HABITAT AND BIOLOGY
The Xenopus laevis is a frog with an entirely aquatic life which is very well appreciated by lovers of aquariums specifically because of this characteristic of not needing to live on land, unlike most amphibians. Its areas of origin include regions of Southern Africa; however, nowadays, as a result of the reckless abandonment of subjects previously kept in captivity as aquarium amphibians or as experimental animals, it is also commonly found in lake and swampy districts of the United States of America, of Central and South America and of Western Europe. The Xenopus frog, despite being a tropical animal, is an extremely adaptable species which can also survive in unfavourable climatic conditions. In nature this amphibian prefers still water basins with no currents and nourishes itself with animal proteins in the form of insects, small fish, larvae and products of decomposition.
Regno: Animalia
Philum: Cordata
Classe: Amphibia
Ordine: Anura
Famiglia: Pipidae
Genere: Xenopus
Specie: Xenopus laevis
The name derives from the amphibian’s characteristic skin, which is smooth and very slippery. The animal’s eyes are small and black, lack palpebrae and look upwards with an antipredatory function, mostly directed against birds; the hind feet are webbed and the toes have rather long claws linked by a very thin membrane which facilitates swimming; the sensory system is completed by the lateral line system. The colour of the skin varies from a pale green to a very dark green or brown with black spots on the back, while the underside is yellowish-white; there is also an albino variety (Fig. 1) with a yellowish-white body and red eyes. The front limbs are short and poorly developed and have a predominantly tactile function, while the hands are used both to carry food to the mouth - which does not have either teeth or a tongue – and (for the male) to encircle the waist of the female during mating. The surface of the skin is covered by abundant mucus, with protective and antibacterial properties, which makes the animal slippery and complicates any manipulation of it. Respiration is mostly pulmonary, however it can also take place through the skin, which is extremely permeable and capable of ensuring a modest percentage of the gas exchange requirements of the animal. To breathe the frog comes to the surface, protrudes its narices and part of its head just above the surface of the water, inhales rapidly and returns to the bottom of the water basin where it passes most of its time motionless or in search of food. The adult male is usually smaller than the female, with a body (legs excluded) about 10 cm long (Fig. 2), compared to the 20 cm of females (Fig. 3). Sexing is quite simple, especially if the assessment is done with the animal in the dorsal position, as the female has a prominent, pointed structure between the rear legs, which is the eminence of the vulvar labia (Fig. 4): this excrescence is absent in males (Fig. 5).
Starting from the embryo the amphibian develops through 65 stages of tadpole, until stage 66 is reached which corresponds to the adult form: at this stage the animal is no larger than 1 cm in length but has all the features, albeit in miniature, of the adult animal. This amphibian is particularly long-lived and its life expectancy can reach 30 years although in captivity the average life span is 15 years. Xenopus laevis is currently extremely popular as an aquarium animal as well as in biological research for ophthalmological studies and in embryology; in the past the species was even used as a “pregnancy test” since inoculation of urine from a pregnant woman, containing human chorionic gonadotropin (HCG), into a female frog triggers the spawning of eggs. Adult animals are contained as illustrated in Fig. 6.
REARING AND HOUSING
Perfectly normal aquariums can be used to house Xenopus laevis, although they should preferably be a good length and width. The height of the aquarium is extremely important as this amphibian requires shallow water, from a minimum of 15 cm to a maximum of 30 cm. It is better to avoid greater depths which could hamper the animal’s attempts to emerge to the surface to breathe. The height of the walls of the aquarium should nevertheless be greater than the water level as Xenopus frogs are capable of making small jumps over the surface of the water and it is, therefore, possible that they could escape from an aquarium, especially one without a lid. When choosing an aquarium it should be considered that each adult requires between 25 and 50 litres of water. A medium sized aquarium, with a base of 150 x 40 cm and filled with water to a height of 30 cm, would contain 180 litres of water and could, therefore, house from three to seven adults. The size of the aquarium could obviously be considerably smaller for younger specimens; however, while it is better not to exceed a depth of 15 cm, there are no limitations to the length and width of the aquarium. Each small frog should have at least 5-10 litres of water. The optimal water temperature is between 20 and 24°C, even if these frogs can adapt to a temperature range between 10 and 30°C; in any case it is advisable to maintain the temperature of the water constant with a day-night excursion of no more than 1-2°C. In general the aquariums used for Xenopus laevis, especially if kept in an apartment, do not require a heater.
