TY - JOUR
AU1 - Kennedy, Melissa D
AU2 - Barberio, Angelo
AU3 - Connaughton, Victoria P
AB - Abstract Intricate adaptations to visual systems have allowed amphipods to thrive in extreme habitats like caves. In addition to rearranging ocular dioptric elements, adaptations have also been made to photobehaviors to accommodate the environmental conditions of their habitats. Given the prevalence or absence of discernible eyes or photoreceptors across species, phototaxis is a commonly used behavior to assess light sensing mechanisms. Amphipod photobehaviors have increasingly been utilized within neuroethological and ecotoxicological studies but the quality of light used during laboratory experiments is often overlooked. We describe a novel experimental chamber that allows for accurate and efficient measurement of phototaxis under precisely controlled light conditions. We used this experimental chamber to test phototaxis in two related amphipod species, one subterranean (Stygobromus tenuis potomacusHolsinger, 1967) and one surface dwelling (Crangonyx shoemakeriHubricht & Mackin, 1940). Our results confirm that the behaviors of these species are indicative of natural photopreferences and that in certain populations, these preferences vary with light quality. This low cost and efficient method could easily be applied to a variety of animal models and light conditions, allowing for a reproducible and high throughput method to measure phototaxis in laboratory trials. INTRODUCTION Studying the visual system often relies on molecular analysis, especially the expression of opsins. Unfortunately, these methods can be cost and time prohibitive and require sacrifice of study organisms. Behavioral studies, particularly those that rely on light-mediated behaviors like phototaxis, provide a valuable alternative. Phototaxis is the directional movement towards or away from a light source. It can be accomplished with as little as a single photoreceptor (Randel & Jekely, 2016) and relies heavily on the light conditions an animal is accustomed to, especially the wavelength of light. For example, the bluefin killifish (Lucania goodei Jordan, 1880) is found in a variety of habitats and their opsin expression reflects this diversity. Killifish that live in swamps have increased expression of rhodopsin-like and long wavelength sensitive (LWS) pigments because murky swamps have reduced transmission of UV/blue wavelengths. By contrast, killifish found in clear springs, have a higher expression of short-wavelength sensitive (SWS) pigments (Fuller et al., 2004). Another example of opsin variation is the crayfish Procambarus clarkii (Girard, 1852), which has 15 known opsins that are differentially expressed at different times of year (Cronin & Hariyama, 2002) and in different retinal structures (Kingston & Cronin, 2015). Two different LWS opsin paralogs are expressed in surface and cave dwelling Gammarus minus (Say, 1818), with greater overall expression in the surface population (Carlini et al., 2013). Amphipods in Lake Baikal, Russia express multiple LWS opsins, with the number expressed negatively correlated with habitat depth (Drozdova et al., 2021). Behaviorally, Eulimnogammarus cyaneus (Dybowski, 1874), a littoral amphipod, displays the greatest response to blue or green wavelengths, whereas Ommatogammarus flavus (Dybowsky, 1874), a deep-water species, did not respond to any of the stimulating wavelengths used (Drozdova et al., 2020). Hyalella azteca (Saussure, 1858), a freshwater amphipod, has a greater avoidance/negative response to lower (versus higher) wavelengths of light, with the greatest response observed with 650 nm light, suggesting a preference for long wavelengths (Benesh et al., 2005). Clearly, there are species and habitat-specific differences in both opsin expression and responses to light. To form an image, crustaceans, including amphipods, rely on compound eyes. Compound eyes are comprised of many ommatidia. Each ommatidium is covered by a cornea and, generally, includes a crystalline cone, a crystalline cone stalk, and a basal retinula, although the crystalline cone is not always present in amphipods (Hallberg et al., 1980). When light enters an ommatidium, it is directed by the cornea to the crystalline cone to light detecting pigments within the rhabdome. The cells then transmit signals through the retinula, nerve tracts, and ultimately onto the optic nerve (Kingston & Cronin, 2016). Most animals can also sense light without the production of an image. Non-visual, or non-image forming, photoreception relays information about characteristics of the light rather than an image itself. These non-image forming photoreceptors are associated with the central nervous system in various invertebrates (Arendt et al., 2004; Velarde et al., 2005; Santillo et al., 2006; Musio & Santillo, 2009; Kingston & Cronin, 2015, 2016), including amphipods (Drozdova et al., 2021), and can also be found in the dermis (Shand & Foster, 1999; Musio & Santillo, 2009; Cronin & Porter, 2014; Kingston & Cronin, 2016) or in the eye but outside the retina (Shand & Foster, 1999). Non-image forming photoceptors, specifically intracerebral ocelli, have been identified in only one amphipod, Talitrus saltator (Montagu, 1808) (Frelon-Raimond et al., 2002) though they are present in Isopoda (Martin et al., 1995). Like image-forming photoreceptors, non-image forming photoreceptors are opsin based and use a similar phototransduction cascade. These types of photoreceptors are responsible for physiological processes such as circadian entrainment, sleep-wake cycles, alertness, and the pupillary reflex (Shand & Foster, 1999; Cronin & Johnsen, 2016; Kingston & Cronin, 2016; Sonoda & Lee, 2016). Non-image forming photoreceptors are also known to be responsible for a wide array of invertebrate behavioral responses, including reflexive and/or kinetic responses (i.e., phototaxis) and circadian rhythms (Musio & Santillo, 2009). Studies examining light responses of amphipods have been performed in both the field and the laboratory. Field studies reveal a positive phototactic response, whereas laboratory studies identify amphipods as largely negatively phototactic/photonegative (Bethel & Holmes, 1973; Guler & Ford, 2010; Stom et al., 2017), with behaviors varying depending on the species tested (Kohler et al., 2018), water quality/toxin exposure (Stom et al., 2017), and/or parasitic infection (Bethel & Holmes, 1973; Maynard et al., 1998; Benesh et al., 2005). Wavelength/spectral quality can also be an important variable affecting phototactic response in amphipods (Benesh et al., 2005). Field studies are typically performed using light traps positioned within the water column (Navarro-Barranco & Hughes, 2015), whereas laboratory studies use clear chambers with controlled levels of light (Bethel & Holmes, 1973; Maynard et al., 1998; Benesh et al., 2005; Guler & Ford, 2010; Fišer et al., 2016; Stom et al., 2017; Kohler et al., 2018). The laboratory chambers are often glass aquaria that may be vertically oriented (but see Fišer et al., 2016), providing quite a large space for the animals to move and the potential for additional cues (such as gravity/geotaxis). We have developed a simple, low-cost experimental chamber for assessing phototaxis in a laboratory setting that adds to the published literature because it 1) allows individual examination of multiple animals at the same time, 2) removes geotactic stimuli, and 3) can easily accommodate different stimulating light intensities and wavelengths. MATERIALS AND METHODS Design of experimental chamber The experimental chamber (Fig. 1) was constructed from an 18 gallon (68.1 l) opaque black plastic bin (internal dimensions: 44.8 cm × 35.2 cm × 36.5 cm; Rubbermaid, Wooster, OH, USA). Six evenly spaced circular holes (or arenas; 30 mm deep × 100 mm diameter) were carved into a Styrofoam tray that fit within the bin. The holes held circular icepacks which were fitted below the six 100 mm petri dishes (arenas 1–6). The Petri dishes in three of the arenas were painted half black (both dish and lid) to allow animals to choose between dark and light sides and the other three petri dishes were divided in half by a single black line. The Petri dishes painted half-black were referred to as “Preference Chambers,” the others are the control or “Non-Preference Chambers.” During each trial, dishes were filled with water that was maintained at a temperature of 7–11 °C by either replacing the icepacks or refreshing the water. Temperature maintenance during recordings was critical, to ensure the animals experienced similar temperature to holding tanks and their natural environment. A remote-controlled GoPro Hero4 camera (GoPro, San Mateo, CA, USA) was mounted to the lid of the chamber to record evoked behaviors. Figure 1. Open in new tabDownload slide The experimental chamber was constructed from an 18-gallon (68.14 l), black plastic bin with a tray to hold six Petri dishes (arenas 1–6) (A). Light was provided by light boxes mounted to the inside of the plastic bin. A Go-Pro Hero4 camera was mounted to the interior of the box lid (white arrow) for recording behavior. Schematic showing individual Petri dishes (arenas 1–6) in the recording chamber (B). Three of the Petri dishes were painted half black (both dish and lid) to allow animals to choose between a dark or light environment during testing (Preference Chambers). Three control (Non-Preference) chambers were divided in half, but not painted. Each animal was tested in both arenas. To obtain different stimulating light intensities, two 17 inch (43.2 cm) LED light boxes (AGPTek, Brooklyn, NY, USA) were fixed to the interior sides of the bin. Light intensity was controlled using settings on the light boxes (low, medium, or high). Neutral density filters were attached to the light boxes for more control of intensity. Light intensity at each intensity setting (high, medium, low), with and without neutral density filters was measured at five different locations in the experimental chamber (A–E; Fig. 2A) using a hand-held light meter (LX1330B Digital Illuminance/Light Meter; Dr. Meter, Union City, CA, USA). Figure 2. Open in new tabDownload slide Light quality (intensity and wavelength) within the experimental chamber was measured at five locations, labeled (positions A–E) to confirm that each arena received the equivalent light conditions (A). We were able to obtain a variety of wavelengths (grey circles, 450 nm to > 700 nm) using various colored gel acetate filters (red, blue, yellow, and green) placed over the light boxes either alone or in combination (B). The primary stimulating wavelengths used were 452 nm, 512 nm, 532 nm, and 612 nm (white arrows). Known wavelengths for short wavelength sensitive (SWS), middle wavelength sensitive (MWS), and long wavelength sensitive (LWS) opsins in invertebrates are indicated on the slide for comparison. The EM spectra shown in B was originally published in The joy of visual perception [http://www.yorku.ca/eye/spectru.htm]. Reprinted with permission. We controlled wavelength by attaching red, blue, green, and/or yellow colored gel acetate filters (Neweer; Shenzhen Neewer Technology, Guangdong, China), either individually or in combination, to the light box surface and the emitted wavelength was measured using a spectroradiometer (PR-670 SpectraScan, Photo Research; JADAK, Syracuse, NY, USA). We were able to obtain wavelengths ranging from 400 nm to > 700 nm (Fig. 2B). Within this range, we chose four wavelengths for our experiments: 452 nm, 512 nm, 532 nm, and 612 nm. We chose the three lower wavelengths because they overlap with wavelengths reported to maximally stimulate the opsins expressed in invertebrates (Jokela-Maatta et al., 2005; Porter et al., 2006; Santillo et al., 2006; Musio & Santillo, 2009; Kingston & Cronin, 2015). The 612 nm wavelength was chosen to determine if a longer wavelength, outside these peak values, could initiate a response (as in Benesh et al., 2005) in our test animals. Behavioral assays The behavioral assay protocol was adapted from Tain et al. (2006). Briefly, for each trial, 12 adult, male individuals from a single population were divided into two testing groups (six individuals each) and tested under a specific light condition. For each assay, the first group of six individuals (Fig. 3) was randomly placed into arenas 1–6, and allowed to acclimate for 1 min. After a recording session, each individual was moved to an adjacent arena of the opposite chamber type. If an individual was initially placed into a Preference Chamber, that same animal would therefore be transferred to a Non-Preference Chamber, which allowed us to test a given individual in both chamber types. This procedure was then repeated with the other six individuals. Each group of 12 individuals was only used for one trial per day to prevent habituation to the experimental chambers. After testing, individuals were returned to their housing containers. Figure 3. Open in new tabDownload slide For each experimental trial, 12 individuals were divided into two groups of six individuals each and tested under a specific light condition. The first six individuals were randomly placed into the dishes in arenas 1–6 and allowed to acclimate prior to video recordings (A). After recording, each individual was moved to an adjacent arena, allowing each to be tested in both Preference and Non-Preference chambers (B). The procedure was repeated with the remaining six individuals, so all 12 were tested (C). Behavior in each trial was video-recorded for 11 min. Video recordings were then watched and the position of the animal noted. The first minute of each video was not scored as that was the acclimation period. Beginning with the second minute, the position of each individual was recorded at 30 sec intervals, resulting in 20 observations per individual. In Preference Arenas, a score of “1” was given if the individual was on the light side at each recording interval and a score of “’’ was given to individuals on the dark side at the recording interval (see also Guler & Ford, 2010). For the Non-Preference Arenas, a randomly chosen half of the arena was assigned as the “light” side and scored identically to the Preference Arenas. Cumulative scores above 10 were considered photopositive, scores of 10 were considered photoneutral, and scores below 10 were considered photonegative. Study animals The behavioral chamber described above is suitable for small organisms with unrestricted movement in the Petri dish. As proof of principle, we chose to assess the phototactic responses of two species of amphipods, Stygobromus tenuis potomacus (Holsinger, 1967) (herein referred to as S. tenuis) and Crangonyx shoemakeri (Hubricht & Mackin, 1940) (Fig. 4). These species were chosen because they are both members of the family Crangonyctidae and are found