Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye–brain-independent movement and arousal

Significance

This study highlights the breathtaking sophistication of form and function possible with eye-independent light-sensory systems. We have discovered a body-wide sensory organization of unique photoreceptor cells that allows even head-removed flatworms to move like intact animals, revealing the mechanistic framework underpinning one of the most sensitive eye–brain-independent photoresponses known. Distinct from the ocular system, the body-wide sensory array matures in adult-like animals, can trigger arousal of intact animals from a “resting-state” and employs “noncanonical” opsins. Our discovery of a body-wide network of photoreceptor cells triggering coordinated movement is intriguing and conceptualizes how “dispersed”-sensory nodes may network to control outputs typically through a centralized brain. Our work illustrates how eye-independent systems can deeply influence animal physiology and behavior.

Abstract

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye–brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin–expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in “adult-like animals.” Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

Light sensing has independently evolved multiple times and has profoundly shaped life. The ability to process light information in distinct ways and respond to a changing light environment can dramatically shape physiology and fitness of life forms. Movement, triggered by light, is one of the most fundamental responses in nature (1). Among metazoans, a wide variety of animals are known to show coordinated motion in response to light stimuli. So far, this is overwhelmingly known to be mediated through the animal eyes. In fact, eye-driven light sensing and taxis has been extensively studied across phyla. Interestingly, motion in metazoans can also be mediated through eye-independent or extraocular (EO) photoreception (2–5). However, our conceptual and mechanistic grasp on how coordinated movement can be triggered and controlled through EO light-sensing systems is extremely limited. Moreover, the few prominent examples of EO phototaxis have all been reported in life forms/developmental stages completely lacking eyes or possessing only rudimentary eyes (2, 5). Almost nothing is known about sensitive EO light-sensing systems capable of triggering coordinated motion that may coexist with sensitive eyes in a single organism.

There are intriguing reports of photoreceptor molecules that are expressed in locations other than conventional eyes, including in unusual structures seen in polyclad flatworms, clitellate segmented worms, crustaceans, cephalopods, and fishes. However, the functions of such structures remain elusive (6–13). A single organism may indeed possess multiple, independent light-responsive systems, both eye based as well as eye independent (13–17), but the functions rarely overlap. “Nonvisual”/EO sensory systems like pineal glands and deep-brain photoreceptors across vertebrates and retinal ganglion cells in mammals (18–21) have been reported. However, these sensory systems are generally known to perform “nonvisual” functions like maintaining circadian rhythms and modulating behavior (22–25). Here, we report an EO phototactic network that can independently trigger coordinated movement just like what the eye-based (ocular) system can, while also having its own distinctive role even when the eyes are present.

Do highly sensitive EO phototactic systems coexist and function in life forms that have prominent eye-based networks as well? How would such a system operate? What would be the mechanistic framework and the functional consequence of such an eye-independent light-sensory system? Planarian flatworms offer a fascinating opportunity to explore such a paradigm. Planarians are highly light aversive and have well-developed ocular cerebral eyes (eyes connected to a centralized ganglion) that process light stimuli and guide behavior like feeding, escape, and predation (26–30). In fact, the planarian ocular network is highly sensitive and capable of surprisingly complex processing (17). These eye-mediated behaviors are reliant on an organized, cephalized bilobed brain, a prominent example of a “primitive” brain in evolution (17, 31, 32). Indeed, the brain is required for most locomotive behaviors including thermotaxis, chemotaxis, and eye-mediated phototaxis including the ability to discriminate closely related light stimuli, shown by these animals (17, 32–34). However, planarians also show dramatic, eye–brain-independent light-induced movements (17, 35). Even after sudden decapitation (removal of both eyes and brain), worms are able to acutely respond to ultraviolet-A (UV-A) light and show seemingly coordinated movement away from light (17, 35). While such eye–brain-independent behavior has long fascinated biologists, almost nothing is known about how this dramatic behavior is mediated (17, 35–39). It is also not clear what would be the physiological role of such an acutely sensitive EO sensory network capable of triggering coordinated movement, especially since planarians do have a well-developed ocular network.

Here, we show how such an acute response to light is mediated by an organism removed of its “primary” light-sensory organ and brain. We report the discovery of photoreceptor molecules as well as a widespread but organized network of photoreceptor cells that are required for this acute eye-independent UV-A light response. Intriguingly, this entire multiscale sensory system from photoreceptors to the network of cells arises and matures postembryonically, in an “adult-like” organism. This developmental trajectory is distinct from that of the cerebral eyes, which develop embryonically. We also demonstrate that while both the eyes as well the EO network led to coordinated movements relying on the same locomotion machinery, the physiological consequences of the ocular and EO sensory responses can be divergent. Intact planarians periodically go into “sleep-like” resting phases, in which their activity diminishes and sensory perception reduces (40). Strikingly, we find that the EO sensory system in these intact animals can override the natural “rest”-activity cycles and is able to acutely photoactivate and arouse even resting worms. This is distinct from the ocular network that becomes dormant during the “rest phase.” Our work illustrates an unprecedented level of organization and complexity in form and function of an acutely sensitive EO light-sensory system that matures and functions in parallel to the ocular network.

Results

Head-Removed Planarians Can Engage Distinct Ciliary and Exclusively Muscle-Based Locomotory Modes for Coordinated Motion away from UV-A Light.

