Photoprotective mechanisms

Protection of freshwater aquatic organisms from UV-induced injury is dependent on a variety of factors that can function as photoprotective mechanisms. When UVR breaches photoprotective mechanisms in sufficient amount, UV-induced injury will occur. Aquatic organisms vary in their tolerance to UV exposure. There is a likely interplay between the ecological niche occupied by an organism and its UV sensitivity. Throughout an organism's life stages, its habitats and habits will likely complement the organism's tolerance for UV. Nocturnal or crepuscular activity regimes would clearly limit UV exposure, as would the organism's selection of UV-limiting habitats. Fish species have adapted to certain levels of UV and probably exhibit different ranges of tolerance to solar UVR. Species naturally adapted to high levels of solar radiation exposure would be more tolerant of high UV-B levels than species adapted to low levels of solar radiation. Indeed, razorback suckers, a fish species naturally adapted to high solar UV levels, were tolerant of simulated UV-B and did not develop sunburn after 21 days of exposure [42]. In contrast, sunburn was observed in rainbow trout and Lahontan cutthroat trout within two days, and Apache trout (Onco-rhynchus apache) after five days exposure to simulated UV-B [2].

The sensitivity to UVR will vary with the fish's acclimation to solar radiation. Increases in water clarity and changes in water chemistry (particularly decreases in organic carbon content) can heighten UV intensity in the water column [43]. Even though fish may detect UV-B when applied in laboratory studies, they probably do not directly avoid solar UV-B [44], but indirectly avoid UV-B by seeking positions lower in the water column or by seeking shade to avoid intense visible or UV-A. Since solar visible and UV-A would not increase with ozone depletion, higher levels of solar UV-B would go undetected by fish and could cause harmful effects [45]. However, fish that exploit surface or shallow water habitats probably evolved adaptations to tolerate the high U V irradiance of such habitats [46],

There are also physiological characteristics that underlie an organism's tolerance to UV-B including the efficiency of photorepair mechanisms for damaged DNA. DNA is particularly vulnerable to UV because it induces the formation of cross-linkages, or dimers, in the pyrimidine base thymine. Such cross-linkages include cyclobutane-type dimers of thymine, cytosine, and uracil; pyrimidine adducts; photohydrates; and DNA-protein cross-links [47] can interfere with DNA replication and protein synthesis necessary for cell division in growth and replacement and can lead to the development of tumors, as well as lesions. Most organisms are capable of repairing the DNA damage induced by UV-B through excision repair, photoreactivation, and post-replication repair ([47], see also Chapter 9). Among these, photoreactivation is promoted by the DNA photo-lyase, an enzyme that binds to the cyclobutane dimer, becomes activated by absorbing photons from UV-A and visible light, then cleaves the dimer from the ring before unbinding [48]. Blaustein et al. [49] found that the amount of this enzyme in embryonic amphibians is directly correlated with the UV-B tolerance of that species. Although this correlation does not demonstrate a higher rate of dimer repair among tolerant organisms compared to sensitive organisms, it does suggest a cellular basis for photorepair efficiency. However, results from studies with the wood frog (Rana sylvatica) indicated variation in response to photolyase depending on environmental conditions and led Smith et al. [50] to conclude that estimating amphibian photorepair is a complicated process and that previous conclusions regarding the relationship between photorepair and amphibian population decline must be reevaluated. Photorepair is likely to be ubiquitous among fish given the range of species for which evidence for photorepair has been found, including goldfish [51], anchovy larvae [52] and fathead minnow [53]. Photorepair efficiency in fish varied by as much as 500% between two closely related species [54].

Excision repair involves damage recognition, incision of the DNA chain near the site of the lesion as DNA is excised and resynthesized around the damaged site, and ligation following detachment of DNA polymerase [48]. Species may be capable of both types of repair mechanisms and may vary as to which one predominates. For example, much of the DNA repair occurred during daylight hours through photorepair, and remaining repair occurred in darkness through excision repair [55].

Regardless of an organism's efficiency for photorepair such mechanisms are not completely efficient, so do not entirely ensure against potential injury. Therefore, additional protective mechanisms are adaptive. These include pigments that sequester the highly reactive oxygen free radicals or other reactive species that are generated by UV and are responsible for DNA damage and other cellular injuries ([56], see also Chapter 8). UV-shielding pigments also provide a means of protection from UV. These can be extrinsic as well as intrinsic filters. Aquatic animals utilize available environmental factors that provide photoprotection, such as DOC, which reduce environmental UV levels, and as a consequence the dose the organism receives. Aquatic organisms also incorporate UV-absorbing or reflective pigments in their integument.

