Direct damage and means of protection lines of defense

The general, direct effects of UVR at the cellular level are rather uniform within the animal kingdom. These include first of all DNA-damage, membrane damage and a range of other cellular injuries that may be caused by intracellular photoproducts. They also include immunosuppression, yet the responses here may be more different across phyla, especially between invertebrates and vertebrates. Finally skin lesion, cancers and eye-damage (cataract) may be common responses in vertebrates. These effects may sum up in reduced fitness of various kinds, ranging from death to a slight decrease in life span, growth or rate of reproduction. These various types of damage will not be reiterated here. They may, however, be used as examples of reported responses when direct effects on different organisms are treated more specifically towards the end of this chapter.

The fact that animals in most regards share the same nucleic acids and other macromolecules, the same set of enzymes and proteins in general and in fact much of the same cellular machinery also generates fairly general action spectra. Although action spectra with sufficient resolution have been provided for a very limited number of aquatic organisms [2-4], they do all have the common property of rapidly increasing effects towards the lower end of the spectrum, and very limited effects in the PAR-area. In fact for DNA damage the relative biological response to wavelengths beyond 310 nm is negligible [5]. Of specific interest for aquatic organisms are thus the optical properties of the water column per se related to the depth distribution of the animals. If there is a strong attenuation in the short-wave area (as would be the case for localities rich in

DOC), this will be the main determinant of the biologically effective dose, i.e., the sum of the action spectrum and spectral irradiance. Especially for freshwaters, but also to some extent for coastal areas, DOC is by far the most important determinant of UVR attenuation [6,7] and changes in DOC could be far more important for pelagic animals than predicted changes in stratospheric ozone.

Animals may cope with the supposed challenges of UVR in different ways, and, with regard to the direct UVR effects, several lines of defense may be identified that play a different role in different organisms. There may either be seasonal or spatial means of UVR avoidance by timing of reproductive season, depth distribution or diurnal migration. A second line of defense is UV screening compounds (e.g. pigments, mycosporine-like amino acids). Third, there are several enzymes and other macromolecules serving as radical scavengers that in various ways cope with ambient or intracellular harmful photo-products. A last major defense would be the various means of enzymatic photo-repair that is a common property of all organisms.

12.2.1 Seasonal and spatial responses

Commonly, such strategies will be a trade-off between positive and negative effects of solar radiation. Since photosynthetically active radiation (PAR) is the key determinant of primary production, the productivity at the base of the food web will be closely related wavelengths in the range of 400-600 nm. The pelagic grazers that harvest this phytoplankton yield will thus invariably expose themselves also to the shorter wavelengths. Planktonic crustaceans do, however, commonly have a pronounced diurnal vertical migration that may range from a few to hundreds of metres. The downward migration during daytime is mostly attributed the risk of predation from visual predators (mostly fish), but in fact short-wave light may serve not only as the proximate cause of diurnal migration, but also as the ultimate cause as demonstrated by migratory behavior also in the absence of predators.

First, zooplankton may adapt or alter their seasonal or spatial distribution to reduce the UV-stress. Seasonal life cycle adaptations to avoid periods of peak solar intensity may very well be a strategy for UV-exposed and sensitive organisms, yet this is not explicitly demonstrated. Diurnal vertical migration is, however, commonly accredited to direct UVR [8,9]. A critical question is whether organisms have a sufficient spectral resolution to separate UVR from, for example, blue light. This may be important to respond to an increased UVR under constant PAR. Such behavioral responses clearly are important evolutionary traits for swimming animals, and could affect both productivity and trophic interactions; the topic will be fully covered in another chapter, and will thus only briefly be touched upon here to illustrate some ecological implications.