Head-removed planarians are able to move away from UV-A light (Fig. 1 A and C and Movie S1), responding to doses as little as 31 µW/cm² but the mechanism is unknown (17). This whole-body light reception (EO) is distinct from eye-based (ocular) photoreception (including distinct wavelength range) and relies on hitherto unknown photoreceptors (Fig. 1B) (17). Intact planarians typically show a gliding motion using a combination of cilia and muscle activities (41–45). Since there are prior examples of local light sensing and actuation of locomotion autonomously originating at ciliary structures (locally acting photoreceptors) (46, 47), we examined planarian EO photoresponse after ciliary removal. This was achieved through RNA-interference (RNAi) mediated knockdown (KD) of IFT88 (Intraflagellar transport protein) (41). Interestingly, decapitated worms (head-removed) lacking cilia (IFT88 KD) show a distinctive contraction-relaxation type locomotion on illumination with UV-A light (Fig. 1E and Movie S2). Similarly, this distinctive contraction-relaxation type of locomotion is also observed if cilia are inhibited (using serotonin receptor-antagonist mianserin hydrochloride) in head-removed planarians and subjected to UV-A photoillumination (Fig. 1D and Movie S3). Interestingly, on cilia removal even intact planarians show a similar switching from a gliding-type locomotion to contraction-relaxation (peristaltic) locomotion solely relying on the muscle network (41, 43, 44). These results here eliminate the possibility that this EO photoresponse is mediated by an autonomous cilia-associated, localized light-sensing mechanism. Furthermore, results here reveal that the planarian EO sensory system (similar to the eye–brain ocular network) has the ability to recruit both ciliary and muscle-based motor systems for coordinated movement (Fig. 1F).

Fig. 1.

Head-removed planarians are capable of coordinated movement and engaging distinct ciliary- and muscle-based modes of locomotion. (A) Intact planarian flatworms typically rely on their dorsal ganglia (brain) to mediate behaviors such as chemotaxis, thermotaxis, and phototaxis, including complex sensing and discrimination of closely spaced light stimuli (17, 32–34). However, even after head removal, planarians are able to show eye–brain-independent, highly coordinated EO photoresponse on UV-A stimulation. (B) Planarian eye-dependent (ocular) and eye-independent (EO) light responses have distinct spectral signatures. Intact S. mediterranea respond to a broad wavelength range (∼365 to 625 nm) through the ocular (eye–brain) sensory network, while showing an eye–brain-independent, whole-body EO response to UV-A light (∼365 to 395 nm) (17). (C) Randomly oriented head-amputated worms show a coordinated directional movement away from a 395 nm light source, also reference Movie S1. (D) Mianserin hydrochloride treatment (60 μM) results in a switch from ciliary- to muscle-based motion in a head-amputated planarian tail piece showing EO response. The regions highlighted with colored squares show muscular contraction, and the dotted yellow arrow indicates the directional peristaltic muscular waves, also reference Movie S3. (Scale bar, 500 μm.) (E) UV-A stimulated head-removed planarians show gliding movement away from light, mediated by a combination of beating cilia supported by underlying muscular activity, similar to intact worms. Strikingly, absence/disruption of cilia (through RNAi KD of protein IFT88, reference Movie S2) or inhibition of the ciliary neural network using mianserin hydrochloride leads to the head-removed worms switching to a distinctive wholly muscle-based locomotion. UV-A–stimulated headless tail pieces are able to move with alternate contractions and relaxation (peristaltic activity) of muscle networks when cilia are removed or disrupted. This is similar to movement modes seen with intact worms. Therefore, even in the absence of a brain, EO photosensing is sufficient to trigger directional coordinated movement in planarian tail pieces through distinctive alternate modes of locomotion. (F) Suggested framework for EO photosensing and response in planarians (44, 48, 49).

Two Noncanonical Rhabdomeric Opsins with Distinct but Overlapping Expression Patterns Mediate Whole-Body EO Photoresponse.

We next focused on the identity and expression of putative photoreceptor molecules underpinning EO whole-body photoresponse. In Caenorhabditis elegans and Drosophila, unusual gustatory receptor related–like photoreceptors have been implicated in EO phototaxis (2, 3, 50). However, no such receptor homologs were found in planarian transcriptome (17). With opsins being the most diverse animal photoreceptor proteins (51) and previously associated with EO photoreception (4, 52), we attempted to bioinformatically identify all planarian (Schmidtea mediterranea) opsins based on known opsin molecular motifs (SI Appendix, Table S1). We found that planarians have five opsins. In order to determine the phylogenetic history of these planarian opsins, we performed the molecular phylogenetic analysis by adapting a method described earlier (53)(Fig. 2A and Dataset S1). This analysis of planarian opsins along with other opsins from a recent animal opsin database (53) showed that planarians have one canonical rhabdomeric (R)-opsin (Smed-eye opsin), one xenopsin (Smed-xenopsin), one peropsin (Smed-RGR/peropsin), and two other noncanonical R-opsins (Smed-NC R-opn 1 and 2). The canonical R-opsin (Smed-eye opsin) was previously reported to be expressed in the eye (17, 54), but the expression of the other opsins was not known. Now, the “spatial” expression profile of all planarian genes has previously been studied, with transcripts localized to thin “salami” sections of the entire body (55, 56). Notably, the analysis of planarian salami-sections transcriptome data (55, 56) revealed that three out of five opsins showed body-wide expression (Fig. 2B). Smed-eye opsin and Smed-xenopsin did not show any expression in rest of the body and were restricted to the head salami sections (Fig. 2B).

Fig. 2.

Phylogenetic classification and body-wide expression profiles of planarian opsins. (A) Phylogenetic tree of 5 planarian (S. mediterranea) opsins along with other 792 opsins showing 9 opsin paralogs across all animal opsins, using methods described in Ramirez et al. (53). Reference SI Appendix for further details. The complete uncollapsed tree along with gene accession number and the node support bootstrap values can be found in Dataset S1. The nine different opsin paralogs are indicated by different colors, and the outgroup is indicated in gray. Out of the five planarian opsins, one belongs to canonical R-opsin (Smed-canonical R-opsin), and it is known to be expressed in the planarian eye. Furthermore, one xenopsin (Smed-xenopsin), one RGR/Peropsin (Smed-peropsin), and two other NC R-opsins (Smed-NC R-opsin 1 and 2) were identified and classified. The planarian opsins are indicated by colored arrow heads in the collapsed tree, and the color indicates the specific opsin paralog. (B) Spatially binned expression pattern of planarian opsins across the planarian body. Opsin transcript levels across planarian “salami” sections (indicated with dotted black line) are plotted. Three out of five planarian opsins show body wide expression. Data from refs. 55 and 56.

