The Cellular Expression and Genetics of Purple Body (Pb) in the Ocular Media of the Guppy Poecilia reticulata

Our study revealed the presence of all major classes of chromatophores (melanophores, xanthophores, erythrophores, violet-blue iridophores, xantho-erythrophores) and crystalline platelets in various combinations in the iris and ocular media (cornea, aqueous humor, vitreous humor, outer lens membrane) of Poecilia reticulata. This novel ocular media study of P. reticulata takes into account the distinct interactions of Purple Body (Pb) based on results of previous Bias and Squire Purple Body (Pb) publications. Taken in conjunction with other researcher’s published results (regarding UV reflected color and pattern, vision, mate choice, individual preferences, and opsin capabilities) this indicates that these ocular chromatophore populations together create a complex ocular filter mechanism. This mechanism in turn provides spectral capabilities into the UV and Near-UV wavelengths in both Pb and non-Pb individuals. The chromatophores in the cornea, aqueous humor, covering membranes of the lens, and the vitreous humor comprise an ocular filter system that could reduce UV damage to the internal structures of the eye. The guppy’s ability to use UVA as a visual component provides a “private signally system” that cannot be detected by some predators. While non-Pb guppies should derive benefit in the near-UV from violet-blue iridophore units, greater benefit will be derived by Pb individuals with more violet iridophores functioning in the lower UV and near-UV wavelengths. To our knowledge little has been published for P. reticulata concerning pigmentation within the guppy eye. Macroscopic and microscopic imagery is presented.


37
The intent of this paper is multifold: 1. To identify phenotypic and microscopic 38 characteristics of the newly described Purple Body trait in ocular media. 2. To provide 39 photographic and microscopic exhibits of Purple Body and non-Purple Body eyes for ease in 40 identification of chromatophore types (Fig 2) and their interactions in the ocular media. 3.

41
To encourage future study interest at a cellular level of populations in which Purple Body

56
Teleost species, including the Guppy, possess a complex eye with the ability to detect 57 color and shape. Like many prey species, positioning of the eye is set for maximum field of 58 view. Most species are considered to have fixed shape with adjustments made by changes 59 in the amount of pupil protrusion; i.e. distance above the plane of the body. Variation in 60 colors and color characteristics such as hue, depth, etc. cannot be important in female-61 based sexual selection unless the female, and male, can detect these color characteristics.

62
Therefore, the evolution of color characteristics must be accompanied by the evolution of 63 the ability to detect these colors. Endler showed that selection for spectral sensitivity 64 variation in both short-wavelength sensitivity (SWS) and long wave sensitivity (LWS) is due 4 ranged from 78°F to 82°F. Fish were fed a blend of commercially available vegetable and 126 algae based flake foods and Ziegler Finfish Starter (50/50 mix ratio) twice daily, and newly 127 hatched live Artemia nauplii twice daily. A high volume feeding schedule was maintained in 128 an attempt to produce two positive results: 1. Reduce the time to onset of initial sexual 129 maturity and coloration, thus reduce time between breedings. 2. Increase mature size for 130 ease of phenotypic evaluation and related microscopic study.

131
All euthanized specimens were photographed immediately, or as soon as possible, after 132 temperature reduction (rapid chilling) in water (H 2 0) at temperatures just above freezing 133 (0°C) to avoid potential damage to tissue and chromatophores, while preserving maximum 134 expression of motile xantho-erythrophores in Pb and non-Pb specimens. All anesthetized 135 specimens were photographed immediately after short-term immersion in a mixture of 50% 136 aged tank water (H 2 0) and 50% carbonated water (H 2 CO 3 ).

137
All dried specimens were photographed immediately after rehydration in cold water 138 (H 2 0). Prior euthanasia was by cold water (H 2 0) immersion at temperatures just above The existence of similar filters has been described and 152 summarized in other teleost fish species and mammals (Douglas 1989(Douglas , 1999 cornea" (Douglas 1989). Prior to this, ocular media was commonly considered to be "clear"

163
for the most part in both freshwater and marine species.

201
P. reticulata cranial structure is bilaterally symmetric when viewed from a high angle.

202
The Left-right axis gently slopes from the dorsal base in even taper to the supraocciptal 203 surface (see S1 for naming and locations of axial planes). Then a slight increase in taper 204 begins and continues to the mouth (Fig 3A-C). Differential between males and females is 205 minimal, though greater between individuals.

206
The dorsoventral axis is also generally bilaterally symmetric. Operculum (gill plate) is 207 observed to consist of fused bony opercle, preopercle, interopercle and flexible subopercle.

208
Dorsal side slopes downward starting at the dorsal base, increasingly past the supraocciptal

209
to the upper jaw. The ventral side axis maintains a more general upward slope to the 210 subopercle, with increasing upward angle past the interopercle through the dentary to the 211 lower jaw (Fig 3 and 4, D-F). Differential between males and females is minimal, though 212 greater between individuals and often appears more consistent among males

213
High-angle macroscopic images reveal lens protrusion well past the plane of the iris to 214 produce a wide field of view (Fig 3 and 4

218
Variability between males and females is minimal, though it often appeared greater between 219 females and more consistent among males. Pupils express no visible aphakic gap (the 220 "lensless" part of the pupil that does not cover the lens, Schmitz 2011) between the 221 protruding lens and iris in perpendicular macroscopic images (Fig 3 and 4, A-C). This

238
The pupillary response to ambient light changes in bony teleosts is generally considered

256
Observations of the visual axis in the Guppy reveal the eye tilts at a downward angle 257 ( Fig 5A) and slightly forward (Fig 5B) from the tapering body structure. Other than 258 occasional "reflex blinking" to adjust the iris and / or lens, movements that are common in 259 teleost species without the benefit of an eyelid, the eyes are static. This reflex movement is 260 assumed to be a mechanism for muscle relaxation and/or refocusing of the eye. Convex

261
shape and curvature in the plane of the iris was detected in high-angle macroscopic images.