A high spectral resolution and sensitivity is probably common in most pelagic organisms, but again special interest has been devoted to the freshwater cladoceran Daphnia. For Daphnia, sensitivity to UVR was suggested in the early works of Koehler [10] and Merker [11] as cited in Smith and Macagno (1990)

[12]. The thorough study of Smith and Macagno [12] revealed four peaks in the spectral sensitivity in D. magna, at 348, 434, 525 and 608 nm. Peak sensitivity in the ultraviolet was found at 340 nm for Daphnia, and UVR gave a strong negative phototaxis as opposed to visible light [13]. Flamarique and Browman [14] demonstrated a wavelength dependent orientation in polarized light among Daphnia, which was also different between different species. Experiments have shown that Daphnia can detect UVR and respond with downward migration [8,9,13,15], and that pigmented clones migrate less than non-pigmented clones [9,15]. Evidence so far do indicate that aquatic invertebrate can detect UV-A but, probably, not UV-B.

In situ observations support the potential direct effect of detrimental radiation on vertical distribution of zooplankton in Arctic localities. Hebert and Emery [16] reported different patterns of spatial distribution among melanic and hyaline clones of D. pulex and D. middendorjfiana in North-American Arctic. Melanic clones ranged freely through the water column, while unpigmented clones were restricted to the pond bottom under high irradiances. In support of this it was observed that in high Arctic localities at Svalbard the diurnal migration pattern depends on levels of pigmentation and weather conditions. Daphnia in these localities are commonly heavily melanized (see below), but some deeper localities may house transparent (hyaline) clones. For a strictly transparent clone it was observed that the entire population was concentrated at, or close to, the sediment surface during days with clear sky (making sampling by net hauls sometimes nearly impossible), while animals were evenly distributed in the water columns on cloudy days [17]. For heavily pigmented animals in a shallow adjacent pond, no sign of downward migration was recorded even under bright sun. A pronounced diurnal migration of Daphnia, Bosmina and copepods in alpine localities devoid of vertebrate predators was accredited to UVR avoidance [18].

One important aspect in this regard is the trade-off between pigmentation, migration, UV-damage and predation. These aspects have some ecological and evolutionary implications, since increased pigmentation allows animals to stay in upper (warm) layers where food is abundant even under high UVR. On the other hand the pigmentation has its costs both in terms of energy expenditure and visibility that may enhance the risk of predation. These strategies will be more closely examined when discussing the role of pigments (below).

The general ability to respond to short-wave radiation by downward migration is also seen in fish [19]. When placed in the quartz cylinders with three replicate treatments of visible, visible plus UV-A, and visible plus UV-A and B, cod larvae distributed themselves evenly throughout the vertical extent of the cylinder (15 cm) under the visible and visible + UV-A treatments. In the treatments exposed to visible + UV-A and B larvae were consistently found at the bottom of the cylinders, particularly at peak solar intensity. This was observable even on the first few days of the treatments when mortality or morbidity was not a factor. Such UVR avoidance also seems, for fish, to rely on UV-A receptors that have been found in a variety of fish species [20-22], yet as with crustacean invertebrates fish also seem unable to detect UV-B.

12.2.2 Photoprotectivepigmentation

A second line of defense is the photoprotective pigments, of which mycosporine-like amino acids (MAAs), carotenoids and melanins are the most prominent in aquatic animals (Chapter 10). The conspicuous presence of pigmentation in light-exposed animals clearly aims at a higher UVR tolerance. In fact the very presence of these UV-protective properties strongly suggests that UV is a potential stress factors, since all protective means have their cost. This is clearly seen as a general decrease in pigmentation with increasing depths, reaching the extreme in deep-water or cave-dwelling animals that may be almost completely devoid of skin pigmentation. Zooplankton, being susceptible to visual predators, face a dual challenge. Strong pigmentation implies high visibility and thus a high risk of predation. Lack of pigmentation, on the other hand, could render the animals more susceptible to UVR damage. There should thus be a selection towards invisible UV-screens.)

