The Crustacean Species as In Vivo Testing Model – Advantages and Possible Applications in the Nutrition or Pharmaceutical Field

SCHRÖDER Verginica1, BUCUR Adriana Laura1*, IANCU Irina Mihaela1, HONCEA Adina2, BUȘURICU Florica1

1Faculty of Pharmacy, Ovidius University, Mamaia Blvd. 124, 900527, Constanta

2CF Hospital, Bulevardul 1 Mai 3-5, 900123, Constanța 

Emails: verginica.schroder@univ-ovidius.ro, adrianabucur@yahoo.com, iryluna@yahoo.com, adina_honcea@yahoo.com, busuricuflori@yahoo.com

*Coresponding author: adrianabucur@yahoo.com

Abstract

     The evaluation of natural compounds with applications in the fields of nutrition or drug development, is going through testing difficulties, as a consequence of the current legislation which limits the use of laboratory animals. The current study is a review of the main advantages regarding the use of two types of aquatic crustaceans, Artemia salina and Gammarus balcanicus, as biotest organisms for the in vivo evaluation of some pharmaceuticals and natural components. The advantages of these crustacean’s animal model for testing are related to the biology of the species, the particularities of the development, the short cell cycle, the transparency of the organisms (which allows the visualization of the structural changes), the sensitivity to chemical compounds and the other adaptive characteristics. Also, these bio tester organisms facilitate: the speed of testing, the replicability, the use of a large number of specimens of the same age and with the same anatomical-morphological features, very low costs and short time required for testing and the relevance of evaluation. The use of these organisms might represent an important step in the preliminary study of potentially bioactive compounds, given the advantages of in vivo examination.

Keywords: animal model, crustaceans, testing model.

Introduction

    A series of biological processes have remained throughout the conserved evolution, so that the fundamental way of organization and functioning can be found in different organisms from an evolutionary point of view [1, 2, 3]. For these reasons, studies on different taxonomic organisms have elucidated a series of biological phenomena extrapolated to humans. As a result, invertebrates have recently begun to be used as alternatives to laboratory animals for testing, [4, 5, 6] given that there are no legislative limitations for these organisms [7]. They have been used in studies related to Parkinson’s disease, endocrine and memory dysfunction, muscle dystrophy, wound healing, cell aging, programmed cell death, retrovirus biology, diabetes and toxicological testing [6]. 

     Although there are significant differences in the structural organization from the human one, these lower organisms offer a significant number of benefits such as short life cycle, small size, simplified anatomy, cellular or molecular system homology, as a result a large number of invertebrates can be studied in a single experiment within a short period with less ethical problems [5].

      More and more studies have been performed on aquatic organisms in order to identify effects induced by chemicals in the environment, [8] plant extracts [9] or active pharmaceutical ingredients [10]. The results have established new approaches to invertebrate testing. These organisms have acquired bio tester value in the field of ecotoxicology [11, 12, 13, 14], but a series of adaptive qualities can be taken into account in testing natural compounds [15, 16]. 

     To understand in detail the mechanisms of reaction to chemicals, strongly conserved in invertebrates, research models have been created. Cima, 2010, highlights by studying crustaceans’ mechanisms for understanding the evolution of the immune system. According to this study, crustaceans have haemocytes like human macrophages [17]. 

      Also, crustaceans are a model for the study of hormonal control of mineralization, calcification-decalcification of epithelia mimics the vertebrate system [18]. The absorption of water at the level of the epithelium and the maintenance of the mineralization process of the new skeleton represent one of the most important processes for the further growth of these organisms, but also models of membrane activity [19]. 

       Another biomedical application is the understanding of neural mechanisms where crustaceans are a useful model for identifying and manipulating neural systems and characterizing how drug abuse can affect the neural activity of these drug-dependent systems [10, 20, 21]. Also, Paula Chaves da Silva, in 2013, identifies similarities between neurodegenerative processes in invertebrates, respectively crustaceans, and cellular and biochemical processes in vertebrates [2]. The possibility of better understanding unknown mechanisms creates the premises of a more adequate therapeutic approach.

      All these resemblances are important given the need to find a test system with morpho structural and functional homologies. The results depend a lot on the model of species chosen in regard to the type of test and the desired outcome.

