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Thread: Slime Coat Musings

  1. #1
    Daihonmei MikeM's Avatar
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    Slime Coat Musings

    There have been a number of different postings around the 'net recently that involved folks doing all sorts of unnecessary things that could harm the slime coat of their koi. So, I decided it was a good time to chatter a bit about the subject.

    One of the slime coat's functions is reduce drag by coating the irregular surface of the scales. This allows fish to move easily through water, which is very important for the fish, but not nearly as important as the main function.... protecting the fish from fungi, bacteria, viruses and parasites. The slime coat traps these pathogens and naturally sloughs them off as water moves around the fish. Within fish slime are a host of immune agents, so if a pathogen does not slough off with the slime, there may well be an antibody that kills it. The slime coat also plays a role in mediating the osmoregulatory system. A slime coating also soothes a fish’s open wounds and promotes healing through diverse mechanisms. There is a substantial amount of medical research occurring regarding isolated components of fish slime in attempts to develop new medications to promote more rapid healing and substitute for current antibiotics. There is also gas exchange occurring on the surface of a fish’s skin, not just in the gills. The slime coat mediates this gas exchange (but I do not have any understanding of how it works).

    For koikeepers, the slime coat presents our best defense against unwanted external parasites, but often captive fish don’t have a slime coat nearly as strong as their wild counterparts. Diet and water quality are the main tools for maintaining a healthy slime coat …and increasing a fish’s overall immune system. Feed a pellet designed for koi and keep water quality high. The slime coat will take care of itself, as long as the koikeeper avoids unsafe surfaces in the pond and does not go doing things that harm the slime coat. Folks who keep themselves busy playing around with the water and the fish are not doing their koi any favors. Netting a fish can break open the slime coat, giving parasites and bacteria entry to the fish’s tissue. Poor water quality can decrease fish’s natural immune system function, resulting in an incomplete slime coat. Radical pH fluctuations can damage slime coat production. Chemical additives and medications can both increase slime coat production temporarily, and damage the slime coat due to the harsh oxidative effects.

    Perhaps the most serious issues concern new acquisitions. Everything about capturing, packing, shipping and unpacking koi damages their natural slime coat. The slime coat is broken by netting. Fish are then placed in close quarters, rubbing the slime coat. During shipping, water conditions change quickly, causing all sorts of immune reactions within the fish and rough handling by cargo workers is common. Restoration of the slime coat should be one of the first goals when a new koi is received. When quarantining a new koi, folks often ignore the importance of allowing the slime coat to be restored as an initial step. Some koikeepers automatically engage in a regimen of medicating new koi placed in quarantine. However, absent some emergency situation, it is generally best to give a new acquisition several days to acclimate in a calm environment before imposing some regimen of chemical treatments. Personally, I seldom have new koi bring parasites into the pond, because I limit acquisitions to a few dealers who take great care to be sure the koi they are selling are ‘clean’. The greater risk for my new acquisition is that the resident varmints in my pond will attack her. A healthy, natural slime coat is the best defense (and offense) against those pathogens. The medications generally used against parasites can ‘burn away’ some of the slime coat. Of course, there are parasites that evolved alongside the fish and can penetrate the slime coat to burrow into fish tissue.When parasites are a problem, those medications are necessary. But, when used unnecessarily, the reduction of the slime coat is exactly what a new koi does not need. Koi treated in quarantine should be given several days post-treatment to restore the slime coat before being exposed to all the critters residing in the main pond.

    If someone wants to delve into the science of the slime coat, do an internet search for "Carp Mucus And Its Role In Mucosal Defense", a 2012 thesis by Maria C. van der Marel. She publishes the results of a series of studies using carp. (The 100+ pages get pretty heavy on the jargon.) More useful IMO is her bibliography of hundreds of articles concerning the broader subject. Those cover a huge range of research into the not so simple subject of fish slime.
    Last edited by MikeM; 05-23-2017 at 09:28 AM.

  2. #2
    Jumbo sacicu's Avatar
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    I have been a proponent of artificial slime coat thru the application of an aloe vera solution. In the national koi show this year where i hold the position of water quality chairman for the past 5 years I applied liberally Stresscoat in almost all the showvat for every water change.

    In a koishow environment, the koi is subjected to a lot of stressors. Netting, handling, bagging, releasing, adjusting to a new water and small vat environment and being subjected to lots of people viewing can be somewhat be a traumatic experience for koi. In such situations, the slime coat is easily damaged which allows pathogen and cross contamination to happen quickly. This is why many show koi that return to owners pond will or may experience some health problems.

    My intention in applying a synthetic slime coat is to reduce stress experience by koi by providing an artificial slime coat during the long period stay in the showvat and thereby lessen koi health problems during and after the show. I know for some show purist, water in the show vat needs to be kept free from chemicals unless there is evident problems arising. However I disagree with that idea because a plastic trophy will never be more important than the health and safety of koi participating in a koishow. The risk of applying a synthetic slime coat is very minimal to safeguard the koi from stress, and possible contamination of bacteria and parasites.

