Ommochromes of the Compound Eye of Arthropods from the Insects and Crustaceans Classes: Physicochemical Properties and Antioxidant Activity

Abstract

The chapter is devoted to the study of the physicochemical properties of the ommochromes of the compound eye of arthropods. Ommochromes are the characteristic pigments of invertebrates. They are believed to function in the eyes as screening and protective pigments that protect photoreceptor cells from the damaging effects of light. Ommochromes were isolated, purified, and obtained in preparative quantities from crustaceans (Crustacea; order Decapoda) and insects (Insecta; families Stratiomyidae, Sphingidae, Blaberidae, Acrididae, and Tenebrionidae). The physicochemical properties of the isolated ommochromes were studied by absorption and fluorescence spectroscopy, electron spin resonance (ESR) and Mossbauer spectroscopy, and high-performance liquid chromatography. The antioxidant activity of ommochromes was studied by methods of inhibiting lipid peroxidation induced by reactive oxygen species and variable valence metal ions and by quenching luminol chemiluminescence. The data obtained are important both for understanding the biological functions of arthropod eye ommochromes and for the development of new pharmacological preparations based on ommochromes for the prevention and treatment of pathologies associated with the development of oxidative stress.

1. Introduction

Numerous species of animals that arose during the “Cambrian Explosion” (540–490 million years ago) had great diversity in the structure of the organs of vision. However, all this diversity can be divided into two main types: the first is the compound eye of most invertebrates (the most typical example is the eyes of arthropods), and the second is the chambered eye of vertebrates. The compound eye of arthropods consists of many small ocelli (ommatidia), each with its own photoreceptor cells (rhabdoms). The eyes contain both light-sensitive visual pigments and screening pigments. The main function of the screening pigments in the eye is light-filtering and light-absorbing. By absorbing and transmitting light in certain areas of the spectrum, preventing its reflection and scattering, screening pigments play an important role in shaping the spectral sensitivity of the eye; due to the absorption of scattered light, they determine the resolution of the eye (contrast and sharpness of the image); a number of other properties of visual perception also depend on them.

The main screening pigments of the compound eye of arthropods are organelles containing ommochromes [1]. Ommochromes are some of the main pigments of the compound eye of arthropods that protect photoreceptor cells from the damaging effects of ultraviolet light, visible light, and reactive oxygen species, both by optical shielding and by chemical neutralization of free radical products. An interesting fact of evolution: although almost all invertebrate species can synthesize and accumulate melanins, their visual organs contain mainly ommochromes, not melanins, as screening pigments.

The ommochromes were apparently first discovered by A. Johansen in 1924 in the primary and secondary pigment cells of the ommatidium of the compound eye of Drosophila, which contained two different types of pigment granules—purple-red and ocher [2]. The term “ommochromes” itself first appeared in the works of the German researcher E. Becker [3, 4, 5]. Becker proposed the general name “Ommochromes” for the pigments contained in the eyes of Drosophila and Calliphora flies, subdividing them into two large groups, “Ommatins” and “Ommines” [6]. Then, in the 1950s and 1960s, the molecular structure and the main physicochemical characteristics of ommochromes from a wide variety of classes and species of invertebrates were studied in detail. These numerous works were carried out by German chemists Butenandt and co-authors [1, 7, 8]. The antioxidant activity of arthropod eye ommochromes (shrimp: Pandalus latirostris, dragonfly: Calopteryx splendens, and butterfly: Pieris brassicae) was discovered in the 1980s [9, 10]. In 1985, it was shown also that arthropod eye ommochromes, like melanins, have a stable ESR signal with a high concentration of paramagnetic centers, which increases when exposed to ultraviolet and visible light [11].