Advantages of in vivo testing on crustaceans

  • It is an easy material to handle in the laboratory; efficiency is also given by the significant reduction of time, testing costs and the possibility of performing many repetitions. The specific laboratory materials are aquariums and water pumping installations, utensils and devices necessary for measuring the physico-chemical parameters of water during the experiment (pH, oxygen, salinity, conductivity, oxygen consumption).
  • The methods and techniques are identical with the applications used on human or mammalian samples (microscopy techniques, spectrometry, enzymatic evaluation, cytotoxicity, genotoxicity). 
  • Testing allows the evaluation of water-soluble substances in which the whole body is in contact with the tested substance. As a result, the effect of some membrane mechanisms, can be tested and appreciated quite easily (membrane transport of the tested compounds, effects on membrane permeability or integrity of cellular signalling through membrane receptors).
  • Assessments can be made regarding sublethal or lethal effects, cytotoxicity measurements, evaluations of lipid or protein, repeated, in a short time. The speed of the test and the possibility to quantify the effects by establishing the benchmarks according to the purpose of the test.
  • If it is desired to follow the way in which the tested compounds are ingested, the functionality of the digestive tract or the amount of damage can be evaluated.
  • The test can be used before testing on mammals as well as before testing on cell cultures, in both cases leading to cost limitations.
  • The possibility of building mathematical models based on many tested organisms. The possible number of tested specimens can be large, up to the hundreds. 

Artemia sp. and Gammarus sp. examples of organisms used as a biological model in laboratory testing

     Our previous studies have highlighted the effects of natural compounds [16, 22, 23] on the crustacean’s animal model. In addition, the effects of exposing the animals to drugs [24, 25], food dyes [15] or other additives [26] were identified in the laboratory. These biomarker species offered the possibility to evaluate some cytological phenomena under the action of plant extracts [27, 16] contributing to estimates regarding the biological activity of the analysed compounds as well as being compared with other testing methods, such as human tumour cell cultures procedures [16].

  1. Artemia sp. is one of the most commonly used crustacean species used as a biological model for testing the toxicity of xenobiotics [28, 29], plant extracts [9, 30] or components of animal origin [31]. It is also a model for the teratogenic actions of some substances [32] or for the evaluation of the toxic activity of nanoparticles [33] in examining the toxic effect induced by bioactive components [34].

     The species has several advantages that qualify it to be a test model such as: small size of body, a short life cycle, the larvae in the first stages are easy to follow, being transparent allowing in vivo cytological evaluations, with / or without staining (Fig.1). The naupliar stage I (larvae in the first 24 h after hatching) are used in short-term tests, having the advantage that they do not feed, and the evaluation of the tested substances will be made based on the mechanisms of penetration through membranes, not interfering with digestion. They allow the evaluation of small quantities of substance, the testing can be done in experimental premises (cell cultures plates) with a test volume of 1-2 mL, and the answer is given by the tested substance at concentrations of ng or µg.

Figure 1. The Artemia sp. and Gammarus sp. model of experimental testing design

  1. Gammarus sp. is an organism adapted to aquatic conditions, being found in clean freshwater with vegetal detritus. These biological and ecological characteristics have allowed the use of this species as a bio-tester [11, 14] which has certain advantages: it is easy to purchase and does not require special conditions of maintenance or feeding. The organisms are accommodated to laboratory conditions for 10 days after which they could be moved into small recipients and proceed the experimentation with the added testing compounds (Fig. 1). It has high sensitivity to environmental chemicals and small dimensions. 

The measurement of the effects and the determination of the acute toxicity (24 – 96h) or of the chronic toxicity (maximum 10 -14 days) can be done by quantifying and analysing the connection between a stimulus (dose) and the responses that could occur to a large number of specimens, with statistical modelling possibilities. The determination of morphological or histological changes does not require special conditions, different from the classical histology methods [35]. The existence of an open circulatory system allows the obtaining of a haemolymph volume and the following of the effects induced by the tested substances on haemocytes, cells like human macrophages. The short life cycle also allows tracking the phenomena of embryonic or organs development.

An important advantage is given, to both species, by the possible measurable effects on the body as well as at cellular and subcellular levels (Table 1). The degree of change in metabolism is highlighted for both species, allowing the evaluation of possible damage: the behaviour of animals (the degree of mobility, swimming behaviour); determination of physiological changes induced by toxic or potentially toxic factors (respiratory consumption, feeding, excretion). Also, the model organisms make possible the completion of some studies [36, 37] with diverse cellular and subcellular evaluations far more efficient.

Table 1 The possible measurable effects on crustacean animal model 

Organism Cellular and subcellular levels
Behaviour

Locomotion

Reproduction behaviour

Fecundity

Mortality

Metabolic activity (excretion, feeding)

Moult 

Organogenesis

 Membrane integrity

Cellular adhesivity

Cytoplasmatic vacuolization

Proteins

Lipids

Carbohydrates

Cellular division

Enzyme 

DNA/RNA evaluation

Receptors and signalling molecules

Conclusions

     Unlike commonly used tests such as cell culture testing, laboratory animal or genotoxicity testing, the use of biological models with invertebrate organisms brings a significant advantage. It provides significant toxicity results, which can be considered preliminary benchmarks for standardized tests, with a minimum consumption of reagents and financial effort as well as high efficiency regarding the duration of the test.