    Everyear I try to study the after health incidence koi show report and study what more can be done to further lessen problems.

    By doing stricter disinfection of nets and vats, better understanding and monitoring of water quality and koi condition, better advise to participants to fast their koi, preventing vat overloading and employing water conditioning chemicals that help restore the slime coat of the koi, I believe the risk involved in joining the annual koi show would be less and therefore more rewarding instead of troublesome.
    coolwon likes this.

  3. #3
    Daihonmei MikeM's Avatar
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    I have never understood how adding an Aloe vera concoction to pond/aquarium water would restore the slime coat. But, I know these additives work in aquaculture to reduce deaths when fish are handled or transported. When I was reading some of the studies there were references to the polymeric proteins in these additives being attracted to exposed tissue and thereby artificially replacing the natural slime coat, but I have not come across literature explaining just how this works. That it seems to work is generally accepted in both industry and science labs. However, the mechanisms involved and the relative effectiveness of the various types of polymer additives has not been studied much. There really is no way today to confirm the claims of the many brand product manufacturers, but numerous aquaculture businesses have experienced the benefits enough to incur the expense. One article (with what I consider a rather misleading title) delves into the need for more studies. Here it is without charts and notes... interesting for the broad review of the literature as of 2010:

    A Review of Polymer-based Water Conditioners for Reduction
    of Handling-related Injury
    Ryan A. Harnish • Alison H. Colotelo •
    Richard S. Brown
    Received: 27 April 2010 / Accepted: 26 October 2010 / Published online: 31 December 2010
    _ Springer Science+Business Media B.V. 2010

    Abstract
    Fish are coated with an external layer of
    protective mucus. This layer serves as the primary
    barrier against infection or injury, reduces friction,
    and plays a role in ionic and osmotic regulation.
    However, the mucus layer is easily disturbed when
    fish are netted, handled, transported, stressed, or
    subjected to adverse water conditions. Water additives
    containing polyvinylpyrrolidone (PVP) or
    proprietary polymers have been used to prevent the
    deleterious effects of mucus layer disturbances in the
    commercial tropical fish industry, aquaculture, and
    for other fisheries management purposes. This paper
    reviews research on the effectiveness of water
    conditioners, and examines the contents and uses of
    a wide variety of commercially available water
    conditioners. Water conditioners containing polymers
    may reduce external damage to fish held in containers
    during scientific experimentation, including surgical
    implantation of electronic tags. However, there is a
    need to empirically test the effectiveness of water
    conditioners at preventing damage to and promoting
    healing of the mucus layer. A research agenda is
    provided to advance the science related to the use of
    water conditions to improve the condition of fish
    during handling and tagging.

