Bacterial deodorant: a symbiotic relationship between tree frogs and Pseudomonas bacteria

The river begins with the blue brook, and the friendship begins with mutually beneficial symbiotic relations between two biological subjects. In the harsh conditions of wildlife, when half of its inhabitants want to eat you, smiles rarely help. But what exactly helps is the aforementioned symbiosis - mutually beneficial cooperation between individuals of different species. Each of the participants in such a relationship gets something different: protection, food, home, vehicle, etc. But the tree frogs of the species Boana prasina symbiosis with certain bacteria is needed for love affairs. Today we will get acquainted with the amazing observations of scientists who have found out that the bacteria that live on the skin of frogs contribute to the selection of the unique aroma for individual sexes, which plays an important role in finding a partner and, accordingly, in reproduction. Why do scientists sniff frogs, what do they smell like (frogs, not scientists), and how did such an unusual symbiosis between amphibian and bacterium arise? The answers are waiting for us in the report of the research group. Go.

About friendship in nature

For a person, the benefits of friendship are usually psychological (emotional). We meet new people, begin to make friends and communicate because it is simply pleasant to us. The same can be said about our pets. Cats, dogs, hamsters, fish, etc. these are friends for a person and moral rather than physical support. Although there are people living on the principle of mom from the cartoon "Prostokvashino":

- Well, you yourself think what the benefits of this cat?
- Well, why do you need a favor? What is the use of this picture on the wall, for example?
- From this picture on the wall is very big benefit - it blocks the hole on the wallpaper!

But back to the flora and fauna. In the wild (we will not consider symbiosis in controlled conditions) relations between representatives of different species are necessary for survival or for a more comfortable life.
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Clownfish in anemone.

Vivid and one of the most famous examples are clown fish and sea anemones (sea anemones). The fishes, at the sight of which many exclaim something like “oh, Nemo!”, Living inside the anemone, are immune to their ciliary cells (stinging / stinging cells), but predators are very susceptible. In other words, clown fish live in a house with an electric fence. Actinia also benefit from such a neighborhood in the form of nutrients from fish waste products (yes, the phrase “de *** marketing relationships” for actinium sounds quite literally).


Another unusual symbiosis, some types of anemone form with hermit crabs.

From the depths of the sea move to a blooming meadow. Who do we always represent next to the flower? Of course, the bees. These workaholics collect nectar from flowers, that is, they get food. At the same time, having flown from flower to flower, they carry out indirect pollination, so to speak, than they help plants in reproduction. In other words, the bee would answer yes to the question “what are you, holding a candle?”, After which it would sting you for such inappropriate questions.


Bees on a sunflower.

In addition to bees, other species of insects (ants, bumblebees, butterflies, etc.), as well as vertebrates (birds, bats, rodents, etc.), help plants in the pollination process.

As we have already understood, symbiotic relationships are not uncommon. We are rather surprised by the participants of this relationship than the fact of their existence.

It is worth noting that the smell is a chemical signal that allows you to get some information about its source. Not only frogs, but also many species of vertebrates use smells as a way to exchange information. Recall what cats do (especially often in the spring) - this way they notify other members of their species of their presence, the right to the territory, etc. If we consider the sources of odors in general, there are several main sources:

• primary (de novo) secretory synthesis in certain glands;
• by-products of excretion, that is, excretions of the vital activity of the organism;
• use of the environment;
• waste products of symbiotic bacteria.

Back in the 70s of the last century, a “theory of fermentation” was stated, stating that symbiotic bacteria in mammals metabolize proteins and lipids found in aromatic glands. Consequently, volatile compounds are produced, which the carrier uses for communication.


Croaking tiger frog.

If we talk about frogs and toads, then sounds (acoustic signals) have always been considered the main method of transmitting information (i.e. communications). But the smells are not given special attention, considering them not so important in this process. It turned out that this is all different. Not only is the smell very important in transmitting information (determining sex, attracting a partner, protecting against predators, etc.), it also arises with the help of symbiotic organisms, namely bacteria.

Scientists found out that the secret secreted by the skin of frogs contains organic sulphides * , sesquiterpenes * and methoxypyrazines * . The source of these chemical compounds were bacteria from the group Pseudomonas , living on the skin of the studied individuals.

Organic sulphides * are sulfur-containing analogues of ethers.

Sesquiterpenes * - a group of organic compounds from the class of terpenes, which consists of hydrocarbons from C ₁₅ H ₂₄ to C ₁₅ H ₃₂ .

Methoxypyrazines * are chemical compounds that produce odors.

As I mentioned earlier, scientists chose Boana prasina species living in the South American region as test subjects. This species has a rather long reproductive period, during which the males use acoustic signals and emit a very strong and characteristic odor. Scientists emphasize that studies on the role of odors in the social activity of tree frogs have already been carried out, but the source of these odors has not yet been investigated.

