Wide-Field Calcium Imaging of Dynamic Cortical Networks during Locomotion.

Motor behavior results in complex exchanges of motor and sensory information across cortical regions. Therefore, fully understanding the cerebral cortex's role in motor behavior requires a mesoscopic-level description of the cortical regions engaged, their functional interactions, and how these functional interactions change with behavioral state. Mesoscopic Ca2+ imaging through transparent polymer skulls in mice reveals elevated activation of the dorsal cerebral cortex during locomotion. Using the correlations between the time series of Ca2+ fluorescence from 28 regions (nodes) obtained using spatial independent component analysis (sICA), we examined the changes in functional connectivity of the cortex from rest to locomotion with a goal of understanding the changes to the cortical functional state that facilitate locomotion. Both the transitions from rest to locomotion and from locomotion to rest show marked increases in correlation among most nodes. However, once a steady state of continued locomotion is reached, many nodes, including primary motor and somatosensory nodes, show decreases in correlations, while retrosplenial and the most anterior nodes of the secondary motor cortex show increases. These results highlight the changes in functional connectivity in the cerebral cortex, representing a series of changes in the cortical state from rest to locomotion and on return to rest.

Additive and epistatic effects influence spectral tuning in molluscan retinochrome opsin.

The relationship between genotype and phenotype is non-trivial because of the often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, comprising an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype (λmax). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket is responsible for fine spectral tuning, or small changes in the λmax of these light-sensitive proteins. Mutations resulted in a blue or red shift as predicted, but with dissimilar magnitudes. Shifts ranged from a 16 nm blue shift to a 12 nm red shift from the wild-type λmax. These mutations do not show an additive effect, but rather suggest the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis.

Medium compensation in a spring-actuated system.

Mantis shrimp strikes are one of the fastest animal movements, despite their occurrence in a water medium with viscous drag. Since the strike is produced by a latch-mediated spring-actuated system and not directly driven by muscle action, we predicted that strikes performed in air would be faster than underwater as a result of reduction in the medium's drag. Using high-speed video analysis of stereotyped strikes elicited from Squilla mantis, we found the exact opposite: strikes are much slower and less powerful in air than in water. S. mantis strikes in air have a similar mass and performance to latch-mediated spring-actuated jumps in locusts, suggesting a potential threshold for the energetics of a 1-2 g limb rotating in air. Drag forces induced by the media may be a key feature in the evolution of mantis shrimp strikes and provide a potential target for probing the braking system of these extremely fast movements.

An Electrophysiological Investigation of Power-Amplification in the Ballistic Mantis Shrimp Punch.

Mantis shrimp are aggressive, burrowing crustaceans that hunt using one the fastest movements in the natural world. These stomatopods can crack the calcified shells of prey or spear down unsuspecting fish with lighting speed. Their strike makes use of power-amplification mechanisms to move their limbs much faster than is possible by muscles alone. Other arthropods such as crickets and grasshoppers also use power-amplified kicks that allow these animals to rapidly jump away from predator threats. Here we present a template laboratory exercise for studying the electrophysiology of power-amplified limb movement in arthropods, with a specific focus on mantis shrimp strikes. The exercise is designed in such a way that it can be applied to other species that perform power-amplified limb movements (e.g., house crickets, Acheta domesticus) and species that do not (e.g., cockroaches, Blaberus discoidalis). Students learn to handle the animals, make and implant electromyogram (EMG) probes, and finally perform experiments. This integrative approach introduces the concept of power-amplified neuromuscular control; allows students to develop scientific methods, and conveys high-level insights into behavior, and convergent evolution, the process by which different species evolve similar traits. Our power-amplification laboratory exercise involves a non-terminal preparation which allows electrophysiological recordings across multiple days from arthropods using a low-cost EMG amplifier. Students learn the principles of electrophysiology by fabricating their own electrode system and performing implant surgeries. Students then present behaviorally-relevant stimuli that generate attack strikes in the animals during the electrophysiology experiments to get insight into the underlying mechanisms of power amplification. Analyses of the EMG data (spike train burst duration, firing rate, and spike amplitude) allow students to compare mantis shrimp with other power-amplifying species, as well as a non-power-amplifying one. The major learning goal of this exercise is to empower students by providing an experience to develop their own setup to examine a complex biological principle. By contrasting power-amplifiers with non-power-amplifiers, these analyses highlight the peculiarity of power amplification at multiple levels of analysis, from behavior to physiology. Our comparative design requires students to consider the behavioral function of the movement in different species alongside the neuromuscular underpinnings of each movement. This laboratory exercise allows students to develop methodology, problem-solving and inquisitive skills crucial for pursuing science.