    Introduction
    Fish are commonly handled by researchers and
    aquaculturists for a variety of reasons (e.g., external
    examination, recording of length and weight, transport,
    surgical implantation of transmitters). Handled
    fish are at risk of acquiring injuries to their epidermal
    layer from contact with capture gear (i.e., nets, hooks)
    and sampling equipment (i.e., measuring boards,
    surgery table). These injuries can be latent (e.g.,
    mucus loss) or visually apparent (e.g., scale loss) and
    can result in sub-lethal and lethal consequences for the
    fish. Some researchers utilize commercially available
    water conditioners (e.g., PolyAqua, Stress Coat) to aid
    in the protection of the epidermal layer of fish, and to
    promote healing if injury does occur. However, the
    effectiveness of these products and any negative
    effects they may have on fish have not been
    thoroughly investigated. The goal of this review is
    to examine the contents, uses, and effectiveness of
    polymer-based water conditioners and provide recommendations
    for future research.
    The epidermal layer of fishes is abundant with
    goblet, Malpighian, and other secretory cells that
    serve as the primary biological interface between
    teleost fish and their environment by producing a
    layer of protective mucus (Shephard 1994; Ottesen
    and Olafsen 1997). Mucus consists mainly of water,
    along with high molecular weight, gel-forming macromolecules
    that are predominated by glycoproteins
    known as mucins (Shephard 1994). The mucus layer
    serves as the primary barrier against infection (Pickering
    1974; Pickering and Macey 1977; Ingram 1980;
    Alexander and Ingram 1992; Nagashima et al. 2001),
    protects against injury (Pickering and Richards
    1980), reduces friction (Rosen and Cornford 1971;
    Pickering 1974), and plays a role in ionic and osmotic
    regulation (Handy et al. 1989; Shephard 1994).
    The mucus layer is easily disturbed when fish are
    netted, handled, transported, stressed, or subjected to
    adverse water conditions such as high particulates
    (Roberts and Bullock 1980; Buermann et al. 1997)
    and contaminants (Muniz and Leivestad 1980; Eddy
    and Fraser 1982). Disturbances to the mucus layer
    may alter the ionic and osmoregulatory abilities of fish
    while also making them vulnerable to scale loss, skin
    abrasions, and a variety of bacterial, fungal, and
    parasitic diseases (Wedemeyer 1996). These disturbances
    can be particularly harmful to juvenile salmonids
    undergoing the parr–smolt transformation
    because they can alter the developing hypoosmoregulatory
    ability that pre-adapts the fish to life in
    seawater (Wedemeyer 1996). The smoltification process
    requires large amounts of energy reserves and is
    stressful to fish (Specker 1982; Virtanen 1987), as
    indicated by outbreaks of disease upon seawater entry
    (Stoskopf 1993). For example, Bouck and Smith
    (1979) reported coho salmon smolts Oncorhynchus
    kisutch experienced 75% mortality when exposed to
    salt water following experimentally induced mucus
    and scale loss, compared to 0% mortality for smolts
    exposed to freshwater following injury. Although the
    mucus layer can be regenerated relatively quickly, the
    compensatory energy required to do so may compromise
    the survival of fish as they enter seawater
    (Wedemeyer 1996).
    For these reasons, it is desirable to prevent or
    minimize the effects of any disturbance to the mucus
    layer that may occur during the netting, handling
    (including surgical tag implantation), and transporting
    of fish. The tropical fish industry has successfully
    used water additives containing polyvinylpyrrolidone
    (PVP) or proprietary polymers to prevent the deleterious
    effects of mucus layer disturbances that can
    occur during the transportation of aquarium fish
    (Wedemeyer 1996). When abrasions and scale loss
    do occur, these polymers temporarily bond to
    proteins on the exposed tissue, forming a protective
    coating that is displaced as healing proceeds and the
    mucus layer is regenerated (Wedemeyer 1996). These
    polymer formulations are being used increasingly by
    the aquaculture industry and state and federal
    conservation hatcheries as a water additive for
    transporting juvenile salmonids and other non-food
    fish (Wedemeyer 1996; Harmon 2009).
    Research related to water conditioners
    Relatively few studies have been published in peer-reviewed
    fisheries journals regarding the usefulness
    of these polymer formulations (summarized in
    Table 1). Some of the studies conducted have shown
    potential benefits of water additives containing synthetic
    polymers for minimizing handling and transport
    mortality. Much of this research was conducted
    using black bass Micropterus species that were held
    in live wells containing water conditioners or other
    additives. For example, survival of angled largemouth
    bass Micropterus salmoides held for 3–9 h in
    water that contained an unspecified commercially
    available water conditioner was significantly higher
    than survival of angled fish held in unconditioned
    water (Plumb et al. 1988). However, there was no
    significant difference in survival between angled fish
    that were released immediately and those that were
    held in the conditioned water. Gilliland (2003)
    explored the effectiveness of different live-well
    operating procedures in reducing mortality of black
    bass and found that the live-well additives significantly
    improved the survival of tournament catches in
    Oklahoma.
    Several studies have indicated that water conditioners
    were used effectively in aquaculture facilities.
    For example, mortality was reduced by 23–43% when
    Polyaqua, a commercially available water conditioner,
    was added to holding tanks while adult steelhead
    Oncorhynchus mykiss were examined repeatedly for
    spawning ripeness over a 3-month period (Wedemeyer
    1996). A concentration of 100 ppm Polyaqua used
    during transport significantly reduced the prespawning
    mortality of adult fall Chinook salmon Oncorhynchus
    tshawytscha and steelhead caused by the freshwater
    fungus Saprolegnia (Wedemeyer 1996). Addition of
    NovAqua, another commercially available conditioner,
    to transport water increased survival of delta
    smelt Hypomesus transpacificus captured using a seine
    net by about 27% over that of the control (Swanson
    et al. 1996). The improved survival was attributed to
    the polymers, which may have reduced physiological
    stress responses, such as osmotic imbalances (Swanson
    et al. 1996).
    Although most studies have found water conditioners
    to be effective at reducing stress and increasing
    survival of handled or transported fish, Cooke
    et al. (2002) found increased cardiac recovery times
    for smallmouth bass Micropterus dolomieu held in
    live wells conditioned with 0.5% Catch-and-Release
    Formula that was gradually flushed with lake water
    compared to fish held in live wells that were flushed
    with only lake water, suggesting that conditioners
    may be detrimental to fish recovery. However, this
    study was limited by sample size (n = 3). Prolonged
    recovery following stress (e.g., handling and surgery)
    could potentially increase the likelihood of mortality
    or behavioral alterations. One of the challenges
    associated with using water conditioners is that many
    are proprietary with claims of effectiveness that often
    are not validated (Cooke et al. 2002).