And now, having become acquainted with the concept of symbiosis, the research participants and the goals of scientists, let us proceed to become acquainted with the results of observations.

Research results

Through SPME (solid-phase microextraction) and GC / MS (gas chromatography / mass spectrometry), scientists found that about 60-80 different chemical compounds are included in the volatile secretion of the skin of males and females of the type B. prasina, including: alcohols, aldehydes , alkenes, esters, ketones, methoxypyrazines, terpenes (hemiterpenes, monoterpenes and sesquiterpenes) and thioesters.


Image No. 1: comparison of the chemical composition of the volatile secretion of the skin of males and females of the species B. prasina.

Designations to the graph above: Mop - methoxypyrazines; Het - hemiterpenes; Mnt - monoterpenes; Sqt - sesquiterpenes; Hyc - hydrocarbons; Toe - thioethers; Eth - esters; Ket - ketones; C * - aromatic compounds also found in terrariums.


Table 1: chemical analysis of volatile secretion of the skin of males and females of the species B. prasina.

At the same time, it turned out that hemiterpenes, ketones and alcohols (49.9–78.3%) make up the majority of this “fragrant soup” in both sexes.

If we compare the chemical composition to determine its differences in males and females, then there are SQT (sesquiterpenes), TOE (thioethers) and MOP (methoxypyrazines), which can be seen in 2A and 2B .


Image number 2: a comparative analysis of the chemical composition of the secretion of males and females.

Dual graph 2A is based on the relative content of volatile compounds. The sample dispersion centroids are depicted as a circle for females and a triangle for males. Figure 2B shows the relative contribution of each class of chemical compounds to the first four main components obtained in the PCA analysis (principal component method). The size of the circle indicates the absolute value of the contribution, and the color (from red to blue) indicates this contribution is negative or positive.

Thanks to the creation of the LDA function (linear discriminant analysis), it became obvious to scientists that the individual’s sex can be distinguished by the chemical profile ( 2C ): the Kruskal-Wallis criterion: DF = 1, P = 0.001.

A little explanation of the terminology in the data:

• the word “interaction” means that both factors were taken into account in the data analysis - the sex of the individual and the method of sampling;
• the word "gender" means analyzing data solely for determining the sex under certain conditions of taking a sample;
• in vivo - analysis on a living organism (not on a sample taken from it);
• sample - analysis of skin samples taken from a living individual (analyzed separately from the individual).

Of the three most distinctive classes (TOE, SQT, and MOP), it was MOP (methoxypyrazines) that were more pronounced in females than in males. This is evident from the results of the likelihood ratio test. Interaction: χ ² ₁ = 1.20; P = 0.2729; gender: χ ² ₁ = 12,778; P = 0.0004.

At the same time, the males had SQT indices (sesquiterpenes) higher than those of females. Interaction: χ ² ₁ = 23,817; P <0.0001; gender (in vivo): χ ² ₁ = 22.933; P <0.0001; gender (sample): χ ² ₁ = 1.317; P = 0.2511.

Finally, analysis of the TOE data (thioethers) showed that the males of this chemical compound have more, but only in the analysis for living individuals (in vivo). Interaction: χ ² ₁ = 10.618; P = 0.0011; gender (in vivo): χ ² ₁ = 10.9505; P = 0.0009; gender (sample): χ ² ₁ = 2.0051; P = 0.1568.

In a more detailed examination of the composition of SQT and TOE, scientists identified two main substances that were expressed the most, namely dihydroedulan II in SQT and C ₆ H ₁₂ S in TOE.

Next, the researchers decided to more thoroughly deal with methoxypyrazines (MOP), of which 4 were already allocated:

• MOP1 - 2-isopropyl-3-methoxypyrazine (C ₈ H ₁₂ N ₂ O)
• MOP2 - 3-isopropyl-2-methoxy-5-methylpyrazine (C ₉ H ₁₄ N ₂ O)
• MOP3 - 2-sec-butyl-3-methoxypyrazine (C ₉ H ₁₄ N ₂ O)
• MOP4 - 3-sec-butyl-2-methoxy-5-methylpyrazine (C ₁₀ H ₁₆ N ₂ O)

Image No. 3: data on all four types of MPAs in males and females.

As can be seen from the data analysis, MOR3 is very pronounced in males, but not in females, regardless of the method of analysis (interaction: χ ² ₁ = 0.1219; P = 0.727; sex: χ ² ₁ = 49.4548; P <0 , 0001).

But MOR4, on the contrary, is strongly expressed in females, but not in males (interaction χ ² ₁ = 5.3977; P = 0.0202; gender (in vivo): χ ² ₁ = 25.5523; P <0.0001; sex (sample): χ ² ₁ = 33.1275; P <0.0001).

MOR1 and MOR2 were rather poorly represented in both sexes and together accounted for approximately 0-12.8% of the total number of methoxypyrazines.