in hypotelminorheic habitats (i.e., seepage springs and associated groundwater) around Washington, DC (Keany et al., 2018). These species, however, 1) are morphologically different and 2) rarely occur in the same location (Keany et al., 2018): C. shoemakeri is surface-dwelling, preferring warmer waters, whereas S. tenuis prefers the cooler, lower conductance water found in shallow subterranean habitats. Stygobromus tenuis, also known as Potomac groundwater amphipod, exhibits pronounced troglomorphic traits including a lack of ommatidia and body pigment, consistent with a subterranean-habitat dweller, whereas C. shoemakeri exhibits large eyes and full, brown body pigmentation. Both were collected in 2017 and 2018 from surface seepage springs associated with the Potomac River in Arlington, VA. Stygobromus tenuis was collected from Pimmit Run and C. shoemakeri from North Seep habitats. Live individuals were maintained in the laboratory in aerated Deer Park (system) water at 4 °C with water changes and food added every 4 d. The same housing conditions were used throughout the duration of the experiment. Figure 4. Open in new tabDownload slide Light responses of two amphipods Stygobromus tenuis (A) and Crangonyx shoemakeri (B), both from the family Crangonyctidae and found within surface seepage springs associated with the Potomac River in Arlington, VA, USA. Stygobroums tenuis exhibits pronounced troglomorphic traits including a lack of ommatidia and body pigment, whereas C. shoemakeri has very large eyes and full, brown body pigmentation. Under each photo, is the regression analysis of preference scores recorded across the four stimulating wavelengths used, 452, 512, 532, and 612 nm. Each symbol represents the score of a single individual. S. tenuis phototaxis/preference scores were significantly affected by wavelength, with a decrease in preference scores (increased photonegativity) with increasing wavelength (A, bottom panel). In contrast, preference scores for C. shoemakeri were not affected by wavelength (B, bottom panel). Given the morphological and habitat differences between the two species, we hypothesized that 1) preference scores would differ between the two populations, 2) preference scores would depend on light quality (intensity and wavelength), and 3) control (non–preference) scores would remain close neutral and be unaffected by changing light conditions. Statistical analysis To verify that preference scores from each species were due to a preference for light or dark, and not the experimental chamber, each preference score was directly compared to the corresponding non-preference score from the same individual using a Related-Samples Wilcoxon Signed Rank Test. We used a Univariate ANOVA to determine if species, brightness and/or wavelength had a significant influence on preference scores. To determine the relationship between wavelength and preference scores, we performed a linear regression independently for each species. All statistics were performed using SPSS software (ver. 26, IBM) and evaluated at α = 0.05. RESULTS There were no differences in light intensity within the experimental chamber (Table 1), indicating all arenas in the chamber experienced the same lighting conditions. Table 1. Light intensity measurements in the experimental chamber. To confirm light was evenly distributed throughout the chamber, intensity measurements (in lux) were collected at various locations (Position) in the experimental chamber. Intensity was measured at each brightness setting (low, medium, high) and with 0, 1, 2, or 3 neutral density filters attached to the light boxes (Lux 0, Lux 1, Lux 2, Lux 3). Values recorded at a given brightness were not significantly different across position within the chamber (see also Figure 2). Position . Box setting . Lux 0 . Lux 1 . Lux 2 . Lux 3 . A Low 79.20 34.00 17.00 9.00 A Medium 314.00 148.00 76.00 42.00 A High 613.00 282.00 155.00 86.00 B Low 90.90 36.00 17.00 8.00 B Medium 384.00 154.00 74.00 38.00 B High 755.00 310.00 150.00 77.00 C Low 87.30 39.00 23.00 17.00 C Medium 350.00 171.00 102.00 81.00 C High 715.00 345.00 197.00 155.00 D Low 101.00 35.00 18.00 9.00 D Medium 376.00 152.00 82.00 40.00 D High 766.00 304.00 166.00 81.00 E Low 90.60 19.00 10.00 E Medium 371.00 154.00 83.00 45.00 E High 663.00 333.00 169.00 89.00 P value 0.998 0.999 0.992 0.756 Position . Box setting . Lux 0 . Lux 1 . Lux 2 . Lux 3 . A Low 79.20 34.00 17.00 9.00 A Medium 314.00 148.00 76.00 42.00 A High 613.00 282.00 155.00 86.00 B Low 90.90 36.00 17.00 8.00 B Medium 384.00 154.00 74.00 38.00 B High 755.00 310.00 150.00 77.00 C Low 87.30 39.00 23.00 17.00 C Medium 350.00 171.00 102.00 81.00 C High 715.00 345.00 197.00 155.00 D Low 101.00 35.00 18.00 9.00 D Medium 376.00 152.00 82.00 40.00 D High 766.00 304.00 166.00 81.00 E Low 90.60 19.00 10.00 E Medium 371.00 154.00 83.00 45.00 E High 663.00 333.00 169.00 89.00 P value 0.998 0.999 0.992 0.756 Open in new tab Table 1. Light