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Restraining small decapods and amphipods for in vivo laboratory studies

In: Crustaceana
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  • 1 Institute of Biologyat Irkutsk State University, Lenin st. 3, Irkutsk 664025, Russia
  • | 2 Baikal Research Centre, Lenin st. 21, Irkutsk 664003, Russia
  • | 3 Institute of Biologyat Karelian Research Centre of Russian Academy of Sciences, Pushkinskaya st. 11, Petrozavodsk 185035, Russia
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Abstract

During physiological studies, it is often required to restrain a small animal for an experiment, as it is typically challenging to perform in vivo imaging or measurements on freely moving individuals. In this article we describe two widely applicable approaches for repeated restraint of small (approx. 0.5-4 cm) decapods and amphipods that are established in our laboratory: immobilization using gentle gluing and suction. Application of both these approaches as well as their advantages and disadvantages are discussed in detail.

Abstract

During physiological studies, it is often required to restrain a small animal for an experiment, as it is typically challenging to perform in vivo imaging or measurements on freely moving individuals. In this article we describe two widely applicable approaches for repeated restraint of small (approx. 0.5-4 cm) decapods and amphipods that are established in our laboratory: immobilization using gentle gluing and suction. Application of both these approaches as well as their advantages and disadvantages are discussed in detail.

Introduction

A number of popular methods in physiological research in crustaceans involve immobilization of the animal for a period of time. Such fixation can be helpful during investigation of gill ventilation dynamics and cardiac activity (Bierbower & Cooper, 2009; Jakob et al., 2016). Other techniques for in vivo studies, such as confocal microscopy (Maza et al., 2016) and application of implanted wire electrodes (Clemens et al., 1998) or optical microsensors (Gurkov et al., 2016) for physiological monitoring, strongly require tight immobilization of the crustacean.

Large crustaceans (>5 cm) have a relatively strong exoskeleton, and it is possible to hold animals with clamps or bands, which do not damage the animal and allow free movements of the legs (Clemens et al., 1998; Maza et al., 2016). In case of smaller crustaceans, such as many species of decapods (Crustacea, Decapoda) and amphipods (Crustacea, Amphipoda), mechanical fixation is often difficult to perform because of their thin exoskeleton and gentle bodies. To reduce activity of crustaceans (i.e., for anaesthesia), hypothermia and a number of chemical agents were suggested and can be chosen depending on the experimental setup (Oswald, 1977; Fregin & Bickmeyer, 2016). However, such treatment may significantly affect biochemical and physiological parameters of the organism.

Probably, the most obvious way of restraint using some rigid frame has a number of limitations and disadvantages. First, such a frame is usually specifically designed for a particular object, and different species or even different size groups of the same species may require slightly different frame designs. Second, if the animal is newly positioned in the frame before each measurement, the repeated fixation procedure can lead to damage of some individuals. Alternatively, if the animal is kept in the frame for a long time and cannot move, such fixation may be stressful and also may impede gill ventilation.

Here we describe two generally applicable approaches to restrain small (body length ∼0.5-4 cm) crustaceans that are established in our laboratory: gentle gluing and suction. The advantages of these techniques include the possibility to use them for different decapod and amphipod species of various sizes and easy fixation/detachment of the crustacean. To take into account a specific body orientation naturally preferred by an animal, we present two types of chambers for immobilization of the crustaceans either vertically or horizontally.

Material and methods

Animals

The following two species were chosen to demonstrate the principles of the suggested immobilization procedures: an amphipod, Eulimnogammarus verrucosus (Gerstfeldt, 1858), endemic to Lake Baikal, and a decapod, Caridina multidentata Stimpson, 1860. For both species, approx. 3 cm long adults were used.

For physiological experiments, amphipods were collected in the coastal zone of Lake Baikal by kick-sampling, transported to the laboratory in temperature-controlled containers and acclimated to laboratory conditions. Amphipods were held in aquaria (approx. 2 l) containing Baikal water with constant aeration and temperature 6-7°C (average annual temperature for the coastal zone of Lake Baikal).

