The visual diversity of living things on planet Earth is due not so much to a craving for beauty and the desire to be different, but rather in the habitat, habits and even gastronomic preferences. Someone prefers to remain unnoticed, merging with the environment due to camouflage color and unusual body shape. Someone, on the contrary, with all their appearance speaks to anyone who dares to approach, about their poisonousness. And someone just loves to show off in front of a partner and trample a competitor into the mud. Any visual feature has a function. For example, the color of clown fish of the species Amphiprion percula, which became world-famous thanks to the cartoon "Finding Nemo", is associated with their territoriality. And so scientists from the Okinawa Institute of Science and Technology (Japan) decided to find out how and where clown fish get their trademark white stripes on the body. When clown fish show their stripes, what mechanisms are involved in this, and what does anemones have to do with it? We will find answers to these questions in the report of scientists. Go.
Basis of research
Clownfish is a genus of marine ray-finned fish from the pomacentral family, numbering about 30 species inhabiting the reefs of the Indian and Pacific Oceans.
Clown fish, despite their hot temper, are not alien to teamwork.
One of the most famous features of these fish is their symbiotic relationship with anemones (anemones) - organisms from the order of escaping coral polyps. They look like a bundle of tentacles (Cthulhu approves) with or without a leg attached to solid sea soil. Anemones lead a sedentary lifestyle and feed on what, due to their stupidity or due to the coincidence of circumstances, falls into their tentacles. However, it will not be possible to see sharp attacks or any other attack, because anemones kill (or paralyze) their prey with the help of stinging cells (cnidocytes). The paralyzed (at best, already dead) victim is then transferred by tentacles to the mouth of the anemone.
But clown fish, looking at anemones, see not danger, but free housing. The tentacles of anemones are covered with mucus, protecting them from the action of their own stinging cells. Clownfish are also covered in this mucus, making them immune to deadly cnidocytes. Fish use anemones as a home and even as a food source (leftover anemones, fallen off tentacles, secretions). In return, they clean the anemones of debris, undigested food and drive water through its tentacles, providing "ventilation".
Many species of clown fish are extremely jealous of their anemones, driving away anyone who approaches a distance that they think is "suspicious". In other words, despite their funny name, these fish do not behave like kind clowns from the Licedea tetra, but like Pennywise.
Many scientists associate the color of clown fish and their territoriality. However, until now it was not known how such a color is formed. In particular, scientists have always been puzzled by the three white stripes on the body of adults, which the young do not have. This indicates that during their life the fish undergo some kind of metamorphosis. It remains to find out which ones.
Scientists note that the main task of biology is to understand speciation. But this is not limited to understanding the difference between species (in exaggerated terms), but also to understanding the causes and mechanisms that cause differences within one species, whether physiological or behavioral.
Diversity within a single species can be expressed as phenotypic variations between individual populations. But even within a separate population, there may be differences caused by the influence of the environment, behavioral characteristics or developmental conditions.
In some cases, phenotypic variability may reflect the plasticity of adaptive development, i.e. the ability of organisms to change their developmental trajectories to create phenotypes that are precisely adapted to environmental conditions. An example of such plasticity is the varied color in animals of the same species.
In words, it looks quite simple, but in fact, such plastic changes are associated with complex changes at the physiological, cellular and molecular levels. Scientists honestly admit that although plasticity is known to science, the mechanisms underlying it are practically not studied.
In this vein, it is worth noting another important and unusual process that occurs during the development of some organisms - metamorphosis. This process is due to serious changes in the structure of the body (or part of it) in the course of individual development. Metamorphosis is regulated by thyroid hormones (TH from thyroid hormones). Consequently, any changes in TH during the metamorphosis caused by these hormones can affect both the process and the result of such a complex process of transformation of the body. TH also plays an important role in the transition of pigmentation from larva to adult. For example, in zebrafish ( Danio rerio ), TH promotes the maturation of specific pigment cells, black melanophores and yellow xanthophores.
Danio rerio is also called a lady's stocking (who said that ichthyologists do not have a sense of humor).
Therefore, there is a high probability that a similar pattern should be observed in clown fish. In the work we are considering today, scientists decided to test the relationship between TH and the metamorphosis of the color of clown fish, and also considered the potential influence of the habitat (two different species of anemones) on this process.
The study focuses on closely related species Amphiprion ocellaris and Amphiprion percula , which live in symbiosis with anemones in the tropical Indo-Pacific region. Scientists have found that juveniles of A. percula show different rates of white stripe formation depending on the species of anemones where they live: in the anemones of the species Stichodactyla giganteastripes appear faster, and slower in the sea anemone Heteractis magnifica . Consequently, the formation of stripes (i.e., certain metamorphic changes) is influenced not only by internal factors (thyroid hormones), but also by external factors (habitat).
Research results
During postembryonic development, Amphiprion individuals gradually acquire stripes on the head, trunk, and caudal peduncle. In Kimbe Bay, Papua New Guinea, A. percula occurs in two different species of anemones: S. gigantea and H. magnifica , but the fish that live there belong to the same population.
