gigas. J Shellfish Res 21:707–714

    Google Scholar 

  • Wales R (1959) The differential staining of human and dog spermatozoa. Aust J Exp Biol Med 37:433–439. https://doi.org/10.1038/icb.1959.44

    Article  Google Scholar 

  • Weeks JM, Svendsen C (1996) Neutral red retention by lysosomes from earthworm (Lumbricus rubellus) coelomocytes: A simple biomarker of exposure to soil copper. Environ Toxicol Chem 15:1801–1805. https://doi.org/10.1002/etc.5620151022

    Article  CAS  Google Scholar 

  • Wijsman JWM, Troost K, Fang J, Roncarati A (2019) Global production of marine bivalves. Trends and challenges. In: Smaal AC, Ferreira JG, Grant J et al (eds) Goods and services of marine bivalves. Springer International Publishing, Cham, pp 7–26

    Google Scholar 

  • Winckler J (1974) Vitalfärbung von Lysosomen und anderen Zellorganellen der Ratte mit Neutralrot. Prog Histochem Cyto 6:III–89. https://doi.org/10.1016/S0079-6336(74)80001-X

    Article  Google Scholar 

  • Wourms J (1987) Oogenesis. In: Reproduction of marine invertebrates, Vol 9: General aspects, seeking unity in diversity. Blackwell Scientifc Publications, Palo Alto, pp 117–124

    Google Scholar 

  • Zetsche EM, Meysman FJ (2012) Dead or alive? Viability assessment of micro-and mesoplankton. J Plankton Res 34:493–509

    CAS  Google Scholar 

Download references

Acknowledgements

We thank Mathilde Clairambault for her patience and perseverance in the laboratory spawning trials. We are indebted to David Berteau and employees of Chellet-Berteau Production for their warm welcome and permission to sample on-site.

Author information

Authors and Affiliations

  1. Laboratoire de Biologie Marine, MMS, Faculté des Sciences, Université de Nantes, 2 rue de la Houssinière, 44322, Nantes, Cédex, France

    Peter G. Beninger & Daphné Chérel

  2. Faculté des Sciences et Technologies, La Rochelle Université, Avenue Michel Crépeau, 17042, La Rochelle, Cédex, France

    Lucie Kessler

Authors
  1. Peter G. Beninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daphné Chérel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lucie Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter G. Beninger.

Ethics declarations

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.

Conflict of interest

The authors declare no competing interests.

Additional information

Handling Editor: Gavin Burnell

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Beninger, P.G., Chérel, D. & Kessler, L. Examining bivalve fecundity: oocyte viability revealed by Neutral Red vital staining. Aquacult Int 29, 1219–1231 (2021). https://doi.org/10.1007/s10499-021-00686-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10499-021-00686-6

Keywords

Search

Navigation