A heterogeneous category of UV-screening compounds are collectively labeled MAAs. These are widespread in shallow water organisms [23,24] absorb radiation primary in the 310-360 nm range and seem to be primarily associated with UV-stress. Probably most of the MAAs in heterotrophs are derived via food from autotrophs [25,26]. MAAs are also found in freshwater zooplankton, yet there may be conspicuous differences among taxa. Ethanol extracts from alpine copepods contained compounds absorbing in the range for MAA (peak absorb-ance at 330nm), while no such were found in Arctic or alpine populations of Daphnia (Figure 1, Borgeraas and Hessen, unpublished data). This is in support of Sommaruga and Garcia-Pichel [27] who reported frequently high concentrations of MAAs in alpine calanoid copepods, moderate levels in sympatric rotifers, while virtually no MAAs were detected in cladocera. This points to some important taxonomic differences in pigmentation strategies, at least between copepods and cladocera, corresponding to what is seen for carotenoids. Helbling et al. [28] also demonstrated the key role of MAAs for copepods exposed to extreme UVR such as Boeckella titicacae, which, as the name hints, is an inhabitant of lake Titicaca (3810 m a.s.l). They also demonstrated a downregula-tion of the MAA synthesis under reduced UVR exposure, suggesting that this is a dynamic trait. The more general properties of MAAs are covered in Chapter 10.

12.2.2.2 Melanin

Melanins are complex macromolecules that serve as highly efficient screens for short wave radiation across the animal kingdom. They are widespread in all groups of metazoans, and have at least two key ecological functions: camouflage and UV-protection. The potential role of melanin in UV-protection is perhaps best illustrated by freshwater zooplankton. Some cladocerans have developed a highly conspicuous carapace melanization that appears to be a unique adaptation to UVR. Highly UV-exposed populations of various species and clones of the Daphnia pulex complex and Daphnia longispina may frequently have a dark

■ - Heteroscope saliens —Daphnia longispina

■ - Heteroscope saliens —Daphnia longispina

Figure 1. Example of absorption spectra of aqueous ethanol extracts of D. longispina and the copepod Heterocope saliens showing the peak of the carotenoid absorption maximum at 476 nm present in both species and of UV-absorbing compounds at 327 nm in the copepod.

appearance that most often is caused by a carapace melanization [16,29]. Within a region these clones often occur in the clearest ponds and are replaced by non-melanic clones when vegetation cover increases or water transparency decreases due to increased concentrations of DOC.

That carapace melanization in zooplankton is, apparently, a unique property of Arctic and alpine cladocera is somewhat puzzling since the ability is shared among different clones and taxa. In fact almost all arthropoda may synthesize melanin for eyes and specific structures. All Daphnia species may synthesize melanin for protection of their resting eggs (ephippia), yet among the crustaceans the ability of carapace melanization seem restricted to Arctic and alpine cladocera and a few other crustacean taxa. Aquatic insects may commonly possess melanization, as do the vertebrates.

The fact that melanized animals are far more tolerant to UVR than their non-melanized relatives, [9,16,29,30] and Figure 2, strongly supports the view of a primarily UV-protective function. This is further supported when comparing UVR transmission through the carapace of hyaline and melanic animals. Melanized carapaces offer a highly efficient UV-absorption, especially with the shorter wavelengths, relative to transparent ones (Figure 3). Another argument for the role of melanins in UV-protection is the fact that melanin synthesis involves great costs both in terms of energy demands and increased risk of predation (see below). The fact that this kind of extensive melanization occurs almost exclusively in alpine and arctic localities could be accredited to the common UV-

Figure 2. Population development of melanized (filled symbols) and hyaline Daphnia under high light intensity (upper panel) medium light intensity (middle panel) or high light intensity plus Mylar sheet (lower panel). Five juvenile individuals were added in triplicate for each treatment, and population development followed for 12 days, covering one reproductive cycle. Light was provided with a 15 W Wilber-Lourmat lamp with peak intensity at 312 nm, and blue-white light (100 ¡xE m-2 was provided for photorepair.

[From Hessen 29.]

Days of exposure

Figure 2. Population development of melanized (filled symbols) and hyaline Daphnia under high light intensity (upper panel) medium light intensity (middle panel) or high light intensity plus Mylar sheet (lower panel). Five juvenile individuals were added in triplicate for each treatment, and population development followed for 12 days, covering one reproductive cycle. Light was provided with a 15 W Wilber-Lourmat lamp with peak intensity at 312 nm, and blue-white light (100 ¡xE m-2 was provided for photorepair.