The three opsins showing body-wide expression in the planarian S. mediterranea were further examined for a functional role in EO UV-A photoresponse. Individual RNAi-mediated KD of either NC R-opn 1 or 2 significantly attenuated the EO UV-A photoresponse at 395 nm. NC R-opn 1 or 2 opsin (single-opsin) KD worms were either unresponsive or showed significantly reduced movement when stimulated with UV-A light (395 nm). On the contrary, KD of Smed-peropsin was not observed to affect the EO photoresponse (Fig. 3 A and B). To address specificity of the KD phenotype, we allowed the NC R-opn 1 and 2 KD worms to regenerate their head (brain and eyes) and examined their ability to respond to visible (500 nm) light (SI Appendix, Fig. S1A). Notably, after head regeneration, either NC R-opn 1 or 2 KD worms are able to move away from light as efficiently as the control worms (SI Appendix, Fig. S1 B and C). Contrastingly, RNAi-mediated KD of the Smed-eye opsin in worms undergoing anterior regeneration leads to a clear defect in phototactic response to visible (500 nm) light, consistent with its role in ocular phototaxis (17). Furthermore, to confirm that the loss of EO photoreception phenotype persisted even after the worms had been subjected to visible (500 nm) light stimuli and phototaxis assay, we reamputated the newly regenerated head and again examined response to UV-A in decapitated worms (SI Appendix, Fig. S1A). Again, either NC R-opn 1 or 2 KD head-removed worms showed a persistent reduction in EO phototactic response (SI Appendix, Fig. S1D), confirming that the NC opsin KD experiment truly presented a specific EO sensing defect. Taken together, RNAi experiments suggest that there are two NC R-opsins that are responsible for EO light sensing in planarians.

Fig. 3.

Two NC R-opsins mediate EO phototaxis and express in two distinct, spatially patterned populations of cells in the planarian body. (A) Schematics showing protocol used for RNAi KD to test for the role of specific opsins in eye–brain-independent EO photoreception. (B) Plots showing time taken by individual head-amputated planarians to move out of 1 cm spot of 395 nm (UV-A) light. Data shown for head-removed worms after RNAi KD of either Smed-peropsin (n = 35), NC R-opn 1 (n = 50), or NC R-opn 2 (n = 50) as compared to respective controls. Maximum time allowed for worms to move out of 1 cm UV-A spot was 300 s, after which they were considered unresponsive. Error bar indicates SEM. Two-tailed, unpaired t test. ***P < 0.0001 (test of significance performed only when SD (SD) ≠ 0, if SD = 0 then results were interpreted based on the difference observed). (C and D) Spatial expression patterns of NC R-opn 1 (C) or NC R-opn 2 (D) examined through FISH followed by confocal imaging (images are representative of results seen in at least 20 animals). Image was captured by imaging two regions of interest of the same animal at 40× magnification and stitched during acquisition by zeiss zen software. (Scale bar, 100 μm.) (E) Dual FISH-based confocal-imaging analysis reveals that both NC R-opn 1 and 2 are coexpressed in an array of single, periodically spaced peripheral cells. Representative confocal images from a double FISH experiment from a peripheral region (as shown in the schematic) shows individual cells expressing NC R-opn 2 (green, marked with yellow dotted line) also express NC R-opn 1 (magenta, marked with yellow dotted circle). On the flip side, all NC R-opn 1–expressing cells do not express NC R-opn 2, with NC R-opn 1–expressing cells comparatively more widespread across the planarian body (SI Appendix, Fig. S4). Even at the periphery, we observe cells that exclusively express NC R-opn 1 but not NC R-opn 2 (marked with white dotted circle). Images are representative of results seen in at least five animals. (Scale bar, 10 μm.)

A comprehensive phylogenetic analysis shows that the two body-wide opsins identified here belong to a distinct diverged branch in the R-opsin class: NC R-opsins (Dataset S1 and SI Appendix, Fig. S2). While the canonical R-opsins have long been known as the principle photoreceptors in the invertebrate eye, nothing definitive is known about the function of this newly identified group of NC R-opsins (53). Here, we offer direct evidence of two NC R-opsins functioning as photoreceptors in acute, eye–brain-independent light responses.

We performed fluorescence in-situ hybridization (FISH) experiments to address the expression pattern of the two body-wide EO photoreceptors (NC R-opn 1 and 2). FISH combined with confocal imaging experiments on NC R-opn 1 showed that this opsin is highly expressed in cells close to both the dorsal and ventral surfaces as well as certain cells at the periphery (Fig. 3C). In contrast, NC R-opn 2 prominently showed expression only in specific cells at the periphery of the organism (Fig. 3D and SI Appendix, Fig. S3).

FISH imaging initially appeared to identify two distinct populations of cells that could contribute to the EO photoresponse. Intriguingly, “double FISH” experiments to examine coexpression of NC R-opsins in the same worm revealed a surprise. Cells in the periphery of the animal that express NC R-opn 2 also expressed NC R-opn 1 (Fig. 3E). We verified this by extensively analyzing images acquired from different locations in the worm (SI Appendix, Fig. S4). Cells expressing both NC R-opsins 1 and 2 are found below the epidermis and are present as distinct single cells at the periphery of the worm body. These dual-opsin cells are clearly different from single-opsin (NC R-opn 1)–expressing cells that may also be found in the same vicinity (SI Appendix, Figs. S4 and S5A). Therefore, there are indeed two distinct cell populations identified here. One population of cells present across both the dorsal and ventral subepidermal region across the planarian body expresses only one opsin, NC R-opn-1, while another cellular population that dots the periphery of the organism expresses both NC R-opn 1 and 2.

Array of Peripheral Cells Expressing Two NC R-Opsins Mediate EO Photoresponse.