2. Ommochromes of the compound eye of crustaceans—chemical nature, pathways of biosynthesis, and main physiochemical characteristics

2.1 Chemical structure and biosynthesis of ommochromes

In the eyes of invertebrates, ommochromes are localized in both pigment and receptor retinular cells. In addition to the eye, ommochromes have also been found in the cuticle, in excrement, and even in cells in the nervous system of insects [12]. In the cell, ommochromes are associated with specific proteins and are located in specialized organelles, ommochrome-containing granules [13]. Since the term “granules” does not imply the presence of an outer membrane, and ommochrome-containing organelles are surrounded by a membrane, it was proposed to call granules containing ommochromes ommochromasomes [14, 15]. The chemical precursors of ommochromes, especially 3-hydroxykynurenine, are thought to be transported into ommochromasomes by ATP-dependent transmembrane transporters of the ABC family [16]. Thus, in Drosophila, ommochrome precursors are transported by heterodimers of the White/Scarlet ABC transporter and its involvement in the transport of ommochrome precursors is believed to be evolutionarily ancient and widespread, particularly in insects.

Organelles containing ommochromes are inherent in almost all cells of the ommatidium. In the eyes of arthropods, these organelles are localized both in primary and secondary pigment cells and in receptor retinular cells. In shape, these are spherical formations 0.2–2 μm long surrounded by a single-layer membrane [13, 17, 18, 19], and in pigment cells, the diameter of ommochromasomes, as a rule, is much larger than in photoreceptor ones [20]. Ommochromasomes usually contain electron-dense osmiophilic material. This material likely contains complexes of ommochrome precursors and ommochromes themselves with ommochrome-binding proteins [15].

The ommochromes are generally divided into two main types—the yellow-red ommatins and the less-studied purple ommins. All ommochromes contain a phenoxazine/phenoxazone ring structure in their composition. The difference between ommatins and ommins is due to the fact that ommins presumably contain an additional phenothiazine ring (Figure 1).

Figure 1.

Basic ring structures of ommochromes.

The third type of ommochromes, ommidins, is much less common in arthropods and was found mainly in Orthoptera (Orthoptera) [21, 22]. According to their chemical structure, ommidins contain sulfur and, presumably, by analogy with pheomelanins, they are products of co-oxidation of two amino acids, in this case, tryptophan and cysteine.

Since ommochromes easily enter into various redox reactions, decarboxylation and esterification reactions, there is a large biological diversity of ommochromes in nature [15, 23]. Of the ommatins, the most common are xanthommatins, which have a characteristic phenoxazine group in their structure (Figure 2) [18, 24, 25]. They are especially common in the eyes of insects.

Figure 2.

Structure of xanthommatin and it uncyclized form.

In addition to xanthommatin, other ommatins, derivatives of xanthommatin, occur in the eyes of arthropods. The most common are rhodommatin, which is a β-glycoside of dihydroxanthommatin, and ommatin D, rhodommatin sulfate. Of the ommins, ommin A is the most common. The eyes of crustaceans and most insect species contain ommins that have maximum absorption in the visible region of the spectrum around 520 nm. The ommochromes are synthesized from tryptophan via a kynurenine intermediate [15, 18]. Figure 3 shows a diagram of the main pathways for the biosynthesis of ommochromes.

Figure 3.

Scheme of the main pathways for the biosynthesis of ommochromes. Enzymes involved in the biosynthesis of ommochromes: (1) tryptophan 2,3-dioxygenase, (2) kynurenine formamidase, (3) kynurenine 3-monooxygenase, (4) phenoxazone synthase.