Spectral absorption of visual pigments in stomatopod larval photoreceptors.

Larval stomatopod eyes appear to be much simpler versions of adult compound eyes, lacking most of the visual pigment diversity and photoreceptor specializations. Our understanding of the visual pigment diversity of larval stomatopods, however, is based on four species, which severely limits our understanding of stomatopod eye ontogeny. To investigate several poorly understood aspects of stomatopod larval eye function, we tested two hypotheses surrounding the spectral absorption of larval visual pigments. First, we examined a broad range of species to determine if stomatopod larvae generally express a single, spectral class of photoreceptor. Using microspectrophotometry (MSP) on larvae captured in the field, we found data which further support this long-standing hypothesis. MSP was also used to test whether larval species from the same geographical region express visual pigments with similar absorption spectra. Interestingly, despite occupation of the same geographical location, we did not find evidence to support our second hypothesis. Rather, there was significant variation in visual pigment absorption spectra among sympatric species. These data are important to further our understanding of larval photoreceptor spectral diversity, which is beneficial to ongoing investigations into the ontogeny, physiology, and molecular evolution of stomatopod eyes.

Seeing double: visual physiology of double-retina eye ontogeny in stomatopod crustaceans.

Stomatopod eye development is unusual among crustaceans. Just prior to metamorphosis, an adult retina and associated neuro-processing structures emerge adjacent to the existing material in the larval compound eye. Depending on the species, the duration of this double-retina eye can range from a few hours to several days. Although this developmental process occurs in all stomatopod species observed to date, the retinal physiology and extent to which each retina contributes to the animal's visual sensitivity during this transition phase is unknown. We investigated the visual physiology of stomatopod double retinas using microspectrophotometry and electroretinogram recordings from different developmental stages of the Western Atlantic species Squilla empusa. Though microspectrophotometry data were inconclusive, we found robust ERG responses in both larval and adult retinas at all sampled time points indicating that the adult retina responds to light from the very onset of its emergence. We also found evidence of an increase in the response dynamics with ontogeny as well as an increase in sensitivity of retinal tissue during the double-retina phase relative to single retinas. These data provide an initial investigation into the ontogeny of vision during stomatopod double-retina eye development.

Out of the blue: the evolution of horizontally polarized signals in Haptosquilla (Crustacea, Stomatopoda, Protosquillidae).

The polarization of light provides information that is used by many animals for a number of different visually guided behaviours. Several marine species, such as stomatopod crustaceans and cephalopod molluscs, communicate using visual signals that contain polarized information, content that is often part of a more complex multi-dimensional visual signal. In this work, we investigate the evolution of polarized signals in species of Haptosquilla, a widespread genus of stomatopod, as well as related protosquillids. We present evidence for a pre-existing bias towards horizontally polarized signal content and demonstrate that the properties of the polarization vision system in these animals increase the signal-to-noise ratio of the signal. Combining these results with the increase in efficacy that polarization provides over intensity and hue in a shallow marine environment, we propose a joint framework for the evolution of the polarized form of these complex signals based on both efficacy-driven (proximate) and content-driven (ultimate) selection pressures.

Morphological and molecular description of the late-stage larvae of Alima Leach, 1817 (Crustacea: Stomatopoda) from Lizard Island, Australia.

Alima pacifica and A. orientalis are stomatopods commonly found at Lizard Island, Great Barrier Reef, Australia. There are currently no descriptions that link the larvae to the adult morphotype despite the frequent occurrence of the last larval stage of these two species. We used DNA barcoding of the cytochrome oxidase I (COI) gene to link the last stage larvae of A. pacifica and A. orientalis to the respective adult morphotype. Detailed morphological descriptions of the late larva of each species are provided and compared to other described last-stage Alima larvae. These data support previous studies that suggest paraphyly of the genus Alima.

Investigation of the ultrastructures and retinal arrangements of larval stomatopod eyes.

Though the transparent apposition eyes of larval stomatopod crustaceans lack most of the unique retinal specializations known from their adult counterparts, increasing evidence suggests that these tiny pelagic organisms possess their own version of retinal complexity. In this paper, we examined the structural organization of larval eyes in six species of stomatopod crustaceans across three stomatopod superfamilies using transmission electron microscopy. The primary focus was to examine retinular cell arrangement of the larval eyes and characterize the presence of an eighth retinular cell (R8), which is typically responsible for UV vision in crustaceans. For all species investigated, we identified R8 photoreceptor cells positioned distal to the main rhabdom of R1-7 cells. This is the first evidence that R8 photoreceptor cells exist in larval stomatopod retinas, and among the first identified in any larval crustacean. Considering recent studies that identified UV sensitivity in larval stomatopods, we propose that this sensitivity is driven by this putative R8 photoreceptor cell. Additionally, we identified a potentially unique crystalline cone structure in each of the species examined, the function of which is still not understood.