    Use of water conditioners
    Despite the lack of research related to the use of
    water conditioners, they appear throughout the literature
    in studies involving the transport and holding of
    fish. Water conditioners are generally not used for the
    transport of food-fish, since they are not approved by
    the FDA. However, water conditioners, such as
    Propolyaqua, Polyaqua, Novaqua, and Start Right,
    have been used in the transportation of a variety of
    species including rainbow trout, channel catfish,
    largemouth bass, walleye, bluegill, brown trout and
    splittail Pogonichthys macrolepidotus (Taylor and
    Kynard 1985; Carmichael and Tomasso 1988;
    Swanson et al. 1996; Helfrich et al. 2001; Danley
    et al. 2002; Weber et al. 2002; Beeman and Maule
    2006; Floyd et al. 2007; Kline and Bonar 2009).
    Commercially available water conditioners are also
    often added to live wells and/or recovery tanks during
    and after angling tournaments, such as those for
    largemouth bass (Plumb et al. 1988; Meals and
    Miranda 1994), as well as in recovery tanks after
    surgical implantation of transmitters (Stress Coat,
    Godinho and Kynard 1993; Stress Coat, Richardson-
    Heft et al. 2000; Polyaqua, Gaines and Martin 2004).
    Polymer-based water conditioners have also been
    used to soak or coat the surgical pad (Stress Coat,
    Hockersmith et al. 2000) or work surface (Stress
    Coat, Peterson and Barfoot 2003; Polyaqua, Mueller
    et al. 2006). In addition, water conditioners such as
    Polyaqua and Vidalife have been added to anesthetic
    solutions used in tagging procedures (Ficke and
    Myrick 2009; Brown et al. 2010, Seitz et al. 2010).

    Contents of water conditioners
    Many water conditioners are commercially available,
    but some are formulated only to dechlorinate water
    and/or bind heavy metals. These conditioners use
    dechlorinating agents such as sodium thiosulfate and
    ascorbic acid, chelating (metal binding) agents such as
    ethylenedianinetetra acetic acid (EDTA), and buffering
    agents such as tris (hydroxymethyl) aminomethane
    that restore acid–base balance. Water additives
    that form a protective ‘‘slime layer’’ will contain a
    polymer (often PVP or carboxymethyl cellulose
    [CMC]) or colloid (Table 2). Some additives contain
    aloe extract from leaves of the Aloe vera plant.
    Manufacturers of these products claim that the Aloe
    vera extract promotes healing of damaged tissue. One
    potential drawback to water additives that contain
    Aloe vera extract or CMC is the addition of organic
    waste load that can reduce the water quality and
    oxygen levels in a closed system. This may not be
    an issue, depending on the density of fish, length of
    time fish are held, and oxygen content of the water.
    However, the effects of these substances on gill tissue
    are unclear. Taiwo et al. (2005) tested the survival and
    behavior of tilapia (Oreochromis niloticus) exposed to
    different concentrations of aqueous extract of A. vera
    for up to 96 h. One hundred percent of tilapia exposed
    to 50 ppm A. vera died within the duration of the
    experiment. Fish used in this experiment exhibited
    severe depigmentation and destruction of organs
    (including gills). The evidence of the toxic effects of
    A. vera on fish solidifies the need to empirically test
    water conditioners, and their chemical components,
    for potential negative effects on fish.

    Research needs
    This review highlights the need for empirical studies to
    examine the effects of water conditioners on fish.
    Comparative studies should be conducted to investigate
    the differences in currently available and
    employed water conditioners such as those highlighted
    in this review (e.g., Polyaqua, Stress Coat). These
    comparative studies should explore the effectiveness
    of multiple water conditioners for reducing mucus loss
    and infection for fish, as well as examining the
    sublethal and lethal effects these water conditioners
    may have on fish.
    Although polymer-based water conditioners are
    commonly used during fish surgery, no known studies
    have been conducted to examine their effectiveness in
    this function. The application of a polymer-based
    water conditioner to the transport water, anesthetic
    solution, and surgery table may prevent the harmful
    effects of mucus layer disturbances that can occur
    during the tagging process. However, research is
    needed to compare the effectiveness of multiple water
    conditioners used in various applications during the
    surgery procedure.
    Because disturbance to the mucus layer of fish is
    often latent, the detection and quantitative measurement
    of mucus loss can be difficult. However, recent
    studies have found fluorescein, a non-toxic dye, to be
    an effective means for identifying latent epithelial
    damage on fish (Noga and Udomkusronsi 2002;
    Davis and Ottmar 2006; Dauble et al. 2007; Colotelo
    et al. 2009). Fluorescein could be used to quantify the
    initial amount of experimentally-inflicted injury on
    fish and to document the healing process of fish
    treated with different water conditioners. It could also
    be used to determine the effectiveness of polymerbased
    water conditioners to prevent damage to the
    mucus layer.

    Conclusion
    Water conditioners containing polymers may reduce
    external damage to fish held in containers during
    scientific experimentation, including surgical implantation
    of transmitters. However, there is a need to
    empirically test the effectiveness of water conditioners
    at preventing damage to and promoting healing
    of the mucus layer. It is unadvisable to use water
    additives that contain Aloe vera extract or CMC in
    closed holding systems due to the potential for these
    additional organic wastes to reduce water quality and
    oxygen levels. However, these organic materials
    likely do not have a negative effect on water quality
    or oxygen levels in open, flow-through holding or
    transport systems. Because no studies have directly
    compared multiple water conditioners, additional
    research is needed to determine which additive best
    protects the mucus layer of fish under different
    conditions. Additionally, to understand all the potential
    applications and the extent of polymer-based
    water conditioner use in fisheries applications, the
    authors encourage others to report their use of water
    conditioners, including the specific brand and concentration
    used.