[From Hessen 29.]

transparency and shallowness in these habitats, rendering the animals vulnerable to high levels of UVR. For Arctic areas there will also be a continuous diurnal UV-exposure during summer. Also, these habitats commonly have low (or no) prédation pressure from fish.

Hyaline

Melanie

280 300 320 340 360 Wavelength (nm)

Figure 3. Spectral transmittance of irradiation of Daphnia carapaces with different levels of melanization. Hyaline: dorsal part of hyaline carapace. Melanie: dorsal part of pigmented animal (Hessen et al. [114]). A hyaline individual of Daphnia is shown in the insert.

Hyaline

Melanie

280 300 320 340 360 Wavelength (nm)

Figure 3. Spectral transmittance of irradiation of Daphnia carapaces with different levels of melanization. Hyaline: dorsal part of hyaline carapace. Melanie: dorsal part of pigmented animal (Hessen et al. [114]). A hyaline individual of Daphnia is shown in the insert.

The role of melanin in other pelagic metazoans has been less well studied. Melanic zooplankton are generally rare both in freshwater and marine areas since this would dramatically increase the risk of predation, yet there are some surface dwelling species that may have a conspicuous dark coloration such as some species of Arctic winged snails (opistobranchs) with high levels of melanin (Hessen unpublished).

The role of melanins in fish may be more obscure. A key function is control of color pattern in the skin that may serve both as sexual signals and camouflage. In teleost fish, a melanin-concentration hormone (MCH) regulates adaptive color change, and the MCH activity may be related to photoperiod [31]. To what extent UVR plays a role in this regulation is not settled. There are probably several components in the outer layers offish skin that protect it against UV, and a direct relation between the amount of these compounds and UVR-induced erythema in fish has been revealed [32,33]. On the other hand, the direct role of melanin in UV-protection may be less important, although the role of melanins in this regard may be highly species-specific. One study comparing albino and pigmented fish (medeka, Oryzias latipes) revealed no difference in UV-B induced mortality, yet both morphs had similar amounts of colorless UVR absorbing compounds in their skin [33]. The presence of MAAs has been verified in this species [25]. This suggests that, at least for this species, melanin does not play a major role in UVR protection. Although the major photoprotective role of melanins is simple sunscreening, melanin precursors may also serve as antioxidants, and in fact the activity of key anti-oxidants like super-oxide dismutase has been linked to the presence of melanin in fish skin [34].

In amphibians, the dark coloration in eggs and tadpoles is chiefly accredited to the presence of melanin, and the presence of melanin together with a suite of other UV-B absorbing compounds renders at least the tadpoles of the common frog, Rana temporaria, rather insensitive to UVR [35]. Nevertheless, UVR is suggested as one (among several) key factors explaining the dramatic decline of amphibians worldwide [36]. Although melanin without doubt plays a key role in some mammals (such as humans), the corresponding role of skin and hair melanins for aquatic mammals is less obvious. Presumably these animals would be more at risk of either eye damage such as cataract or indirect (food web) effects.

12.2.2.3 Carotenoids

Carotenoids may serve a dual role in photoprotection in organisms, serving as either anti-oxidant or radical scavenger, and offering protection from direct photon flux by quenching. The conspicuous red coloration of alpine and highly light-exposed plankton organisms was recognized in early work such as that of Merker [11] and Brehm [37]. This coloration is caused by high levels of tissue carotenoids, and the role of carotenoids in photoprotection in clear low-land localities has been also convincingly demonstrated [38,39]; yet carotenoids, apparently, are aimed more towards the UV-A and blue. Carotenoids are present in all groups of crustaceans; however, at highly variable levels and carotenoid composition. The major groups of carotenoids identified in calanoid copepods are astaxantin, cryptoxanthin, echinenone and hydroxyechinone-like fractions, all probably derived through food from algal /î-carotene precursors [39]. Partali et al. [40] recorded a total of 11 different carotenoids in Daphnia magna, some in trace amounts only. They also demonstrated how the carotene profiles in Daphnia could vary with food source.

