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Two Horizontal Visual Streaks, a Temporal Area Centralis and Comparatively Larger Cone Diameters in the Retina of the Torquigener pleurogramma

Lillian Toomeya, Jern Cabrala, Marisa Fewstera, Dani Fynna, Anita-Marie Marbecka, Jack Zimdahla aNeuroscience Discipline, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009

Purpose: Retinal ganglion cells and photoreceptors have a strong relationship with a species’

habitat and feeding behaviour. Though many teleost retinas have been studied, the retina of the

common blowfish, Torquigener pleurogramma, has never been examined. This study aimed to

determine the presence of a relationship between morphological features, ganglion cell distribution

and photoreceptors, and the blowfish’s environment.

Methods: Four retinal wholemounts were prepared using blowfish caught in the Swan River,

Western Australia. Two of the retinal wholemounts were stained using 0.5% cresyl violet. The

ganglion cell densities were then mapped onto an isodensity contour map. The remaining two

retinas were gently teased apart, and photoreceptor dimensions were measured.

Results: There was a temporal area centralis, with a peak density of 5.38 x 103 cells/mm2, which

was encompassed by a horizontal visual streak around the retinal meridian. There was another

ventro-temporal horizontal streak with a peak density of greater than 1.50 x 103 cells/mm2. The

cones were found to have a diameter of 7.65 x 10-3mm ± 0.93 x 10-3, whilst rods had a diameter of

1.80 x 10-3mm ± 0.52 x 10-3.

Conclusion: All the findings in this study were consistent with the known habitat and feeding

behaviour of the T. pleurogramma. The presence of two visual streaks was highly important, as this

has only been observed in two other species. Future studies should determine if there are any

ontological changes in the blowfish retina, and investigate the presence of two visual streaks in

shallow water teleost retinas.

1. Introduction

1.1 Retinal studies

No two animals see the world alike, with various mechanisms and adaptations altering visual

perception. Retinal studies can reveal a plethora of details about a species and their environment.

The topographic location, density, and integration between both retinal ganglion cells and

photoreceptors determines how an animal perceives their surroundings. These cell types specialize

and evolve to best equip a species to survive in their particular environment. Through studying

these two cell types, a deeper understanding of the habitat and feeding behaviour of a species is

developed, and comparative assessment of how different animals see the world can be performed.

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1.2 Retinal ganglion specialisations in teleost fish

Vertebrate retinal ganglion cells form regions of increased density that correspond with increased

visual acuity (Rolls and Cowey, 1970). In teleost fish, retinal ganglion distribution is correlated to

both feeding behaviour and habitat type (Williamson and Keast, 1988). Species with a continuous

view of the horizon have an increase in the ganglion cell density across the retinal meridian,

forming a horizontal visual streak (Munk, 1970). This allows them to scan their environment for

predators and look for plants or other static food (Collin and Partridge, 1996). An area centralis is a

concentric pattern of a high density of ganglion cells on the retina (Collin and Pettigrew, 1988a). A

temporal area centralis specialisation is critical to the feeding behaviour of species that eat plankton

and algae, as they need high frontal acuity to catch their prey (Shand et al., 2000).

For example, the collared sea bream, Gymnocranius bitorquatus, inhabits open areas of shallow

sand banks, and finds food by sifting through the surrounding sand and coral rubble with its

prominent mouth (Carcasson, 1977). Correspondingly, Collin and Pettigrew (1988b) found the

collared sea bream possessed two retinal specialisations, a strong dorso-temporal area centralis and

a weaker horizontal streak. The strong area centralis provides acuity in the lower frontal visual

space, and assists the fish in finding possible food sources, whilst the horizontal streak is useful for

both navigation and predator detection.

1.3 Teleost photoreceptors

It is through the integration of these ganglion cells with photoreceptors that complex visual

perception begins to flourish. Teleost fish possess rods, cones, and double cones, giving them

diverse colour vision mechanisms (Fernald, 1988). Most shallow water teleost fish can easily detect

colours and patterns (Gurthrie and Muntz, 1993). Fish have a range of visual pigments in their

cones, which allows them to see from ultraviolet through to red on the visual spectrum (Land and

Nilsson, 2012). Many fish, such as the Australian lungfish, Neoceratodus forsteri, have multiple

types of oil droplets present in the cones, acting as a filter for incoming light and increasing colour

sensitivity (Bailes et al., 2006).