  4. #4
    Jumbo sacicu's Avatar
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    When you are tasked to take care koi in a show vats valued from 1000 to as high as 50000 dollars in 120vats with no filtration whatsoever for 2 to 3 days, there is tremendous responsibility to keep these koi safe. Relying only on just water changes and just test kits to keep ammonia from going up is simply not enough. In a another recent koishow, a 97cm kohaku perished when the koi kept on getting agitated and producing excess mucus slime after every several water changes performed in a span of a few hours. Eventually the koi died. I will not comment on the reason why it died but I would have approached the situation differently.

    Personally the way I see it too much water change in a short period of time when the slime coat is compromise is not good unless of course one wants to get rid of poison contaminant. Ammonia blockers and artificial slime coat have useful purpose that just cannot be dismissed.

  5. #5
    Daihonmei MikeM's Avatar
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    I agree with you, Sacicu. And, I am surprised to learn there are shows relying solely on water changes to maintain water quality in a show setting. Anyone who reads my postings know how much I favor lots of water changes... for ponds. When it comes to caring for koi in a show tank, you are dealing with an entirely different environment. In most U.S. shows reliance is placed on ammonia binders, with water testing occurring multiple times each day on a regular schedule to assure there is no harmful ammonia present. Some use ammonia binders with artificial slime coat. Some shows have worked with zeolite filtration, but the cost of individual systems for each tank is cost prohibitive for most. Water changes in a show environment seem risky to me. It will not be effective unless a huge percentage is changed out, and it likely would be required multiple times per day with large koi or 'heavily' stocked tanks.... and it does not take many small koi to be heavily stocked in a few hundred gallons of unfiltered water! The risks of dramatic pH shifts, temperature differentials, etc., etc. are high and the whole process would add great stress to a fish already stressed by being netted, transported and placed in a strange environment with people peering at them from short distances for hours on end. The fish already suffer from adrenaline-rush flight response.

  6. #6
    Daihonmei MikeM's Avatar
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    Mycosporine-like Amino Acids

    In the course of delving into literature concerning the slime coat, I have come across studies concerning the ability of the slime coat of some fish to protect against UV radiation. Most fish studied have a UV-protective slime coat to some degree, but not all. I have not found a study focused on the slime coat of carp. So, whether the information gleaned relates to our koi is not determined, but until it is found that carp do not have a UV-protective slime coat, it seems reasonable to assume that they do not differ from the bulk of fish species. Thus, one of the health benefits provided by the slime coat is UV protection, resulting in reduction in skin damage.

    The UV protection comes from carotenoids and especially certain amino acids called mycosporine-like amino acids (MAAs). The level of MAAs in the slime coat is directly related to the diet of the fish. High concentrations of MAAs are found in various algae, diatoms and cyanobacteria. (I suspect the MAAs found in diatoms come from symbiotic cyanobacteria living in the diatoms.) MAA rich organisms include copepods, shrimp, etc. that consume MAA rich algae, etc. Fish with a high MAA diet have been shown to have greater resistance to UV than fish on a diet lacking in MAAs.

    These findings have me wondering about Hikkui and the diets we feed our koi. It is known from experience that there is some genetic base to certain individual koi being prone to Hikkui. Perhaps this relates to an inability to utilize MAAs as efficiently as other koi, resulting in lowered UV resistance. Could such koi be benefitted by a diet rich in MAAs? Many of the greenwater algae contain high levels of MAAs. Could the oft-observed temporary cure of placing Hikkui-infected fish in greenwater be caused not only by the reduction of UV exposure, but also increased MAA consumption? These questions (and more in a similar vein) are beyond my ability to study, but it does cause me to think about the level of MAAs in the koi pellets we feed. I am not aware of any pellet being analyzed for MAA content. Fishmeal-based pellets could vary in MAA content from batch to batch according to whatever the ground-up fish had been eating. Since MMAs are generally higher in algae, cyanobacteria and invertebrates that consume algae and cyanobacteria, it would seem that foods made with spirulina, krill and shrimp would be higher in MMAs than foods that are just fishmeal and grains. However, this is just speculation given the absence of MMA analyses.

    A recent (2016) study gives a general overview of UV resistance and MMAs in fish. The introduction and discussion portions are reproduced in the following post....
    Last edited by MikeM; 06-06-2017 at 10:25 AM. Reason: typo

  7. #7
    Daihonmei MikeM's Avatar
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    For those willing to take on fairly technical writing, the following is taken from a 2016 study. Only the introduction and discussion are reproduced.... which are lengthy enough! I have eliminated footnote references, which are incredibly numerous, because they interfere with readability in this format. For more, do a search using the full title of the article.