intensity measurements in the experimental chamber. To confirm light was evenly distributed throughout the chamber, intensity measurements (in lux) were collected at various locations (Position) in the experimental chamber. Intensity was measured at each brightness setting (low, medium, high) and with 0, 1, 2, or 3 neutral density filters attached to the light boxes (Lux 0, Lux 1, Lux 2, Lux 3). Values recorded at a given brightness were not significantly different across position within the chamber (see also Figure 2). Position . Box setting . Lux 0 . Lux 1 . Lux 2 . Lux 3 . A Low 79.20 34.00 17.00 9.00 A Medium 314.00 148.00 76.00 42.00 A High 613.00 282.00 155.00 86.00 B Low 90.90 36.00 17.00 8.00 B Medium 384.00 154.00 74.00 38.00 B High 755.00 310.00 150.00 77.00 C Low 87.30 39.00 23.00 17.00 C Medium 350.00 171.00 102.00 81.00 C High 715.00 345.00 197.00 155.00 D Low 101.00 35.00 18.00 9.00 D Medium 376.00 152.00 82.00 40.00 D High 766.00 304.00 166.00 81.00 E Low 90.60 19.00 10.00 E Medium 371.00 154.00 83.00 45.00 E High 663.00 333.00 169.00 89.00 P value 0.998 0.999 0.992 0.756 Position . Box setting . Lux 0 . Lux 1 . Lux 2 . Lux 3 . A Low 79.20 34.00 17.00 9.00 A Medium 314.00 148.00 76.00 42.00 A High 613.00 282.00 155.00 86.00 B Low 90.90 36.00 17.00 8.00 B Medium 384.00 154.00 74.00 38.00 B High 755.00 310.00 150.00 77.00 C Low 87.30 39.00 23.00 17.00 C Medium 350.00 171.00 102.00 81.00 C High 715.00 345.00 197.00 155.00 D Low 101.00 35.00 18.00 9.00 D Medium 376.00 152.00 82.00 40.00 D High 766.00 304.00 166.00 81.00 E Low 90.60 19.00 10.00 E Medium 371.00 154.00 83.00 45.00 E High 663.00 333.00 169.00 89.00 P value 0.998 0.999 0.992 0.756 Open in new tab We first compared preference scores when the amphipods were in the Non-Preference (control) Chambers to obtain baseline phototactic responses. We found the mean scores were comparable and close to photoneutral (score of 10) for both species, with C. shoemakeri having an average score of 8.73 ± 3.57 (N = 45) and S. tenuis averaging 8.33 ± 4.41 (N = 66). These scores were not significantly different from each other (P = 0.348), and they were not impacted by either wavelength (P = 0.795) or light intensity (P = 0.284). We next compared the control scores when the amphipods were in the Non-Preference Chambers with the scores obtained when the amphipods were in the Preference (experimental) Chambers. We found both species displayed photonegative behaviors characterized by significantly lower mean scores (P < 0.001) in the Preference Chambers. In these chambers, S. tenuis had an average score of 5.41 ± 4.43 (N = 78) and C. shoemaker an average score of 3.37 ± 2.61 (N = 51). Further, the preference score of C. shoemakeri was significantly lower than that of S. tenuis (P = 0.002), indicating a greater light avoidance/photonegative response by C. shoemakeri. Light intensity did not affect these scores (P = 0.481), indicating the presence of light, not its brightness, induced the response. When we examined responses to different wavelengths of light, however, we identified species-specific differences in photobehavior (P = 0.026). For S. tenuis, wavelength significantly predicted the preference score (P = 0.032; Fig. 4) and accounted for 4.7 % of the variation. More specifically, S. tenuis displayed decreased preference scores (increased negative phototaxis) when long stimulating wavelengths were used and increased preference scores (positive phototaxis) in response to short wavelength stimuli. In contrast, wavelength was not found to be a predictor of preference score/phototactic response for C. shoemakeri (P = 0.378; Fig. 4). While there appears to be a trend of decreased preference score (or increased photonegativity) at longer stimulating wavelengths for C. shoemakeri, this relationship was not significant. DISCUSSION We describe a low-cost experimental chamber that can be used to assess phototactic responses in aquatic invertebrates and report data from two different amphipod species as proof-of-principle. As a method, our investigation has three important implications. Firstly, the conditions in the chamber can be easily controlled, providing the ability to isolate and examine the effect of light quality, i.e., color (wavelength) and intensity, on phototaxis. Secondly, the chamber allows multiple individuals to be tested at the same time while also distinguishing among individuals. The horizontal orientation of all dishes within the chamber removes geotactic stimuli, allowing purely light-induced behaviors to be recorded. Thirdly, the overall chamber design is robust as evidenced by constant lighting conditions throughout and photoneutral behaviors when the amphipods were placed in the non-preference chambers. The control behavior indicates amphipods spent equal amounts of time on both sides of the Petri dish in constant light conditions, identifying a lack of bias within the Petri dishes themselves. This is an important finding