Also, fish that achieve deeper depths have comparatively larger rod diameters, whereas those who

live in shallower waters tend to have comparatively larger cone diameters (Busserolles et al., 2014).

This is due to deeper water requiring strong scotopic vision, and thus a greater rod contribution to

vision. Similarly, shallow water requires stronger photopic vision, with a bigger demand on cone

activity.

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1.4 Torquigener pleurogramma

The common blowfish, Torquigener pleurogramma, is a teleost fish that lives primarily in estuaries.

A high density of the population inhabits estuarial sand banks with a water depth of less than 1.5m

(Potter et al., 1988). The species has a benthic-macroinvertebrate/algae feeding pattern, as a bottom-

feeder along the sand (Miller and Skilleter, 2006). Blowfish live to a maximum age of 6 years old,

reaching a maximum size of 230mm, and are relatively low on the estuarial food chain (Potter et al.,

1988).

Despite many studies focusing on shallow-water teleosts, the retina of the T. pleurogramma has not

yet been analysed. This study aimed to determine any relationship between the blowfish habitat and

feeding pattern and general morphology, ganglion cell specialisation, and photoreceptor types and

dimensions.

A similar retinal ganglion distribution was expected as that found in the collared sea bream, as

discussed in 1.2. It was hypothesised that the blowfish would display a strong temporal area

centralis, in accordance with its algae-based feeding behaviour. Also, since the blowfish lives at the

sand-water horizon, it was predicted that a horizontal visual streak would occur across the retinal

meridian. Furthermore, it was expected that the blowfish would exhibit rods, cones and double

cones, with oil droplets, similar to other teleost fish. Finally, it was hypothesised that the blowfish

would have comparatively larger cone diameters and smaller rod diameters than species that inhabit

deep-sea scotopic environments.

2. Materials and Methods

2.1 Specimens

Eyes were acquired from two blowfish, T. Pleurogramma. They were collected from the Swan

River, Western Australia using a mesh net with a 10-12mm gauge a week before sacrifice. The

average fish length was 122.5mm ±13.4, making the fish three years old and sexually mature

(Potter et al., 1988). The fish were dark adapted for at least 30 minutes before being terminally

anaesthetized using clove oil (0.1%). The University of Western Australia Ethics Committee

approved all procedures in this study (RA/03/200/534).

2.2 Preparation of retinal wholemounts

The eyes were removed from the head using a pair of forceps, placed in saline, and a dorsal cut was

created for orientation. By cutting around the limbus, the cornea and lens were removed. The

pigment epithelium and sclera were gently detached. To remove the vitreous, a few crystals of

hyaluronidase were placed on the retinal cup for 10 minutes, before being gently washed off with

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0.1M phosphate buffered saline. Small cuts were made around the retinal margins, to make the

retinal cup flatten onto the slide.

2.3 Nissl staining and analysis of retinal ganglion cells

Two of the retinas were fixed with 4% buffered formalin for 5 minutes. Excess fixative was

removed using filter paper. After being air dried, the retina was then stained for Nissl substance in

0.5% cresyl violet (Ullmann et al., 2012). Retinal images were taken using an Olympus OM

compound research microscope attached to a Moticam 2.0x megapixel camera. The retina were

divided into 1mm2 grids, and each image was regularly sampled every 2mm across the retina. Each

sample image had an area of 0.04mm2. All ganglion cells were manually counted in each sample

region, while glial cells and cell nuclei were not counted. There were a total of 34 sample regions

counted. Each count was then multiplied by 25 to determine the cell density within the 1mm2 grid.

The densities were then mapped onto the corresponding areas of the retina, and an isodensity

contour map was constructed by grouping areas of similar density.