Which of the two cellular populations (SI Appendix, Fig. S5B) identified here are responsible for mediating the EO photoresponse? Lack of transgenesis and limited genetic tools in planarians make this a challenging question to address. However, recent availability of single-cell transcriptomic data in planarians provided a unique opportunity (57, 58). Our analysis of two independently generated single-cell transcriptome datasets indicated that NC R-opn 1 is expressed primarily in body-wide “pigment cells” (SI Appendix, Fig. S6 A–E). Pigment cells are present in the subepidermal regions and are the cells solely responsible for skin pigmentation (59, 60). We showed pigment-cell expression of NC R-opn 1 by performing “double FISH” imaging experiments, examining spatial coexpression of NC R-opn 1 with a known pigment cell marker, Smed-lysoLP-1 (61). Imaging data confirms that NC R-opn 1–expressing cells were observed to express Smed-lysoLP-1 (SI Appendix, Fig. S6F).

A recent study on planarian pigment cells showed that these cells could be ablated through low light exposure with visible light (60). Interestingly, analysis of the available transcriptomic data from such depigmented animals showed an ∼twofold down-regulation of NC R-opn 1 expression but no change in NC R-opn 2 expression levels upon loss of pigment cells (61). FISH imaging experiments performed after light-induced ablation of pigment cells showed no change in the spatial expression pattern of NC R-opn 2 (Fig. 4 A and B). Strikingly, however, the expression of NC R-opn 1 in such worms is only limited to distinct peripheral cells (SI Appendix, Fig. S7). No body-wide expression in the dorsal and ventral subepidermal region is retained, vis-a-vis control worms. In fact, in worms that have lost pigment cells, NC R-opn 1 expression begins to resemble that of NC R-opn 2 (SI Appendix, Fig. S7). Overall, our experiments collectively indicate that cells that exclusively express NC R-opn 1 are indeed pigment cells while another distinct peripheral population of cells express both the NC R-opn 1 and 2 (Fig. 4E).

Fig. 4.

Peripheral cell population expressing both NC R-opn 1 and 2 is responsible for EO photoresponse in head-removed planarians. (A) Prolonged low-level light exposure gives rise to depigmented planarians (De-P) through an ablation and loss of pigment cells. Shown here are the representative transmitted light images of pigment control (P) and depigmented (De-P) worms. (Scale bar, 500 μm.) (B) Light-induced ablation/loss of pigment cells does not affect the expression of NC R-opn 2 in peripheral opsin-expressing cells in the planarian midplane (see schematics, Far Left, Right). Shown are confocal micrographs of NC R-opn 2 expression analyzed through FISH after ablation of pigment cells (De-P) compared to control, pigmented worms (P). Images are representative of results seen in at least five animals. (Scale bar, 100 μm.) (C) Head-amputated planarians show an enhanced EO UV-A photoresponse after ablation of NC R-opn 1–expressing pigment cells (De-P), compared to their pigmented controls (P). Data showing time taken by head-removed worms to move out of a 395 nm light spot after photoillumination (n = 16). (D) KD of NC R-opn 1 attenuates EO photoresponse even in planarians subjected to ablation of pigment cells. While the pigment cells expressing NC R-opn 1 are ablated (SI Appendix, Fig. S7), it is the NC R-opn 1 expressed in peripheral cells that likely contributes to EO photoresponse. Data showing time taken by head-amputated, pigment-cell ablated (De-P) planarians to move out of a 395 nm light spot, with either additional KD of NC R-opn 1 or control treatment (n = 15). Error bars indicate SEM. Two-tailed, unpaired t test. ***P < 0.0001. (E) Schematics showing the overall expression pattern of two distinct populations of cells expressing the NC R-opsins in pigmented as well as pigment cell-ablated worms. There is a body-wide expression of single-opsin (NC R-opn 1)–expressing cells. Pigment-cell ablation also leads to a loss of single-opsin–expressing cells (SI Appendix, Fig. S7). There is a second population of cells lining the periphery of the worm, wherein NC R-opn 1 and 2 are coexpressed. These dual-opsin–expressing cells are not affected by pigment-cell ablation, and the depigmented worms show enhanced UV-A photoresponse.

Are the peripherally located cells expressing both NC R-opn 1 and 2 the elusive photoreceptor cells responsible for the dramatic eye-independent light responses? To address this, we tested the response of worms that have lost NC R-opn 1–expressing pigment cells (light-induced pigment-cell ablation) to UV-A stimulation. Strikingly, not only did such worms show a clear response to 395 nm UV-A light, the photoavoidance response was significantly stronger. Worms lacking pigment cells expressing a single opsin (NC R-opn 1) were able to move out of UV-A light spot significantly faster compared to controls (Fig. 4C). This suggests that pigment cells may act like a shield for UV light and upon their removal, more UV light reaches EO phototactic receptors leading to this enhanced photoresponse. Our results also indicate that even though pigment cells express opsin NC R-opn 1, these cells may not contribute to the EO phototaxis shown by head-removed planarians.

Taken together, our results (including robust whole-body photoavoidance in worms lacking pigment cells, Figs. 3E and 4E) now indicate that single cells organized along the periphery of the worm body are responsible for body-wide EO photoresponse. Consistent with the peripheral cells being the EO photosensory array in planarians, a physical ablation of the worm periphery completely attenuates any EO photoresponse to low-dose UV-A (63 to 69 µW/cm²) stimulation (Movie S4). Furthermore, localized UV-A stimulation of the periphery is sufficient to produce localized peripheral contraction (Movie S5) again consistent with idea of a peripheral sensory array mediating UV-A EO photoresponse.

