Over the week-long period it took for the flatworms to regenerate their heads, the team monitored how quickly their brains and eyespots regrew, and when they began responding to visible light again. After four days, the eyespots had grown back, but the worms continued to react more strongly to UV than to visible light.
Only after seven days did they regain their stronger preference to slither away from visible light — suggesting that their eyespots and brains were retaking control. It was not until the 12th day that their sensitivity to such light increased to the point that they reacted more strongly to light at the bluer end of the visible spectrum. They move away from light.
Planarians are hermaphroditic , that is, they contain both male and female sex organs. They can reproduce asexually simply by pinching in half; each half grows a new half.
The reproductive cycle typically involves two host species, a primary host and a secondary or intermediate host. Adults live in the primary host and larvae develop in the secondary host. The life cycle often alternates between sexual and asexual reproduction. Nearly half of people in the tropics have blood flukes. Schistosomiasis is a blood fluke that afflicts million people in the world. The secondary host is a snail.
Figure 1. Left: Planarian anterior end X Middle: Planarian digestive tract mid section X Right: Planarian c. Observe either a preserved liver fluke or a slide of a liver fluke using a dissecting microscope.
Figure 2. Several of the identified genes are known to have versions that play a role in the vertebrate eye but have not been found in the fruit fly eye. Among these are genes involved in eye development and others associated with age-related macular degeneration and Usher syndrome, a disorder that causes progressive retinal degradation. One of the key genes identified in planarian eye development is the transcription factor ovo , which activates the expression of many other genes as the eye forms.
Until now ovo had been associated with neural tube and germ cell development in other organisms, but not with the eye. In planarians, ovo is vital for eye for regeneration and eye maintenance in the adult, and is also active in eye development in the embryo. In fact, when ovo is experimentally turned off, planarians with head amputations cannot regenerate their eyes and eyes of otherwise normal adult planarians vanish after a couple months.
Planarian eyes are very different from fly eyes, and we're already seeing the benefits of studying diverse model species, like the discovery of a critical role for ovo. Lapan, S. Although this unimodal action spectrum is consistent with a single eye opsin, further microspectroscopic analysis would be required to precisely define the absorption profile of the eye. We propose that worms sense small wavelength changes by comparing the effective intensity of light inputs sensed at the eye.
In the simplest scenario, the signal sensed would scale with the actual absorption of light by the eye. For any given wavelength, the light absorbed would be a function of the intensity of incident light and extinction coefficient of the photoreceptors.
Given two equal intensity photon fluence rate light inputs of different wavelengths even with a single opsin photoreceptor type , there will be differences in the amount of light sensed at the eye, reflecting the intrinsic absorption spectrum of the opsin. Remarkably, when illuminated with closely spaced spectral inputs, the animals are able to convert small differences in effective intensities into virtually binary behavioral choices. Differences in effective intensities likely lead to changes in aggregate PRN response and signaling to a putative downstream processing center.
The networks are able to parse these differences in effective intensities through comparative processing, leading to phototactic choice. This choice can also be described as acute gradient sensing. Gradient sensing is seen in other organisms, such as Platynereis 42 and Drosophila larvae Although planarians appear too acutely sensitive, this ability to sense light gradients likely represents a widespread evolutionary advance.
Further work would be required using RNAi and imaging methods to look for mechanistic and structural underpinnings of acute gradient sensing. Unlike with ocular response, the extraocular response times scale strongly with light intensity; also, wavelength choice patterns, seen with eye-mediated sensing, are absent. The extraocular response network appears to integrate whole-body sensing events, finally leading to directional phototaxis Fig. Even the photoreceptor s mediating the extraocular response is distinct from the eye opsin, with different spectral ranges.
High sensitivity to long UV proxy for filtered sunlight is consistent with the environmental niche of many planarians. Being highly light-aversive, a whole-body response may be beneficial.
Many planarians can also propagate through fission 41 , A robust, phototactic extraocular response would allow newly fissioned, vulnerable tail pieces to avoid direct or bright light. How is UV-A sensed and processed? Opsins are some of the most widely expressed light sensing proteins in the animal kingdom Extraocular sensing using opsins has been seen in several organisms spread across different phyla However, novel photoreceptors cannot be discounted. For instance, Drosophila larvae 27 and C.
To list the possible extraocular photoreceptor molecules in Schmidtea , we conducted a preliminary investigation using bioinformatics. A pipeline designed to look for opsin-like proteins in the existing planarian transcriptome yields six candidates fig. S9 , excluding the previously identified eye opsin.