2.4 Preparation and analysis of photoreceptor wholemounts

After the preparation of the retina as described in 2.2, small pieces of the remaining two retinas that

were completely free from pigment epithelium were cut off and placed onto separate microscope

slides. The retinal fragments were then gently teased apart using a pair of fine forceps, to separate

the retinal layers and make the photoreceptors easier to detect under a microscope. Using the same

digital setup as explained in 2.3, retinal images were taken of the photoreceptor layers.

Measurements of the photoreceptors were made through the program ImageJ, using a photo of the

graticule for reference of scale. Rod length (RL) and diameter (RD) and cone length (CL) and

diameter (CD) were measured, and averages and standard deviations were evaluated.

3. Results

3.1 Fish morphology

The blowfish had a dorso-ventrally flattened body, with a body height of 27.5mm ± 0.71. The

posterior of the body was the flattest portion of the body. The body width was 27.0mm ± 1.41, and

each fish had five fins; one dorsal, one ventral, two pectoral and one tail. Additionally, the fish

possessed small spikes along the dorsal surface of the body, which faced in a posterior direction.

The open mouth height was 5mm, with a width of 10.2mm ± 0.28. The blowfish had four large

teeth, two in the upper jaw and two in the lower jaw, with large, thick lips. Proportionally, the

mouth and teeth were large for the body size.

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The eyes of the fish bulged dorsally outwards, giving an upward field of view. The eyes had a

spherical shape, with a fixed circular shaped pupil. The width between the eyes in the head was

19mm ± 1.41. The lens diameter was 3.6mm. The pupil diameter was 4.4mm, and the pupil height

was 4.3mm. The eye height was 7.6mm and the length was 8.2mm. There was an axial eye length

of 6.7mm. The general morphology of the T. pleurogramma and the pigmentation pattern of the eye

is displayed in figure 1.

Figure 1. General morphology of the T. pleurogramma. (A) Lateral view of the blowfish, prior to the preparation of

the retinal wholemounts. The fish had a distinct, contrasting colouring, with the dorsal surface of the body appearing a

weedy green, and the ventral surface a sandy yellow. R, rostral; V, ventral. Scale bar = 1cm. (B) Frontal view of the

right eye after removal from the head. The eye was removed from the head using a pair of forceps and was bathed in

saline. The pupil was highly refractive and had a blue glaze filter, with a small dorsal green-pigmented area. The

surrounding scleral cornea appeared orange. V, ventral; T, temporal. Scale bar = 2mm.

3.2 Ganglion cell distribution and measurement

Only one retina had sufficient Nissl staining for use in ganglion cell counts. Five ganglion cell

counts were excluded from the final distributional analysis, due to shrinkage distorting the cell

density. These regions were all at the nasal or ventral retinal margins. There was a range of

ganglion cell densities from 425 to 5375 cells/mm2 across the retina. There was a total retinal

ganglion cell count of approximately 180,028 cells. The resulting isodensity contour map is shown

in figure 2.

A

B A

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Figure 2. Isodensity contour map of ganglion cells over the right retina of a T. pleurogramma. Numbers indicate

retinal ganglion cell density x 103 . The retinal area was 89mm2. There was a strong temporal area centralis (AS), with a

peak cell density of greater than 5.00 x 103 cells/mm2. There was a strong dorso-temporal horizontal visual streak (VS)

encompassing the AS. There was also a weaker ventro-temporal horizontal VS, with a peak density of greater than 1.5 x

103 cells/mm2. D, dorsal; T, temporal. Scale bar = 1mm.

Average ganglion cell diameter (mm) (n=10) was 6.60 x 10-3 ± 1.80 x 10-3. There was a range of

ganglion cell diameters (mm) of 4.00 x 10-3 to 1.00 x 10-2. The ganglion cells were, on average,

16.1% smaller in the areas of low density.

3.3 Photoreceptor measurements

The retinas analysed contained both cones and rods, however did not possess any double cones or

oil droplets. Average cone diameter (CD) (n=20) was 7.65 x 10-3 mm ± 0.93 x 10-3. Average cone

length (CL) (n=6) was 1.18 x 10-2 mm ± 0.075 x 10-2. Average rod diameter (RD) (n=20) was 1.80 x

10-3 mm ± 0.52 x 10-3. Average rod length (RL) (n=20) was 1.64 x 10-2 mm ± 0.33 x 10-2.

