Fish in the Bay – Summer 2024. Clupeiform Color Special – Riddle of the Guanine Crystal.

This is a special report to consolidate recent observations and discoveries regarding light refracting-properties we see so often in Clupeiform fish dorsal shading.   

“Clupeiform Colors” has been a long-running thread in Fish in the Bay reports. The recent adoption of a “Clupeiform Color Scale,” mentioned in the previous blog post and discussed again below, allows us to further refine thoughts about this phenomenon. 

1. Guanine Crystal Science: It’s more than just fish scales.

Crystal collage from three sources as indicated.

Guanine crystals in fish scales both reflect and refract light.  They are a key component of the Clupeiform “stealth suit of invisibility.”  The crystals provide a mirror-like sheen on the sides and belly of the fish …  and a colorful diffractive iridescence across the dorsum: https://www.ogfishlab.com/2020/12/13/fish-in-the-bay-december-2020-anchovy-rebound-blue-fish/ 

Why Fish Scales Shine – “Guanine forms crystals, and it alone of the nucleic acids is widely used outside of the DNA in all sorts of organisms – almost always for manipulating light. In addition to shiny skin and scales, it is sometimes found in the eyes of animals,” says Weiner.  https://wis-wander.weizmann.ac.il/chemistry/why-fish-scales-shine 

Hard science:

• Levy-Lior et al. (2008) Biogenic Guanine Crystals from the Skin of Fish May Be Designed to Enhance Light Reflectance https://pubs.acs.org/doi/10.1021/cg0704753 
  “The metallic luster from the skin of fish is due to a photonic crystal system composed of multilayer stacks of cytoplasm and crystals. The crystals are described as thin (50−100 nm) plates of guanine, with no reference to their hydration state. … We established through X-ray diffraction that their crystal structure is that of anhydrous guanine. … which is composed of densely packed H-bonded guanine molecules.”
• Hirsch et al. (2017) Biologically Controlled Morphology and Twinning in Guanine Crystals https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201704801
  “Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror.”
• Kimura et al. (2020) Guanine crystals regulated by chitin-based honeycomb frameworks for tunable structural colors of sapphirinid copepod, Sapphirina nigromaculata https://www.nature.com/articles/s41598-020-59090-4 
  “Sapphirinid copepods … exhibit tunable structural colors originating from a layered structure of guanine crystal plates. …  the presence of chitin-based honeycomb frameworks that are composed of flat compartments regulating the guanine crystal plates. The structural color is deduced to be tuned from blue to achromatic via yellow and purple by changing the interplate distance according to vital observation and optical simulation using a photonic array model.”
• Wagner et al. (2023) Macromolecular sheets direct the morphology and orientation of plate-like biogenic guanine crystals https://www.nature.com/articles/s41467-023-35894-6
  “Guanine crystals are the most widespread molecular bio-crystal. The crystals are constructed from π-stacked, H-bonded layers, and exhibit exceptional optical properties, due to the extreme in-plane refractive index … By precisely controlling the shape and assembly of these crystals, organisms produce an array of different optical phenomena used in camouflage, display, and vision. …  All these crystals share a common feature—they preferentially express the highly reflective (100) face (parallel to the high-index, H-bonded layers) to maximize reflection.”

2. Fun Science: Guanine Crystals in Cosmetics.

Guanine – from Wikipedia https://en.wikipedia.org/wiki/Guanine   “The word guanine derives from the Spanish loanword guano (‘bird/bat droppings’), which itself is from the Quechua word wanu, meaning ‘dung’.

“In 1656 in Paris, a Mr. Jaquin extracted from the scales of the fish Alburnus alburnus so-called “pearl essence”, which is crystalline guanine.  In the cosmetics industry, crystalline guanine is used as an additive to various products (e.g., shampoos), where it provides a pearly iridescent effect. It is also used in metallic paints and simulated pearls and plastics. It provides shimmering luster to eye shadow and nail polish. Facial treatments using the droppings, or guano, from Japanese nightingales have been used in Japan and elsewhere, because the guanine in the dropping makes the skin look paler.”

Uguisu no fun – https://en.wikipedia.org/wiki/Uguisu_no_fun#cite_note-sny-6  “literally meaning “nightingale faeces” in Japanese, … also called the “Geisha Facial.”

