James A. Murray

Frequency as a releaser in the courtship song of two crickets, Gryllus bimaculatus (de Geer) and Teleogryllus oceanicus: a neuroethological analysis

Abstract1.The courtship behavior of male field crickets, Gryllus bimaculatus (De Geer) and Teleogryllus oceanicus, is a complex, multimodal behavioral act that involves acoustic signals (a courtship song; Fig. 1A,B). The dominant frequency is 4.5 kHz for T. oceanicus song (Fig. 1A) and 13.5 kHz for G. bimaculatus (Fig. IB).2.When courting males are deprived of their courtship song by wing amputation, their courtship success declines markedly but is restored when courting is accompanied by tape-recordings of their courtship songs or a synthetic courtship song with only the dominant frequency of the natural song; other naturally occurring frequency components are ineffective for restoring mating success (Figs. 4, 5).3.It has been suggested that an identified auditory interneuron, AN2, plays a critical role in courtship success. Chronic recordings of AN2 in an intact, tethered female show that AN2s response to the natural courtship song and synthesized songs at 4.5 and 13.5 kHz is similar in T. oceanicus. By contrast, in G. bimaculatus, AN2s response to the natural courtship song and synthesized song at 13.5 kHz, but not at 4.5 kHz, is similar (Figs. 2,3).4.In behavioral experiments, playback of a 30 kHz synthetic courtship song in G. bimaculatus does not restore courtship success, yet this same stimulus elicits as strong a response from AN2 as does the normal courtship song (Fig. 6). Thus, contrary to earlier work by others, we conclude AN2 is not, by itself, a critical neural link in the courtship behavior of these two species of crickets.

Computer-assisted visualizations of neural networks : expanding the field of view using seamless confocal montaging

Microscopic analysis of anatomic relationships within the neural networks of adult and developing tissues often requires sampling large spatial regions of neuronal architecture. To accomplish this, there are two common imaging approaches: (1) image the entire area at once with low spatial resolution; or (2) image small sections at higher magnification/resolution and then join the sections back together by mosaic reconstruction (photomontaging). Low magnification imaging is relatively rapid to perform, resulting in a visualization that encompasses a large field of view with an extended depth of field. However, for fluorescence microscopy, low magnification visualizations are often plagued by poor spatial resolution. High magnification imaging possesses superior spatial resolution, but it produces an image with limited depth of field. When creating a larger field of view, the final image is also fragmented at the boundaries where multiple images are stitched together. Using confocal microscopy as well as features of common image processing programs, we outline a new method to transform individual, spatially contiguous z-series into a montage with a seamless field of view and an extended depth of field. In addition, we show that the manual alignment of images our method requires does not introduce significant errors into the final image. We illustrate our method for visualizing neural networks using tissues from the adult gastropod mollusc, Tritonia diomedea, and the developing zebrafish, Danio rerio.

Function of identified nerves in orientation to water flow in Tritonia diomedea.

We determined which sensory and motor nerves mediate orientation to flow in the marine slug Tritonia diomedea, and tested the hypothesis that the slug orients to water flow by comparing the intensities of water flow stimulation on each side of its body. Lesion experiments revealed which nerves carried information necessary for flow orientation. The lateral branches of Cerebral Nerve # 2 were the only cerebral nerves necessary for flow orientation. Cutting all cerebral nerves except the lateral branches of Cerebral Nerve # 2 did not eliminate flow orientation. Thus, the lateral branches of Cerebral Nerve # 2 were both necessary and sufficient (among the cerebral nerves) for flow orientation. Denervation of one side of the head by cutting Cerebral Nerves # 1–4 on one side did not eliminate normal flow orientation. We have revised our model of how Tritonia diomedea orients to flow to allow for this unilateral determination of flow direction. Unilaterally cutting Pedal Nerve # 3, which contains many pedal motor axons, reduced turning toward that side, but did not affect final orientation to flow. The ability to detect flow direction was not compro mised by the inability to initially turn towards flow.

Pedal neuron 3 serves a significant role in effecting turning during crawling by the marine slug Tritonia diomedea (Bergh)

The marine nudibranch Tritonia diomedea crawls using its ciliated foot surface as the sole means of propulsion. Turning while crawling involves raising a small portion of the lateral foot margin on the side of the turn. The cilia in the lifted area no longer contribute to propulsion, and this asymmetry in thrust turns the animal towards the lifted side. Neurons located in the pedal ganglia of the brain contribute to these foot margin contractions. T. diomedea has a natural tendency to turn upstream (rheotaxis), and pedal flexion neuron Pedal 3 elicits foot margin lift and receives modulatory input from flow receptors. To assess the contribution of this single cell in turning behavior, two fine wires were glued to the surface of the brain over left and right Pedal 3. We determined that Pedal 3 activity is correlated with subsequent ipsilateral turns, preceding the lift of the foot margin and the change in orientation by a consistent interval. Both Pedal 3 cells show synchronous bursts of activity, and the firing frequency of the ipsilateral Pedal 3 increased before turns were observed to that side. Stimulation of the electrode over Pedal 3 proved sufficient to elicit an ipsilateral turn in Tritonia.