13.4.1 Photoprotective substances in aquatic organisms

There are several types of photoprotective substances found in aquatic organisms (see Chapter 10). Photoprotective pigments have certain general characteristics as outlined below [57]. Briefly, most photoprotective substances share the rc-electron systems that occur in conjugated bond structures such as alternating single and double bonds in linear molecules, and in aromatic and cyclic compounds containing electron resonance. Overlapping orbits of 7r-electrons have absorption maxima in the UV region that causes an energetic transition of 7r-electrons to anti-bonding 7i*-electron orbits. Alteration in the structure of a conjugated molecule changes the absorbance characteristics and therefore the irradiance spectra that are attenuated. Longer wavelengths are absorbed by larger molecules and shifts in wavelengths absorbed occur as the number of conjugated bonds or number of substituents is increased. Absorption increases as more side chains and substitutions are added to the molecular structure. The non-bonding electrons of oxygen, nitrogen, and halogen atoms become a part of the resonance of the ring structure and shift the absorption maximum. Over the course of evolution, changes in the molecular structure have developed for specific absorbance characteristics. An organism can screen a broad spectrum of UV-B and UV-A wavelengths by synthesizing a range of photo-absorbing molecules.

Mycosporine-like amino acids, another type of photoprotective substance, have been found in a diversity of organisms ranging from bacteria to fish [57]. Up to 19 kinds of mycosporine-like amino acids have been identified. Certain organisms contain several of these substances that broadly screen UV [58]. The concentrations of these substances increase proportionately with the intensity of UV irradiance they are exposed to [58-60]. Mycosporine-like amino acids are probably not synthesized by fish and invertebrates, but acquired through the diet, especially from grazing on algae [61]. Gadusol, also believed to be photo-protective, is structurally related to mycosporine-like amino acids and is found in the eggs of cod and Mediteranian fish [62] and in brine shrimp [63].

Photoprotective melanins are found in a diversity of vertebrate and invertebrate organisms and are polymers formed from 5,6-dihydroxyindole, a phenolic and indolic compound [64]. Melanins are complex molecules that broadly absorb UV and visible radiation, but show no specific absorption maximum; however, their absorption increases with decreasing wavelength [65,66]. Melanin is produced in melanophores that then deposit melanin on subcellular organelles called melanosomes, which are often positioned above the nucleus [67]. Exposure to UV can cause an increase in melanin production and the number of melanosomes during long-term exposures to UV [57]. Fish typically have no melanin in the epidermis leaving this skin layer relatively unprotected from UVR, and appear to rely on a colorless compound(s) secreted in the mucus covering the body to provide epidermal photoprotection [68]. In frogs (Figure 4), however, melanin does occur in the epidermis, where photoprotection appears to be related to the amount of melanin as well as its distribution [69]. UV-B-tolerant boreal toads (Bufo boreas) have a distinct double layer of melanin. Related Woodhouse's toads (Bufo woodhousii) that live in lower altitude habitats also have a double melanin layer, but the melanin appears to be diffuse and less concentrated. Nocturnal gray tree frogs (Hyla versicolor) have a single layer of melanin, and are sensitive to UV-B. Tiger salamanders (Ambystoma tigrinum) have a diffuse and limited distribution of melanocytes and are highly sensitive to UV-B [Carey personal communication].

Carotenoid pigments occur widely among crustacean zooplankton, though the composition and quantities vary with species [55]. Carotenoids are produced by algae, bacteria and plants, and transferred to invertebrate and vertebrates through the food chain [70]. Carotenoids are thought to be the main photoabsorbing pigment in copepods. Copopods show greater concentrations of carotenoids than do cladocerans, and those living in UV intense environments have higher concentrations than organisms from low UV habitats [71]. Caro-

Bufo bóreas

Hyla versicolor

Figure 4. Distribution of melanin in the larval skin of (A) the boreal toad (Bufo boreas), (B) Woodhouse's toad (Bufo woodhousii), (C) gray tree frog (Hyla versicolor), (D) tiger salamander (Ambystoma tigrinum). Note dense layer of melanin below skin (single arrow) in the toad and frog larvae, and the melanin distribution in the epidermal layer (double arrow) which is most dense in the UV tolerant boreal toad, and least dense in the nocturnal (and UV sensitive) gray tree frog. Melanin distribution in tiger salamander larvae is diffuse throughout the dermis and epidermis (arrows) and far less dense compared to the frogs and toads. [(A,B) modified from Little et al. [69]; (D) from Carey unpublished data.]

^ _ Ambystoma tigrinum

Bufo bóreas ffl

Hyla versicolor

Figure 4. Distribution of melanin in the larval skin of (A) the boreal toad (Bufo boreas), (B) Woodhouse's toad (Bufo woodhousii), (C) gray tree frog (Hyla versicolor), (D) tiger salamander (Ambystoma tigrinum). Note dense layer of melanin below skin (single arrow) in the toad and frog larvae, and the melanin distribution in the epidermal layer (double arrow) which is most dense in the UV tolerant boreal toad, and least dense in the nocturnal (and UV sensitive) gray tree frog. Melanin distribution in tiger salamander larvae is diffuse throughout the dermis and epidermis (arrows) and far less dense compared to the frogs and toads. [(A,B) modified from Little et al. [69]; (D) from Carey unpublished data.]

^ _ Ambystoma tigrinum tenoids are often distributed throughout the body of copepods, whereas cladocerans tend to concentrate carotenoids in ovarian lipids and eggs [55]. In addition to carotenoids, melanin is also present in the cuticle of cladocerans and tends to be more pronounced among species living at high altitudes [72,73]. Carotenoids have limited UV-filtering capacity, however, and in cladocerans the carotenoids are thought to play an important photoprotective role by sequestering oxygen free radicals [55,56].