Image # 1
However, the young A. percula living in the sea anemone S. gigantea have more white stripes than the young A. percula living in the sea anemone H. magnifica . In 33% of 148 individuals (200 to 250 days old) in anemones S. gigantea had three white stripes, while only 5% of 118 individuals of the same age in H. magnifica had three stripes ( 1A and 1B ).
Observations suggest that the species of anemones (i.e. the habitat) affects the timing of the formation of white stripes in young A. percula . Multiple regression analysis helped to check the correctness of this statement, allowing to establish the dependence of one variable on two or more independent variables. As expected, the analysis confirmed that juveniles living in S. gigantea always had more stripes than individuals from the sea anemone H. magnifica( 1C and 1D ).
Image β2 The
formation of stripes is directly related to the process of postembryonic development, in particular with metamorphosis. Therefore, by tracing the stages of this process, it is possible to establish a connection between the bands and the level of thyroid hormones (TH).
View A. ocellaris shows two schemes of pigmentation in the development of time before the step β5 (about 9 days after hatching), the larvae have a yellow ksantofory * with a set of star melanophores which form two horizontal stripes covering myotomes * (red arrows on the 2A- 2D ).
Chromatophores * - pigment-containing or light-reflecting cells, divided into subclasses depending on the color:
xanthophores - yellow;
erythrophors - red;
leukophores - white;
melanophores - black / brown;
cyanophores - blue;
iridophores - silvery.
Miotome * is a paired rudiment of skeletal muscles in the embryos of chordates.Beginning at the fifth stage, the larvae acquire three white vertical stripes (white arrows at 2E - 2G ), orange xanthophores outside the future white stripes (orange arrows at 2E), and melanophores distributed throughout the body (black arrows at 2E and 2F ). These melanophores are present throughout the body and have a higher density at the border of the white stripes ( 2F and 2G ).
For a better understanding of the changes in the pattern on the body of individuals occurring at the fourth stage, it was necessary to assess the expression of pigmentation genes at the postembryonic stages. For this, RNA was extracted from the larvae at each stage of development for transcriptome analysis. The main emphasis was placed on the study of pigmentation genes ( 2H ), in particular on iridophore genes, since they are responsible for the formation of white stripes.
It was found that stages 1-3 are strikingly different from stages 4-7 in the main component 2 (PC2; 2H and 2I ).
(PCA principal component analysis) β , .Among the genes related to PC2, the fhl2b, pnp4a and prkacaa genes were more pronounced in stages 5-7 compared to stages 1-3. On the other hand, the gbx2, trim33, gmps, and oca2 genes were more pronounced in stages at stages 1-3 compared to stages 5-7 ( 2J ).
Also, during the analysis, a clear division of stages was observed for all functional categories: specification of pigment cells, development of xanthophores, synthesis of pteridine pigment of xanthophores, development of melanophores, regulation of melanogenesis and biogenesis of melanosomes.
These observations suggest that at the fourth stage there is some important shift in color development, involving all three types of pigment cells.
Given that thyroid hormones (TH) play an important role in the process of metamorphosis, which controls the pigmentation pattern in many teleost fish, it is logical that TH regulates the timing of white stripe formation during metamorphosis of clown fish.
To test this hypothesis, tests were carried out in which larvae of the third stage of development were exposed to various concentrations (10 -6 , 10 -7 and 10 -8 M) of the active thyroid hormone T3. Three days after treatment with T3, subjects showed an earlier appearance of white stripes than individuals from the control group.
Image No. 3
This acceleration effect was dose dependent on T3. Three days later, two bands appeared in the following proportion of experimental subjects: 0% - control group; 50% - at 10 -8 M T3; 78% - at 10 -7 M and 73% - at 10 -6 M ( 3A - 3E ).
Then a similar test was carried out, but with the blocking of TH by means of a mixture of MPI (methimazole, potassium perchlorate, and iopanoic acid). The treated larvae of the third stage of development showed a nine-day delay in the formation of white stripes compared to the control group (test group at 3H and control group at 3G). About 75% of individuals from the control group had stripes on the head and trunk, from the test group only 15% of individuals had stripes, the rest were left without pigmentation ( 3F ). White stripes in the fish from the test group were all formed in spite of TH blocking, but only after 25 days ( 3I ).
The manipulations with TH also affected the pigment cells. The number of melanophores increased significantly within 48 hours after the application of 10-6 M T3 ( 3J ). On the other hand, blocking TH with MPI led to an almost imperceptible decrease in the number of melanophores 48 and 72 hours after treatment ( 3J ).
Taken together, the results of these tests suggest that TH does indeed control the timing of white stripe formation and also affect pigment cells (iridophores and melanophores).
To more accurately determine how exactly TH affects iridophores, we analyzed the expression of the iridophore genes (fhl2a, fhl2b, apoda.1, saiyan, and gpnmb) after treatment of larvae with exogenous TH.