Photomicrographs showing example photoreceptor dimensions are displayed in figure 3.

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Figure 3. Photomicrographs at x40 magnification of photoreceptor types in the retina of the T. pleurogramma.

Both cones and rods were identified and measured. Example diameter (A) and length (B) measurement dimensions for

both types of photoreceptors are illustrated. CD was taken from cones at a dorsal view, and CL was taken from a lateral

view. RD and RL were both taken from a lateral view. Scale bar = 20µm.

4. Discussion

4.1 Morphological findings

The morphological findings of the T. pleurogramma support the habitat and feeding patterns

described in 1.4. The dorso-ventrally flattened body supports a shallow water estuarial habitat, as

this shape allows the blowfish to lie flat along the bottom of the sand banks. Furthermore, the

distinct colouring of the body, also confirms this, as it would allow the fish to camouflage into its

sandy environment. The position of the eyes supports this sandbank habitat, as they bulge upwards,

looking up to the water above.

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The proportionally large mouth with four large teeth confirms that the blowfish has a benthic-

macroinvertebrate/algae feeding pattern (Miller and Skilleter, 2006). Additionally, the dorsal spikes

support that the blowfish is low on the estuarial food chain, as they could be a defense mechanism

against predators who attack from above.

The blue glaze present on the pupil could act as a filter for light, removing the excess blue light

from the blowfish’s surroundings. This would allow the fish to see more red in the environment,

which would be beneficial for survival in a blue light filled environment such as the shallow

watered estuarial sandbanks. The presence of the orange pigment within the scleral cornea was

consistent with the body of work surrounding the blowfish cornea, as it has been found that T.

pleurogramma has an occulsable cornea. This means that the species has the ability to regulate their

corneal pigment cover in response to illumination, and in the blowfish, the yellow pigment is stored

in the scleral cornea (Siebeck et al., 2003).

4.2 Ganglion cell distribution implications

The hypothesis that there would be strong temporal area centralis observed in the T. pleurogramma

was supported. Since the eyes were relatively far apart on the head, the temporal area centralis

would correspond with high acuity in the frontal field of view. There was the greatest density of

ganglion cells in the centre of the area centralis, suggesting that the frontal visual sampling was of

greatest importance. This temporal area centralis would be useful for detection of algae and

macroinvertebrate food sources within the environment, corresponding with the feeding behaviour

supported by the mouth morphology discussed in 4.1. This is highly consistent with the body of

work surrounding bottom-feeder fish retinal specialisations (Shand et al., 2000; Collin and

Pettigrew, 1988b).

However, there were two temporal visual streaks present in the blowfish retina, one dorsal and one

ventral. The presence of two temporal horizontal streaks is highly significant, as there are only two

other known species to possess this trait, Aplocheilus lineastus and Epiplatys graham (Collin and

Pettigrew, 1988b). It has been theorized that the central band is used for the detection of predators

whereas the ventral band is important during feeding (Thewissen and Nummela, 2008). However,

in relation to the blowfish, arguably the central dorsal band is used for increased sampling for

detection of food sources, assisted by the temporal area centralis. The ventral band therefore would

be useful for scanning for predators, as predators are more likely to attack from above since the fish

lives on the sandbank. This threat of an overhead attack is confirmed by the presence of the

defensive dorsal spikes, as discussed in 4.1. This example of convergent evolution in relation to

different streak functions is interesting, and further studies should focus on determining if this is

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unique to the blowfish. This could be achieved by comparing this retinal specialisation with species

that both inhabit a similar region and exist on a similar diet.

The ganglion cells being smaller in areas of low density is inconsistent with the body of research, as

there is usually a crowing effect that makes ganglion cells smaller in high density areas (Perry and

Linden, 1982). This is because ganglion cell size is a function of their dendritic field size, which

increases towards the periphery as they become responsible for more photoreceptors (Collin and

Pettigrew, 1988c). The significance of this divergence away from the findings of other studies is as

yet unknown, and further research should determine whether this was a sampling issue, or whether

there is functional implication for this pattern of ganglion cell size.