     Evaluations allow statistical modelling for short-term (24-48 h) or long-term (1-2 generations) reactions or modelling of effects on several types of mechanisms (physiological, behavioural, cellular).

     The use of biological models of the crustacean type in the field of testing pharmaceutical or natural compounds, further develops the premise of an interdisciplinary research with significant advantages in terms of streamlining analyses and modelling phenomena in vivo.

REFERENCES

    1. Buchmann, K. (2014). Evolution of innate immunity: clues from invertebrates via fish to mammals -Frontiers in Immunology. Molecular Innate Immunity, September 2014.Volume5.  Crustacean Central Nervous System in Focus: Subacute Neurodegeneration Induces a Specific Innate Immune Response. PLoS ONE 8 (11): e80896. doi: 10.1371/journal.pone.0080896. 
  • National Research Council. (1999). Marine Organisms as Models for Biomedical Research In:  From Monsoons to Microbes: Understanding the Ocean’s Role in Human Health. Washington, DC: The National Academies Press. P.144. https://doi.org/10.17226/6368.
  1. Berger, J. (2009). Preclinical testing on insects predicts human haematotoxic potentials. Laboratory animals, 43, pp. 328-332.
  2. Wilson-Sanders, S.E. (2011). Invertebrate models for biomedical research, testing, and education, ILAR J., 52, pp. 126-152.
  3. Lagadic, L., T. (1998). Caquet Invertebrates in testing of environmental chemicals: are they alternatives? Environ. Health Perspect., 106, pp. 593.
  4. Sonali, K. D., Shashikant, C. D. (2015). Alternatives to animal testing: A review, Saudi Pharmaceutical Journal, Volume 23, (3), pp. 223-229.
  5. Sheehan, D. (2003). Applications of in vitro techniques in studies of biomarkers and ecotoxicology. In vitro Methods in Aquatic Ecotoxicology. Edited by Carmel Mothersill and Brian Austin, Springer, 465 p.
  6. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L. (1982). Brine Shrimp: A convenient general bioassay for active plant constituents. Plant med, 45:31-34.
  7. Alcaro, A., Panksepp, J., & Huber, R. (2011). D-Amphetamine stimulates unconditioned exploration/approach behaviours in crayfish: towards a conserved evolutionary function of ancestral drug reward. Pharmacology, biochemistry, and behaviour, 99 (1), 75–80. https://doi.org/10.1016/j.pbb.2011.04.004
  8. EPA (1996). Gammarid acute toxicity test-8501020 Guideline. EPA 712-C-96-130, http://www.epa.gov.
  9. Dixon, R.D., Aundrey M. P., Dixon, Linda, R.J., Awadesh, N., Jha. (2002). Marine invertebrate eco-genotoxicology: a methodological overview. Mutagenesis, vol 17, (6), pp.495-507. 
  10. Ross, V.H., and William A. M. (2003). Invertebrate biomarkers: links to toxicosis that predict population decline, Ecotoxicology and Environmental Safety 54, pp.366–374.
  11. Gerhart, A. (2011) GamTox: A low Cost multimetric Ecotoxicity Test with Gammarus spp. for in and ex situ Application. International Journal of Zoology, ID 574536, 7pg.
  12. Busuricu, F., Schröder, V., Margaritti, D., Nadolu, D., Anghel, H.A. (2019). Preliminary study regarding sodium benzoate and other food dyes synergic action using BSLA cytotoxicity test. Series D. Animal Science, Bucharest, Vol. LXII, (1), pp. 410-415.
  13. Popovici, V., Bucur, L. A., Schröder, V., Gherghel, D., Mihai, C.T., Caraiane, A., Badea, F. C., Vochița, G. and Badea, V. (2020). Evaluation of the Cytotoxic Activity of the Usnea barbata (L.) F. H. Wigg Dry Extract, Molecules, 25, 1865; doi:10.3390/molecules25081865. 
  14. Cima, F. (2010). Microscopy methods for morpho-functional characterisation of marine invertebrate haemocytes Microscopy: Science, Technology, Applications and Education Méndez-Vilas and J. Díaz (Eds.), pp 1100–1107.
  15. Luquet, G., Marin, F. (2004). Biomineralisations in crustaceans: storage strategies, C.R. Palevol, 3, pp. 515-534.