    Ultraviolet Absorbing Compounds Provide a Rapid Response Mechanism for UV
    Protection in Some Reef Fish [Journal of Photochemistry and Photobiology]
    Braun, C., Reef, R., Siebeck, U.E.
    School of Biomedical Sciences, The University of Queensland, Brisbane, Australia.
    Department of Geography, University of Cambridge, United Kingdom.

    Introduction

    Ultraviolet radiation (UVR, 280 - 400 nm), specifically short-wavelength UVB radiation (280
    - 315 nm) causes damage to DNA. The formation of cyclobutane pyrimidine dimers (CPDs)
    between adjacent pyrimidine bases is one of the key consequences of UVB exposure
    leading to structural changes in the DNA double helix, which can inhibit polymerases thus
    arresting replication and transcription of the DNA sequence. If left unrepaired, UV induced
    DNA damage can lead to mutations and apoptosis of affected cells. In fish,
    the effects of UVR exposure include behavioural changes (e.g.in trout and salmon),
    damage to tissues of the skin (Japanese medaka ) and the brain (Northern Pike ), DNA
    damage MAAs [mycosporine-like amino acids] can be found in fish eggs, larvae and in the ocular media as well
    as the external mucus of reef fish and can lead to increased mortality (Zebrafish,
    Atlantic cod and Sea Bream).

    In the tropics, where levels of UVR are among the highest on Earth, the clear and
    shallow waters around coral reefs allow UVR to penetrate farther than in other aquatic
    ecosystems leading to a high risk of UVB induced DNA damage. Due to changes in
    ozone levels, aerosols, greenhouse gases and cloud cover as well as loss of coral
    complexity due to increased cyclone intensity and severe coral bleaching, UVR
    around coral reefs is likely to continue to increase. These changes can be mediated wither [whether]
    directly by increases in irradiance, or indirectly by the increases in water clarity and
    loss of shelter.

    Protection from harmful UVR in marine organisms can arise from physical barriers (e.g.
    shells and scales) as well as from UV-absorbing compounds (UVACs) like carotenoids
    and Mycosporine-like amino acids (MAAs). Over twenty MAAs with absorbance
    maxima between 309 and 360 nm have been found in the tissues of hundreds of marine
    species from all trophic levels and all latitudes, and together with Gadusol (absorbance
    maximum ~ 290 nm) cover the UVB and UVA spectrum. This variety of MAA
    compounds is synthesized by microbes, fungi and plants via the shikimate pathway
    and alternatively the pentose phosphate pathway. Although some of the genes from
    the shikimate pathway have been found in the sea anemone Nematostella vectensis and corals,
    MAAs cannot be synthesized by animals and are likely of dietary origin. In reef fish, over
    100 species (of 137 studied) show UV absorbing mucus, and the
    tissues where MAAs can be found are as varied as the number of compounds. MAAs
    have been detected in fish eggs, larvae and in the ocular media as well as the
    external mucus of reef fish, all tissues which are vulnerable and exposed to UVR.
    MAAs as part of all UVACs in fish mucus are widely recognized to act as sunscreens due to
    their absorbance properties, the tissues in which they are found, and due to
    their ability to prevent sunburn when topically applied to the skin of mice. MAAs have
    been shown to protect against cleavage delay in sea urchins and have recently been
    linked to reduced DNA damage in an intertidal gastropod. In corals and other marine
    organisms, MAA concentrations in exposed tissues are linked environmental levels of UVR,
    as reviewd by Shick and Dunlap. There is circumstantial evidence that MAAs may also
    have a protective function in reef fish. The MAA concentration in the external mucus of
    reef fish correlates with the levels of UVR in their habitat. In captivity, the UVR
    absorbance of mucus of Hawaiian wrasse that were provided with an MAA-rich diet
    decreased under conditions that lacked UVR, suggesting that there is an energetic cost to the
    maintenance of MAA protection in the external mucus. In the presence of UVR and
    under the same dietary conditions, MAA levels in mucus remained at pre-capture levels. The
    MAA profiles detected using laboratory methods (HPLC) and the absorbance of whole mucus
    samples measured in the field both vary between species and geographical locations.
    Mucus absorption has been established as a proxy for MAA concentration in the external
    mucus of reef fish, and can be easily quantified in the field by measuring UV absorbance
    in mucus samples.

    Here, we address the sunscreen hypothesis, specifically that a higher level of UVACs lead to
    reduced UV- induced DNA damage. Therefore, fish with different known mucus absorbances
    were exposed to a high pulse of UVB radiation in order to induce UV-specific DNA damage
    in the skin. If UVACs, of which MAAs are an integral part, indeed acted as sunscreens, it is
    expected to find higher DNA damage (CPDs) in fish that have lower levels of UVACs in their
    mucus. Consequently, we tested for a sunscreen function of UVACs shortly after capture,
    assuming unchanged mucus absorbance, and after a period of captivity, which is shown to
    reduce mucus absorbance.

