Killer whale innovation: teaching animals to use their creativity upon request.

Thinking flexibly is a skill that enables animals to adapt to changing environments, which enhances survival. Killer whales, Orcinus orca, as the ocean apex predator display a number of complex cognitive abilities, especially flexible thinking or creativity when it comes to foraging. In human care, smaller dolphins and other marine mammals have been trained to think creatively while under stimulus control. The results of these previous studies have demonstrated that bottlenose dolphins, Tursiops truncatus, can create original behaviors in response to an innovative cue. We trained and tested a total of nine killer whales from two different facilities on the innovate concept, using the same methodology. The killer whales ranged in age from 5 to 29 yrs with 4 females and 5 males. The results indicate that the killer whales demonstrated high fluency, originality, some elaboration, and flexibility in their behaviors. Individual variability was observed with younger animals demonstrating more variable behaviors as compared to the older animals. Males seemed to display less complex and lower energy behaviors as compared to females, but this impression may be driven by the age or size of the animal. These results support existing evidence that killer whales are dynamic in their thinking and behavior.

Caenorhabditis elegans recognizes a bacterial quorum-sensing signal molecule through the AWCON neuron.

In a process known as quorum sensing, bacteria use chemicals called autoinducers for cell-cell communication. Population-wide detection of autoinducers enables bacteria to orchestrate collective behaviors. In the animal kingdom detection of chemicals is vital for success in locating food, finding hosts, and avoiding predators. This behavior, termed chemotaxis, is especially well studied in the nematode Caenorhabditis elegans. Here we demonstrate that the Vibrio cholerae autoinducer (S)-3-hydroxytridecan-4-one, termed CAI-1, influences chemotaxis in C. elegans. C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant defective for CAI-1 production. The position of the CAI-1 ketone moiety is the key feature driving CAI-1-directed nematode behavior. CAI-1 is detected by the C. elegans amphid sensory neuron AWC(ON). Laser ablation of the AWC(ON) cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. These analyses define the structural features of a bacterial-produced signal and the nematode chemosensory neuron that permit cross-kingdom interaction.

Drosophila larval NMJ immunohistochemistry.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila larval NMJ dissection.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

Genome-wide P-element screen for Drosophila synaptogenesis mutants.

A molecular understanding of synaptogenesis is a critical step toward the goal of understanding how brains "wire themselves up," and then "rewire" during development and experience. Recent genomic and molecular advances have made it possible to study synaptogenesis on a genomic scale. Here, we describe the results of a screen for genes involved in formation and development of the glutamatergic Drosophila neuromuscular junction (NMJ). We screened 2185 P-element transposon mutants representing insertions in approximately 16% of the entire Drosophila genome. We first identified recessive lethal mutants, based on the hypothesis that mutations causing severe disruptions in synaptogenesis are likely to be lethal. Two hundred twenty (10%) of all insertions were homozygous lethal. Two hundred five (93%) of these lethal mutants developed at least through late embryogenesis and formed neuromusculature. We examined embryonic/larval NMJs in 202 of these homozygous mutants using immunocytochemistry and confocal microscopy. We identified and classified 88 mutants with altered NMJ morphology. Insertion loci in these mutants encode several different types of proteins, including ATP- and GTPases, cytoskeletal regulators, cell adhesion molecules, kinases, phosphatases, RNA regulators, regulators of protein formation, transcription factors, and transporters. Thirteen percent of insertions are in genes that encode proteins of novel or unknown function. Complementation tests and RT-PCR assays suggest that approximately 51% of the insertion lines carry background mutations. Our results reveal that synaptogenesis requires the coordinated action of many different types of proteins--perhaps as much as 44% of the entire genome--and that transposon mutageneses carry important caveats that must be respected when interpreting results generated using this method.

Early alpha/beta interferon production by myeloid dendritic cells in response to UV-inactivated virus requires viral entry and interferon regulatory factor 3 but not MyD88.

Alpha/beta interferons (IFN-alpha/beta) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-alpha/beta are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-alpha/beta production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-alpha/beta in mDCs while fusion-defective virus did not induce IFN-alpha/beta. The response to replication-defective virus was largely intact in MyD88-/- mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-alpha/beta in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-alpha/beta in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

Cigarette smoke induces anaphase bridges and genomic imbalances in normal cells.

Exposure to cigarette smoke has long been linked to carcinogenesis, but the emphasis has been placed on mutational changes in the DNA sequence caused by the carcinogens in smoke. Here, we report an additional role for cigarette smoke exposure in contributing to chromosomal aberrations in cells. We have found that cigarette smoke condensate (CSC) induces anaphase bridges in cultured human cells, which in a short time lead to genomic imbalances. The frequency of the induced bridges within the entire population decreases with time, and this decrease is not dependent upon the p53-mediated apoptotic pathway. Additionally, we show that CSC induces DNA double stranded breaks (DSBs) in cultured cells and purified DNA. The reactive oxygen species (ROS) scavenger, 2' deoxyguanosine 5'-monophosphate (dGMP) prevents CSC-induced DSBs, anaphase bridge formation and genomic imbalances. Therefore, we propose that CSC induces bridges and genomic imbalances via DNA DSBs. Furthermore, since the amount of CSC added to the cultures was substantially less than that extracted from a single cigarette, our results show that even low levels of cigarette smoke can cause irreversible changes in the chromosomal constitution of cultured cells.