Surf and turf vision: Patterns and predictors of visual acuity in compound eye evolution.

Eyes have the flexibility to evolve to meet the ecological demands of their users. Relative to camera-type eyes, the fundamental limits of optical diffraction in arthropod compound eyes restrict the ability to resolve fine detail (visual acuity) to much lower degrees. We tested the capacity of several ecological factors to predict arthropod visual acuity, while simultaneously controlling for shared phylogenetic history. In this study, we have generated the most comprehensive review of compound eye visual acuity measurements to date, containing 385 species that span six of the major arthropod classes. An arthropod phylogeny, made custom to this database, was used to develop a phylogenetically-corrected generalized least squares (PGLS) linear model to evaluate four ecological factors predicted to underlie compound eye visual acuity: environmental light intensity, foraging strategy (predator vs. non-predator), horizontal structure of the visual scene, and environmental medium (air vs. water). To account for optical constraints on acuity related to animal size, body length was also included, but this did not show a significant effect in any of our models. Rather, the PGLS analysis revealed that the strongest predictors of compound eye acuity are described by a combination of environmental medium, foraging strategy, and environmental light intensity.

Using larval barcoding to estimate stomatopod species richness at Lizard Island, Australia for conservation monitoring.

Stomatopods (Crustacea, Stomatopoda) are well studied for their aggressive behavior and unique visual system as well as their commercial importance in Asian and European countries. Like many crustaceans, stomatopods undergo indirect development, passing though several larval stages before reaching maturity. Adult stomatopods can be difficult to catch due to their inaccessible habitats and cryptic coloration. By sampling larvae from the planktonic community, less effort is required to obtain accurate measures of species richness within a region. Stomatopod larvae were collected between 2006 and 2015 from the waters around the Lizard Island reef platform in Eastern Australia. Cytochrome oxidase I (COI) mitochondrial DNA sequences were generated from each larval sample and compared to a database of COI sequences tied to adult specimens. Of the 20 species collected from Lizard Island as adults which have COI data available, 18 species were identified from larval sampling. One additional species identified from larval samples, Busquilla plantei, was previously unknown from Lizard Island. Nine larval OTUs were found not to match any published adult sequences. Sampling larval stomatopod populations provides a comparable picture of the adult population to benthic sampling methods and may include species richness beyond what is measurable by sampling adult populations.

Long-Wavelength Reflecting Filters Found in the Larval Retinas of One Mantis Shrimp Family (Nannosquillidae).

Both vertebrates and invertebrates commonly exploit photonic structures adjacent to their photoreceptors for visual benefits. For example, use of a reflecting structure (tapetum) behind the retina increases photon capture, enhancing vision in dim light [1-5]. Colored filters positioned lateral or distal to a photoreceptive unit may also be used to tune spectral sensitivity by selective transmission of wavelengths not absorbed or scattered by the filters [6-8]. Here we describe a new category of biological optical filter that acts simultaneously as both a transmissive spectral filter and narrowband reflector. Discovered in the larval eyes of only one family of mantis shrimp (stomatopod) crustaceans (Nannosquillidae), each crystalline structure bisects the photoreceptive rhabdom into two tiers and contains an ordered array of membrane-bound vesicles with sub-wavelength diameters of 153 ± 5 nm. Axial illumination of the intrarhabdomal structural reflector (ISR) in vivo produces a narrow band of yellow reflectance (mean peak reflectivity, 572 ± 18 nm). The ISR is similar to several synthetic devices, such as bandgap filters, laser mirrors, and (in particular) fiber Bragg gratings used in optical sensors for a wide range of industries. To our knowledge, the stomatopod larval ISR is the first example of a naturally occurring analog to these human-made devices. Considering what is known about these animals' visual ecology, we propose that these reflecting filters may help improve the detection of pelagic bioluminescence in shallow water at night. VIDEO ABSTRACT.

Selection of the intrinsic polarization properties of animal optical materials creates enhanced structural reflectivity and camouflage.