Restraint with gluing

The first approach is to stick a small adapter to the animal (fig. 1A-C). By this adapter a decapod or an amphipod can be quickly and easily attached to a larger holder in an appropriate position, and at the same time the adapter does not significantly affect movements of the crustacean when it is detached. A thin piece of sponge can be used as the adapter. The sponge contacts with the glued surface at a number of points, and the contact area at each point is small. As a result, fixation is simultaneously strong and flexible, unlike if a piece of plastic or tissue is used. It should be noted that the sponge adapter can hold many air bubbles that can increase buoyancy of the animal. Therefore, in order not to interfere with the “natural-like” swimming activity of animals in acclimation tanks, it is recommended to make a piece of sponge as small and thin as possible (no more than 50% of the body area), but large enough for sufficient contact with the chitinous exoskeleton.

Fig. 1.
Fig. 1.

Vertical and horizontal restraint of crustaceans with gluing to a sponge adapter. The glued sponge adapter A-C allows to quickly attach a decapod to a larger piece of sponge A, D or to fix an amphipod to a soft base by pinning of the adapter E. Arrows indicate inflow and outflow of water.

Citation: Crustaceana 91, 5 (2018) ; 10.1163/15685403-00003778

To glue the sponge adaptor to the animal, we took the individual out of the aquarium, placed it into an incision inside a large wet sponge (to reduce stress for the animal) and carefully wiped its dorsum to dry. A drop of cyanoacrylate adhesive was placed on a dry piece of sponge adapter and spread over the sponge surface. Excess glue was absorbed into the sponge and thus did not contact with chitin. The adapter was quickly attached to the exoskeleton and we waited 1 min until the glue was polymerized. The sponge adapter was washed well with water to remove unpolymerized chemicals and the crustacean was returned to the aquarium for recovery. It is recommended to change the aquarium water after approximately 1 h to remove any residual chemicals and acclimate animals after the procedure (Clemens et al., 1998).

Fig. 1A, D-E depicts how to attach the animal with adapter to the holding facility. For vertical positioning, the sponge adapter was placed in an incision in a larger piece of the sponge (of the same material), which can be squeezed between two glasses of a narrow glass chamber. For fixation in the lateral position, the adapter can be pinned to a soft base (sponge or paraffin) lining the bottom of a flat chamber (e.g., a Petri dish) or hooked with an attachment clip and immobilized using a strong magnet underneath the chamber. In both cases, the inflow and outflow of water into the cell can be carried out by peristaltic pumps, in order to control conditions (temperature, oxygen, etc.) in the chamber. When the horizontally immobilized animal is observed under a microscope with high magnification inside the chamber with circulating water, the chamber consisting of communicating vessels (as described in the next section) is useful to reduce water fluctuations. If the animal requires a strict temperature regime, an aquarium chiller can be inserted between the chamber and peristaltic pump when using both this and another method of immobilization (for example, it is important for the cold-adapted E. verrucosus).

Restraint with suction

For fixation by suction, the water outflow from the chamber (fig. 2) can be created by a peristaltic pump (a velocity up to approx. 0.5 l/min is usually enough). If the animal is to be observed from the top of the chamber (e.g., under a microscope), we recommend using a chamber consisting of two communicating vessels that allow to significantly reduce water fluctuations caused by water inflow (fig. 2B, D). For horizontal fixation, two Petri dishes of different sizes can be used as parts of the chamber, while a tank for vertical fixation can be made of glass microscope slides or plastic pieces (e.g., sawn from plastic Petri dishes).

Fig. 2.
Fig. 2.

Horizontal A-B and vertical C-D restraint of crustaceans with suction. Arrows indicate inflow and outflow of water.