Advances in the neural bases of orientation and navigation

The ability to locomote in one direction (oriented movement), and the ability to navigate toward a distant goal are related behaviors that are phylogenetically widespread. Orientation behaviors include finding the source of an odor or acoustic signal, using a sun-compass for guidance, and moving relative to fluid-dynamic cues. Such abilities might require little more than directionally selective sensors coupled to a turning mechanism. This type of behavior, therefore, can be implemented by relatively simple circuits. In contrast, navigation involves both the ability to detect direction, as well as a map-sense that provides position. Navigation is less common and arguably requires greater brain computation than does simple orientation, but may be present in arthropods as well as in vertebrates. Great progress has been made in exploring the biophysical and sensory bases for these behaviors, and in recent years the locations and the identity of the cellular transducers of the sensory stimuli (for example, geomagnetic fields) have been narrowed in some taxa. Similarly, neurons within the central nervous that most likely function in higher order computational processes have been identified. For example, direction-selective and position-responsive brain cells have been located in the brains of mammals and birds, and these cells might contribute to a cognitive map that can enable navigation. One model organism in which orientation and navigation has been extensively studied is the sea slug Tritonia diomedea. This animal orients its crawling to chemical, hydrodynamic, and geomagnetic cues. The brain of Tritonia has ∼7000 relatively large neurons that facilitate circuit analysis. Recent work has characterized both peripheral and central neural correlates of orientation signals, as well as the control of thrust and turning, and studies of their field behavior have suggested how these disparate orientation systems may be integrated. These findings provide the basis for future studies to determine how the nervous system combines multiple sensory cues into a complex hierarchy of signals that can direct motor output and therefore guide navigational tasks.

The Mitochondrial Genomes of the Nudibranch Mollusks, Melibe leonina and Tritonia diomedea, and Their Impact on Gastropod Phylogeny

The phylogenetic relationships among certain groups of gastropods have remained unresolved in recent studies, especially in the diverse subclass Opisthobranchia, where nudibranchs have been poorly represented. Here we present the complete mitochondrial genomes of Melibe leonina and Tritonia diomedea (more recently named T. tetraquetra), two nudibranchs from the unrepresented Cladobranchia group, and report on the resulting phylogenetic analyses. Both genomes coded for the typical thirteen protein-coding genes, twenty-two transfer RNAs, and two ribosomal RNAs seen in other species. The twelve-nucleotide deletion previously reported for the cytochrome oxidase 1 gene in several other Melibe species was further clarified as three separate deletion events. These deletions were not present in any opisthobranchs examined in our study, including the newly sequenced M. leonina or T. diomedea, suggesting that these previously reported deletions may represent more recently divergent taxa. Analysis of the secondary structures for all twenty-two tRNAs of both M. leonina and T. diomedea indicated truncated d arms for the two serine tRNAs, as seen in some other heterobranchs. In addition, the serine 1 tRNA in T. diomedea contained an anticodon not yet reported in any other gastropod. For phylogenetic analysis, we used the thirteen protein-coding genes from the mitochondrial genomes of M. leonina, T. diomedea, and seventy-one other gastropods. Phylogenetic analyses were performed for both the class Gastropoda and the subclass Opisthobranchia. Both Bayesian and maximum likelihood analyses resulted in similar tree topologies. In the Opisthobranchia, the five orders represented in our study were monophyletic (Anaspidea, Cephalaspidea, Notaspidea, Nudibranchia, Sacoglossa). In Gastropoda, two of the three traditional subclasses, Opisthobranchia and Pulmonata, were not monophyletic. In contrast, four of the more recently named gastropod clades (Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia) were all monophyletic, and thus appear to be better classifications for this diverse group.