These animals adapt well to all types of water although they prefer hard water containing reasonable amounts of calcium salts. They do, however, suffer in the presence of nitrites and nitrates and hence the values of these should be as close to neutrality as possible. In the same way chlorine and its salts are a threat to this species and it is, therefore, a good rule to use only water that has previously been decanted and to avoid direct, abrupt contact with tap water which often contains small amounts of sodium hypochlorite. Alternatively, water bioconditioners and reverse osmosis systems may be used. Pumps may be used for water filtering and treatment although they should not be too powerful to avoid water movements that could be a source of stress and disturbance to the animals: in natural conditions this amphibian prefers still water. The use of an ultraviolet steriliser on the pump may be useful to sanitise water and to decrease the bacterial load. The alternative to filtering systems is to change the water periodically, which should be done two or three times a week. It is good practice to perform regular chemical and microbiological analyses of the water so as to take the necessary steps should the bacterial load exceed the warning threshold: it should be remembered that the Gram-negative bacteria Pseudomonas and Aeromonas are the cause of many pathologies affecting Xenopus laevis. The tank for adults should never contain particularly small objects, as Xenopus frogs tend to ingest anything that is small enough to enter their mouths (so the size of the mouth should be taken as the parameter determining what is too small). They will also eat small fish, so it is not advisable to house both frogs and small fish in the same aquarium.
As far as concerns the material to place at the bottom of the tank, a few centimetres of river-bed gravel can be used. Although this does make cleaning of the tank difficult, it allows nitrifying bacteria to develop and the resulting substrate is more similar to that which the animal would encounter in nature. Decorative objects for aquariums and large stones may be used together with rooted or free-floating plants, even if these are often devastated by the frogs. With regards to illumination, light can be supplied for a period of 10-12 hours. In tanks used for small frogs which have just completed their metamorphosis or for reproduction, it is better not to introduce tank furniture or a gravel bottom in order to make management easier. It is also a good rule to avoid keeping large adult frogs together with very small ones in the same aquarium as this could lead to episodes of cannibalism. The use of ultraviolet light is indispensable for the amphibian’s synthesis of vitamin D and for the fixation of calcium.
DIET
The Xenopus laevis ingests more or less anything that fits into its mouth and its diet is based on animal proteins. Young specimens of around 1 cm are usually fed with Chironomus, Artemia and Tubifex but also with aquarium fish food flakes or worms cut into segments of a couple of millimetres. As the size of the animal increases larger pieces of food can be used, going up to pieces of 1 cm2 as for example in the case of animals fed with chicken hearts and livers or bovine, horse or turkey muscles which must all be cut down to the right size. Other foods which are greatly appreciated are moths, whole worms, adult insects (crickets, butterflies, flies, locusts) and fish breeding food pellets but even small fish or minnows which may also contribute to maintaining the predatory instinct of these amphibians. It is better to avoid fly larvae because of their cuticle which is particularly indigestible and can cause intestinal injury.
The Xenopus laevis does not chew, as it is toothless, and hence it simply swallows the food intact. As for the frequency of food administration, newly metamorphosed animals should be fed once a day; subsequently food can be administered once or twice a week. Should there be the desire to feed the adults daily as well, the amount of food administered should be reduced. In most cases if the animal is given a varied diet it should not require supplementary mineral salts and vitamins; however, the use of such supplements once a week, injected directly into the food, may help to prevent nutritional deficiencies (e.g., the so-called rubber jaw).
REPRODUCTION
The Xenopus laevis reaches sexual maturity at around 12 months of age. In a controlled environment four to eight reproductive cycles can take place per year, at least theoretically, independently of the season. In reality X. Laevis requires special conditions for reproduction and in captivity it does not always breed or spawns eggs spontaneously, since factors such as overcrowding, water currents and the absence of a nursery may have a negative impact on the reproductive success. In order to increase the likelihood of success, each couple must be isolated in special tanks with a double metal mesh flooring so as to allow the eggs to reach the bottom of the tank under the force of gravity thereby protecting them from their parents, which could ingest them or damage them by crushing them under their own bodies (Fig. 7). The depth of the water, measured from the higher of the two floors, should be no greater than 15 cm so as to allow the animals to breathe during mating without them getting too tired, also in view of the fact that the female is burdened by the weight of the male’s body. Insemination is external and the male inseminates the eggs as these are expelled from the female: for the entire duration of the mating, which can last even up to a couple of days, the male remains attached to the female circling her waist with his front limbs (Fig. 8).
The eggs laid are enveloped in a mucilaginous gel which allows them to adhere in clusters to the surfaces of the aquarium, although a good number succeeds in reaching the bottom of the tank (Fig. 9). During mating the animals should not be fed and they should be separated after 48 hours. At the end of this period the two frogs will be particularly stressed and weakened and they must be kept in conditions of maximum tranquillity and adequately nourished.