Citation: Crustaceana 91, 5 (2018) ; 10.1163/15685403-00003778

An adapter with drain hole can be fixed tightly in the centre of the horizontal chamber, while in the vertical tank it is advisable to glue the adapter to a magnet and fix it on the tank wall using an outer magnet (magnets from HDDs are strong enough for this purpose). Common fittings for aquarium aeration can be used as such adapters. It is recommended to apply aquarium sealant for sealing and gluing most parts of the chambers, except the side of the inner magnet that is in contact with the tank wall. To isolate the magnet from circulating water and also make it slippery (to easily move the animal along the tank wall), we recommend covering the magnet with yacht varnish.

The negative pressure created by the pump allows fixation of decapods and amphipods by the chosen part of their exoskeleton. As an option, a small metal grid may be attached to the fixing adaptor in order to prevent possible damage of smaller species (<1 cm) with thin exoskeleton. In case of using the tank for vertical immobilization (especially with quickly moving crustaceans), we recommend to first suck the animal swimming in the chamber to the adapter and only then fix the adapter to the tank wall by the external magnet.

Heart rate measurements

The heart rate of E. verrucosus amphipods was visualized using fixation with gluing under a Mikmed-2 (LOMO, Russia) microscope with an EOS 1200D (Canon, Japan) camera during 25 min. After fixation under the microscope and before visualization of heart rate, the individual was held in the chamber for at least 2 min. Contractions of the central haemolymph vessel were counted in the third body segment during 15 s each 3 min of observation for 5 individuals separately. The head of the animal was kept from the illuminating light of the microscope as much as possible. The temperature inside the chamber with an amphipod was maintained and controlled with the aquarium chiller. After 6 min of video recording, temperature was increased from 5.5-8°C (nearly equal to acclimation temperature range) to 16-18.5°C during 10 min.

In vivo pH measurements with implanted fluorescent microsensors

Implantable pH-sensitive fluorescent microsensors were prepared as described previously (Gurkov et al., 2016). The working substance inside the microsensors was the fluorescent probe SNARF-1-Dextran (D3304, Thermo Fisher Scientific, U.S.A.) encapsulated into semipermeable shell consisting of two oppositely charged polymers poly(allylamine hydrochloride) (PAH; Sigma No. 2832315) and poly(sodium 4-styrenesulfonate) (PSS; Sigma No. 243051) and the outmost layer of poly(l-lysine)-graft-poly(ethylene glycol) co-polymer (PLL-g-PEG; SuSoS, SZ34-67). The microcapsule shell had the following structure: (PAH/PSS)6/PLL-g-PEG with SNARF-1-Dextran inside.

Microsensors in saline (5 μl, about 300,000 microsensors) were injected into the central haemolymph vessel of E. verrucosus using insulin syringes. For pH measurements, amphipods were immobilized using suction under a Mikmed-2 (LOMO, Russia) fluorescent microscope connected to a QE Pro (Ocean Optics, U.S.A.) spectrometer. Microsensors were visualized in the central haemolymph vessel in the red channel, and their fluorescence spectra were recorded. The spectra were decomposed to reference spectra of protonated SNARF-1, deprotonated SNARF-1 and amphipod autofluorescence spectrum to subtract the autofluorescence spectrum as described previously (Gurkov et al., 2016). After spectral decomposition, haemolymph pH was calculated using the calibration curve for the microsensors obtained in buffers with different pH by the ratio of fluorescence intensities at 605 and 640 nm.

Additionally, amphipod haemolymph was extracted to verify the obtained measurements using a pH-meter with microelectrode InLab Nano (Mettler Toledo, U.S.A.).

Results

The proposed approaches for immobilization of small crustaceans were previously used on decapods (Volkova et al., 2017) and amphipods (Gurkov et al., 2016), but were not described in detail. In this study the presented restraint techniques were applied to amphipods Eulimnogammarus verrucosus for two types of physiological measurements to demonstrate feasibility of both approaches.