At the first stage of synthesis, tryptophan is oxidized to formylkynurenine. This process is catalyzed by an enzyme, tryptophan 2,3-dioxygenase (TDO) (tryptophan pyrrolase; EC 1.13.11.11). This enzyme, which was first identified in Drosophila (“vermilium” gene required for the synthesis of brown eye pigment), is a tetrameric complex containing a heme required for the oxidation of tryptophan [26, 27]. At the second stage of synthesis, kynurenine is formed. This process can be either spontaneous or catalyzed by the enzyme kynurenine formamidase (KFase; EC 3.5.1.9) [28]. Since N-formylkynurenine is known to be unstable and is rapidly converted to kynurenine in vitro, the KFase enzyme may not be required for this process. The third step in biosynthesis is the hydroxylation of kynurenine to 3-hydroxykynurenine by the enzyme kynurenine-3-monooxygenase (KMO; EC 1.14.13.9). KMO synthesis is encoded by the cinnabar gene in Drosophila melanogaster. KMO contains FAD as a cofactor. FAD is reduced to its active form FADH2 by NADPH. Then FADH2 oxidizes kynurenine in the presence of oxygen, which leads to the formation of 3-hydroxykynurenine [29]. In the last step, xanthommatin is formed by the condensation of two molecules of 3-hydroxykynurenine. It is still unclear whether the enzyme phenoxazone synthase (PHS; EC 1.10.3.4) is involved in this process. Thus, several works [30, 31, 32, 33] have shown that ommochrome-containing organelles can accelerate the formation of xanthommatin in both enzymatic and non-enzymatic ways. It is assumed that the precursor of xanthommatin may be a labile uncyclized xanthommatin (Figure 2), which is initially formed by the condensation of two molecules of 3-hydroxykynurenine. This uncyclized xanthommatin, which has been extracted from crustaceans and insects, can spontaneously form xanthommatin at room temperature [34, 35, 36, 37].

The processes leading to the biosynthesis of reduced xanthommatin, as well as the reactions of formation of ommatin D, rhodommatin, as well as the biosynthesis of ommins, have not yet been sufficiently studied.

2.2 Physical and chemical properties of ommochromes

2.2.1 Redox properties

Ommochromes are colored substances responsible for coloration of many invertebrates. Xanthommatin and its derivatives, such as ommatin D and decarboxylated xanthommatin, are known to determine color and its changes in arthropods [38, 39, 40]. Changes in the coloration of ommochromes are usually associated with redox transitions in the pigment molecule. In this case, the reduction leads to a shift of the absorption maximum to the longer wavelength region of the spectrum (bathochromic shift). For example, during puberty in some species of dragonflies, the body color changes from yellow to red, which is associated with the appearance of a larger amount of reduced ommochromes [41].

The chemical properties of ommochromes contribute to their participation in electron transport, in reactions with oxidizing agents, reducing agents and free radicals. It is well known [6, 42, 43] that arthropod ommochromes are easily oxidized by hydrogen peroxide or potassium superoxide. In this case, a significant shift of the absorption maximum to the shorter wavelength region (hypochromic shift) is observed. The reaction with hydrogen peroxide proceeds in at least two stages. First, the transition of ommochromes to the oxidized form occurs, which is probably followed by a gradual destruction of the pigment during prolonged incubation with an oxidizing agent, which manifests itself in a further decrease in the absorption of the pigment in the visible range [43].

Ommochromes can also act as a reducing agent in redox reactions. For example, we have previously shown that shrimp eye ommochromes easily oxidize ferrous ions to ferric ions [44]. In this work, ⁵⁷Fe sulfate salt was used and the Mössbauer spectra of the formed iron complexes with ommochromes were studied. The obtained gamma resonance spectra were characteristic of high-spin Fe³⁺ complexes, which indicated complex formation and simultaneous oxidation of Fe²⁺ ions by the ommochromes. The absence of relaxation spectra for Fe³⁺ − ommochrome complexes indicated their cluster nature. The Fe³⁺ ions bound in a complex with ommochromes were located close enough to each other, which ensured an effective spin-spin interaction, which led to a rapid relaxation of the electron spin and “collapse” of the magnetic hyperfine structure [44].

The coordination of iron ions apparently occurs with the carboxyl, amino and imino groups of the ommochrome molecules. This can be represented within the structure of xanthommatin by the formation of a six-membered metallocycle with a system of conjugated bonds due to the coordination of Fe³⁺ with the imine nitrogen atom (1) and oxygen atom (2) of the neighboring ring according to the scheme (Figure 4):

Figure 4.