Our preliminary analysis, based on molecules that have been implicated in extraocular photoreception in other organisms opsins, gustatory receptor—like, and TRP channels , yields some candidate photoreceptor s. Further work would be required to identify the light receptor s.
Planarian tails show coordinated movement away from UV, suggesting an independent neuronal signaling—dependent locomotor response. Where does the photosensing occur? In some organisms showing extraocular sensing, photoreceptor molecules have been reported in neurons 23 , 27 , Classical nervous system studies and immunostaining have showed the existence of subepidermal nerve plexus in planarians 46 — Therefore, it is conceivable that light sensing molecules present in these neurons lead to extraocular response.
There have been reports on ectopic eyes present in polychaetes 49 ; however, there is little or no information on these photoreceptive structures in planarians.
Investigating the structural and functional underpinnings of this high-sensitivity extraocular response would be fascinating. Because planarians show full eye-brain regeneration after injury, it is possible to address the nature of light sensing and processing in unprecedented ways. Examining functional recovery of light sensing revealed a hierarchical, multilayered sensing paradigm.
For eye-mediated light sensing, our data from wavelength choice experiments performed over eye-brain regeneration showed how gross sensing could be temporally delineated from finer sensing Fig. There is a period of time when regenerating worms unlike intact worms can sense light but cannot show clear behavioral choices in binary wavelength assays.
Thus, the effective intensity—based acute gradient sensing is layered on a basic ability to detect light, with a progressive buildup of sensing and processing capacity. This reveals that functional recovery continues much after gross regeneration of the eye-brain structures is complete.
Full functional recovery likely requires fine-tuning of sensing and processing capabilities, involving pruning and patterning of the neural networks. Regeneration data support our model that acute sensing likely involves significant post-sensory comparative processing. Confocal imaging of planarian brain using a synaptic marker over regeneration is consistent with progressive buildup of neural capacity Fig.
Although gross regeneration of basic brain structures appears to be complete by about day 5 after decapitation, significant enrichment of synaptic density coincides with the recovery of acute sensing and processing abilities. A Schematic showing a timeline of return of different phototactic abilities during head regeneration in S. By day 5 after amputation, worms sense light but have no ability to finer intensity discrimination assayed through wavelength choice , which is acquired gradually significantly later.
See B for ocular control. B Model for hierarchical relationship between networks regulating ocular and extraocular sensing, including switching during regeneration. During days 1 to 4 after amputation, worms show only extraocular response anchored in ventral nerve cord VNC and peripheral neurons.
By about day 5, ocular response recovers in worms, but signaling flux from cerebral visual networks is unable to override signals resulting from extraocular photosensing. Ocular and extraocular photosensing coexist, but extraocular sensing is dominant in competition assays. Day 7 onward, flux from visual network strengthens, allowing the ocular response to be dominant, as in intact worms. Black arrow indicates visible light input, whereas violet arrow indicates long UV light input.
Existence of two independent light sensing networks in planarians, along with eye-brain regeneration capacity, again allows unique lines of inquiry. For intact worms, in competition experiments, the cerebral ocular response can override the reflex-like extraocular response, setting up a baseline hierarchy among the responses Fig.
Ocular-extraocular choice experiments during regeneration reveal remarkable plasticity in these hierarchies. Thereafter, following a clear transition, the dominance of the ocular or brain-mediated response is reestablished Figs. If the extraocular response is a more rudimentary, ancient response predating the eye and a processive brain, then does regeneration coincidently mimic aspects of evolutionary trajectories?
Ocular-extraocular choice experiments over regeneration also provide new insights into how the eye-brain network interacts with the whole-body light sensing network Figs. A headless worm can undergo locomotion in response to UV. By about day 5 in regeneration after head removal , a cerebral eye and a minimal brain are established, which link to the ventral nerve cord, whole-body nervous system, and the locomotion machinery.
This link of the visual network to the locomotor network is sufficient to generate movement in response to light. However, it appears that the signaling flux from the cerebral eye and brain at this point is still insufficient to override the extraocular or reflex-type response anchored in the whole-body neural network. After day 7, we propose that this signal from the eye-brain to the whole-body system strengthens, allowing overriding of the extraocular response Fig.
Appearance of a threshold wherein brain-mediated response begins to dominate a whole-body response has implications for how the eye and the cephalic ganglion regenerate and how connectivity between two distinct neural networks, the cephalic ganglion and the ventral nerve cord in this case, is established and fine-tuned during regeneration.
To our knowledge, this may be the first report of its kind, describing such clear and dynamically interchanging hierarchies between two distinct light sensory systems, all revealed through relatively simple phototactic choice assays performed over regeneration.