The larvae of the third stage of development were treated with T3 at various concentrations (10-6, 10-7 and 10-8 M) for 12, 24, 48 and 72 hours. The expression of the studied genes was controlled using nanostrings in RNA isolated from whole larvae.
After treatment with T3, much more transcripts of these genes were detected than in the control group (without T3 treatment). In some cases (apod1a and gpnmb) this effect appeared after 12 hours, and in others (fhl2a, fhl2b and saiyan) only after 24 or 48 hours. It follows that TH affects the expression of genes that are expressed in the iridophores of clownfish.
Next, the scientists decided to test whether TH promotes iridophore differentiation. For this, larvae of the third stage of development were treated with T3 (dose 10 -6 M) for a long time. This was a comparison of juveniles at the sixth stage, when stripes on the head and body begin to develop in clown fish.
Curiously, the youngsters from the test group (T3 treatment) did not fully form bands, in contrast to the control group. A detailed examination of individuals from the test group revealed numerous ectopic iridophores on the sides. These fish also had a less vibrant orange color.
Processing with MPI resulted in normal stripes. However, the color was duller due to a lack of iridophores or crystalline guanine deposition within the iridophores, usually responsible for the white (or iridescent) color of the stripes ( 3I ).
These results suggest that exogenous TH leads to a decrease in orange coloration and defects in white streak formation.
Image No. 4
However, the TH treatment accelerated the streaking process. Recall that fish of the A. percula species living in the sea anemone S. gigantea form white stripes faster than their counterparts living in anemones of another species. Therefore, there must be some connection between stripe formation, TH and habitat.
To check this, scientists selected 12 individuals 12-27 mm in size (one white stripe is either forming or already formed) living in S. gigantea (n = 6) and in H. magnifica (n = 6). Next, the scientists measured the TH level of each individual.
Comparison showed that T3 concentrations were significantly higher in young animals from S. gigantea versus those of H. magnifica ( 4A ). Next, a comparison was made of gene expression in young animals from H. magnifica (n = 3) and from S. gigantea (n = 3).
Of the 19063 genes analyzed, only 21 were significantly more expressed in individuals from S. gigantea , while 15 were significantly more expressed in individuals from H. magnifica ( 4B ).
Among the differentially expressed genes, the duox gene was found, which encodes a double oxidase involved in TH production. This gene was overexpressed in S. gigantea compared to H. magnifica . Therefore, the rate of white stripe formation in A. percula is associated with differential T3 level, which, in turn, is associated with differential duox expression.
At the final stage, the scientists checked whether duox is required to form the iridophore pattern. For this test, zebrafish were used, in which the maturation of iridophores depends on TH.
In the larvae of the control group, densely packed iridophores formed one wide intermediate row, then the second intermediate row began to form ventrally. In duox-deficient larvae, only one wider intermediate zone ( 4C ) has developed .
As a result, the majority of fish from the control group developed two complete intermediate rows, while in fish with duox deficiency only one ( 4D and 4E ). This suggests that duox, presumably acting through TH, contributes to the timing of the appearance of the interband regions of the iridophore and to the formation of the pattern between the bands in zebrafish.
For a more detailed acquaintance with the nuances of the study, I recommend that you look into the report of scientists and additional materials to it.
Epilogue
In this work, scientists decided to reveal the secret of the white stripes on the body of clown fish. It turned out that this uncomplicated color (especially compared to some other fish) is the result of the joint work of several important factors: genes, hormones and even anemones, in which clown fish live.
The most unusual observation was that fish from the same population, but living in two different species of anemones, form their color differently (in terms of time). Comparison of the genes of these fish showed that only 36 genes differ. Among them, scientists have isolated the duox gene, which encodes a protein of the same name, which is involved in the formation of double oxidase. This gene plays an important role in the production of thyroid hormones (TH), which in turn affect the rate at which pigment patterns are formed on the body of clown fish.
But why do fish from one sea anemone βcolorβ faster than others? Scientists are not yet ready to give an exact answer, but they have a theory. Actinia, in which body streaks form faster, is probably more toxic. Therefore, thyroid hormone levels rise in response to this toxicity.
Scientists themselves believe that the difference in the rate of strip formation in individuals from the same population is just one of many signs of different adaptive tactics that fish use to increase effective coexistence with anemones. In the future, they intend to conduct an even more thorough comparative analysis of fish living in different anemones in order to identify additional differences.
Nature seems simple and uncomplicated: trees are growing, birds are singing, bees are buzzing, etc. But upon closer examination, we begin to notice how many complex processes occur in the life of even the smallest insect or the most widespread plant. All properties, characteristics and qualities that an organism possesses are the result of a long and meticulous evolution, the main goal of which is survival. And if an organism wants to survive in a constantly changing world, it must use the entire arsenal of adaptive talents, from the most primitive to the most complex.
Thanks for your attention, stay curious and have a great weekend, guys! :)
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