Hypothetical structure of iron-xanthommatin complex.

This exceptionally efficient binding of Fe²⁺ by ommochromes (with subsequent oxidation to Fe³⁺) into prooxidant inactive complexes may be one of the important mechanisms of the antioxidant action of shielding pigments in the arthropod eye [45, 46].

2.2.2 Free-radical properties of ommochromes

Previously, we showed that the ommochromes of the eyes of dragonflies and shrimps, similarly to melanins, exhibit a stable paramagnetic resonance signal [11]. These results were confirmed for the eye ommochromes of insects of various families [43]. All the studied ommochromes had a pronounced singlet electron spin resonance (ESR) signal with values of g factors close to the g factor of a free electron (2.0045–2.0048) and a fairly high concentration of unpaired electrons (> 10¹⁷ spin/g dry weight). Figure 5A shows the ESR spectra of ommochromes for 4 insect species.

Figure 5.

ESR spectra of ommochromes. (A) ESR spectra of ommochromes of the black soldier fly (1), butterflies tobacco hawk moth (2), marbled cockroach (3), and desert locust (4). (B) Oxidation of ommochromes with hydrogen peroxide (curve 2) result in drop of ESR signal; (1) original spectrum.

Irradiation of ommochromes with ultraviolet or visible region of the spectrum at liquid nitrogen temperature leads to a significant increase in the amplitude of the ESR signal (up to 5–9 times greater than the original dark signal) without changing the nature of the signal. The signal amplitude reaches saturation after approximately 40–50 min of exposure to light at the temperature of liquid nitrogen. When the light is turned off and the temperature is 1–3°C, the intensity of the ESR signal returns to its original level within 30–60 seconds. The ESR signal of ommochromes also turned out to be sensitive to the action of hydrogen peroxide (Figure 5B, curves 1 and 2). Oxidative destruction of the ommochromes by hydrogen peroxide led to a sharp drop in the ESR signal and, ultimately, to a complete loss of paramagnetism, which is probably due to the destruction of the phenoxazine ring in the structure of ommochrome molecules [47], which initially exhibits free radical properties. Destruction of ommochromes from the Drosophila eye with hydrogen peroxide has been shown previously [6].

The high concentration of stable free radical centers makes it possible to consider ommochromes as scavengers of active free radicals. The value of g factor of ommochromes, which is in the range between 2.004 and 2.005, is typical for phenoxy radicals [48]. It is known that the intermediates of phenoxazine, which is part of the structure of the ommochrome molecule, exhibit a stable ESR signal [49, 50] and can, apparently, cause the ESR signal that we found in ommochromes of insects. Moreover, it is possible that phenoxazine determines the antiradical activity of ommochromes [47].

2.2.3 Antioxidant activity of ommochromes

The ommochromes of all studied arthropod species exhibit high antioxidant activity by inhibiting the lipid peroxidation reaction induced by various reactive oxygen species and the action of ultraviolet and visible radiation [15, 43, 45, 51, 52]. Figure 6 demonstrates the inhibitory effect of fly eye ommochromes on the peroxidation of the outer segments of photoreceptors (POS) of the bull eye (Figure 6A) and shrimp eye ommochromes on the process of peroxidation of POS of the frog eye (Figure 6B). Fly eye ommochromes at a concentration of 350 μg/mL reduced the POS peroxidation rate by more than threefold. The antioxidant activity of ommochromes was most pronounced when the process of POS peroxidation was induced under conditions of hyperoxia. Hyperoxia was induced by intense bubbling of pure oxygen in the POS suspension (Figure 6B).

Figure 6.

Inhibitory effect of fly ommochromes (A) and shrimp ommochromes (B) on lipid peroxidation. (A) Inhibition of POS peroxidation induced by Fe²⁺-ascorbic acid. (B) Inhibition of POS peroxidation induced by hyperoxia.

