An efficient low-cost laboratory workflow for the study of blood cells and RNA extractions in marine invertebrates

Marine invertebrates are model organisms in several areas of biological sciences, being a source of massive biological information. Although, the scientific relevance of marine invertebrates, the research with them can be limited for their tissue characteristics and troubles for the replication of physical and chemical properties of seawater. Thence, the main goal of this laboratory workflow is to provide a useful methodological approach to reduce the experimental limitations during the study of marine invertebrates. The present study describes experimental methodologies for the collection, transport, and maintenance of sessile tunicates. Also, an approach to observe and characterize, a diverse population of blood cells in marine invertebrates, by several cytological stains and electron microscopy. Lastly, suggestions and protocols to extract quality RNA from samples with high concentrations of salts, pigments, secondary metabolites, and polysaccharides. This methodological approach can be easily adapted to other marine invertebrates, moreover uses low-cost reagents and Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 March 2021 doi:10.20944/preprints202103.0030.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. widely available laboratory equipment. Making possible the study of different types of marine animals in diverse locations.


Introduction
The ocean is 70% of our planet's surface. Life originated and diversified in the oceans, and a large proportion of biodiversity is concentrated in oceanic environments (Barnes & Hughes, 1982; Kaiser et al., 2010) . A relevant proportion of marine biodiversity and biomass is conformed by marine invertebrates. These animals are also preponderant in marine ecosystems, being keystone species in some cases, such as the coral reefs (Barnes & Hughes, 1982) . Accordingly, it is possible to obtain massive biological information by the study of marine invertebrates.
Marine invertebrates have been the model for biological research for centuries, being a source of massive biological information. Cephalochordates and hemichordates have been studied regarding their evolutionary aspects (Swalla & Smith, 2008). Sea urchins, tunicates, mollusks, and polychaetes, are model organisms to study gametogenesis, embryogenesis, cell signaling, and developmental processes (Gilbert, 2003) . Cephalopods have been studied for innovations in nanomaterials (Phan et al., 2013) . Sea urchin and tunicate embryos are used to understand the toxicity of chemical compounds in marine environments (Bellas et al., 2005). Molluks and crustaceans are biondicators for the study of plastic pollution in the marine food webs (Setälä et al., 2018). The marine invertebrates are a crucial source of biological knowledge. Although, the relevance of marine invertebrates in biological science, the research with them is limited for their tissue characteristics and troubles during replication of marine environment.
The research with marine invertebrates required specific conditions (e.g. salinity, pH, and temperature), related to the type of organism, or the kind of methodological procedure. Some marine ecosystems are hard-to-reach places, making difficult and expensive the transport of the specimens and samples (Calado & Leal, 2015) . In vivo experiments required, the maintenance of Thus, the main goal of this laboratory workflow is to provide a useful and efficient experimental approach, to solve troubles during the extraction and manipulation of blood cells, and RNA extractions in marine invertebrates. Also, taking into count the use of simple and low-cost reagents and equipment.

Methodologies
These procedures were developed and standardized during the study of the colonial tunicates Symplema brakenhielmi and S.rubra. These marine invertebrates have a dense and elastic tunic composed of cellulose. The small zooids are inside this hardly breakable tunic. Moreover, the internal tissues are surrounded by blood with a high concentration of pigments and secondary metabolites. These characteristics of Symplegma species restrict the quantity and quality of tissues, available for nucleic acid extractions, cytological techniques, and electron microscopy. The resultant protocols were successfully tested in other tunicates such as Botryllus sp., Styela plicata, and the cnidarian Cassiopea sp.

Taxon sampling
Symplema brakenhielmi and S.rubra are benthic animals, living attached to hard marine substrates. Rocky shores, mangroves, and Yacht port are locations with abundant colonial tunicates.
Yacht ports are ideal places to collect because these locations provide colonial animals in abundance and ease of collection. Colonial tunicates are usually located in floating buoys, cords, and pilots ( Fig.1 Process 1). Pieces of colonies between 1-2 cm 2 are carefully removed from the substrate, cleaned, and placed in recipients with fresh seawater. Colony pieces are attached to microscope glass slides with a thread and stored in microscope slide boxes with perforations for water circulation. Boxes are attached to floating structures in Yacht port for three weeks. The attached colonies are cleaned, to be subsequently used in experiments ( Fig.2 A, B).

