Cobalt chloride treatment reduces DASPEI staining in the olfactory epithelium of the cichlid <i>A</i>. <i>burtoni</i>.

<p><b>A</b>) Representative photomicrographs of DASPEI-stained olfactory epithelia from <i>A</i>. <i>burtoni</i> immersed in untreated-control water, low-calcium water, low-0.1mM CoCl₂, and high-2mM CoCl₂ immediately after treatment and 18-hours post-treatment. <b>B</b>) Quantification of DASPEI fluorescence intensity of the olfactory epithelium. Fluorescence intensity was normalized to untreated-controls such that a value = 1 indicates the same fluorescence, >1 indicates greater fluorescence, and <1 indicates reduced fluorescence relative to the untreated-controls. Different letters indicate statistical significance at P<0.05. Tukey’s box plots were used to represent data (N = 6 fish per group): data median is represented by a line and data mean by an open circle, the box extends to the furthest data points within the 25th and 75th percentile, and whiskers extend to the furthest data points not considered outliers. Absence of whiskers indicates absence of data points outside of the 25th/75th percentile. Outliers are represented by closed circles. Scale bars in A represent 200μm. <i>Abbreviations</i>: C: caudal, L: lateral, M: medial, R: rostral.</p

Cobalt chloride-treated <i>Astatotilapia burtoni</i> have reduced <i>cfos</i> staining in the olfactory bulb.

<p><b>A</b>) Animals treated with cobalt chloride have reduced <i>cfos</i> staining in the internal cellular layer of the olfactory bulb compared to untreated-control and low-calcium fish (N = 4–6 fish for each group). Photomicrographs of <i>cfos</i> staining (purple label) in the olfactory bulbs of representative untreated-control (<b>B</b>), low-calcium (<b>C</b>), low-0.1mM CoCl₂-treated (<b>D</b>), and high-2mM CoCl₂-treated (<b>E</b>) fish. Arrows indicate internal cellular layer (ICL) of the olfactory bulb. Different letters represent statistical significance at P<0.05. Scale bars in B-E represent 100μm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159521#pone.0159521.g002" target="_blank">Fig 2</a> legend for box plot descriptions.</p

CoCl₂-treated <i>Astatotilapia burtoni</i> had similar <i>cfos</i> staining in a control brain region that is not known to process olfactory information.

<p><b>A</b>) <i>A</i>. <i>burtoni</i> immersed in untreated-control, low-calcium, low-0.1mM CoCl₂, and high-2mM CoCl₂ solutions had similar numbers of <i>cfos</i>-stained cells in the Dl-dr indicating that CoCl₂ treatment did not affect overall brain activation levels (N = 4–6 fish for each group). Photomicrographs of <i>cfos</i> staining in the Dl-dr of untreated-control (<b>B</b>), low-calcium (<b>C</b>), low-0.1mM CoCl₂-treated (<b>D</b>), and high-2mM CoCl₂-treated (<b>E</b>) fish allowed to recover for 18-hours Same letters represent no statistical significance at P<0.05. Scale bars in B-E represent 10000B0035m. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159521#pone.0159521.g002" target="_blank">Fig 2</a> legend for box plot descriptions. <i>Abbreviations</i>: Dl-dr, rostral portion of the dorsal part of the lateral zone of the dorsal telencephalon; Dl-v, ventral part of the lateral zone of the dorsal telencephalon; ICL, internal cellular layer of the olfactory bulb.</p

Goldfish and cavefish treated with CoCl₂ have reduced DASPEI staining of the olfactory epithelium.

<p>Representative photomicrographs of the olfactory epithelium of control (<b>A</b>) and CoCl₂-treated (<b>B</b>) goldfish, <i>C</i>. <i>auratus</i>. <b>C</b>) Quantification of DASPEI fluorescence intensity of the olfactory epithelium of control and CoCl₂-treated <i>C</i>. <i>auratus</i> (N = 7–8 fish per group). Representative photographs of the olfactory epithelium of control (<b>D</b>) and CoCl₂-treated (<b>E</b>) Mexican blind cavefish, <i>A</i>. <i>mexicanus</i>. <b>F</b>) Quantification of DASPEI fluorescence intensity of the olfactory epithelium of control and CoCl₂-treated <i>A</i>. <i>mexicanus</i> (N = 6–7 fish per group). Different letters indicate statistical significance at P<0.05. Scale bars in A, B, D, and E represent 200μm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159521#pone.0159521.g002" target="_blank">Fig 2</a> for box plot descriptions. Abbreviations: C: caudal, L: lateral, M: medial, R: rostral. Orientation in A also applies to images in B, C, and D.</p

Cobalt Chloride Treatment Used to Ablate the Lateral Line System Also Impairs the Olfactory System in Three Freshwater Fishes