It is important to note that in this case, the oxidation conditions were closer to those in vivo. It can be seen that under hyperoxia there is an increase in the rate of accumulation of TBA-reactive products in the POS suspension by about twofold. The presence of ommochromes at a low concentration (40 μg/mL) completely suppresses this effect. The antioxidant activity of ommochromes may be associated with the removal of free radicals due to the antiradical properties of these molecules [15, 52, 53, 54].

The redox balance in the cell is determined by the ratio of free radicals and antiradical molecules. When the concentration of free radicals is exceeded, oxidative stress occurs. The presence of antiradical molecules capable of neutralizing free radicals contributes to the control of oxidative stress. Apparently, there are two mechanisms of interaction of ommochromes with free radicals—electron transfer and hydrogen atom transfer [54]. It is known that ommochromes easily neutralize superoxide anion radicals [45, 52, 55, 56]. We have previously shown that shrimp eye ommochromes enhance the breakdown of superoxide radicals in both aqueous and anhydrous media (Figure 7) [55].

Figure 7.

Acceleration of the decomposition of superoxide radicals by shrimp eye ommochromes in anhydrous (A) and in aqueous (B) media.

Superoxide anion radicals obtained by electrochemical reduction of oxygen on a mercury cathode (at a current of 2 mA) in anhydrous medium (a solution of tetrabutylammonium iodide in dimethylformamide) slowly decompose by reacting with each other (superoxide concentration was measured by the degree of reduction of nitro blue tetrazolium or cytochrome c). The addition of shrimp eye ommochromes (170 μg/ml) led to a significant acceleration of the process of decomposition of superoxide radicals (Figure 7A). Ommochromes also accelerate the destruction of superoxide radicals in an aqueous medium (Figure 7B). Superoxide radicals were generated in the oxidation of xanthine to uric acid catalyzed by the enzyme xanthine oxidase. In the presence of shrimp ommochromes (50 μg/mL) a significant decrease in the rate of adrenaline oxidation to adrenochrome (480 nm) by superoxide radicals is observed [55].

The antiradical activity of ommochromes is apparently due to the presence of phenoxazine and phenothiazine rings in their structure. Phenoxazine-based compounds are known to be good antioxidants [47]. The antiradical properties of ommochromes can be directly linked to the N–H group of the phenoxazine/phenothiazine ring, which means that only reduced ommochromes can act as powerful antiradical and antioxidant molecules in cells [47]. It has been shown that phenoxazines and phenothiazines under in vivo conditions are strong inhibitors of autoxidation and ferroptosis (iron-dependent oxidative stress) due to the neutralization of lipid radicals [57]. Recent studies have shown that phenoxazine-based compounds can be used to protect against oxidative stress [47, 54, 57].

Thus, arthropod ommochromes have a pronounced antioxidant activity, inhibiting peroxidation processes even at relatively low concentrations. The antioxidant activity of insect ommochromes was comparable to that of natural melanins [42] and synthetic antioxidants of the oxypyridine series [58]. On this basis, we can confidently assume that the ommochromes of the arthropod eye at physiological concentrations have antioxidant effects.

2.2.4 Light sensitivity of ommochromes

The ommatins are known to be sensitive to irradiation. They readily undergo methylation, methoxylation, and decarboxylation under the influence of light in acidified methanol [15, 59, 60, 61]. Some studies have reported that during the incubation of the extracted ommochromes in acidified methanol for several hours at room temperature, the formation of ommochromes was observed that differed in spectral characteristics from the original pigment [37]. Under the influence of light on the ommochromes, the generation of superoxide anions is observed [62]. In particular, it was shown that illumination of a system containing a suspension of shrimp eye ommochromes in the presence of detergent cetyltrimethylammonium bromide (CTAB) and nitro blue tetrazolium reduces the latter with the formation of formazan (Figure 8).

Figure 8.

Light-dependent reduction of nitro blue tetrazolium by shrimp eye ommochromes (A) and the inhibitory effect of superoxide dismutase (B).