as it verifies both the methods described for the assay and the efficacy of the experimental chamber. Other laboratory studies assessing phototaxis typically use clear aquaria with one side light and one side darkened (Bethel & Holmes, 1973; Maynard et al., 1998; Guler & Ford, 2010), or tall, vertical chambers with light positioned at either the top or the bottom (Benesh et al., 2005). A more recent report examined amphipod phototaxis in a chamber somewhat similar to ours (Fišer et al., 2016). In all designs, amphipods display photonegative/negatively phototactic behaviors (Maynard et al., 1998; Guler & Ford, 2010; Stom et al., 2017; Kohler et al., 2018). This baseline response is altered by parasitic infection (Bethel & Holmes, 1973; Maynard et al., 1998; Benesh et al., 2005; Tain et al., 2006), changed water conditions (Stom et al., 2017), exposure to pharmaceuticals (Tain et al., 2006; Guler & Ford, 2010), and/or whether the amphipod is from a surface or a subterranean habitat (Fišer et al., 2016). Our experimental chamber could be applied to these exposure studies, or others, with a variety of small organisms. We tested the phototactic responses of C. shoemakeri, a large surface-dwelling species with well-developed eyes and S. tenuis, a smaller, subterranean-dwelling species, with no obvious eye structures. Both were found to be negatively phototactic when exposed to white light regardless of the intensity level. Crangonyx shoemakeri, however, had a significantly larger light avoidance response than S. tenuis, revealing species-specific differences. This result was somewhat surprising as a previous report identified consistent photophobic responses across subterranean species and more variable responses from surface species (Fišer et al., 2016). When different wavelengths of light were used, S. tenuis was the more sensitive, displaying increased photonegativity in response to long wavelengths of light and increased positive phototaxis in response to shorter wavelengths. These species differences are likely due to adaptations to their different habitats. As a surface-dwelling species found in warm water, shallow seeps (Keany et al., 2018), light intensity would be more of a stimulus for C. shoemakeri, as it denotes not only day-night cycles, but visibility to predators and, in summer months, potential desiccation of the habitat due to evaporative water loss (Gilbert et al., 2018). By contrast, less light reaches subterranean habitats and, depending on water depth, only certain wavelengths of light will be present. Consequently, subterranean organisms have evolved reduced eyes (as is the case for S. tenuis) and/or reduced (as found in Gammarus minus (Carlini et al., 2013) and in Lake Baikal amphipods (Drozdova et al., 2021)) or absent (as in Allobathynella bangokensis Park & Cho, 2016 (Kim et al., 2017)) opsin expression, suggesting a reduced response, or an inability to respond, to light stimulation (Drozdova et al., 2020). We, nonetheless, observed a clear photonegative response by S. tenuis. Given the morphology of S. tenuis it is likely that this species is detecting light using non-visual, extra-ocular photoreceptors. In other invertebrates, including amphipods, non-visual photoreceptors are distributed throughout the nervous system (Arendt et al., 2004; Velarde et al., 2005; Santillo et al., 2006; Musio & Santillo, 2009; Kingston & Cronin, 2015, 2016; Drozdova et al., 2021). These receptors express LWS and SWS opsins in crayfishes (Kingston & Cronin, 2015), which may also be the case for S. tenuis given the strong responses for 612 nm (avoidance) and 452 nm (preference) light. These results contrast the behavior of the freshwater amphipod Hyalella azteca, which, if given a choice between two colors of light, prefers the longer wavelength (Benesh et al., 2005). The preference for short wavelength light by S. tenuis likely reflects transmission of blue light and emission of short wavelengths by substances in groundwater (Baker & Genty, 1999) and, as a result, would be the only wavelength that is “seen”. Species, and/or habitat-specific, differences in photobehaviors have been reported in other amphipod species, however, not in the two distantly related species used here. We report that both species display negative phototaxis, with spectral wavelength determining the response S. tenuis, but not C. shoemakeri. Crangonyx shoemakeri displayed greater photonegativity, with the presence of light, rather than its qualities, sufficient to evoke a response. These behaviors were documented in a low-cost, easily adaptable experimental set-up that can be applied to other studies examining the impact of compound exposure, parasitic infection, or different water samples on light-driven responses. ACKNOWLEDGEMENTS Dan Fong collected the amphipods used in the study. We thank Dan Fong and Chris Tudge for comments on earlier versions of the manuscript. Thanks also to the anonymous reviewers for their