Methods for dynamic and whole volume imaging of the zebrafish heart.

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.

Odor coding of nestmate recognition in the eusocial ant Camponotus floridanus.

In eusocial ants, aggressive behaviors require the ability to discriminate between chemical signatures such as cuticular hydrocarbons that distinguish nestmate friends from non-nestmate foes. It has been suggested that a mismatch between a chemical signature (label) and the internal, neuronal representation of the colony odor (template) leads to aggression between non-nestmates. Moreover, a definitive demonstration that odorant receptors are responsible for the processing of the chemical signals that regulate nestmate recognition has thus far been lacking. To address these issues, we have developed an aggression-based bioassay incorporating highly selective modulators that target odorant receptor functionality to characterize their role in nestmate recognition in the formicine ant Camponotus floridanus Electrophysiological studies were used to show that exposure to either a volatilized antagonist or an agonist eliminated or dramatically altered signaling, respectively. Administration of these compounds to adult workers significantly reduced aggression between non-nestmates without altering aggression levels between nestmates. These studies provide direct evidence that odorant receptors are indeed necessary and sufficient for mediating aggression towards non-nestmates. Furthermore, our observations support a hypothesis in which rejection of non-nestmates depends on the precise decoding of chemical signatures present on non-nestmates as opposed to the absence of any information or the active acceptance of familiar signatures.

Age and Task Modulate Olfactory Sensitivity in the Florida Carpenter Ant Camponotus floridanus.

Age-related changes in behavior and sensory perception have been observed in a wide variety of animal species. In ants and other eusocial insects, workers often progress through an ordered sequence of olfactory-driven behavioral tasks. Notably, these behaviors are plastic, and workers adapt and rapidly switch tasks in response to changing environmental conditions. In the Florida carpenter ant, smaller minors typically perform most of the work needed to maintain the colony, while the larger majors are specialized for nest defense and rarely engage in these routine tasks. Here, we investigate the effects of age and task group on olfactory responses to a series of odorant blends in minor and major worker castes. Consistent with their respective roles within the colony, we observed significant age-associated shifts in the olfactory responses of minors as they transitioned between behavioral states, whereas the responses of majors remained consistently low regardless of age. Furthermore, we have identified a unitary compound, 3-methylindole, which elicited significantly higher responses and behavioral aversion in minor nurses than in similarly aged foragers suggesting that this compound may play an important role in brood care. Taken together, our results suggest that age- and task-associated shifts in olfactory physiology may play a critical role in the social organization of ant colonies.

Intramyocardial Injection for the Study of Cardiac Lymphatic Function in Zebrafish.

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The present protocol describes a method for injecting fluorescent tracers into the zebrafish myocardium to study interstitial fluid and debris uptake into cardiac lymphatic vessels. To do so, microspheres (200 nm diameter) and quantum dots (<10 nm diameter) are introduced into the myocardium of live zebrafish, which can be tracked using ex vivo confocal microscopy. These tracers are then tracked intermittently over several hours to follow clearance from the myocardium into cardiac lymphatic vessels. Quantum dots are transported through cardiac lymphatic vessels away from the heart, while larger microspheres remain at the injection site for over three weeks. This method of intramyocardial injection can be extended to other uses, including the injection of encapsulated MS or hydrogels to locally release cells, proteins, or compounds of interest to a targeted region of the heart.

Advances in the Study of Olfaction in Eusocial Ants.

Over the past decade, spurred in part by the sequencing of the first ant genomes, there have been major advances in the field of olfactory myrmecology. With the discovery of a significant expansion of the odorant receptor gene family, considerable efforts have been directed toward understanding the olfactory basis of complex social behaviors in ant colonies. Here, we review recent pivotal studies that have begun to reveal insights into the development of the olfactory system as well as how olfactory stimuli are peripherally and centrally encoded. Despite significant biological and technical impediments, substantial progress has been achieved in the application of gene editing and other molecular techniques that notably distinguish the complex olfactory system of ants from other well-studied insect model systems, such as the fruit fly. In doing so, we hope to draw attention not only to these studies but also to critical knowledge gaps that will serve as a compass for future research endeavors.