Does NC R-opn 1 still contribute to EO photoresponse in worms where pigment cells have been ablated? This was tested by RNAi-mediated KD of NC R-opn 1 in pigment-cell ablated worms. Head-removed worms lacking pigment cells show a clear attenuation of EO response to UV-A light on KD of NC R-opn 1 (Fig. 4D). The contribution of both the NC R-opsins is further seen from the wavelength-dependent behavior of opsin KD worms. At 395 nm (5.03 × 10¹⁴ photons cm⁻² s⁻¹), the KD of either NC R-opn 1 or NC R-opn 2 is sufficient to abolish EO photoresponse in head-removed worms (Fig. 3C and SI Appendix, Fig. S8). However, at 340 nm (5.03 × 10¹⁴ photons cm⁻² s⁻¹), it requires the KD of both NC R-opn 1 or NC R-opn 2 to abolish the EO photoresponse. The behavior at 365 nm (5.03 × 10¹⁴ photons cm⁻² s⁻¹) is intermediate between 395 and 340 nm (SI Appendix, Fig. S8). Overall, our results indicate that both NC R-opn 1 and NC R-opn 2 expressed in peripheral photosensory cell populations contribute to EO photoresponse, with an additive effect seen at certain UV-A light wavelength.

How would an array of opsin-expressing cells lining the periphery of the worm mediate coordinated movement on acute light stimulation? Intriguingly, we find that the opsin-expressing cells are juxtaposed with the body-wide nerve net that runs across the worm. Flatworms are unique in having evolved a more centralized (cephalized) nervous system comprising a brain and a pair of ventral cords, while still retaining a more diffuse nerve net reminiscent of that seen in Cnidarians (62–66). The planarian nerve net has also been described as a subepidermal and submuscular plexus and eventually connects to the pair of ventral nerve cords (64–66). To examine the location of photoreceptor cells relative to the nervous system, we screened multiple antibodies and identified a G-α11 antibody that stains both the nerve net as well as the centralized nervous system in planarians (SI Appendix, Fig. S9 A–C). Through dual-antibody FISH staining and confocal imaging, we find that the NC R-opn 2–expressing cells are consistently proximal to the body-wide nerve net (SI Appendix, Fig. S9D). This opens the possibility that the opsin-expressing sensory array may co-opt the nerve net along with the ventral nerve cords to transduce light-mediated coordinated motion.

Distinct from Development of the Eye–Brain Ocular Network, Functional Maturation of the EO Light-Sensing System Is Postembryonic.

Our work confirms the coexistence of at least two distinct but interlinked light-sensing networks with separate photoreceptors and sensory organization. Generally, EO photoreception is thought to be a primitive form of light sensing compared to the eye (67). Since development can offer a window into evolutionary trajectories, we asked how the developmental time scales for the eye and body-wide array of sensory cells compare. For this, we collected newly hatched planarians (“hatchlings,” ∼14 d postegg deposition) and examined light sensing. Interestingly, hatchlings (even on the day of hatching) have well-developed eyes and show a clear response to visible (500 nm) light, similar to adults (Fig. 5A) (17). Newly hatched worms move toward dark when stimulated with 500 nm light. More so, newly hatched worms are even able to discriminate between closely spaced visible light stimuli, through their cerebral eyes. We have previously shown that just an ∼25 nm change in visible-light (ocular stimuli) wavelength completely switches behavior in adult worms (17). Similarly, hatchlings also show such wavelength-dependent photoswitching. When presented with two light stimuli (500 and 545 nm), newly hatched worms are able to show clear binary choices, similar to that seen with “adult” worms (Fig. 5B). Therefore, newly hatched worms possess a developed eye–brain network capable of acute sensing and comparative processing. However, when newly hatched worms are decapitated, tail pieces are unable to respond to UV-A (395 nm) light stimuli, showing that the EO light sensing is not functional in newly hatched worms (Fig. 5 C and D). Interestingly, after 1 wk of development posthatching, juveniles appear to develop EO photoreception and respond. Head-removed juveniles (1 wk posthatching) are able to sense and respond to UV-A stimuli, and this EO photoresponse further strengthens until 4 wk posthatching (Fig. 5 C and D). We then examined if this postembryonic appearance of EO-sensing behavior coincides with the maturation of the cellular network underpinning the response. FISH analysis and confocal imaging shows that newly hatched planarians do not appear to have a substantial presence of NC R-opn 2–expressing cells (Fig. 5E). This is consistent with lack of EO sensing response. However, FISH and confocal imaging reveals a clear appearance of peripheral NC R-opn 2–expressing cells over 2 wk posthatching. Again, this appearance and maturation of the EO cellular network coincides with the strengthening of EO-sensory behavior (Fig. 5E). This is indeed striking, showing clearly that the ocular- and EO-sensory networks develop across very distinct timescales. Our work shows that the body-wide EO light-sensing network undergoes significant maturation in an adult, acquiring functional maturation only weeks after hatching. On the other hand, the cerebral eye (eye–brain ocular system) is functional at the time of birth.

Fig. 5.

Distinct from cerebral-eye development, functional maturation of the EO light-sensing system is postembryonic. (A) Freshly hatched worms (“hatchlings,” white arrowhead indicates eyes) show a functional, eye-mediated visible (500 nm) light-mediated phototactic response. “Hatchlings” were scored for ability to discriminate a visible light spot (500 nm light input) from dark (n = 20 worms). Plotted is the discrimination index (D.I.) based on the final location of the worms when subjected to a “choice” between a 500 nm spot of light and dark. The percentages of the worms in regions R1 (500 nm), R2 (“no choice”), and R3 (dark) at the end of the assay are also tabulated along with the schematic of the binary-choice assay. (B) Newly hatched worms (“hatchlings”) are able to discriminate closely matched ocular light stimuli similar to the intact worms. In binary-choice assays, adult planarians are able to show clear choices when presented with light inputs of distinct wavelengths, indicative of eye–brain-mediated acute light-gradient sensing and comparative processing (17). Here, newly hatched worms are examined for such ability. Plotted is the response of worms when subjected to two equal photon-flux light inputs of 500 and 545 nm (n = 67 worms). The percentages of the worms in regions R1 (500 nm), R2 (“no choice”), and R3 (545 nm) at the end of the assay are displayed along with the schematic of the binary-choice assay. (C) Schematic representing the experimental design to address postembryonic EO light sensing in newly hatched worms (hatchlings) as well as in “juvenile” worms 1, 2, and 4 wk posthatching. (D) Functional maturation of the EO light-sensing system is postembryonic. Newly hatched planarians (“hatchlings”) fail to respond to UV-A post-head removal. Data shows time taken by either newly hatched worms (“hatchlings,” n = 10) or “juvenile” worms (1, 2, or 4 wk posthatching; n = 7, 10, 10, respectively) to move out of a UV-A (395 nm) spot, after head removal. Error bar indicates SEM. ***P < 0.0001. (E) Gradual, postembryonic appearance of peripheral EO photoreceptor cells in “juvenile” planarians. Shown are confocal micrographs of worm tail pieces, following FISH analysis of NC R-opn 2 expression. Micrographs show either freshly hatched worms (hatchlings) or juvenile worms 1 and 2 wk posthatching. Violet squares highlight the peripheral NC R-opn 2–expressing EO photoreceptor cells. Images are representative of results seen in at least five animals. (Scale bar, 100 μm.)