Illumination of the systems in control samples containing either only nitroblue tetrazolium and CTAB, or only ommochromes in the presence of CTAB did not lead to any spectral changes. The ommochromes reduced nitro blue tetrazolium in the presence of a detergent and in the dark, but the reaction rate was an order of magnitude slower than in the case of illumination. Photogeneration of superoxide radicals was observed when ommochromes were illuminated with both ultraviolet and visible light. The superoxide photogeneration process was inhibited by superoxide dismutase (Figure 8B). The concentration of superoxide dismutase causing 50% inhibition was 25 μg/ml.

Thus, arthropod eye ommochromes are capable of photogeneration of the superoxide radicals. When illuminated, ommochromes are able to reduce oxygen to its anion-radical form. This process, apparently, is inherent only in the reduced form of ommochromes, since oxidized ommochromes reduce NBT with much less efficiency under illumination. The physiological significance of this process is unclear. On the one hand, it is possible that this process proceeds in vivo at an insignificant rate and does not pose a risk for cell photodamage. On the other hand, it is known that oxidized phenoxazine derivatives can be reduced by glutathione during irradiation [63]. Superoxide anion radicals are an intermediate product of this reaction. Based on the structural similarity of ommochromes to phenoxazine derivatives, it can be assumed that they can also be reduced in light. Moreover, the reduced form of ommochromes can catalyze this process. Such a mechanism in the tissues of the eyes of invertebrates would be particularly effective, since oxidized ommochromes formed in the course of interaction, for example with superoxide, would be constantly renewed in light.

3. The main functions of ommochromes in the eyes of arthropods

It is generally accepted that ommochromes in the cells of the compound eye of arthropods perform the function of optical shielding of light-sensitive elements of retinular cells and individual ommatidia from each other, as well as the function of regulation of spectral sensitivity of photoreceptors [23]. Recently, more and more facts have appeared showing that ommochromes, along with carotenoids that quench singlet oxygen, can protect eye cells from oxidative stress.

3.1 Optical screening function

In light, the pigment organelles of photoreceptors and pigment cells change their position, tending to group around the rhabdom (Figure 9). During dark adaptation, ommochromasomes migrate away from the light-sensitive rhabdom, opening it to light. During light adaptation, they migrate back to the light-sensitive rhabdom, protecting it from excess light. Such retinomotor activity is one of the mechanisms of light and dark adaptation of the compound eye of arthropods.

Figure 9.

Ommochromasomes migrate during light (A) and dark adaptation (B).

Another striking example of the influence of the retinomotor effect on visual acuity is the highly developed compound eye of insects, such as the superposition eye of Diptera. The rhabdom in these eyes acts as a light guide or optical fiber. The position of the screening pigment granules that surround the microvilli of the rhabdomer containing rhodopsin changes during the daily cycle. During daylight hours, the granules surround the microvilli in such a way that the light is concentrated on them and not scattered. Therefore, dipterous day vision is highly acuity. In the dark time of the day, the granules migrate from the rhabdomers, the light is scattered, and visual acuity decreases, but the sensitivity of the eye increases.

3.2 Adjustment of the spectral sensitivity of the eye

Screening pigment-containing organelles can regulate the spectral sensitivity of the arthropod compound eye. One example is the adjustment of the spectral sensitivity of the fly’s compound eye. Normally, there are two types of screening pigments in the eyes of flies: red ommochromes and yellowish pterins. First ones absorb light in the visible range, passing it only in the red region of the spectrum, the second ones absorb mainly in the ultraviolet region. Therefore, all light passing to the rhabdom through its own facet, that is, unshielded, is absorbed by the visual pigment rhodopsin with λ_max ≈ 500 nm. The light passing to the rhabdom from the side, through the neighboring facets, is filtered by ommochromasomes. As a result, only red light reaches the rhabdom. Thus, the fly’s eye turns out to be endowed with a peculiar additional red-sensitive (λ_max = 620 nm) light detector of a “filter” nature. Combined with the main green-sensitive (λ_max ≈ 500 nm) light detector, this additional “filter” light detector, based on the same rhodopsin, allows flies to distinguish colors [64, 65, 66].