comments and suggestions. REFERENCES Arendt , D. , Tessmar-Raible , K., Snyman , H., Dorresteijn , A. & Wittbrodt , J. 2004 . Ciliary photoreceptors with a vertebrate-type opsin in an invertebrate brain . Science , 306 : 869 – 871 . Google Scholar Crossref Search ADS PubMed WorldCat Baker , A. & Genty , D. 1999 . Fluorescence wavelength and intensity variations of cave waters . Journal of Hydrology , 217 : 19 – 34 . Google Scholar Crossref Search ADS WorldCat Benesh , D. , Duclos , L. & Nickol , B. 2005 . The behavioral response of Amphipods harboring Corynosoma constructum (Acanthocephala) to various components of light . Journal of Parasitology , 91 : 731 – 736 . Google Scholar Crossref Search ADS WorldCat Bethel , W. & Holmes , J. 1973 . Altered evasive behavior and responses to light in amphipods harboring Acanthocephalan cystacanths . Journal of Parasitology , 59 : 945 – 956 . Google Scholar Crossref Search ADS WorldCat Carlini , D. , Satish , S. & Fong , D. 2013 . Parallel reduction in expression, but no loss of functional constraint, in two opsin paralogs within cave populations of Gammarus minus (Crustacea: Amphipoda) . BMC Ecology and Evolution , 13 : [https://doi.org/10.1186/1471-2148-13-89]. Google Scholar OpenURL Placeholder Text WorldCat Cronin , T. & Hariyama , T. 2002 . Spectral sensitivity in crustacean eyes. In: The crustacean nervous system ( K. Wiese, ed.), pp. 499 – 511 . Springer-Verlag , Berlin & Heidelberg , Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Cronin , T. & Johnsen , S. 2016 . Extraocular, non-visual,and simple photoreceptors: an introduction to the symposium . Integrative and Comparative Biology , 56 : 758 – 763 . Google Scholar Crossref Search ADS PubMed WorldCat Cronin , T. & Porter , M. 2014 . The evolution of invertebrate photopigments and photoreceptors, In: Evolution of visual and non-visual pigments ( D. Hunt, ed.), pp. 105 – 135 . Springer Science+Business Media , New York . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Drozdova , P. , Kizenko , A., Saranchina , A., Gurkov , A., Firulyova , M., Govorukhina , E. & Timofeyev , M. 2021 . The diversity of opsins in Lake Baikal amphipods (Amphipoda: Gammaridae) . BMC Ecology and Evolution , 21 : [https://doi.org/10.1186/s12862-021-01806-9]. Google Scholar OpenURL Placeholder Text WorldCat Drozdova , P. , Saranchina , A. & Timofeyev , M. 2020 . Spectral sensitivity of the visual system of endemic Baikal amphipods . Limnology and Freshwater Biology , 4 : 781 – 782 . Google Scholar OpenURL Placeholder Text WorldCat Fišer , Ž. , Novak , L., Luštrik , R. & Fišer , C. 2016 . Light triggers habitat choice of eyeless subterranean but not of eyed surface amphipods . The Science of Nature , 103 : [https://doi.org/10.1007/s00114-015-1329-9]. Google Scholar OpenURL Placeholder Text WorldCat Frelon-Raimond , M. , Meyer-Rochow , V., Ugolini , A. & Martin , G. 2002 . Intracerebral ocelli in an amphipod: extraretinal photoreceptors of the sandhopper Talitrus saltator (Crustacea, Amphipoda) . Invertebrate Biology , 121 : 73 – 78 . Google Scholar Crossref Search ADS WorldCat Fuller , R. , Carleton , K., Fadool , J., Spady , T. & Travis , J. 2004 . Population variation in opsin expression in the bluefin killifish Lucania goodei: a real-time PCR study . Journal of Comparative Physiology Part A , 190 : 147 – 154 . Google Scholar Crossref Search ADS WorldCat Gilbert , H. , Keaney , J. & Culver , D. 2018 . Response of shallow subterranean freshwater amphipods to habitat drying . Subterranean Biology , 28 : 15 – 28 . Google Scholar Crossref Search ADS WorldCat Guler , Y. & Ford , A. 2010 . Anti-depressants make amphipods see the light . Aquatic Toxicology , 99 : 397 – 404 . Google Scholar Crossref Search ADS PubMed WorldCat Hallberg , E. , Milsson , H. & Elofsson , R. 1980 . Classification of amphipod compound eyes - the fine structure of the ommatidial units (Crustacea, Amphipoda) . Zoomorphologie , 94 : 279 – 306 . Google Scholar Crossref Search ADS WorldCat Holsinger J.R . 1967 . Systematics, speciation, and distribution of the subterranean amphipod genus Stygonectes (Gammaridae) . United States National Museum Bulletin , 259 : 1 – 176 . Google Scholar OpenURL Placeholder Text WorldCat Hubricht , L. & Mackin , J. G. 1940 . Description of nine new species of fresh-water amphipod crustaceans with notes and new localities for other species . American Midland Naturalist , 23 : 187 – 218 . Google Scholar Crossref Search ADS WorldCat Jokela-Maatta , M. , Pahlberg , J., Lindstrom , M., Zak , P., Porter , M., Ostrovsky , M., Cronin , T. & Donner , K. 2005 . Visual pigment absorbance and spectral sensitivity of the Mysis relicta species group (Crustacea, Mysida) in different light environments . Journal of Comparative Physiology Part A , 191 : 1087 – 1097 . Google Scholar Crossref Search ADS WorldCat Keany , J. , Christman , M., Milton , M., Knee , K., Gilbert , H. & Culver , D. 2018 . Distribution and structure of shallow subterranean aquatic arthropod communities in the parklands of Washington DC . Ecohydrology , 12 : e2044 [https://doi.org/10.1002/eco.2044]. Google Scholar Crossref Search ADS WorldCat Kim , B.