<div><p>Fishes use multimodal signals during both inter- and intra-sexual displays to convey information about their sex, reproductive state, and social status. These complex behavioral displays can include visual, auditory, olfactory, tactile, and hydrodynamic signals, and the relative role of each sensory channel in these complex multi-sensory interactions is a common focus of neuroethology. The mechanosensory lateral line system of fishes detects near-body water movements and is implicated in a variety of behaviors including schooling, rheotaxis, social communication, and prey detection. Cobalt chloride is commonly used to chemically ablate lateral line neuromasts, thereby eliminating water-movement cues to test for mechanosensory-mediated behavioral functions. However, cobalt acts as a nonspecific calcium channel antagonist and could potentially disrupt function of all superficially located sensory receptor cells, including those for chemosensing. Here, we examined whether CoCl₂ treatment used to ablate the lateral line system also impairs olfaction in three freshwater fishes, the African cichlid fish <i>Astatotilapia burtoni</i>, goldfish <i>Carassius auratus</i>, and the Mexican blind cavefish <i>Astyanax mexicanus</i>. To examine the impact of CoCl₂ on the activity of peripheral receptors, we quantified DASPEI fluorescence intensity of the olfactory epithelium from fish exposed to control and CoCl₂ solutions. In addition, we examined brain activation in olfactory processing regions of <i>A</i>. <i>burtoni</i> immersed in either control or cobalt solutions. All three species exposed to CoCl₂ had decreased DASPEI staining of the olfactory epithelium, and in <i>A</i>. <i>burtoni</i>, cobalt treatment caused reduced neural activation in olfactory processing regions of the brain. To our knowledge this is the first empirical evidence demonstrating that the same CoCl₂ treatment used to ablate the lateral line system also impairs olfactory function. These data have important implications for the use of CoCl₂ in future research and suggest that previous studies using CoCl₂ should be reinterpreted in the context of both impaired mechanoreception <i>and</i> olfaction.</p></div

Pearson correlation of OE DASPEI-staining and brain <i>cfos</i>-staining in <i>A</i>. <i>burtoni</i>.

<p>Pearson correlation of OE DASPEI-staining and brain <i>cfos</i>-staining in <i>A</i>. <i>burtoni</i>.</p

Cobalt chloride-treated <i>Astatotilapia burtoni</i> have lower densities of <i>cfos</i>-stained cells in the posterior part of the dorsal telencephalon (Dp) at 18-hours post-treatment.

<p><b>A</b>) <i>A</i>. <i>burtoni</i> treated with high-2mM CoCl₂ have fewer <i>cfos</i>-stained cells in the Dp 18-hours post-treatment but not immediately following treatment (N = 4–6 fish for each group). Photomicrographs of <i>cfos</i> staining in the Dp of untreated-control (<b>B</b>), low-calcium (<b>C</b>), low-0.1mM CoCl₂-treated (<b>D</b>), and high-2mM CoCl₂-treated (<b>E</b>) fish allowed to recover for 18 hours. Different letters represent statistical significance at P<0.05. Scale bars in B-E represent 100m. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159521#pone.0159521.g002" target="_blank">Fig 2</a> legend for box plot descriptions. <i>Abbreviations</i>: E, entopeduncular nucleus; Dp, posterior part of the dorsal telencephalon; Vp, postcommissural nucleus of the ventral telencephalon.</p

Example of auditory evoked potential (AEP) traces recorded from <i>A. burtoni</i>.

<p>Averaged AEP traces from a representative subordinate male in response to a 200 Hz stimulus at several different intensities. An averaged trace from a control dead fish at 120 dB in response to a 200 Hz stimulus shows no response. Bottom trace shows the actual stimulus waveform recorded by the hydrophone at the position of the fish head. Threshold at this frequency was 105 dB_rms re: 1 µPa based on the repeatable waveform and the presence of an FFT peak at twice the stimulus frequency.</p

Dual Roles of an Algal Farming Damselfish as a Cultivator and Opportunistic Browser of an Invasive Seaweed

<div><p>Herbivory is a fundamental process determining reef resilience, and while algal farming damselfishes can help shape benthic assemblages, an understanding of their contribution to areas outside of defended territories is relatively unexplored. Here, we demonstrate how the farming damselfish <i>Stegastes marginatus</i> plays a dual role in benthic structuring by 1) contributing to persistence of the invasive macroalga <i>Acanthophora spicifera</i> within a Hawaiian marine protected area, where the macroalga occurred exclusively inside <i>Stegastes</i> territories, and 2) behaving as an opportunistic browser of the exotic alga outside their territorial borders. Greater than 50% of the biomass of tethered <i>A. spicifera</i> was consumed within one-hour when placed outside <i>Stegastes</i> territories, compared to <5% lost from tethers within territories or herbivore exclusion cages. <i>In situ</i> remote video revealed that tethered <i>A. spicifera</i> located outside territories was grazed primarily by the surgeonfish <i>Acanthurus nigrofuscus</i> (∼68% of total bites) and, surprisingly, by <i>S. marginatus</i> (∼27% of total bites) that left their territories to feed on this resource on 107 occasions during 400 min of filming. Further, for over half of those occurrences where <i>S. marginatus</i> grazed on the tethered macroalga outside of territories, they fed alongside conspecifics and other species, displaying little of the aggressiveness that characterizes this damselfish. These results show that <i>S. marginatus</i> plays a wider role in determining benthic assemblages than previously recognized, acting both as cultivators of a canopy-forming invasive macroalga within their territories, and as opportunistic browsers in neighboring sites. Consequently, <i>S. marginatus</i> can affect benthic species composition across their territory borders. These results provide a rare example of interspecific facilitation of an exotic alga by an indigenous marine fish. Accounting for fish behaviors more broadly is important to further our understanding of ecological processes that shape reef ecosystems to improve management of MPAs that often support extensive farming damselfish populations.</p></div

Characteristics of courtship sounds produced by male <i>A. burtoni</i> during quiver behaviors.

<p>A) Relationship between mean peak frequency (Hz) of sounds and male body size (standard length) shows that larger males produce lower frequency sounds. Each point represents the mean±SE of several sounds produced by an individual fish. B) There is a positive linear relationship between the number of pulses per sound and total sound duration (ms).</p