Many animals use structural coloration to create bright and conspicuous visual signals. Selection of the size and shape of the optical structures animals use defines both the colour and intensity of the light reflected. The material used to create these reflectors is also important; however, animals are restricted to a limited number of materials: commonly chitin, guanine and the protein, reflectin. In this work we highlight that a particular set of material properties can also be under selection in order to increase the optical functionality of structural reflectors. Specifically, polarization properties, such as birefringence (the difference between the refractive indices of a material) and chirality (which relates to molecular asymmetry) are both under selection to create enhanced structural reflectivity. We demonstrate that the structural coloration of the gold beetle Chrysina resplendens and silvery reflective sides of the Atlantic herring, Clupea harengus are two examples of this phenomenon. Importantly, these polarization properties are not selected to control the polarization of the reflected light as a source of visual information per se. Instead, by creating higher levels of reflectivity than are otherwise possible, such internal polarization properties improve intensity-matching camouflage.This article is part of the themed issue 'Animal coloration: production, perception, function and application'.

A shape-anisotropic reflective polarizer in a stomatopod crustacean.

Many biophotonic structures have their spectral properties of reflection 'tuned' using the (zeroth-order) Bragg criteria for phase constructive interference. This is associated with a periodicity, or distribution of periodicities, parallel to the direction of illumination. The polarization properties of these reflections are, however, typically constrained by the dimensional symmetry and intrinsic dielectric properties of the biological materials. Here we report a linearly polarizing reflector in a stomatopod crustacean that consists of 6-8 layers of hollow, ovoid vesicles with principal axes of ~550 nm, ~250 nm and ~150 nm. The reflection of unpolarized normally incident light is blue/green in colour with maximum reflectance wavelength of 520 nm and a degree of polarization greater than 0.6 over most of the visible spectrum. We demonstrate that the polarizing reflection can be explained by a resonant coupling with the first-order, in-plane, Bragg harmonics. These harmonics are associated with a distribution of periodicities perpendicular to the direction of illumination, and, due to the shape-anisotropy of the vesicles, are different for each linear polarization mode. This control and tuning of the polarization of the reflection using shape-anisotropic hollow scatterers is unlike any optical structure previously described and could provide a new design pathway for polarization-tunability in man-made photonic devices.

Additions to Philippine Slender Skinks of the Brachymeles bonitae Complex (Reptilia: Squamata: Scincidae) I: a new species from Lubang Island.

A new species of slender skink is described from the Philippines. The species is endemic to Lubang Island, and is assigned to the Brachymeles bonitae Complex based on phenotypic and genetic data. Specimens were collected from Lubang Island between 1991 and 2012, and were examined based on morphological data (qualitative traits, meristic counts, and mensural measurements). Published genetic sequence data from phylogenetic studies of the genus reveal the new species to be highly divergent from congeners. Brachymeles ligtas sp. nov. is differentiated from other members of the genus based on a number of distinct morphological features, including small body size (SVL 60.7-79.6 mm), bidactyl fore-limbs, digitless hind limbs, high number of presacral vertebrae (50), and the absence of auricular openings. Additionally, the new species has diagnostic, distinct dorsal head scale patterns. This new species becomes the only member of the genus known to occur on the deep-ocean island of Lubang.

Additions to Philippine Slender Skinks of the Brachymeles bonitae Complex (Reptilia: Squamata: Scincidae) III: a new species from Tablas Island.

Studies of the diversity of Philippine amphibians and reptiles have resulted in the continued description of cryptic species. Species formerly thought to range across multiple recognized faunal regions are now considered to be assemblages of multiple unique species, each restricted to a single faunal region. This pattern continues to hold true when considering Philippine skinks of the genus Brachymeles. Recent studies have resulted in the description of numerous unique species with many exhibiting various degrees of digit loss or limb reduction, as well as suggesting that unique lineages are still present in the B. bonitae Complex. In this paper, we describe a new species of fossorial skink within this species complex from Tablas Island based on collections made nearly 50 years ago. Although no genetic data are available for the new species, examinations of morphological data (qualitative traits, meristic counts, and mensural measurements) support its distinction from all other members of the genus. Brachymeles dalawangdaliri sp. nov. is differentiated from other members of the genus based on a suite of unique phenotypic characteristics, including a small body size (SVL 66.0-80.9 mm), bidactyl fore-limbs, digitless, unidactyl, or bidactyl hind limbs, a high number of presacral vertebrae (49), the absence of auricular openings, and distinct dorsal head scale patterns. The description of the new species increases the diversity of endemic vertebrates recognized to occur in the Romblon Island Group in the central Philippines.