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Croaking Science: How glass frogs make themselves (almost) invisible

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Written by Roger Downie

Froglife and University of Glasgow

Back in July 2020, Isabel Byrne, Chris Pollock and I wrote a Croaking Science article that described the Centrolenidae, a neotropical family of 158 species known as glass frogs because of their transparency. In that article, we focused on reproduction in glass frogs. Here I discuss new work on their transparency and its contribution to their survival.

The importance to animals of body surface colours and patterns has long been studied. A key work is Hugh Cott’s book Adaptive Coloration in Animals (1940). Broadly, animals have evolved two different strategies: a) aposematic coloration, where they possess some protection, such as toxicity, and advertise this by being conspicuously coloured and patterned: in amphibians, poison-arrow frogs are an obvious example; b) cryptic coloration, where colours and patterns in some way blend with the background, making the animal difficult to detect: such methods of camouflage may match the background, or disrupt edges, or mimic background features. Cott undertook his doctoral research while lecturing at the University of Glasgow and was supervised by John Graham Kerr, the regius professor of Zoology who had developed an interest in the military uses of camouflage during World War 1. Cott’s thesis (1938) was titled The problem of adaptive coloration with special relevance to the Anura, and he moved to the University of Cambridge soon after completing it. Both he and Kerr were frustrated by the military authorities being unwilling to accept scientific advice on camouflage during both World Wars.

Transparency as a means of camouflage is fairly common in aquatic habitats, but is rare on land. For vertebrates, the red coloration of the haemoglobin in blood is a particular problem. In water, ice fish and eel larvae avoid this problem by having no red blood cells. In glass frogs, new research by Taboada et al. (2022) demonstrates a different strategy: when at rest and requiring concealment from predators, they hide their red blood cells.  

Glass frogs have highly transparent muscles and ventral skin, and the major internal organs: heart, liver and digestive organs, lie within mirrored sacs containing reflective guanine crystals which reflect the incident light. However, when frogs are active, their blood vessels are easily visible because of the circulating red blood cells. Taboada and colleagues compared active, awake glass frogs with individuals asleep and resting on leaves. They found that asleep frogs transmitted 34-61% more light than active individuals, making them more transparent, and that red blood cells were essentially absent from most of the circulatory system when the frogs were asleep. To find where the red cells were, they had to use the technique of Photo-acoustic microscopy, which can penetrate into solid tissue and visualise its contents. They found that the red cells were located in extensible sinuses in the liver, increasing liver volume by 40%. The transition from asleep to active in terms of red cell distribution took about 60 minutes. Measurements of the oxygen content of the blood showed that this decreased by 31% during sleep, suggesting a possible negative aspect of the red cell hiding process. The research team carried out comparative measurements on three other kinds of arboreal frog: none of them showed the transparency found in glass frogs, or the concealment of red blood cells when at rest. The authors speculate that the mechanism underlying red cell hiding in glass frogs may exploit a widespread ability in frogs, where other species have been shown to regulate respiration by temporary storage of red cells in the liver.

In another report, Barnett et al. (2020) question the use of the word ‘transparency’ as applied to glass frogs, since their dorsal green pigmentation renders them ‘translucent’, rather than transparent. Barnett and colleagues showed that translucent model frogs offered protection against avian predators and that translucency made detection of individuals slower. 

This work is a reminder of the many ways in which the physiology of amphibians is unusual, such as the ability of some species to survive freezing, helping them to survive in a changing world.

Acknowledgement

Thanks to Malcolm Kennedy for drawing my attention to the Taboada et al. paper.

References

Barnett, J.B. et al. (2020). Imperfect transparency and camouflage in glass frogs. PNAS 117, 12885-12890.

Byrne, I., Pollock, C. and Downie, J.R. (2020). See-through frogs are worth a look. Froglife eNewsletter Croaking Science July 2020.

Taboada, C. et al. (2022). Glass frogs conceal blood in their liver to maintain transparency. Science 378, 1315-1320.

Croaking Science: See-through frogs are worth a look

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Glass frogs comprise a family (Centrolenidae) of 158 species found in the forests of the neotropics: Central America and northern South America. They get their name from the transparency of their belly skin, which allows the internal organs to be easily seen. They are adapted to life in the trees, possessing pads on the tips of their digits that allow them to adhere to leaves. These structures appear to have evolved independently in several lineages of frogs, since molecular phylogenetic results show that the main families where these pads occur (Hylidae, Rhacophoridae, Centrolenidae) are not closely related.