Do chitons have a compass? Evidence for magnetic sensitivity in Polyplacophora

Several animals and microbes have been shown to be sensitive to magnetic fields, though the exact mechanisms of this ability remain unclear in many animals. Chitons are marine molluscs which have high levels of biomineralised magnetite coating their radulae. This discovery led to persistent anecdotal suggestions that they too may be able to navigationally respond to magnetic fields. Several researchers have attempted to test this, but to date there have been no large-scale controlled empirical trials. In the current study, four chiton species (Katharina tunicata, Mopalia kennerleyi, Mopalia muscosa and Leptochiton rugatus, n = 24 in each) were subjected to natural and artificially rotated magnetic fields while their movement through an arena was recorded over four hours. Field orientation did not influence the position of the chitons at the end of trials, possibly as a result of the primacy of other sensory cues (i.e. thigmotaxis). Under non-rotated magnetic field conditions, the orientation of subjects when they first reached the edge of an arena was clustered around 309–345° (north–north-west) in all four species. However, orientations were random under the rotated magnetic field, which may indicate a disruptive effect of field rotation. This pattern suggests that chitons can detect and respond to magnetism.

Daily tracking of the locomotion of the nudibranch Tritonia tetraquetra (Pallas 1788 = Tritonia diomedea Bergh 1894) in nature and the influence of water flow on orientation, crawling, and drag

We observed orientation and locomotion of the nudibranch Tritonia tetraquetra in its natural habitat using SCUBA over many sequential days, in three different months. The slugs oriented significantly headfirst to tidal currents. Nevertheless, the direction of locomotion of the slugs over hours was not usually correlated with tidal flow direction (i.e. not indicative of consistent rheotaxis). We did not find evidence of consistent body axis orientation to the geomagnetic field, but the direction of locomotion of some groups of slugs over hours was significantly correlated with geomagnetic direction. Independent of direction, each slug changed position by an average of ∼2 m during a single tidal phase (∼6 h), and changed position by an average of ∼4 m over a full tidal cycle (∼25 h). Orientation to flow reduced drag, and reduced the probability that a slug will be dislodged from the soft bottom, in laboratory experiments. Slugs deprived of olfactory and flow cues exhibit a search-like pattern of multiple and frequent turns. †The species described here is Tritonia tetraquetra (Pallas 1788). In the recent modern literature, it has been referred to as Tritonia diomedea (Bergh 1894), but this name now has junior synonym status (Martynov 2006).

Introduction to the Symposium—Chemicals that Organize Ecology: Towards a Greater Integration of Chemoreception, Neuroscience, Organismal Biology, and Chemical Ecology

Ecology as a process is shaped by biotic and abiotic influences. In some cases, a single type of chemical can have effects on an ecosystem, effects that are disproportionate to its relative mass, such that the ecosystem would organize differently in the absence of this type of chemical. The hallmark examples are those ‘‘molecules of keystone significance’’ (Ferrer and Zimmer 2012), such as the biosequestered alkaloid tetrodotoxin. Keystone molecules are analogous to keystone species in their capacity to mediate large ecological effects disproportionate to their mass. Some predators/grazers may evolve the ability to sense toxins being released from potential prey, making the toxin a semiochemical, molecules that serve as information-bearing cues among organisms (Ferrer and Zimmer 2012). Not all semiochemicals may be of keystone significance but they do have a greater effect on ecology that would be predicted, based on their abundance relative to all biomolecules, and could be said to ‘‘organize ecology.’’ Zimmer and Derby (2011) created a conceptual framework relating chemical defenses and neurobiological function in the SICB 2011 symposium entitled ‘‘Neural Determinants of Ecological Processes from Individuals to Ecosystems.’’ They advocated for an approach to defensive chemicals that integrated the isolation and identification of chemicals with study of behavioral responses, sensory and neuronal mechanisms of action, and ecological effects and adaptation. Their goals included learning how defensive chemicals affect the abundance and distribution of organisms and species. An integrative approach would include not only analytical chemistry, illustrating distributions of chemical within individual organs and across trophic levels, but would also attempt to link the chemicals to behavioral and physiological effects, and, ultimately, to reproductive success so as to define the mechanisms of the selective forces that shape ecology. Defensive chemicals can be released and repel competitors or predators/herbivores (van Alstyne et al. 2015, this issue), and one might predict that these competitors would eventually evolve an insensitivity to such cues unless the released chemicals were toxic. Alternatively, defensive chemicals can be accumulated or sequestered and they can affect trophic interactions upon contact or after partial ingestion. Some of these accumulated chemicals, such as saxitoxin (Ferrer et al. 2015, this issue) can be biosequestered over multiple trophic levels and keystone molecules are prime examples (Ferrer and Zimmer 2012). Also, chemicals with less comprehensive roles can also contribute to organizing ecological interactions. Other effects of defensive chemicals include the evolution of counter-measures such as avoidance of, or resistance to, toxins, and evolution of mechanisms of sensation (Lunceford and Kubanek 2015, this issue). One goal of this symposium was to extend the causes of ecology to the exchange of semiochemicals (pheromones, kairomones, allomones, and synomones) (Sbarbati and Osculati 2006). Thus, Integrative and Comparative Biology