UV-A Arousal of Resting Worms Shows EO System but Not the Eye Can Override the Natural Rest–Activity Cycle of the Animal.

Highly sensitive EO light sensing may significantly influence the physiology, life cycle, behavior, and evolutionary trajectory of an organism. Since EO photoreception matures postembryonically after the complete development of the eye–brain-mediated ocular system, can this photoreception distinctively influence physiology of intact worms? To address this, we examined the behavior of worms over time. We observed that planarians during prolonged resting periods go into a physiologically inactive or a “resting” state; this is consistent with a recent report in this regard (40). Interestingly, during this inactive or “sleep”-like state in intact worms, worms do not respond when stimulated with 500 nm light (Fig. 6A and SI Appendix, Fig. S10). This is completely distinct from the behavior of active worms, which are reported to be highly light aversive (17, 32, 38). This implies that the planarian eye–brain-based ocular network fails to respond to visible (500 nm) light stimuli, when in the inactive state. This is consistent with the inactive state as being a “sleep-like” state with reduced sensory responsiveness (68). Notably, however, when “inactive” or resting intact worms are stimulated with UV-A (395 nm, equal or lesser photon flux than 500 nm) “extraocular” input, worms respond instantaneously and move away from the light source (Fig. 6A and SI Appendix, Fig. S10). This finding is significant and suggests that the EO light-sensing network has the ability to bypass the physiologically inactive state and respond to light, whereas the eye–brain-mediated ocular network stops functioning during the period of inactivity. We then asked if the UV-A photostimulation of intact resting worms is indeed dependent on the NC body-wide photoreceptors we have identified and if this is independent of the eyes. We first confirmed that an RNAi-mediated KD of OVO, a master regulator of eye patterning and development/regeneration (69) fully removes the eye, and these eyeless worms are still able to go into periods of rest/inactivity, just like control worms (SI Appendix, Fig. S11). Similarly, NC R-opn 1 and 2 dual-KD intact worms are also able to move into the resting/inactive state. Furthermore, either eye removal (OVO KD) or KD of both NC EO opsins does not change the insensitivity of inactive worms to visible (500 nm) light stimuli. Like control worms, eyeless as well as NC R-opn 1 and 2 dual-KD worms show no response to 500 nm light when resting (Fig. 6B). On the contrary, while UV-A is able to acutely photostimulate control or eyeless (OVO KD) worms that are inactive, KD of the two NC R-opsins (NC R-opn 1 and 2) significantly attenuates the UV-A photostimulation of resting worms (Fig. 6C). These results are indeed striking and show that UV-A photostimulation of resting worms is dependent on the NC R-opsins (EO photoreceptors) and are not dependent on the presence of eyes. Therefore, indeed, the body-wide EO light-sensing network is able to override the physiology of rest in an animal, unlike the ocular system. Overall, these findings allude to a specific function associated with an EO network even in an intact worm, with eye–brain-based ocular network present.

Fig. 6.

NC R-opsin–mediated EO sensing directly influences physiology of intact animals, independent of cerebral-eye circuit. (A) EO light-sensing interferes with natural rest–activity cycles, distinct from ocular stimuli (visible light). Intact worms in the “resting” or physiologically inactive state do not respond to visible light but respond acutely to UV-A stimulus. Intact planarian worms (in dark) were left undisturbed for 4 h, and the worms observed in transition into a physiologically inactive state (contracted and immobile) were then provided with either visible light (500 nm) or UV-A (395 nm) light stimuli (equal photon flux) and their response measured. Plotted is the time taken (in seconds) by the previously “resting” intact worms to move out of a spot with equal photon flux of either visible (500 nm) or UV-A light (395 nm) (n = 20). Response was observed for 300 s; this time point here indicates unresponsive worms. (B) Eyeless and NC opsin KD worms in inactive state fail to respond to ocular light stimuli. Plotted is the time taken (in seconds) by the previously “resting” intact worms to move out of a spot of visible light (500 nm). Response time of either control, eyeless (OVO KD), or NC R-opn 1 and 2 double-KD worms is plotted. Worms were observed to be inactive for 4 h prior to light stimulation (n = 10). (C) UV-A–specific photostimulation of previously inactive worms is dependent on NC R-opsins and not on eyes. Plotted is the time taken (in seconds) by the previously “resting” intact control, eyeless (OVO KD), or “NC R-opn 1 and 2 double KD worms” to move out of a spot of UV-A light (395 nm). Worms were observed to be inactive for 4 h prior to light stimulation. While previously inactive eyeless (and control) worms were acutely photoactivated and showed movement away from UV-A light, inactive worms deficient in NC R-opsins 1 and 2 showed severely attenuated photoresponse (n = 10). (A–C) Error bar indicates SEM. ***P < 0.0001 (A) ***P < 0.0004 (C) (test of significance performed only when SD ≠ 0, if SD = 0 then results were interpreted based on the difference observed).