The fixation with the glued sponge adapter shows good applicability for long (>1 min) immobilization of small crustaceans and was used for heart rate monitoring (fig. 3A). The obtained results demonstrate pronounced reaction of amphipods to an acute temperature increase by 10°C. Amphipod heart rate was stable at the same temperature and raised about 1.8-fold under the heat shock with slight a delay in reaction after the temperature increase.

Fig. 3.
Fig. 3.

Examples of in vivo measurements with the proposed restraint techniques in amphipods Eulimnogammarus verrucosus (Gerstfeldt, 1858). A, Heart rate monitoring under acute temperature increase using restraint with gluing. B, In vivo measurements of haemolymph pH using implanted fluorescent microsensors directly in circulatory system of amphipods immobilized with suction in comparison with pH values obtained for extracted haemolymph using pH-meter. The implanted microsensors are based on the fluorescent molecular probe SNARF-1. Dots represent individual measurements; solid horizontal lines indicate medians.

Citation: Crustaceana 91, 5 (2018) ; 10.1163/15685403-00003778

Restraint with suction is recommended for applications involving fluorescent microscopy and short (approx. 1 min) measurements. It was applied to measure haemolymph pH in vivo directly in amphipod circulatory system using implanted fluorescent microsensors (fig. 3B). The median pH value obtained in vivo is equal to 8.15, in good concordance with measurements by pH-meter for extracted amphipod haemolymph.

Discussion

To the best of our knowledge, this article contains the first comprehensive description of restraint techniques that were designed specifically for small crustaceans of approx. 0.5-4 cm in size. Even though both described approaches for repeated immobilization of crustaceans are applicable to a wide range of species with different body sizes, they have disadvantages that make them preferred for different experimental designs.

Restraint with a small glued sponge adapter allows quick and convenient attachment of the animal and leads to less anxiety of the crustacean, especially in the case of an amphipod immobilized horizontally on a sponge. However, the main disadvantages of this procedure are contact with gluing chemicals and potential influence of the adaptor on the animal behaviour. A possible non-toxic alternative to cyanoacrylate glue is dental cement, but this was not tested. Another important property of sponge adapters is their fluorescence, which can be a significant problem when the animal is visualized using fluorescent microscopy.

In case of fixation with suction, decapods and amphipods have no contact with any potentially toxic chemicals, and their behaviour cannot be influenced in any way in the holding aquaria. This approach is preferable for fluorescent microscopy due to the absence of sponge fluorescence, but such immobilization leads to higher anxiety of the animal (and requires more time) than fixation with a glued adapter, especially in case of amphipods. To reduce amphipod anxiety during such restraint, we suggest covering the head of the animal with a piece of tissue or sponge. As an option, the cover can be enhanced with a small metal core (made of an attachment clip) and pressed to the bottom using a magnet under the Petri dish. In our experience, combination of restraint using suction and such holding down head cover gives the best result for horizontal fixation of amphipods. It is also important to mention some complications related to the attachment of quickly moving decapods to the sucking adapter. Certain species (like Caridina multidentata) can even jump out of the small tank when a researcher tries to fix the individual. If it is appropriate for the experimental design, short mild hypothermia until the decapod is attached can be suggested to decrease moving activity of the animal.

In spite of some degree of stress to the animals that can be related to both fixation procedures, we believe that our approaches will be useful for researchers who need to restrain small crustaceans for in vivo visualization or continuous recording of physiological parameters without anaesthesia. Our design leaves maximum freedom of movement of all parts of the crustacean body relative to each other and allows free movement of the legs for natural ventilation.

Acknowledgements

Authors are greatly thankful to Dr. Polina Drozdova (Saint Petersburg University) for her help in improvement of the manuscript. This study was supported by the Russian Science Foundation (No. 15-14-10008, 17-14-01063), the Russian Foundation for Basic Research (No. 15-29-01003), Ministry of Education and Science of Russia (“Goszadanie”, No. 6.1387.2017/4.6) and a Grant of the President of Russia (No. MK-6804.2018.4).

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