Another example of adjusting the spectral sensitivity of the compound eye is associated with the study of screening pigments in crustaceans—glacial-relict shrimps of the Mysis genus. Subspecies of the relict shrimp Mysis relicta in the postglacial period (about 10,000 years ago) were in different lighting conditions. It was shown that in the subspecies inhabiting the slightly saline waters of the Baltic Sea at shallow depths, the maxima of the spectral eye sensitivity curves, as a rule, are shifted by 20–30 nm to the short-wavelength region of the spectrum compared to subspecies of M. relicta inhabiting in fresh lake waters [67]. Studies have shown that the lake shrimp M. relicta, living at great depths in a brown-red light environment, has a long-wave spectral sensitivity with a maximum of about 600 nm. In contrast, the sea shrimp M. relicta, inhabiting at a shallow depth of the Baltic Sea, has a shorter wavelength spectral sensitivity with a maximum of about 570 nm. It was shown [68, 69] that both populations contain two types of rhodopsins in two types of rhabdoms, one with an absorption maximum at 525–530 nm, and the other with an absorption maximum at 565–570 nm. However, in the lake population of shrimp, the content of longer-wavelength rhodopsin significantly predominates, while in the sea population, on the contrary, the content of shorter-wavelength rhodopsin significantly predominates [70, 71]. It is important to emphasize that both in the sea and lake populations of M. relicta, the spectral sensitivity of the eye is noticeably shifted to the long-wavelength region of the spectrum with respect to the absorption spectra of their visual pigments. Using microspectrophotometry, we studied the absorption spectra of pigment-containing organelles in the eyes of both populations of M. relicta shrimp [67, 69]. Most pigment organelles had absorption spectra in the blue region of the spectrum, which is typical for xanthommatins (maximum at 455 nm). A smaller number of pigment organelles had absorption spectra characteristic of a mixture of ommochromes—ommatins and ommins (unpronounced maxima at 440 and 555 nm) (Figure 10A and B).

Figure 10.

Absorption spectra of eye ommochromasomes of Sea (1) and Lake (2) Mysis relicta populations. (A) Ommochromasomes with xanthommatin-like spectra; (B) Ommochromasomes with ommatin-ommin-like spectra.

In addition, in the eyes of sea mysids, but not lake, populations were recorded in a small number of spectra characteristic of ommins (maximum about 580 nm). Calculations of the spectral sensitivity of the eye of M. relicta, formed by the absorption spectra of visual pigments, taking into account the filtering of light by ommochrome-containing organelles, made it possible to construct a spectral sensitivity curve completely identical to the real spectral sensitivity of the eye of M. relicta. Thus, the spectral features of ommochromes in the eyes of the lake mysid population led to a shift of the maximum spectral sensitivity of the eye by 30 nm to the long wavelength region of the spectrum, which is very important for vision at great depths with low illumination.

3.3 Oxidative stress protection function

It is known that the lake population of relict shrimp M. relicta is much more sensitive to the damaging effects of light compared to the sea population—even moderate illumination of animals, for example, on the surface of the water in the evening in the absence of sun, leads to complete loss of vision [72]. It was suggested that one of the possible reasons for the high sensitivity of the lake shrimp population to light may be associated with a lower level of protection of eye cells from the action of free radical products induced by irradiation. This means that the system of antioxidant defense in the shrimp of the lake population is significantly lower than in the shrimp of the sea population. Indeed, a study of the comparative activity of antioxidant systems and their components in the eyes of two M. relicta shrimp populations showed that the sea shrimp population has a significantly more active antioxidant defense system compared to the lake population [73]. As can be seen from Figure 11, the homogenates obtained from the eyes of the shrimp of the lake population practically did not inhibit the process of accumulation of hydroperoxides during oxidation of cardiolipin liposomes, while the homogenates obtained from the eyes of the shrimp of the sea population were active in this respect.