-M. , Kang , S., Ahn , D.-H., Kim , J.-H., Ahn , I., Lee , C.-W., Cho , J.-L., Min , G.-S. & Park , H. 2017 . First insights into the subterranean crustacean Bathynellacea transcriptome: transcriptionally reduced opsin repertoire and evidence of conserved homeostasis regulatory mechanisms . PLoS One , 12 : e0170424 [https://doi.org/10.1371/journal.pone.0170424]. Google Scholar Crossref Search ADS PubMed WorldCat Kingston , A. & Cronin , T. 2015 . Short- and long-wavelength-sensitivite opsins are involved in photoreception in both the retina and throughout the central nervous system of crayfish . Journal of Comparative Physiology Part A , 201 : 1137 – 1145 . Google Scholar Crossref Search ADS WorldCat Kingston , A. & Cronin , T. 2016 . Diverse distributions of extraocular opsins in crustaceans, cephalopods and fish . Integrative and Comparative Biology , 56 : 820 – 833 . Google Scholar Crossref Search ADS PubMed WorldCat Kohler , S. , Parker , M. & Ford , A. 2018 . Species-specific behaviours in amphipods highlight the need for understanding baseline behaviors in ecotoxicology . Aquatic Toxicology , 202 : 173 – 180 . Google Scholar Crossref Search ADS PubMed WorldCat Martin , G. , Jaros , P.P., Chaigneau , J. & Meyer-Rochow , V.B. 1995 . Intracerebral ocelli in the Giant Antarctic Slater Glyptonotus antarcticus (Isopoda: Valvifera) . Journal of Crustacean Biology , 15 : 228 – 235 Google Scholar Crossref Search ADS WorldCat Maynard , B. , Wellnitz , T., Zanini , N., Wright , W. & Dezfuli , B. 1998 . Parasite-altered behavior in a Crustacean intermediate host: field and laboratory studies . Journal of Parasitology , 84 : 1102 – 1106 . Google Scholar Crossref Search ADS WorldCat Musio , C. & Santillo , S. 2009 . Non-visual photoreception in invertebrates . In: Photobiological Sciences Online ( J. Lee & K.C. Smith, eds.) [http://photobiology.info/]. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Navarro-Barranco , C. & Hughes , L. 2015 . Effects of light pollution on the emergent fauna of shallow marine ecosystems: amphipods as a case study . Marine Pollution Bulletin , 94 : 235 – 240 . Google Scholar Crossref Search ADS PubMed WorldCat Porter , M. , Cronin , T., McClellan , D. & Crandall , K. 2006 . Molecular characterization of crustacean visual pigments and the evolution of pancrustacean opsins . Molecular Biology and Evolution , 24 : 253 – 268 . Google Scholar Crossref Search ADS PubMed WorldCat Randel , N. & Jekely , G. 2016 . Phototaxis and the origin of visual eyes . Philosophical Transactions of the Royal Society B: Biological Sciences , 371 [https://doi.org/10.1098/rstb.2015.0042]. Google Scholar OpenURL Placeholder Text WorldCat Santillo , S. , Orlando , P., De Petrocellis , L., Cristino , L., Guglielmotti , V. & Musio , C. 2006 . Evolving visual pigments: hints from the opsin-based proteins in a phylogenetically old “eyeless” invertebrate . BioSystems , 86 : 3 – 17 . Google Scholar Crossref Search ADS PubMed WorldCat Shand , J. & Foster , R. 1999 . The extraretinal photoreceptors of non-mammalian vertebrates . In: Adaptive mechanisms in the ecology of vision ( Archer , S.E.A., ed.), pp. 197 – 222 . Kluwer Academic Publishers , Dordrecht, The Netherlands . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Sonoda , T. & Lee , S. 2016 . A novel role for the visual retinoid cycle in melanopsin chromophore regeneration . Journal of Neuroscience , 36 : 9016 – 9018 . Google Scholar Crossref Search ADS PubMed WorldCat Stom , D. , Zhdanova , G., Saksonov , M., Balayan , A. & Tolstoy , M. 2017 . Light avoidance in Baikalian amphipods as a test response to toxicants . Contemporary Problems in Ecology , 10 : 77 – 83 . Google Scholar Crossref Search ADS WorldCat Tain , L. , Perrot-Minnot , M. & Cezilly , F. 2006 . Altered host behavior and brain serotonergic activity caused by acanthocephalans: evidence for specificity . Proceedings of the Royal Society London B , 273 : 3039 – 3046 . Google Scholar OpenURL Placeholder Text WorldCat Velarde , R. , Sauer , C., KKO , W., Fahrbach , S. & Robertson , H. 2005 . Pteropsin: a vertebrate-like non-visual opsin expressed in the honey bee brain . Insect Biochemistry and Molecular Biology , 35 : 1367 – 1377 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2022. Published by Oxford University Press on behalf of The Crustacean Society. All rights reserved. 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TI - Novel experimental apparatus for laboratory measurements of phototaxis: A comparison between amphipod species
JF - Journal of Crustacean Biology
DO - 10.1093/jcbiol/ruab085
DA - 2022-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/novel-experimental-apparatus-for-laboratory-measurements-of-phototaxis-LCF0vcDjDN
VL - 42
IS - 1
DP - DeepDyve
ER -