Based on the practical experience of the author there are two systems to induce ovulation: one environmental and one hormonal. The “environmental” technique requires enclosure of the animals at a low temperature (10-15°C) for about 6-8 weeks with a short period of daily illumination (4-8 hours) and a subsequent sudden increase of lighting and temperature so as to return to the optimal rearing conditions and simulate the changing of seasons from winter to spring and in so doing stimulate the production of sexual hormones. The other method, the “hormonal” approach, involving injection of HCG, is certainly more rapid and effective: the male is isolated in the reproduction tank and HCG is injected once a day for 3 days; on day 3 the female also receives the injection and is put together with the male. At this stage the animals should be left in peace with only dim lighting; within a few hours mating and egg laying starts. The number of eggs varies from a few hundred to a few thousand. The hormone injection must be done with the animal perfectly immobilised and with a 21G, 4 centimetres long needle: the suspension must be injected into the cisterna placed dorsally in the head region. The needle is introduced posterior to the oblique fibrous line over the pelvis and advanced subcutaneously, in a cranial direction, until it reaches the desired site (Fig. 10); this approach is used to prevent the hormone from spilling from the injection site. With both methods of induction the temperature of the water in the reproduction tank must be kept at 23-24°C and within 24 hours (maximum 48 hours) the adults must be removed, because the egg segmentation starts and even the water currents produced by the movement of the adults could cause the death or malformation of the embryos.
The Xenopus laevis does not take any care of its offspring. Within 72 hours from egg spawning the first swimming tadpoles can be observed. The water of the reproduction tank must be as still as possible. Filtering procedures should be avoided, because the water aspirator, even if working at low power, could suck up the tadpoles that are still not capable of swimming against the current: at most, a small porous stone oxygenator, with an extremely low air flow, could be used. The water of the reproduction tank must be adequately decanted as the presence of chlorine would kill the developing embryos. The reproduction tank should contain at least 80-100 litres of water and every day, with great care and attention, about one quarter of the volume of water should be changed. The eggs which start to develop are easily visible already 24 hours after being laid as they assume a “banana” shape, in contrast to the spherical shape of eggs just laid. After two more days the first small tadpoles, almost motionless and adhering in a vertical position to the walls of the aquarium, can be observed. For the first 6-7 days the small tadpoles should not be fed, as their energy source is the reabsorption of the embryonic membranes. In the subsequent stage the tadpoles are herbivorous and have filtering gills. Their diet is based on an infusion of nettles which is prepared by boiling some nettle powder for 10-15 minutes: the liquid is then filtered through gauze so as to allow suspension of the smallest particles. The infusion should be administered with a Pasteur pipette every day or at the most every other day (Fig. 11). The tadpoles will feed themselves and the material which progressively deposits on the bottom of the aquarium should be removed periodically so as to avoid polluting the water. The tank can be cleaned by aspirating the material deposited on the bottom of the tank with great care and substituting the water removed or delicately “fishing” out the tadpoles and putting them into another previously prepared tank. Thermal shocks must be avoided and the temperature of the water should be kept constant at around 23-24°C.
The larval development lasts on average just over 2 months. However, not all tadpoles reach stage 66 at the same time, consequently – as this takes place – the young, newly metamorphosed animals should be transferred into another tank containing no more than 4-5 centimetres of water. During the larval stages the young animals breathe through their gills while when they reach stage 66 they start pulmonary respiration and a diet based on animal proteins.
At spawning the eggs (zygotes) are 1 mm in size and consist of a single cell which then starts dividing. Within 3 hours the initial cell has divided into 32 cells (stage 6) and the typical morphology of a blastula appears (stage 8) (Fig. 12). At the age of about 26 hours the embryo assumes the typical banana shape (stages 24 to 27) (Figs. 13, 14 and 15) and at stage 45 (Fig. 16) – reached about 48 hours after spawning – the embryos have already taken on the typical conformation of a tadpole.
The following stages are characterized by progressive alterations and the appearance of the buds of the front and rear limbs (stage 50) and the subsequent detachment of these from the body of the tadpole (stages 57 and 58). At stage 60 the tadpole starts taking the shape of an amphibian (even if still with an exclusively aquatic life) with well-defined front and rear legs which can be distinguished from the rest of the body (Fig. 17). From stages 62 onwards (Figs. 18, 19 and 20) the tail progressively decreases in size to become a small appendage at stage 65 (Fig. 21) and then completely disappears at stage 66, when the development is completed.