3. Another Reminder:  Anchovies are oddballs amongst Clupeiforms.

Anchovies are confusing.  We always find many colorless young and even some colorless adults at all times of year.  Unlike other members of the Clupeiform order, Anchovies are slower to generate guanine crystal color across the dorsum when young.  They also slowly lose crystals when exposed to salinity below roughly (??) 20 to 25 ppt.  Additionally, unlike the other Clupeiforms, Anchovies lose guanine iridescence within about 5 minutes when they die. For some reason, guanine coloring is much more fragile in Anchovies.

Anchovies are different in many other ways as well. On the fish family tree (within the Clupeiform order), Anchovies are distant cousins; see https://www.britannica.com/animal/clupeiform/Classification

• Family Clupeidae: Herrings, Sardines, Shads, Pilchards, Menhadens, and relatives.
• Family Engraulidae: Anchovies. 

Adding to the confusion, at least a few of the Anchovies we see in SF Bay are likely members of a different subspecies or hybrids from them.

• Food and Agriculture Organization of the United Nations – Species Fact Sheet. https://www.fao.org/figis/pdf/fishery/species/2107/en?title=FAO%20Fisheries%20%26%20Aquaculture%20-%20Species%20Fact%20Sheets%20-%20Engraulis%20mordax%20(Girard%2C%201856) 
  “Three subpopulations [of Northern Anchovy] were proposed by McHugh (1952), but Miller (1956:23) doubted this. Hubbs (1925) and Hildebrand (1943) recognized two subspecies:
  • (a) E. mordax mordax: body more elongate … vertebrae usually 45 or 46; adults to 14 cm standard length or more; British Colombia to Baja California.
  • (b) E. mordax nanus: body rather deeper … vertebrae usually 43 or 44; adults generally to less than 8 cm standard length; Bays of California.” 

Dark Anchovies in the Bay.   A few older ‘Jumbo-sized’ Anchovies occasionally show up in Lower South SF Bay (LSB).  At 110 to 130 mm in length, they stand out amongst our midget-sized Bay residents.  They are dark and opaque gray across the dorsum. 

• We have long guessed that these extreme dark ones may be part of an “ocean-migrating” cohort. The darkness may develop only after some time at sea.
• Dark Anchovies show iridescent deep blue or green colors. Like our typical Bay Anchovies, their color fades away in lower-salinity Bay water, but their dorsal darkness and opaqueness remains.
• However, this is not a strictly ‘black or white’ issue: Many adult Bay Anchovies also show varying degrees of grayish darkness.   Nothing is ever simple with Anchovies.

Dark Anchovy examples from Luca.  Luca Sartori, our intern from the 2022/23 season, just finished another summer internship aboard the NOAA ship Reuben Lasker performing fish trawls along the California coast.  He emailed me three photos of Anchovies they caught in July.  These ocean Anchovies look larger and darker than the Anchovies we usually see in the Bay. 

• Anchovies caught off the California coast are definitely E. mordax mordax types.
• The extent to which ‘Dark Jumbo-sized’ ocean-going Anchovies interbreed with our colorful midget-sized E. mordax nanus variety remains unknown.

4. Reevaluation of Four Clupeiform Species against a fixed Color Scale.

American Shad – the honest fish.

• In November-December 2019, we first noted that American Shad dorsal color varied consistently with salinity. Limited “bucket-tests” established that Shad dorsal color changes dramatically in less than three minutes as the fish is exposed to varying salinity concentrations.   Unlike Anchovies, Shad do not lose guanine in chromatophore cells in low salinity. Shad colors persist even hours after death. 
• Herring and Sardines also experience salinity-induced color changes roughly corresponding to those in Anchovies and Shad. …

Until recently, we had not developed a workable color scale.  Instead, Clupeiform salinity-induced dorsal hues were casually described as “blue,” “green,” or “brown/gold.”  Intermediate shades were described as “greenish-brown” or “bluish-green” etc.  That approach was NOT sufficiently scientific.  A quantifiable color scale was needed.

Reevaluation of older photos and data yields a more accurate picture of Anchovy, Shad, Herring, and Sardine color changes.  