Freshwater fish vary in their tolerance of UV-B [2]. Tolerance offish exposed to simulated UV-B appeared to be unrelated to melanin pigmentation but related to the amount of an unidentified colorless photoprotective substance in the skin [42,74]. This photoprotective substance appeared to be localized in the outer dorsal skin layers (Table 1), which includes the epidermis and overlying mucus, but was also detected in the eyes and gills of UV-tolerant razorback suckers (Xyrauchen texanus) [68]. The following examples illustrate how the amount of this photoprotective substance in a given fish species is related to the UV tolerance of that species. Channel catfish were found to be extremely sensitive to simulated UV-B, darkening within 24 h of exposure and having no detectable photoprotective substance [17]. Razorback suckers exposed to solar simulated UV-B did not develop sunburn after 21 days of exposure, while rainbow trout sunburned within 48 h [68]. When sections of the dorsal skin were removed from unexposed fish and methanol extracts scanned in a spectrophotometer, there was more photoprotective substance in the extracts of razor-back suckers than rainbow trout [42]. Thus, there was a direct relationship between the amount of photoprotective substance and the period of time in which these fish developed sunburn (Table 2). The photoprotective substance appeared to have functioned as a natural sunscreen and protected razorback suckers from the harmful effects of simulated solar UV-B. In contrast to melanin, which offers some photoprotection at any given wavelength, the photoprotective

Table 1. Absorption maximum (Amax) and amount of photoprotective substance in methanol extracts of various tissues from rainbow trout (Oncorhychus mykiss) and razorback suckers (Xyrauchen texanus). [from Fabacher and Little 68]

Tissue

Species

LN)1

Amountb

Outer dorsal skin layers

rainbow trout

294.0 [0.0]

0.10 [0.0]

razorback suckers

294.5 [0.2]

0.44 [0.05]c

Inner dorsal skin layers

rainbow trout

290.2 [0.4]

0.03 [0.01]

razorback suckers

292.3 [0.6]

0.12 [0.01]c

Eyes

rainbow trout

not detected

-

razorback suckers

293.5 [0.3]

0.02 [0.003]

Gills

rainbow trout

not detected

-

razorback suckers

293.5 [0.3]

0.02 [0.002]

Liver

rainbow trout

not detected

-

razorback suckers

not detected

aMean Amaj [standard error] for six fish.

bMean absorbance units/milligram wet weight (au/wt) [standard error] of tissue for six fish. cPhotoprotective factor of tissue differs significantly between species.

aMean Amaj [standard error] for six fish.

bMean absorbance units/milligram wet weight (au/wt) [standard error] of tissue for six fish. cPhotoprotective factor of tissue differs significantly between species.

Table 2. Relative amount of sunscreen present in fish skin, average UV exposure time to induce sunburn, and vulnerability of fish species to UV injury; compiled from Little and Fabacher [2], Ewing et al. [17], Fabacher and Little [68,74]

Days of exposure to Vulnerability to U V

Species Amount of sunscreen sunburn injury

Razorback sucker 100 >21 low

Pigmented Medaka 59 10 medium

Albino Medaka 59 10 medium

Rainbow trout 23 2 high

Channel catfish ND 1 very high

Wavelength (nm)

Figure 5. Spectrophotometric UV absorbance of photoprotective substance from the skin of razorback sucker (Xyrauchen texanus). Absorbance maximum occurs at 294 nm, the broad shoulders of the absorbance curve would provide protection from UV wavelengths less than and greater than 294 nm. [From Fabacher and Little 68.]

Wavelength (nm)

Figure 5. Spectrophotometric UV absorbance of photoprotective substance from the skin of razorback sucker (Xyrauchen texanus). Absorbance maximum occurs at 294 nm, the broad shoulders of the absorbance curve would provide protection from UV wavelengths less than and greater than 294 nm. [From Fabacher and Little 68.]

substance would offer a large amount of protection in the UV-B wavelength range because the absorption maximum is around 294 nm and the slope of the shoulders of this peak covers many of the UV-B wavelengths (Figure 5).

When cutthroat trout and razorback suckers were exposed to simulated UV-B, cutthroat trout (Oncorhynchus clarki henshawi) showed grossly visible signs to exposure (dorsal skin darkening) by 48 h [15], Razorback suckers, however, did not show any visible signs of sunburn during the entire 72 h of exposure. Cutthroat trout had considerably less of the photoprotective substance than did razorback suckers. Histologic examination of cutthroat trout dorsal skin showed sloughing of mucous cells, necrosis and edema in both epidermis and dermis, and, in some cases, secondary fungal infection (Figure 6). Conversely, histologic observation of razorback sucker skin revealed that necrosis had occurred, but the severe sloughing and necrosis observed in cutthroat trout skin had not occurred (Figure 7). There was an increase in razorback sucker epidermal thickness, apparently resulting from hypertrophy and hyperplasia of large cells containing large central regions of low electron density. These

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