In summary, this study significantly affects our fundamental understanding of light sensing responses. Our work highlights the remarkable diversity and plasticity in light sensing and processing that remain unexplored in nature. Because planarians have eye structures and neural networks that appear simple yet similar to those observed in other animals, these acute and distinct light sensing and processing abilities may be more widespread in nature.
This work also sets the stage for a comprehensive examination of light-induced behavior in flatworms wherein new questions based on comparative visual processing and regeneration of neural networks can now be addressed. Our results demonstrating the interplay between ocular and extraocular light sensing networks are likely without precedent and reveal the rich and multilayered light responses in planarians that can now be examined further. Each LED is referred by its peak wavelength in the study.
This allowed precise control of LED light intensities. S11 was also used for extraocular-mediated behavior experiments. Light power measurements were made using Newport power meter R and D detector. For the experiments involving neutralization and overturning of wavelength choice, the unit of light measurement and intensity was photon flux or photon fluence rate number of photons per unit time per unit area.
This was done to ensure consistency with the theory of action spectroscopy and to build a wavelength-dependent response function using light intensity required for neutralization of choice behavior.
The stability of the signal was checked for the duration of the experiment in all cases. Worms were fed beef liver extract once in 3 days. Worms were starved for at least 2 days before the start of any experiment. Earlier work shows that planarians respond broadly to visible light, with subtle differences in response to colors Using single-wavelength phototaxis assays, we confirmed that planarians S.
The setup for the single-input, light-dark phototaxis has been illustrated in figs. S2A and S Planarians were starved for at least 2 days for all phototaxis experimentation on intact or regenerating worms.
About 5 ml of medium was added to the slide, and a single planarian was placed in the center region R2, 10 mm wide; fig. S2A using a Pasteur pipette. After 2 min, the region fig. S2A in which the planarian is present is determined.
This is repeated for several worms, and a DI is calculated. Phototaxis during regeneration was also performed as described above, except that the position of the worm was determined after 3 min. All planarian choice assays were performed according to the schematic shown in Fig. LED lights were used as above. Similar to the single-input phototactic assay described above, each worm was placed in the center region and allowed to make a choice with a cutoff time of 2 min.
Outcomes can be movement to region 1, region 2, or no choice. Similarly, binary choice experiments were performed during regeneration, and the position of the worm for calculation of DI was determined after 3 min. For experiments testing overturning of wavelength choice through light intensity dosage Fig.
Increasing dosage of the less-aversive input was provided as 2 X , 4 X , or 8 X as indicated in the figures. For building a behavioral response profile to address intensity-based wavelength choice neutralization Fig. Choice neutralization experiments were performed with , , , and nm as second wavelengths. According to the principle of a classical action spectroscopy, at the point of choice neutralization, the photoresponse R ; proportional to the product of extinction coefficient and number of photons incident for the two inputs should be identical 33 and can be used to build an action spectrum.
As described above, the photon fluence rate at nm was kept constant 1. For determining wavelengths that affect extraocular phototaxis, planarian tail pieces 24 hours after decapitation; fig. S2B were subjected to phototaxis assays similar to the single-input phototaxis assays. We measured the extraocular response using LEDs that showed their peak response at the following wavelengths from to nm.
To examine the dosage light intensity dependence of extraocular photoreception Fig. A 1-cm light circle was made to fall on the worm, and the time it took the worm to escape from the circle of light was calculated and plotted against the intensity of light used. To understand the attenuation of extraocular response on the visible side of the spectrum, we performed photoavoidance experiments with , , and nm wavelengths at a photon flux of 2.
Similarly, to study the ocular-extraocular choice over regeneration, we subjected anterior regenerates tail-forming head and posterior regenerates head-forming tail to similar binary phototactic choice assays over a period of 8 days after head amputation.
The heads of asexual planarians 5 to 10 mm in length were cut and processed for FISH as per the protocol described by Pearson et al. The reduction step after fixation was omitted, and the worms were stored in methanol after dehydration.
Bleaching was performed for 2 hours using a formamide-based solution 1. The post-hybridization washes, antibody blocking, incubation of peroxidase-conjugated anti-digoxigenin antibody, and post—antibody incubation washes were performed as per the protocol by King and Newmark The development of the fluorescent signal was performed using a tyramide signal amplification TSA reaction as per the protocol by King and Newmark The tyramide conjugates were synthesized according to the protocol described by Hopman et al.
For experiments involving immunostaining followed by FISH, the protocol mentioned below was followed.
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