While the role of carotenoid photoprotection seems well justified in copepods, it is more obscure in the cladocera [16,41]. Sub-Arctic alpine copepods (Hetero-cope) were found to have ten times more carotenoids than sympatric populations of cladocerans, and even low-land transparent copepods have higher carotenoid levels than highly light-exposed Daphnia [41]. Carotenoids are also widespread in fish, notably anadromous salmonids, yet the role of carotenoids in photoprotection in these species is not settled.

12.2.2.4 Other UVR screening compounds

The above-mentioned major UV screening molecules or pigments are not an exhaustive list of potentially UV-blocking or UV-absorbing compounds. Most organic and inorganic structures in exuvia, skin or epidermis may serve such a role although not particularly evolved for this purpose. There might be, however, several unidentified substances aimed specifically at UV protection. For instance the egg shell or chorion of cod eggs do apparently offer some UV protection, since fewer cyclobutane pyrimidine dimers (a kind of DNA damage caused only by UV-B, Chapter 9) are found in eggs compared with recently hatched embryos [42]. This is apparently not a common feature of all species, however, since no corresponding difference between eggs and hatched embryos was detected in Northern anchovy [42,43].

12.2.2.5 UV, pigments, prédation and evolutionary trade-offs Pelagic metazoans commonly face multiple challenges with regard to photo-protective strategies. Since the adaptive role of pigments is to allow for a surface dwelling life, this is indeed in conflict with the risk of prédation, which normally is highest under high light conditions (surface layers). The synthesis of pigments is an energy demanding process. This is particularly well demonstrated for zooplankton with a melanized exuvia, where the melanin needs to be re-synthesized for each moult, and where growth rates are lower in pigmented animals as compared with their hyaline conspecifics [29]. Thus the melanized clones are assumed to be competitively inferior under moderate or low UV-stress. Moreover, these dark animals are more visible to visual predators, and when co-occurring with hyaline clones, as may occur in some alpine lakes, there is a strong selective preference among fish for the melanized individuals [44].

Similarly, Hansson [45] found that the level of carotenoid pigmentation in copepods was up to ten times higher in lakes without predatory fishes than where fishes are present. Moreover, animals from the same population exposed to either UVR or predators displayed a 10% difference in pigmentation after only four days, suggesting that pigmentation is an inducible trait. The latter is a particularly intriguing observation, since it demonstrates that adaptations to either UV or prédation in terms of pigmentation do not necessarily require evolutionary time scales. For melanized Daphnia, most clones may rapidly shut down their melanin synthesis in the absence of UV [19]. The extent to which the presence of fish or fish kairomones may induce shifts in pigmentation strategies (as commonly observed for morphological features) is not known. Non-mela-nized Daphnia are apparently not capable of switching into melanized under prevailing UVR, however [19], suggesting that this in a non-inducible property that probably depends on a slight genetic modification.

Also, zooplankton without a conspicuous pigmentation may show a depth distribution and adaptation to different UV regimes at different depths. Comparison of a large number of epipelagic (15 species) and mesopelagic (19 species) organisms revealed pronounced differences in their tissue transparency to different wavelengths, yet all species were considered transparent [46]. In general, the tissues from epipelagic species had lower UV transparency as compared with mesopelagic species, demonstrating that more subtle regulation of intracellular UV-exposure may occur without increasing the risk of prédation.

12.2.3 Anti-oxidants

A third defense mechanism are intracellular processes such as repair of DNA damage and the production of quenching agents and anti-oxidant enzymes that neutralize reactive oxygen species (ROS) produced by UV [47]. Examples include carotenoids (CAR), involved in quenching of activated photosensitizers and singlet oxygen, superoxide dismutase (SOD), which eliminates the superoxide radical, catalase (CAT), which detoxifies hydrogen peroxide to oxygen and water, and glutathione transferase (GST), which neutralizes peroxidized macro-

molecules and detoxifies breakdown products after lipid peroxidations [48]. Antioxidants are linked with resistance against UVR in plants, microorganisms and mammalian cells and skin tissue [47,49,50], but little is known about antioxidant protection in pelagic metazoans.