Starting at the blue end: High salinity ~ 25 ppt.  Bay Anchovies turn “blue” above salinity around 18 ppt.  Herring are still “bluish-green” even at salinity above 25 ppt! 

Using a Color Scale is more scientific:  The best matches for each species at 25.6 ppt:

• Anchovy dorsal color = Bosporous (#12),
• Herring color = Thermal Spring (#9)

Medium-high salinity ~ 18 ppt (25 ppt at bottom) December 2023.  Anchovies were “blue” and Herring were “green.”

Using the Color Scale:  The best dorsal color matches for each species:

• Anchovy dorsal color = Bosporous (#12),
• Herring color = Lounge Green (#7)

The Anchovy exception:  Baby Anchovies have No color / no guanine in Chromatophores. 
Chromatophore Score = 0. 

Medium-high salinity ~ 18 to 19 ppt in January.  Again, Anchovies were still just above their “blue” threshold.  Herring browned-down to a more robust shade of “green.”

Using the Color Scale:  The best matches for each species at ~18 ppt:

• Anchovy dorsal color = Bosporous (#12),
• Herring color = Lounge Green (#7)

Herring brown-down from green to brownish at around 17 ppt: 

• Herring dorsal color at ~17 ppt = Lounge Green (#7). The Herring shown at top appears to be browning-down to Different Gold (#4) at near 17 ppt.   
• Herring color at ~25 ppt was bluer = Thermal Spring (#9).

Example from February. Anchovy shown above was described at the time as “slightly bluer.” The Herring was “slightly browner.”

Using the Color Scale:  

• Anchovy dorsal color is “Green Bay” (#11).
• The Herring shown here appears to be greening-up from “Bengal Grass” (#2) to “Lounge Green” (#7).

Shad “blue-up” from brown to green at around 13 ppt salinity.  This blog collage from December showed brown & greenish Shad that blued-up to “greener” in the tray.

Using the Color Scale:  

• Shad can be described as “greening-up from brownish-green colors (numbers 4 & 6) to “Lounge Green” (#7).

Herring are browner than Shad at any given salinity.  We can now measure the additional brownness.

Using the Color Scale at just above 13.6 ppt:  

• Shad dorsal colors are “Nurture Green” (#6)
• Herring colors range from “Bengal Grass” (#2) to “Best Bronze” (#1).  

Sardines – the brownest of the Clupeiforms. Only three Sardines have been caught in Lower South Bay trawls in recent years (N=3).  Each Sardine was much browner per salinity level than has been observed in any other Clupeiform fish.   

• A single individual caught in 2020 indicated that the Sardine brown-to-green transition occurs near 27 ppt!   Anchovies, Shad, and even Herring are blue at that level of salinity.

Anchovies at adjacent stations on same day.  At salinity below 10 ppt Anchovies usually brown-down to golden.  Not all do.  What is going on here?

The lingering Anchovy mystery: “Dark Anchovies” break this predictable pattern. 

In the photos above, dozens of Anchovies in Pond A19 were either bright gold or colorless where salinity was well below 10 ppt – as expected.  In contrast, the Anchovy to the right continued to display “Green Bay” (#11) color where salinity was even lower!   

• This dark Anchovy will likely lose guanine and color, but will never brown-down to a golden hue. The dark suntan never disappears.
• Is she merely an older Anchovy who has made one or more tours in the ocean? Or, is she a hybrid from the ocean-going E. mordax mordax lineage?

5. Conclusion.

Clupeiform Salinity-induced Color Change – a graphical depiction.

Graphing Clupeiform colors.  Even a crude numeric color scale can be graphed and calculated into means and modes.

By stacking specific Clupeiform dorsal hues by light wavelengths against salinity (in ppt) a “Color Curve” can be charted for each species. 

• Anchovies are “bluest” at any given salinity.
• Shad are slightly greener & browner than Anchovies.
• Herring are browner still.
• Sardines are brownest of all.

The life history of “Dark Anchovies” remains a mystery.

Zardoz, Zed discovers the true nature of the Tabernacle https://www.youtube.com/watch?v=m4EESnKyRIY

• 1974 Sci-Fi movie “Zardoz” – This is strangest movie role ever played by Sean Connery.
• This quirky futuristic 1970s film barely broke even at the box office. Much later, it became a minor cult classic.