Figure 11.

Effect of eye homogenates shrimp Mysis relicta on Fe²⁺-ascorbate-induced peroxidation of cardiolipin liposomes.

The time interval required to reach the maximum concentration of hydroperoxides in the presence of the homogenate of the eyes of shrimp from the sea population was almost three times longer than in the control. At the same time, during the initial period of the reaction (up to 20 min), the content of hydroperoxides in the liposome/homogenate system of the lake population was more than 4 times higher than that for the liposome/homogenate system of the sea population. This means that the shrimp of the sea population have a higher activity of antioxidant defense systems than the shrimp of the lake population. The study of the content of the main systems of antioxidant defense—the activity of antioxidant enzymes (superoxide dismutase and glutathione peroxidase), as well as the concentration of α-tocopherol in the homogenates of both types of eyes, showed no significant difference for the homogenates of sea and lake shrimp populations [74]. At the same time, analysis of homogenates for the content of ommochromes showed that the concentration of ommochromes in the homogenate of the eyes of shrimp in the sea population was almost 2.5 times higher than in the lake population (Table 1).

Population M. relicta	Eyes number	Ommochrome content
		μg of dry weight per eye	μg of dry weight per mg protein
Lake population	150	14.3 ± 1.8	90.5 ± 11.8
Sea population	150	39.3 ± 2.9	218.6 ± 17.5

Table 1.

The content of ommochromes in the eyes of marine and lake shrimp Mysis relicta populations.

Table 1 shows that the concentration of ommochromes in the eyes of sea population shrimp reaches almost 40 μg per eye. If we assume that the volume of one eye does not exceed 0.5 μl, then the concentration of ommochromes in them reaches 80 mg of dry weight per 1 ml. In shrimp of the lake population, the low concentration of ommochromes may be associated with the loss of pigments in the process of adaptation to a deep-sea lifestyle in very low light conditions. At the same time, the ommochromes of sea and lake populations are identical in their spectral and antioxidant characteristics. These data indicate that the higher antioxidant activity of the eye homogenate of shrimp from the sea population can indeed be associated with a higher content of ommochromes in them.

Another example of the antioxidant function of ommochromes is related to the suppression of oxidative stress in triatomine bugs (Hemiptera: Reduvidae). For example, the action of ultraviolet radiation on red-eyed mutants bugs lacking ommochromes led to severe oxidative stress and damage to ommatidia, especially in the case of abundant blood feeding, when a high concentration of heme, a powerful generator of reactive oxygen species, is formed in the body [51]. While in wild-type bugs, exposure to ultraviolet caused an increase in the intensity of the synthesis of ommochromes and protection of eye structures from light and oxidation.

4. Conclusion

Ommochromes, which are found in large numbers in the cells of the compound eye of arthropods, are necessary for these animals to perform a wide variety of functions. These are optical shielding of individual ommatidia from each other, increasing the resolution of the eye, expanding the range of color perception, and regulating the spectral sensitivity of photoreceptors. In addition, ommochromes can protect the eyes of arthropods from oxidative stress caused both by ultraviolet or intense visible light irradiation and by excessive generation of reactive oxygen species. But ommochromes may be necessary not only for arthropods. Recently, there have been works pointing to the antimicrobial, antifungal, and antiglycation properties of ommochromes [75, 76, 77]. Due to their biological activity, ommochromes can be promising pharmacological preparations for prevention and treatment of pathologies associated with the oxidative stress development.

Acknowledgments

This research was funded by the Ministry of Science and Higher Education of the Russian Federation, grant number 075-15-2020-773. The authors are grateful to the Russian Academy of Sciences and the Tvärminne Zoological Station of the Helsinki University for many years of assistance in carrying out these studies.

Conflict of interest

The authors declare no conflict of interest.