Sample transporting and culture
Transportation of alive colonies requires a thermal container with an oxygen source. Alive colonies need time to adapt from ocean conditions to culture system tanks. For the first twelve hours after transportation, new colonies need to be maintained with 50% of original seawater and system seawater, without food. A feeding regime with living phytoplankton is useful to maintain healthy colonies (Fig. 2 C-D). The mixture of Isochrysis, Thalassiosira, Pavlona, Nanochlorpsis has nutrients for growing colonies ( Fig.1 Process 2).
Samples for nucleic extraction need to be cleaned to remove other organisms. This activity is more efficient with the aid of a stereo-microscope. Specimens must be kept without food in filtered seawater (FSW) overnight before tissue extraction. Samples collected in remote areas can be cleaned as best as possible and stored in ethanol 70% for DNA extraction, or RNAlater for RNA extraction. The immersion of tissues in the preserving solution is a key step to guarantee tissue preservation and successfully nucleic acid extractions. Thus, in the cases of animals with hard external tissues (e.g hard tunics ), is necessary to make orifices to the external part or dissect internal organs, to put them in contact with the preserving solution ( Fig.1 Process 3B).

Histology in Symplegma colonies
Tunicate colonies attached to slides must be cleaned with a soft brush and let without food overnight in filtered seawater (FSW). Tissues colonies are relaxed in FSW with menthol crystals covering the water surface for 15 minutes, to be fixed with 4% paraformaldehyde (diluted in FSW), at 4 °C overnight. After fixation colonies are washed three times for 15 minutes with PBS and distilled water. Tissues are dehydrated by ethanol series (25%, 50%, 70%, 80%, 90%, 100%) and two xylol washes for 30 minutes. Tissues are embedded in paraffin to cut in sections (6 um).

Blood cell extraction
Blood cell extraction follows a previously described protocol (Cima, 2010), with some changes to improved obtained results. The main limitation to work with blood cells is the constant coagulation and the preservation of blood cell integrity. The constant use of anticoagulants during all the blood extraction is useful to reduce blood clots. Blood cell integrity is preserved by, maintaining the sample in ice, and preparing reagents in FSW for cellular osmoregulation.
Clean attached colonies are immersed in anticoagulant solution (10 mM L-cysteine and 0,38 % sodium citrate, diluted in FSW) for 5 minutes, then the colony is dried with a soft paper towel.
The blood vessels edgs (i.e. Ampullae) are gently cut, the blood is collected with a micropipette, which is rinsed constantly with anticoagulant ( Fig. 3A). Following the blood collection, the anticoagulant is washed by centrifuging for 15 minutes at 3000 rpm. Hemocytes are re-suspend in 500 μl of a solution with 1/3 Anticoagulant and 2/3 FSW. This solution prevents posterior coagulation, and maintain the integrity of the cells. The blood cell solution is mixed gently with a micropipette. Drops from this blood solution are left in slides coated with Poly-L Lysine to attach the blood cells. Slides are maintained in a humid chamber to prevent cell desiccation. After blood cells attachment, liquid excess is discarded placing the slides vertically (Fig. 3B).

Cytological stains
The cytological stains are used to observe the cellular size, content, and conspicuous organelles such as vacuoles, vesicles, and pseudopodia. The general cellular morphology characteristics (i.e. nucleus size, nucleus-cytoplasm ratio, acid and basic contents) are described with hematoxylin and eosin stain. Blood cell populations are characterized with Giemsa stain, used for blood cytology. The lipid content is described with Sudan Black stain. These stains can be abrasive for big vacuolated cells and cells with pseudopodia, and change the cellular morphology.
Thence, the Neutral Red (vital stain) is used to observe living cellular morphology and cellular behaviors.

Hematoxylin and eosin stain
Blood cells attached to slides are fixed for 15 minutes, with 4% Paraformaldehyde (diluted in FSW). Fixed cells are washed with PBS, and stained with Meyer hematoxylin for 10 minutes and eosin for 5 minutes. Blood cells are mounted using glycerin and sealed with a coverslip.