4.3 Photoreceptor analysis

The hypothesis that rods, cones, double cones and oil droplets would be observed was not

supported. There was only evidence of rods and cones, which did not fit in with previous studies of

research teleost fish retinas (Hunt et al., 2015). One explanation for the lack of oil droplets could be

the presence of the blue pigment glaze as discussed in 4.1. Since oil droplets function as a light

filter, and the blue glaze performs the same function, perhaps evolutionarily speaking this left oil

droplets redundant in the blowfish retina.

However, the hypothesis that the diameters of cones would be larger and the diameters of rods

smaller compared to scotopic fish species was supported by the data. The average cone diameter

was over twice the size of those found in the retina of the Makaira nigricans, which is adapted to a

low light level environment (Fritsches et al., 2003). Similarly, rod diameter was on average 0.9µm

smaller than the rods of the Nannobrachium phyllisae, a species of deep-sea lanternfish

(Busserolles et al., 2014). This suggests that cone activity is of greater importance than rod activity

to the T. pleurogramma when compared with low light adapted fish. These findings were consistent

with past research into photoreceptor dimension differences between shallow water teleost fish and

deep-sea mesopelagic fish (Warrant, 2000). Overall in the blowfish retina, the cone diameter was

larger than the rod diameter, which suggests there would be a higher demand on cone function then

rods. This all supported the notion that blowfish live in and have adapted to primarily photopic

environments.

4.4 Limitations and future directions

A major limitation of this study was that only 1.58% of the area of the retina was sampled for

ganglion cell counts. Although this method allowed for an overview approach of cell distribution, it

did not allow for an accurate description of density. To be consistent with previous retinal studies,

the retina should be sufficiently sampled to achieve a Schaeffer coefficient of error (CE) of less

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than 0.1 (Coimbra et al., 2012; Coimbra et al., 2013). This will make the stereological estimates

more reliable across the retina and give a better insight into the true intricacies of ganglion cell

distribution.

Additionally, the Nissl staining was faint around the falciform process, which made focusing on the

ganglion cells difficult in this region. The staining of the inner nuclear layer in the peripheral retina

also made it difficult to focus on the correct retinal layer. This was emphasised by the fact there was

only one retina stained acceptably for ganglion cell analysis. Therefore, this contributed to

decreased accuracy in the final isodensity map. Future studies should aim to have a larger sample

size of at least three retinal wholemounts to increase the reliability of the results (Arrese et al.,

1999; Coimbra, et al., 2009; Coimbra et al., 2013).

Furthermore, there were difficulties visualizing the photoreceptors for analysis. The teasing of the

fragments displaced the cones to be in the dorso-ventral plane rather than the rostral-caudal plane.

This meant that photoreceptors were primarily viewed dorsally, making it difficult to measure

length and determine different types. This reduced the reliability of the photoreceptor dimensions

and the lack of double cones in the blowfish retina. Further research should aim to use a more

accurate method of sampling and analysing photoreceptor dimensions and to determine if double

cones are present in the blowfish retina.

A direction for future study could be determining if there are any ontogenetic changes in either

ganglion cell distribution or photoreceptor types in the T. pleurogramma. The black bream,

Acanthopagrus butcheri, has been found to have an area centralis that moves from dorso-temporal

to dorsal during development as feeding behaviour changes (Shand et al., 2000). Perhaps this could

also be the case with the blowfish, and further studies should examine this as an effect on visual

specialisations. Furthermore, since juvenile fish have been found to have lower photoreceptor

densities and length, a longitudinal study may also discover variations in photoreceptors (Pankhurst,

1987). The blowfishes used in this study were approximately three years old, which, despite being

sexually mature, still were not at their maximum length. Due to the limited research on the T.

pleurogramma, it is unknown whether there are any behavioural ontological changes, and future

research should aim to clarify the species’ development further. Given a wider age range, perhaps

both ganglion cell distribution and the types and dimensions of photoreceptors present would have

been variable.

Retinal specialisations and adaptations to the environment have helped the T. pleurogramma persist

and survive in their estuarial environments. Looking to the future, it’s important to determine the

strength of their similarities with other shallow water teleost fish, but also to further establish the

extent of the features that set it apart.

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