One of the special features of glass frogs is their mode of reproduction. Clutches of eggs, as flat round sheets, each egg encased in jelly, are laid on the surfaces of leaves overhanging streams. Once the eggs have developed into larvae, they hatch and fall into the stream below, often from a considerable height. Glass frogs are divided into two main sub-families: the Centroleninae (121 species) which lay on the upper sides of leaves and then leave the eggs to develop on their own; and the Hyalinobatrachinae (35 species) which lay their eggs on the lower sides of leaves; in these species, the father cares for the eggs, sometimes up till the point of hatching.

Figure 1: Several egg clutches on a single leaf

We studied the glass frog Hyalinobatrachium orientale which has distinct populations in northern Venezuela and northeast Tobago: this distribution is a bit of a puzzle. Northeast Tobago is quite distant from Venezuela and between these two locations is the large island of Trinidad, which has abundant forests, but no glass frogs. Walking along the forest streams of Tobago at night, once the rainy season (June to December) has started, you soon hear the high-pitched peeping of male glass frogs, located on the huge leaves of Heliconia bihai that overhang the water. With the aid of a good torch, you can locate the calling frogs; often, near them, you can spot the little patches of eggs. If you are lucky, you may locate a mating pair. We observed the behaviour of a mating pair. It took them about four hours to complete their clutch of around 30 eggs, laid as a spiral pattern, starting at the centre, with the pair turning as they proceeded. Once egg-laying was complete, the female departed, but the male stayed close to the clutch. Often, when searching for egg clutches, we found the father sitting on top of his eggs. We also found that some fathers, presumably good quality males, were looking after more than one egg clutch at a time. These are at different stages of development, so clearly produced on different nights. It is not entirely clear what functions male egg attendance performs, particularly given that it is not constant. However, observers have seen males driving away egg predators such as wasps and ants; it is also likely that males keep the eggs hydrated by reducing evaporation, simply by sitting on them, or by emptying their bladders over the eggs (this is established in some cases of frog parental care). But this raises another mystery. If paternal care is helpful to incubation success in the Hyalinobatrachinae, why does it not occur in other glass frogs, especially when they lay their eggs on leaf upper surfaces, where you would guess that desiccation and predation would be higher risks.

In the Tobago glass frog, hatching occurs around nine days after laying, although the actual time is variable, allowing tadpoles to be earlier or later stages of development when entering the water. Such variability may be quite common in frog development and represents a trade-off. Early hatchers are less well developed and more vulnerable when they enter the water; but it may be better to risk this than to be predated while still in the nest. It therefore pays the developing larvae to monitor conditions: if the father has deserted his clutch, or hot sun and no rain are risking desiccation, better to hatch early and hope for a stream with few predators.

Figure 2: Metamorphosing glass frog tadpole on a leaf; with pen for scale

The streams where glass frog tadpoles are found are fast-flowing when it rains, and are heavily populated with predatory fish and crustaceans. They are also shaded, making plant productivity low. As a consequence, glass frog tadpoles spend much of their time hidden under rocks, reducing the risks of predation and of being swept away by currents (unlike the tadpoles of some species that inhabit fast streams, glass frog tadpoles lack the kind of suctorial mouthparts that can help cling on to rocks). The tadpoles have long muscular tails, indicating an ability to move rapidly, and are relatively unpigmented, associated with a concealed lifestyle. Their behaviour limits foraging opportunities, so growth is slow. Glass frog tadpoles can take a year to reach metamorphosis, very slow by tropical frog standards, where many species reach that stage in a few weeks. We have been able to locate metamorphosing glass frogs near the edges of streams. They climb up on to the upper surfaces of leaves close to the ground, and take about four days to complete the process, reducing their tail to a stump. To our surprise, we found that they do not remain in one place through this process, but occasionally move around, possibly to confuse potential predators.

The transparency of glass frogs has long puzzled biologists. Recently, a research team from Bristol, Canada and Ecuador has tested an explanation. Their evidence suggests that it is not so much transparency that matters, but translucence. When a glass frog is at rest with its limbs tucked in, the translucence of the limbs blurs the edges of the body, and makes detection by predators more difficult.

Further reading

Barnett et al. Imperfect transparency and camouflage in glass frogs. PNAS (2020).

Byrne et al. The behaviour of recently hatched Tobago glass frog tadpoles. Herpetological Bulletin 144, 1-4 (2018).

Byrne et al. Observations of metamorphosing tadpoles of the Tobago glass frog. Phyllomedusa (in press) (2020).

Delia et al. Glass frog embryos hatch early after parental desertion. Proceedings of the Royal Society B 281, 2013-2037 (2014).

Downie et al. The tadpole of the glass frog Hyalinobatrachium orientale from Tobago. Herpetological Bulletin 131, 19-21 (2015).

Nokhbatolfoghahai et al. Oviposition and development in the glass frog Hyalinobatrachium orientale. Phyllomedusa 14, 3-17 (2015).

Contributing authors : Roger Downie, Isabel Byrne, Chris Pollock.