  16. Kuballa, A., Abigail, E. (2007). Novel molecular approach to study moulting in crustaceans, Bull. Fish. Res. Agen, 20, pp. 53-57.
  17. Wolf, F.W., Heberlein, U. (2003). Invertebrate models of drug abuse. J. neurobiol. 54, (1), pp.161-178.
  18. Kathryn S. Richards, David J. Simon, Stefan R. Pulver, Barbara S. Belts, Eve Marder (2002). Serotinin in the developing stomatogastric system of the lobster Hommarus  americanus. Wiley Periodicals.Inc.
  19. Bucur, L., Schröder, V. (2014). Artemia sp. biotester and the UV shield factor of flowers soft extract of Elaeagnus angustifolia L. International Multidisciplinary Scientific Conferences on Social Sciences and Arts, vol. II, pp. 81-88.
  20. Apetroaei, M., Paduretu, C., Rau, I., Schröder, V. (2018). New-chitosan characterization and its bioassay in different salinity solutions using Artemia salina as bio tester – Chemical Papers, vol. 72, (8), pp. 1853-1860.
  21. Schröder, V., Iancu, I. M., Pavalache, G. (2018). Effects of diazepam on biotester organisms 1st International Health Sciences and Life Congress, 02-05 May, Burdur/ Turkey, pag. 1301-1309.
  22. Schröder, V., Pavalache, G., Apetroaei, M. R, Bucur, L., Tomos, S. (2016). The evaluation of the effects which are induced by the exposure of Gammarus balcanicus (Amphipoda, Crustacea) to cephalexin and doxycycline. In: 3-th International Multidisciplinary Scientific Conferences on SOCIAL Sciences and ARTS SGEM-Section Sociology and Healthcare, 995-1002.
  23. Schröder, V., Bucur, L., Pavalache, G. (2014). The cytotoxicity study of the common pharmaceutical or food additive International Multidisciplinary Scientific Conferences on Social Sciences and Arts, vol. II, pp. 913-920.
  24. Schröder, V., Arcus, M., Anghel, A. H., Busuricu, F., Lepadatu, A.C. (2019). Cell differention process of Artemia sp. larvae tools for natural products testing. Scientific Papers. Series D. Animal Science, Bucharest, Vol. LXII, (1), pp. 149-153. 
  25. Acey, R.A., Tomlison, D.W. (1988). Artemia salina as a model system for assessing the effects of xenobiotics on embryonic development. FASEB J, 2(6):8463.
  26. Libralato, G. E., Prato, L., Migliore, A.M., Cicero, L. M. (2016). A review of toxicity testing protocols and endpoints with Artemia spp., Ecological Indicators, Volume 69, pp. 35-49. https://doi.org/10.1016/j.ecolind.2016.04.017.
  27. Lewis GE: Testing the toxicity of extracts of Southern African plants using brine shrimp (Artemia salina) (1995). S Afr J Sci, 91:382.
  28. Carballo, J.L., Hernández-Inda, Z.L., Pérez, P. et al. (2002). A comparison between two brine shrimp assays to detect in vitrocytotoxicity in marine natural products. BMC Biotechnol 2, 17, pp. 5. https://doi.org/10.1186/1472-6750-2-17
  29. Kersetter, H.W., Schaffer, D.J. (1983). Brine shrimp (Artemia salina) nauplii as a teratogen test system. Ecotoxicol Env Safety, (7), pp. 435-446.
  30. Busi, S., Madhu, D., Kaviyarasu, K. (2020). In: Model organisms to study biological activities and toxicity of nanoparticles. Ed. Medical.466 pages.
  31. Sarah, Q. S., Anny, F.C., Misbahuddin, M. (2017). Brine shrimp lethality assay. Bangladesh J. Pharmacol. 12: 186-189.
  32. Schröder V., Doicescu D., Arcuş M. (2017). The cytomorphology of the haemocytes at Gammarus pulex balcanicus (Amphipoda, Crustacea) and their changes after colchicine exposure. The Romanian Society for Cell Biology, Annals of R. S. C. B., Vol. XXII, (1), pp. 10–16.
  33. Busuricu, F., Negreanu-Pârjol, T., Popescu, A., Margaritti, D., Lupu C., Schröder V., Tomoş, S. (2013). Evaluation of the antioxidant of certain Romanian wine type. JEPE, 14, (4), pp. 1828-1836.
  34. Balaban, D.P., Natalia, R., Busuricu, F., Sava, D. (2004). Etude preliminaire concertant la separation d’un melange d’alginates de l’algue brune Cystoseira barbata. Rapp. Comm. Int. Mer. Medit 37, pp. 485.