Giemsa stain
Blood cells attached to slides are fixed for 30 minutes at 4 °C, with the solution 1 g NaCl and 1 g sucrose in 1 % glutaraldehyde in FSW. Fixed cells are washed with PBS, and stained with 10% Giemsa for 5 minutes. Blood cells are mounted using glycerin and sealed with a coverslip.

Neutral Red for stain acid compartments
Neutral red solution ( 8m/L in FSW) is added to the attached blood cells.

RNA extractions
DNA and RNA extraction is an essential methodology in biological studies. The biological information from RNA has (DNA transcription and expression) diverse applications in biological research, such as transcriptome studies, in situ hybridization, and quantitative RNA expression (Mehra, 1996).  (Fig. 4A). In which the dissections and the previous recommendations are insufficient to prevent the quick degradation of RNA. In the case of S. brakenhielmi and S.rubra the unique protocol that worked for RNA extraction, after test several protocols and kits for RNA extraction (Fig. 4B), is the present protocol with lithium chloride used generally for plants (Barlow & Gammack, 1963) . Thus, this methodological approach can be an appropriate procedure for the RNA extraction in marine invertebrates with similar characteristics.

Tri-Reagent modified protocol
The Tri-Reagent protocol follows a previously described protocol (Sambrook & Russell, 2001). Fresh tissues, samples stored in RNA later and frozen in liquid nitrogen, are used for RNA extraction with satisfactory results. Samples are washed with ice-cold 1X PBS. Tissue homogenization is with liquid nitrogen and a ceramic mortar, or with a plastic homogenizer with TRIzol solution. After homogenization, the solution is live for 5 minutes at room temperature, posterior steps are at 4°C. The homogenate is centrifuge by 12,000 rpm for 10 minutes, to remove the insoluble material (extracellular membranes, polysaccharides). The supernatant is transferred into a new tube, for organic separation with chloroform. Supernatant and chloroform are mixed with a vortex shaker and stand for 5 minutes, then centrifuge at 12,000 rpm for 15 minutes. The organic separation is repeated, to reduce salt and proteins excess, and polysaccharide residues from the tunic. RNA is with isopropanol and an RNA precipitation solution (1.2M NaCl and 0.8M disodium citrate), for 10 minutes. Liquid excess is removed after 10 minutes of centrifugation at 12,000 rpm.
Pellet is dried by air-drying for 5 minutes and washed with 75% ethanol. Ethanol is removed by 5 minutes of centrifugation at 12,000 rpm and evaporation of residues. The clean pellet is diluted in free RNAse water and storage at -80°C. Pellet is resuspended in free RNAse water and incubate for 15 minutes at 55°C, to dilute the RNA precipitate, then stored at -80°C (Fig. 4C).

Conclusions
The methodological approach used for blood characterization was efficient to observe different types of blood cells (e.g hyaline amebocytes, vacuolated cells, granular cells, precursorlike cells). In the case of Symplegma brakenhielmi and S. rubra, was possible to identify and describe about eleven types of blood cells. Therefore, the proposed workflow increases the capacity to detect diverse blood cell populations, allowing a better understanding of blood cells and hematopoiesis in marine invertebrates.
The two methods of RNA extractions are useful to obtain high-quality RNA. The modified method of Trizol is faster and required simple reagents and equipment. The modifications in the organic separation and precipitation of RNA improves the obtained results, being this method a good option to extract RNA in marine organisms.
The method of extraction with lithium is useful to extract RNA from samples with pigments, secondary metabolites, and polysaccharides. This method takes two days for the overnight precipitation with LiCL, however, the results are very satisfactory, allowing high-quality RNA extraction from difficult samples.
The proposed workflow is efficient for the transportation, nucleic acid extractions, and blood cell studies with tunicates. Also, the presented protocols can be easily adapted to other marine invertebrates. This methodological approach uses low-cost reagents and widely available laboratory