    ****

    Discussion

    The UV-absorbing function of reef fish mucus has been relatively well studied, however
    direct evidence for a protective, sunscreen function that actually reduces or
    mitigates the negative effects of UVR exposure in fish, specifically DNA damage, is missing
    from the literature. The results show that UVACs, of which MAAs are a crucial component,
    do indeed act as a first line of defence against UV- induced DNA damage in two species of
    reef fish, Pomacentrus amboinensis and Thalassoma lunare. When fish were exposed to a
    high dose of UVB radiation, the level of DNA damage in the skin of the fish was negatively
    correlated with the integrated mucus absorbance, which is an accepted proxy for the amount
    of MAAs present in the external mucus. Fish exposed to the “UVB –“ treatments had
    variable levels of UVACs levels but overall low DNA damage. Additionally, it was
    discovered that in P. amboinensis, but not T. lunare, mucus absorbance was significantly
    higher in UVB exposed fish compared to control groups, therefore potentially increasing the
    amount of UVAC and MAA protection in their mucus. The changes in mucus absorbance
    observed in this experiment were species-specific, and restricted to fish that were exposed to
    increased UVB light.

    Similar protective functions of natural sunscreens have previously been shown only in
    invertebrates. Adams showed that in larvae of the sea urchin Strongylocentrotus
    droebachiensis, lower MAA levels lead to longer delays in cleavage induced by acute UV
    exposure. Carefoot observed reduced hatching in UV exposed Aplysia eggs, and higher
    MAA levels in spawn from UV exposed adults, however a definite protective function of
    MAAs could not be confirmed. Although we did not directly measure MAA content in
    mucus, our observations of mucus absorbance in relation to DNA damage strongly support a
    protective function of UVACs, and since MAAs are an integral part of fish mucus, also the
    hypothesis that MAAs serve as natural sunscreens.

    The experiments were designed to manipulate mucus absorbance levels found in the external
    mucus of fish to achieve high variability in UVACs levels prior to exposure to UV radiation.
    In particular, to lower the mucus absorbance of one group of fish relative to that of another
    group through the exclusion of UVR light while keeping all other factors constant between
    the groups, including the MAA-rich diet. After ten days, lower mucus absorbance was indeed
    achieved in T. lunare that were held under the exclusion of UVR, which was in agreement
    with previous studies [43, 47] and the suggestion that UVR exposure is necessary for MAAs
    to be sequestered into the external mucus layer. However, no reduction in mucus
    absorbance was observed in P. amboinensis over time, neither in fish that were held in
    conditions lacking UV, nor in fish exposed to natural sunlight for the ten day holding period.
    In the latter group, the increase in mucus absorbance due to the 1 hr UVB pulse possibly
    could have masked a decrease of mucus absorbance caused by the holding conditions. This is
    unlikely however, as the mucus absorbance of fish from the control group held in captivity for
    ten days was no different from that of the control group which was measured within 24 hrs of
    capture. A previous study showed that a reduction of MAAs can be induced in this species by
    using a MAA- free diet. It seems that this species, which has naturally low MAA levels,
    maintains a minimum level of UV- protection in the presence of MAA-rich food, irrespective
    of ambient UVR levels.

    The increase of mucus absorbance in P. amboinensis can be attributed to exposure to the
    UVR pulse which contained a high proportion of UVB light and therefore has a high potential
    to inflict DNA damage, rather than just an exposure to light with high intensity. This
    conclusion follows the fact that there was no significant increase in the integrated mucus
    absorbance, nor a visible change in the shape of the absorbance curves in fish that were
    exposed to the brightness control treatment.To our knowledge, the observed increase of
    mucus absorbance in P. amboinensis within one hour of exposure is the fastest change of such
    nature described in a reef fish. Changes in mucus absorbance, mediated by UVACs and/or
    MAAs after changes to the UVR regime have been documented before, but are
    usually only detectable after several days or weeks. Similarly, the accumulation of MAAs in
    algae, diatoms and corals due to UVB exposure occurs over longer time
    periods.

    Most certainly, UV exposure influenced the mucus absorbance. The mucus absorbance curves
    of P. amboinensis appeared to be similar to the profiles detected by Eckes, who also
    documented a strong absorbance of mucus in the UVA range. Interestingly, the peaks of the
    UV lamps used in the treatments (310 and 360 nm) do not correspond with the mucus
    absorbance peaks (280 – 290 nm, representing most likely the MAA precursor Gadusol, and
    320 – 340 nm, most likely representing the MAAs Palythine-threonine and Porphyra). This
    indicates that a defensive response using UV absorbing compounds is not directly matched to
    the environmental spectrum. However, the light spectrum used in these experiments does not
    recreate a natural exposure, like the one used by Zamzow. Nevertheless, we did observe
    mucus absorbance increases in UVB exposed groups of fish, as did Zamzow, despite the
    different light sources that were used in the experiments.