Discussion

This study highlights the functional complexity and mechanistic underpinnings of an EO light-sensing system and associated behavior. Despite eye–brain removal, planarian body pieces are able to show coordinated motion when stimulated with UV-A light. Here, we have uncovered a sensory framework that is able to actuate such a photoresponse in an animal removed of its eyes and brain. We have identified opsin photoreceptors as well as a unique sensory array of single cells at the periphery of the worm that work in concert to mediate this photoresponse. This unique sensory array is proximal to the body-wide nerve net, is able to engage with multiple modes of locomotion, and is able to trigger movement like an intact worm. We also show that the developmental trajectory as well as the physiology of the EO light-sensing machinery is completely distinct from the cerebral eye. The single-cell array that mediates EO response shows postembryonic maturation. Furthermore, the EO system can bypass the sleep-inactivity cycles in intact worms when the ocular system has become dormant.

Identifying the photoreceptors was key to uncovering the underlying mechanistic framework of the EO photoresponses. Two opsins expressed in the body of planarians were found to be the EO photoreceptors. These EO photoreceptors are distinct from the Smed-eye opsin, with a different spectral signature supporting a parallel and independently functional EO system uncovered here. Interestingly, the two opsin EO photoreceptors fall in a newly classified group of opsins called the NC R-opsins (53). NC R-opsins are a small class of opsins that diverged from a group that gave rise to the canonical R-opsins (53) (SI Appendix, Fig. S2). Canonical R-opsins are commonly recognized as the major photoreceptors in the invertebrate eye (53, 70). Contrastingly, other than their recent phylogenetic classification, almost nothing is known about the function of any NC R-opsins. Our work provides evidence of NC R-opsins functioning as photoreceptors and specifically as EO photoreceptors. Interestingly, expression patterns of a few other NC R-opsins are known. Each of these reports show the expression of NC R-opsins in locations other than the eye, suggesting a more widespread role of these proteins in EO photoreception (SI Appendix, Fig. S2). In fact, there are opsins belonging to all opsin classes that are found to be expressed in locations other than the eye in multiple organisms (52). Intriguingly, such “noneye”-expressing opsins have been previously described as belonging to “early” branching lineages of their respective opsin classes, including “classical” eye opsins (canonical C and R-opsins) (52). Taken together, these findings suggest an intriguing possibility that the EO opsins, especially the NC R-opsins, may have been the “early” photoreceptors that then diverged to yield the ocular opsins.

Planarians and other flatworms have a centralized brain (bilobed dorsal ganglion) that helps mediate a wide diversity of behaviors (17, 29, 31–34, 71). Not surprisingly, on head removal (decapitation), worms lose the ability to feed, responsiveness to chemical cues (chemotaxis), temperature changes (thermotaxis), as well response to visible light (17, 32–34). However, decapitated worms display the most striking and sensitive of EO responses reported among metazoans, with even low doses of UV-A light (≤31 uW/cm²) able to trigger coordinated motion resembling movement in intact worms. Our identification of a sensory array of single cells lining the periphery of the worm allows us to conceptualize how a head-removed organism may acutely sense light and show coordinated movement that normally relies on a brain. Interestingly, we initially identified two putative photosensory cell populations. One cell population was present throughout the planarian body and expressed only one opsin (NC R-opn 1) while the other population was present at the periphery of the worm and expressed two NC R-opsins (NC R-opn 1 and 2). Notably, the lack of known transcription factors regulating these newly identified cell populations as well lack of transgenesis in planarians, made this problem challenging. However, taking advantage of newly identified single-cell transcriptome data/atlas from two independent sources, we were able to identify cells expressing only one opsin (NC R-opn 1) as pigment cells. This was important in eventually discovering the EO sensory architecture. We were able to ablate the single-opsin–expressing pigment cells that led to an enhancement in EO photoresponse rather than a reduction. These experiments ruled out the opsin-expressing pigment cells as photoreceptor cells and helped show the cellular array lining the periphery of the animal body as the EO photosensory network. It is possible that NC R-opn 1 plays a distinctive, hitherto unknown role in the physiology of pigment cells independent of light-driven locomotion. Opsin molecules have been implicated in dispersal of pigmentation in other organisms (7, 72, 73), but this would need to be examined separately. We note here that current lack of transgenesis in planarians preclude targeted genetic ablation of specific photoreceptor/cell populations and limit direct functional analysis. What also remains to be done is to heterologously express the newly identified EO opsins and directly examine their individual photosensory responses.

Our work also touches on the long-standing interest in exploring the nature and evolution of sensory cells and networks in the animal kingdom. While this question remains to be fully addressed, it is intriguing that an analysis of the single-cell transcriptome data sets shows that NC R-opn 2 expressing single cells cluster as either belonging to “glial cell lineages” (58) or as cathepsin⁺ cells (57) (SI Appendix, Fig. S12). The cathepsin⁺ cells are a heterogeneous group that express the human cathepsin-like gene and consist of previously defined cell types like the pigment cell (61) and glial cell (74, 75) as well as multiple unidentified cell types of unknown function (57). While this link between the NC R-opsin–expressing cells and the “glia-like lineages” need to be probed further, it is particularly interesting that NC R-opn 2–expressing cells did not cluster with any known class of planarian neuronal cells (SI Appendix, Fig. S12). This is notable, since currently all the known examples of EO photoreceptor cells that can actuate movement are clearly described as neurons (2–4).

Our work opens lines of inquiry on how an array of “unusual” opsin-expressing photoreceptor cells wire together to actuate coordinated locomotion. The proximity of the EO photoreceptor cells to the body-wide nerve net/plexus suggests the intriguing possibility that these cells may directly couple to the diffuse nerve net and then eventually produce coordinated movement through the more-cephalized ventral nerve cords, considered part of the more “centralized” neural network along with the brain. How animals progressed from a simple nerve-net architecture to a more defined centralized nervous system remains one of the profound questions of animal evolution. Planarians not only have an example of what is referred to as a “primitive brain” in evolution (linked to “cephalized” ventral cords) but also have a diffuse nerve net or plexus (31, 62–66). Light sensing in planarians and related organisms offers an opportunity to explore the interplay/transition between the “diffuse” nerve net and the more “centralized” networks, especially since a body-wide network (just like the brain) retains the ability to trigger coordinated motion.