262
Visual observations, in two forms, of live specimens and photographic images indicate

288
Our study revealed that all major classes of chromatophores (melanophores,  were reduced in heterozygous Pb condition and removed in homozygous condition.

306
Clustered xanthophores, found in all parts of the body and fins in "wild-type" Pb and non-

307
Pb, remained intact in both heterozygous and homozygous Pb condition within the eye.

308
Microscopic "penetration" of the cornea and past the pupil (iris and lens juncture) by 309 ocular focusing was more difficult to achieve in non-dissected specimens, especially in 310 homozygous Pb condition, due to a proliferation of iris melanocytes producing a "reflective

323
Thus, the presence of all chromatophore types should be considered the "normal" in Guppy 324 ocular media, just as they are in the body and finnage.

325
The dense layer of violet-blue iridophores (Pb with a higher ratio of violet to blue, and Endler). We take this a step further to include sensitivity to UV and near-UV wavelengths 332 as being heritable through Pb (Bias and Squire 2017a).

333
Results are presented in the following format and order: A. Non-dissected pupil and iris 334 (Fig 6-8), partial dissection of eye (non-enucleated) with orbit, operculum and dentary 335 intact viewed from high angle (Fig 9-10) and perpendicular (Fig 11), horizontal axis 336 dissection of the eye with lower portions of orbit, operculum and dentary intact viewed from 9 high-angle (Fig 12), B. Protruding lens intact with cornea removed (Fig 13-14), C. Corneal

339
Humor Fluid Extraction (Fig 26-28), F. Lens complete extraction (Fig 29-36). All non-340 dissected images were taken from the right side and all dissection was done on the left side,

358
Higher violet iridophore reflective sheen in the pupillary region, producing a more "purple" The removal of a portion of the scleral skin and the entire true cornea was performed on 412 frozen specimens (Fig 13-14) to preserve the integrity of the structure and maximize the

452
In images (Fig 15, 17-18  against the others for vitreous contamination. The clearest of these were utilized in the 538 microscopy study (Fig 22-25). The aqueous humor is generally clear under transmitted

614
Micro-dissection of the lens was performed on several enucleated eyes (Fig 29-36).

615
After complete lens extraction each lens was saline rinsed multiple times in ethyl alcohol 616 with prolonged soaking to remove potential loose surface contaminants adhering during

636
Several lenses were intentionally crushed with compression between the glass slide and 637 the cover slip. A clear distinction was revealed between ruptured tissue fragments (with 638 chromatophore populations) and the fractured rigid crystalline epithelial cells (Fig 30A-B).

639
Further distinction was visible between fractured epithelial cells forced into underlying 640 reflective crystals within the cortex and the lens itself. Reflective qualities, both yellow and 641 blue, were detected in epithelial cells from the germinative zone of the lens (Fig 31). . We hope that Purple will be mapped to its linkage group.

739
The reflectance value of drosopterin starts dropping as its absorbance value increases. The

740
absorbance value of guppy carotenoid starts increasing at lower wavelengths.

741
Actual reflectance spots showed the highest values for violet spots (Kemp 2009)

771
Further, a number of reports indicate that both the cornea and the lens act as UV filters in 772 many if not most species of vertebrates (Nelson 2001). It is generally held that when UV 773 filtration occurs the cornea is the first UV filter, and the lens is the second.  774 found that the guppy cornea (of an unidentified population) transmits 50% of incident UV at 775 315nm. This indicates that the guppy lens is not a major filter of UVA wavelengths (320-776 400nm). But it may be a significant blocker of UVB rays (280-320nm).

777
Judging from Fig. 3 of Grether (2001) the absorbance values of drosopterin become 778 significant by 525nm and increases to a peak around 480nm and gradually diminishing.

779
Likewise the absorbance values for guppy carotenoids seem to become significant around 780 490nm and extend to below 400nm (the limit of their figure). They point out that 781 absorbance values for intact cells will vary from those of the extracts used in their study.

821
This does not infer that UVA is not potentially harmful to guppies! All UV radiation is

841
The presence of large numbers of iridophores in this system is noteworthy. Violet-blue 842 iridophores were present in the cornea, the outer lens membrane was saturated with violet-843 blue iridophores, and they were present in high numbers in both the aqueous and vitreous

874
For purposes of this study low resolution photos were often preferred over higher

886
All euthanized specimens were photographed immediately, or as soon as possible, after 887 temperature reduction (rapid chilling) in water (H 2 0) at temperatures just above freezing

888
(0°C) to avoid potential damage to tissue and chromatophores, while preserving maximum 889 expression of motile xantho-erythrophores in Pb and non-Pb specimens. All anesthetized 890 specimens were photographed immediately after short-term immersion in a mixture of 50% 891 aged tank water (H 2 0) and 50% carbonated water (H 2 CO 3 ).

892
All dried specimens photographed immediately after rehydration in cold water (H 2 0).