With the data, numbers, comparisons "who has more," we figured out, and now to the most interesting.

The fact is that TOE and MOP are chemical compounds that are produced (as a rule) by microorganisms. Therefore, scientists needed to check whether this is so in the case of tree frogs. Samples were taken from which bacteria associated with the skin were isolated, cultured and identified.

Of, attention, 128 volatile metabolites in bacteria, scientists identified 16, which were also in frogs. And also 5 compounds were found that were only available from bacteria.


Image No. 4: 4A - MOP found in frog skin (1-4) and in the bacteria Pseudomonas (5-8), 4B - GC / MS full ion current chromatogram showing the production of MOR 1-8 (left) of Pseudomonas cultured in Müller – Hinton agar (right).

And here is another very interesting fact - all 4 types of MPAs (we considered them earlier) were produced by a single bacterial isolate. In addition to those present in the MOPs 1–4, the bacterium produced several additional ones (5–8 in image 4A ), which were not found in frogs. It is also worth noting that bacteria had the most “massive” MPA number 5 (C ₇ H ₁₀ N ₂ O or 2-methoxy-3,5-dimethylpyrazine), which is about 82.4 ± 16.7% of the total number of methoxypyrazines.

Comparison of MOR1-4 in bacteria and frogs showed that MOR2 in bacteria was better expressed (5.9 ± 6.9%), while MOP3 and MOP4 were worse than frogs (2.6 ± 5.0% and 2). , 7 ± 2.1%, respectively).


Image No. 5: A population of bacteria on the skin of subjects taken from different regions and at different times.

Individuals for the study were taken from the wild, more precisely from 4 different regions ( 5A ). The number of bacteria differed in individuals from different regions and depended on the time spent in captivity ( 5B ).

Despite variations in more than half the number of active taxonomic units (OTU) observed in skin bacterial colonies at four sites (60–63%) and in five different sampling periods (56–66%), can be identified as representatives of the following bacterial families: Enterobacteriales , Pseudomonadales , Methylophilales , Oceanospirillales , Actinomycetales , Bacillales , Alteromonadales and Burkholderiales . The most pronounced among this variety were Klebsiella ( Enterobacteriales ) and Pseudomonas ( Pseudomonadales ). This is shown in graphs 5A and 5B .

There were no particular differences between the tested individuals from different regions. But the effect of time in captivity was palpable. Thus, the diversity of bacteria and their numbers decreased with time (in captivity): 1st day - 163 ± 26, 3rd: 145 ± 35, 10th: 100 ± 9, 2 weeks: 104 ± 41 and 8 months : 155 ± 34.

The difference, depending on the region of habitat and time in captivity, was also seen among the bacteria Pseudomonas . Some OTUs were found only in frogs from a certain region ( 5C ) or were already manifested in captivity ( 5D ). But 4 species of bacteria from this genus did occur in most samples, that is, they did not depend on geographical factors and time in a controlled laboratory environment. At the same time, there was a difference in the number of bacteria Pseudomonas in males (1.7% on average) and females (2.4% on average). These indicators have decreased during their stay in captivity in both sexes, especially during the first 3 days: from the maximum on the first day 8.8 ± 4.3% to the maximum on the 3rd - 0.4 ± 0.2%.

Considering the data analyzed, it can be concluded that it is Pseudomonas bacteria that are actively involved in the secretion of volatile compounds that are used by the carrier, i.e. frogs, for determining sex, attracting a partner during the breeding season and as a protection against predators.

For more detailed acquaintance with the nuances of the study I recommend to look into the report of the research group and additional materials to it.

Epilogue

The fact that odors play an extremely important role in the life of many living organisms, including amphibians, has been known for quite some time. However, such studies, the purpose of which is to identify the original source of these odors and the mechanisms for the production of volatile chemical compounds, have not yet been carried out.

Scientists have been able to find out exactly which chemical compounds are important for the formation of odor and what produces them. The skin of tree frogs is home to a number of bacteria from different families. Scientists suggest that biomolecules synthesized by the skin of an amphibian serve as a filter by means of which unique combinations of different types of bacteria can inhabit the skin. And volatile compounds secreted by MOP-produced Pseudomonas bacteria in frog skin can act as infochemical substances, that is, a way of communication between the bacteria themselves.

In turn, tree frogs use this as one of the types of signals given to other individuals during the mating season along with acoustic signals, which is confirmed by behavioral tests in the wild and in the laboratory.

In the future, researchers are going to continue their work in order to study in more detail the interaction of bacteria and tree frogs. Scientists call this work the first steps in understanding the symbiotic relationship between amphibians (and not only) and various microorganisms.

Yes, such studies may not bring any practical benefit to humanity, although it is too early to talk about it, for one discovery can lead a series of new ones that somehow can affect our lives. But what exactly these studies give is the knowledge and understanding of aspects of the world around us, about which we do not yet know everything.



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