The postembryonic maturation of the newly discovered EO sensory architecture in an adult-like animal is intriguing. This is in contrast to an ocular network (cerebral eyes) that develops embryonically and is functional at the time of hatching. Embryonic development of the eyes is observed in a large variety of species (76, 77); this is also seen in planarians (78, 79). What may be the significance of the EO sensory network developing after the eye–brain ocular system has already developed? A classical reading of developmental trajectories (von Baer’s law) would suggest that the EO sensing may be a more “specialized or derived trait” that develops significantly after the development of the more conserved/generalized trait (eye–brain-based sensing). This would point to the flatworms evolving the highly sensitive EO sensory array in response to specific evolutionary pressures as part of their adaptation to a low-light environment. This is intriguing and even counterintuitive since EO sensing has generally been thought to be a more primitive form of light-sensing system. Along those lines, even the EO photoreceptors identified here (NC R-opsins) appear to belong to an opsin subclass that has diverged from the canonical R-opsins. Of course, the EO photosensing may indeed be an ancient trait that unexpectedly shows a later development compared to the eye. Alternatively, the acute EO light sensitivity may actually be a more specialized/derived trait that evolved by co-opting distinct photoreceptors in response to the unique selective pressure faced by organisms living in low-light environment. Overall, these findings offer a unique and stunning insight into coexistence, development, and evolution of independent light-sensing systems in a single organism.

Acute light sensing in head-removed planarians can be thought to provide a selective advantage to species that can propagate through fission. Several species of planarians propagate asexually through fission (80, 81), where the worm undergoes fission to yield a headed piece and a tail piece (SI Appendix, Fig. S13). Since each of these pieces can regenerate to yield a complete animal, the ability to sense light in the tail piece would be advantageous during the process of head regeneration (∼7 d). This may be particularly important as planarians are dark dwelling and may use light sensing for navigation, avoidance of light-exposed areas, and possible exposure to predators. However, many planarian species also reproduce sexually (80, 81). Can the ability to sense light independent of the eye also be important in other ways for all worms, independent of whether worms show sexual or asexual reproduction? Notably, here we provide direct evidence of how the eye-independent EO light-sensing network can influence the animal physiology, even in intact worms that have eyes. We find that planarians show clear phases of inactivity. Interestingly, in the “inactive” or “resting” state, intact worms tend to be insensitive to ocular light stimuli. However, resting or inactive worms can be photostimulated by UV-A light; this photostimulation of resting worms is dependent on NC R-opsin EO photoreceptors. Such UV-A sensing (through the EO network) may play an important role as an acute, reflex-like light aversive response that may become particularly critical as a fail-safe sensory mechanism when the eye–brain sensory axis is dormant during periods of rest. Such a mechanism may be distinctly advantageous to a water-dwelling, light-aversive organism that is likely nocturnal and would rest during the day. Resting worms may be carried by currents to exposed areas. Since UV-A can be considered a proxy for filtered sunlight, UV-A photostimulation of resting worms may help exposed worms to move back to dark niches. Overall, the body-wide UV-A photoresponse appears to be akin to a reflex-like response, whereas the eye-based sensing is a more nuanced, processive mode of light sensing.

In summary, our work showcases the fascinating complexity of form and function of an eye–brain-independent light-sensing network, with ability to regulate the physiology of the organism in multiple, unique ways (SI Appendix, Fig. S14). In head-removed worms, this EO system can trigger and control coordinated movement reminiscent of what the eye–brain-mediated ocular system can achieve. Furthermore, in intact animals, when the eye–brain system becomes dormant during the resting phase of the animal, the EO system is able to photostimulate these resting worms (a function distinct from the ocular system). Both the EO and ocular network are coupled to the same locomotion machinery, but the EO system develops after the ocular network, with development occurring in adult-like animals. Along with our discovery of the sensory architecture that underpins this eye–brain-independent light-sensing, our findings represent a major advance spanning virtually all facets of photosensory biology.

Materials and Methods

Planarian Maintenance.

S. mediterranea was maintained at standard laboratory conditions (82). For planarian developmental studies, hatchlings were generated using the protocol described by Davies et al. (79).

Light-Induced Depigmentation.

Planarian worms are known to show depigmentation on prolonged exposure to light (60, 61, 83). For this purpose, we standardized light mediated depigmentation protocol based on the worm size. Details are provided in SI Appendix.

Light Assay.

EO UV-A light-spot assay for head-removed worms, single-input negative phototaxis, and binary-choice light assays on intact worms were all performed using protocols described in Shettigar et al. (17). Please reference SI Appendix for details on light source and measurements.

RNAi-Mediated KD.

Double-stranded RNA was synthesized as described by Rouhana et al. (84) with modifications. The primer sequences and sequence identifier for each of the planarian genes used in this study are mentioned in SI Appendix, Table S2.

Whole-Mount FISH and Immunostaining.

Whole planarians of varying sizes were processed for FISH per the protocol described in Shettigar et al. (17) with the following modifications mentioned in SI Appendix. For experiments in which immunostaining was carried out after FISH/Double FISH, the protocol used in Shettigar et al. (17) was followed. The primary antibody used in these experiments was an anti–G-α11 (sc-365906, Santa Cruz Biotechnology) antibody that was detected using an AlexaFluor 488 conjugated mouse secondary antibody (A11001, Thermo Fisher Scientific).

Data Set.

The generated alignment data and resultant tree files used in this study are available at https://github.com/VairavanLakshmanan/Extraocular_Opsin_Dataset. The complete uncollapsed IQ-TREE opsin phylogeny IQ-Tree is available in Dataset S1. A detailed description of materials and methods is available in SI Appendix.

Data Availability

Generated alignment data and resultant tree files used in this study data have been deposited in Github (https://github.com/VairavanLakshmanan/Extraocular_Opsin_Dataset). All other study data are included in the article and/or supporting information.