    Although UVB exposed P. amboinensis showed increased mucus absorbance, higher levels of
    DNA damage compared to a group of fish not exposed to UVR were still observed. However,
    it is difficult to know how much more DNA damage would have been induced by the
    exposure to the UVB pulse if this increase had not taken place, or mucus measurements in the
    same individuals were taken at the beginning of the treatment. However, such measurements
    require repeated handling of fish to sample mucus, which has been linked to disease in this
    species. Our exposure regime was on a much smaller scale, and we intended to avoid any
    additional handling effects due to repeated sampling. The formation of CPDs, the most
    common type of UV- induced DNA lesions, occurs nearly instantaneously upon
    irradiation with UV light. Any protection by UVACs in the mucus of P. amboinensis
    also did not prevent higher DNA damage levels (up to 58%) than in T. lunare and indicates
    interspecific variation in the susceptibility to UVR, which has been shown in both marine and
    freshwater fish. The fish used in this study differ in their diet and lifestyles, with
    T. lunare being a known diurnals piscivore, foraging for large amounts of time away from
    cover. In contrast, P. amboinensis is a diurnal omnivore, and strongly associated with
    sheltering habitats. Both factors, diet and UV exposure are known to influence the levels
    of UVACs and may have led to the observed susceptibility to DNA damage observed in
    the present study.

    The mechanism of the regulation of MAA content in the mucus and their transport from the
    gut into the mucus layer is unknown. Potential storage locations in tissues such as the gonads
    and gut, followed by transport to the mucus producing goblet cells in the epidermis
    seem possible, and could be responsible for the swift increase in mucus absorbance observed
    in the present study. Whether MAAs could also be stored in the mucus producing goblet cells
    for an even faster release than from the gut is unclear and needs further investigation.
    UVACs in the mucus could also originate directly from the MAA rich food items in the gut,
    without previous storage in other tissues. Gut turnover rates in fish are possibly fast enough to
    process some MAAs to the mucus but certainly not fast enough to prevent the
    formation of CPDs. MAAs originating from bacteria with a functioning shikimate pathway
    transferred into fish mucus cannot be discounted, and could potentially be responsible for
    the changes in mucus absorbance observed in this study. However, mucus is water soluble
    and constantly sequestered and replaced and also possesses antibacterial properties, therefore
    making an external source of MAAs in fish mucus a less likely explanation.

    The modulation of mucus absorbance could be visually triggered, since P. amboinensis is able
    to see UV light in contrast to T. lunare [67, 68], where mucus absorbance modulation must be
    triggered otherwise [42, 43]. For P. amboinensis, the ability to modulate the UV absorption
    properties of their external mucus layer may be essential in order to successfully send and
    receive their UV signals for communication [46, 69]. The ability for fast UVAC modulation
    could be the result of a trade-off, which allows P. amboinensis to communicate in the UV as
    long as the UV damage can be kept at low levels but is induced once damage levels are too
    high, or UV exposure reaches critical levels. Initially at day zero, T. lunare had an up to 63%
    higher mucus absorbance than P. amboinensis, confirming previous findings of family
    differences in MAA levels [13]. At a later stage in the experiment, the high levels of mucus
    absorbance in T. lunare were lost and were up to 50 % lower than in P. amboinensis (cohorts
    exposed to UVB). Insofar as this presents a trade-off between communication with UV
    signals (at times of low UV and low UVAC levels) and protection from UV radiation at times
    of high UV and high UVAC levels is unknown and needs to be examined in detail.

    Variable sunscreen protection could also provide an important selective advantage for reef
    fish to react quickly to increases in UV radiation over a short period of time with tidal
    movements and the movement of fish across a habitat. Changes in the UV regime over
    larger timescales and magnitudes facilitated by climate change are currently ongoing on
    the Great Barrier Reef, with less cloud cover increasing solar radiation and hence UV
    exposure. In this study, the dose of UVB radiation in the “UVB +” treatment (13.1 W*m-2)
    was more than double the amount of UVB radiation measured at Lizard Island at midday in
    the austral summer (6 W*m-2). However, the implications of increased UVR as an additional
    stressor in an ocean that is already impacted by warmer temperatures, higher acidity and a less
    complex habitat are currently poorly understood. The relatively low levels of DNA
    damage in fish that were not UV challenged, compared to the sharp increase in DNA damage
    in fish exposed to the UVR pulse, indicate that at present, the level of UVR in their
    environment does not pose a significant threat and the negative effects of UV exposure are
    being held at bay by the protective function of their MAA sunscreens and other hypothesized
    UV protection mechanisms such as UV specific avoidance behaviour and DNA repair.

  8. #8
    Sansai
    Join Date
    Jun 2010
    Location
    Michigan, USA
    Posts
    249
    My apologies for breaking into a thread, ......but

    I have been out of the country, and having returned to the country I find Koi Bito almost inactive. Has it moved or is it almost inactive. I dont find any of the files either.

    Any info would be appreciated.

  9. #9
    Daihonmei MikeM's Avatar
    Join Date
    Dec 2003
    Location
    Orlando, Florida
    Posts
    11,128
    Still active, Jake.... but not as active as in the past. None of the forums are very active. But, that is a subject for a different thread.

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