A survey of bulk carotenoids, as well as the enzymes SOD, CAT and GST, in populations of various Daphnia species, ranging from coastal rock-pools in Southern Norway to high Arctic populations [51] did not reveal any strong pattern in enzyme activities that could be attributed to species affinities, habitat, or tentative UV-exposure of pigmentation (Figure 4). Since a far higher UV-susceptibility was revealed among hyaline clones relative to melanic clones [19], an a priori assumption would be that the lack of carapace melanization should be compensated for by other means of photoprotection like increased activity of essential anti-oxidants, but in general this was not the case. Studies of natural alpine and Arctic populations of Daphnia did, however, reveal diurnal patterns in levels of anti-oxidants that could be related to solar exposure (Hessen and Borgeraas unpublished). Tissue concentrations of GST and SOD, less so for CAT, displayed a pattern with low levels in the afternoon, following after UV-exposure, and a gradual build-up during the night. Correspondingly a number of anti-oxidants have also been screened in fish skin [34], yet their bearing on UVR susceptibility is not settled.

The above tested anti-oxidants do not provide an exhaustive list of potential scavengers of oxidants in aquatic animals, however, and there may be other UV-protective enzymes that play a key role.

12.2.4 Recovery and DNA repair

A last major defense would be the various means of enzymatic photorepair that is probably a common property of all organisms. These general effects on DNA, proteins and membranes and the corresponding cellular repair mechanisms will not be reiterated here. It has long been known that longer wavelengths, notably in the UV-A and blue, can counteract UVR damage by repair of DNA. This effect is, among others, one reason why laboratory experiments tend to overestimate UVR damage if sufficient spectral quality or quantity is accounted for. There are a number of studies that point to the role of photorepair or photoreac-tivation in marine animals from copepods [52-54], shrimps and eupausides [55,56] to fish [56-58]. Also, for eggs of cod, a pronounced photorecovery was found [2]. There are comparatively fewer studies for fresh waters but, for Daphnia, Siebeck and Böhm [59] not only demonstrated a pronounced species-specific difference in UV-susceptibility, they also provided data on spectral effects and the effects of recovery radiation. While spectral sensitivity for Daphnia followed a general CIE action spectrum, peak recovery radiation was in the blue region (420-440 nm). They found that recovery radiation (light repair) strongly increased when recovery radiation was provided after UV-exposure. This positive effect was further increased when recovery radiation was provided also under UV-exposure. The effect of recovery radiation rapidly decreased with

D. magna

S 200

D pulex/ middendorffiana

D. longispina D. magna

D. magna b

Lab, clonc Humic

Rock-pool Alpine Arctic

QSOD

D longispina b

D. magna

D. pulex/ middendorffiana

D. longispina

Lab. clonc Humic

Rock-pool Alpine

Arctic (12)

B)GST

D. magna D. longispina

D. longispina

D. pulex/ middendorffiana

Lab. clonc

D)CAR

Humic (16)

Alpine Arctic

T3 J

D. longispina b

D. magna

D. longispina c D. pulex/

middendorffiana

Lab. clonc

Rock-pool Alpine

Arctic (13)

Figure 4. Specific activities (U mg-1 protein) of (A) catalase, (B) glutathione transferase, (C) superoxide dismutase and (D) bulk carotenoids in Daphnia. from five different areas of Norway. The lab clone had not received UVR for several generations, Kj. Putten is a humic lake with high levels of DOC and low UVR; Hvaler represents shallow and highly UVR exposed rock-pool populations, Finse represents oligotrophic alpine ponds, and Svalbard represents high Arctic ponds at 79 °N. [From Borgeraas and Hessen, 51, with permission of Kluwer Publishers.]

increasing post-exposure darkness. For freshwater copepods, a pronounced species-specific variability in photorecovery was found [60],

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