Process 1: Taxon sampling
-Using knifes and spatulas collect the tunicates carefully to do not damage the organisms.
-Transport the samples in sea water to laboratory. The samples are classified by morphological characteristics, like color, form and shape. Each sample is labeled with a number and collect place.
-In colonial specimens sample is dividing for different analysis, in solitary specimens, more than one individual is collected. One sample fragment (or individual) is storage in 70% Ethanol to molecular analysis, other part of tissue is fix in 4% formaldehyde to morphological observations. The remaining tissue is using to settle in glass slides. For RNA analysis samples can be preserve in RNAlater, frozen in liquid nitrogen and storage at -80°C.
6.2 Process 2: Transporting and maintenance of living tunicates 6.2.1 Settled colonial tunicates -Colonies are attached with thread on 5cm X 7.5cm glass slides.
-During attachment process microscope slides are maintained in slide boxes with apertures to permit water circulation. Usually this process occurs in one or two weeks in the ocean (attach boxes to port structures) or two and three weeks in a culture system.
-When tunicate is attached to slide, the thread is remove with tweezers. It is important that the attached tunicates are floating or in a surface superior to tank bottom. This allows filtration and prevent contamination with organic wastes. Every day tunicates are feeding with living phytoplankton. The food mixture is prepare from ¼ of each living algae (Isochrysis, Thalassiosira, Pavlona and Nanochlorpsis), in a approximated concentration of 170,000 cell/ml.

Precautions with culture system: A. Ammonium and organic wastes:
One of the crucial aspect in recirculating systems is the accumulation of ammonium and other toxic compounds for animals. The first way to reduce the quantity of these compounds is to prevent the accumulation of organic material pieces. The rest of food and feces begin a process of decomposition affecting the quality of the water. The biofilter is the principal way to reduce the accumulation of ammonium, because the microorganisms in the biofilter processing the ammonium in a sequence of chemical reactions that decompose the ammonium in non toxic forms (Losordo et al.,1998;Wright, 2011) Consequently is necessary maintain the biofilter in good conditions controlling the variables of the system (pH, salinity and temperature). Also add probiotics to the biofilter to maintain system stability (Marion et al., 2011;Wright, 2011) .

B. Oxygen levels
The levels of oxygen are usually a problem in recirculating systems, because the water is maintain in the system for a long period and it is necessary provide oxygen to the system. Also the process to reduce the ammonium in the biofilter consume oxygen, reducing the levels of oxygen in the system. Thus oxygen sources are critical to maintain healthy conditions to the animals. Specially in systems with a high density of animals, or with animals that require large food quantity and produce more organic wastes (e.g solitary tunicates) (Grøttum et al.,1997).

C. Rotten eggs smell => hydrogen sulfide presence
Rotten eggs smell in the seawater is a indicator of hydrogen sulfide presence. This compound is produced by anoxic bacterias, for the increment of organic decomposition. Resulting in anoxic areas in the culture system, usually in are with less water movement and less oxygen. A constant water movement and pipe cleaning, are strategies to control the increment of hydrogen sulfide (Grøttum et al., 1997;Losordo et al., 1998) .
6.3 Process 3A: Blood cell characterization 6.3.1 Blood cell extraction follows a previously described protocol (Cima, 2010),with some changes to improved obtained results -Anticoagulant solution: -10 mM L-cysteine (which binds the thiols of plasma proteins ) -0.38% sodium citrate (a calcium chelating agent) -The solution is in filtered and sterilized sea water adjusted at pH 7.5. *Note that, before blood collection, glass micropipette must be repeatedly rinsed with the anticoagulant solution, in order to prevent hemocytes from adhering to its glass walls. -Hemocyte collection solitary ascidians: -The haemolymph containing hemocytes can be obtained by cutting the tunic and puncturing the heart. -Hemocyte collection colonial ascidians: 1. Put the colony by 5 minutes at r.t in 10 cm petri dish containing 50 ml of an anticoagulant solution. -0,2 M Tris-Hcl pH 7,5 -0,1 M LiCl -5mM EDTA -1/10 of the total volume of SDS 10% -Autoclave the final solution Tunicates samples -For fresh material maintain the tunicate one day before in filtered sea water.
-For colonial tunicates clean the glass slide and the colony with a soft paintbrush.
-For solitary tunicates remove the tunic and use muscle for the mantle avoiding the digestive structures. Day 1 1. Put the tissues in the 2 ml eppendorf and freeze intermediately in liquid nitrogen.

2.
Put one bead metal bead in the eppendorf and shake the tube using a Tissues lysser machine during 40 seconds at a frequency of 30 revolution per second.