Soldier-producing aphids have evolved at least nine separate times. The larvae of soldier-producing species can be organized into three general categories: monomorphic larvae, dimorphic larvae with a reproductive soldier caste, and dimorphic larvae with a sterile soldier caste. Here we report the discovery of a novel soldier type in an undescribed species of Pseudoregma that is morphologically similar to P. bambucicola. A colony of this species produced morphologically monomorphic first-instar larvae with a defensive behavioral dimorphism. These larvae attacked natural predators, and larval response to a simple assay, placing the tips of forceps in front of larvae, was correlated with this attacking behavior. Approximately one third of the first-instar larvae in the colony attacked and this proportion was uncorrelated with the time of day, the ambient temperature, or the diel migratory behavior of the aphids. Migrating larvae rarely attacked. Attacking behavior was correlated with another defensive behavior, hind-leg waving. Attackers were more likely to possess the next-instar skin, suggesting that they were older than non-attackers. This is the first example of a possible within-instar age polyethism in soldier-producing aphids. Canonical variates analysis of seven morphological measurements failed to discriminate between attacking and non-attacking larvae. The monomorphic larvae share some morphometric characteristics in common with the soldiers of P. bambucicola and other characteristics in common with normal larvae. We discuss these results with respect to the evolution and loss of soldier castes in the tribe Cerataphidini.

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

The anatomical and electrophysiological properties of neurons in the stratum lucidum of the CA3 subfield of the hippocampus were examined by using patch-pipette recordings combined with biocytin staining. This method facilitated the analysis of the morphological features and passive and active properties of a recently described class of spiny neurons in the stratum lucidum, as well as aspiny neurons in this region. Some, but not all, synaptic inputs of both types of neurons were found to arise from the mossy fiber system. The axons of spiny neurons in the stratum lucidum were heavily collateralized, terminating primarily in the stratum lucidum and stratum radiatum of CA3, and to a lesser extent in the stratum pyramidale and stratum oriens. Only a few axonal projections were found that extended beyond the CA3 region into CA1 and the hilus. Aspiny neurons fell into two classes: those projecting axons to the stratum lucidum and stratum radiatum of CA3 and those with axon terminations mainly in the stratum pyramidale and stratum oriens. The electrophysiological properties of spiny and aspiny neurons in the stratum lucidum were similar, but on average, the aspiny neurons had significantly higher maximal firing rates and narrower action potential half-widths. The results demonstrate that a diverse population of neurons exists in the region of mossy fiber termination in area CA3. These neurons may be involved in local-circuit feedback, or feed-forward systems controlling the flow of information through the hippocampus.

During low-frequency firing, action potentials actively invade the dendrites of CA1 pyramidal neurons. At higher firing rates, however, activity-dependent processes result in the attenuation of back-propagating action potentials, and propagation failures occur at some dendritic branch points. We tested two major hypotheses related to this activity-dependent attenuation of back-propagating action potentials: (1) that it is mediated by a prolonged form of sodium channel inactivation and (2) that it is mediated by a persistent dendritic shunt activated by back-propagating action potentials. We found no evidence for a persistent shunt, but we did find that cumulative, prolonged inactivation of sodium channels develops during repetitive action potential firing. This inactivation is significant after a single action potential and continues to develop during several action potentials thereafter, until a steady-state sodium current is established. Recovery from this form of inactivation is much slower than its induction, but recovery can be accelerated by hyperpolarization. The similarity of these properties to the time and voltage dependence of attenuation and recovery of dendritic action potentials suggests that dendritic sodium channel inactivation contributes to the activity dependence of action potential back-propagation in CA1 neurons. Hence, the biophysical properties of dendritic sodium channels will be important determinants of action potential-mediated effects on synaptic integration and plasticity in hippocampal neurons.

In Drosophila, pattern formation at multiple stages of embryonic and imaginal development depends on the same intercellular signaling pathways. We have identified a novel gene, eyelid (eld), which is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, eld mutations have effects opposite to those caused by wingless (wg) mutations. eld encodes a widely expressed nuclear protein with a region homologous to a novel family of DNA-binding domains. Based on this homology and on the phenotypic analysis, we suggest that Eld could act as a transcription factor antagonistic to the Wg pathway.

In insects, the shedding of the old cuticle at the end of a molt involves a stereotyped sequence of distinct behaviors. Our studies on the isolated nervous system of Manduca sexta show that the peptides ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP) elicit the first two motor behaviors, the pre-ecdysis and ecdysis behaviors, respectively. Exposing isolated abdominal ganglia to ETH resulted in the generation of sustained pre-ecdysis bursts. By contrast, exposing the entire isolated CNS to ETH resulted in the sequential appearance of pre-ecdysis and ecdysis motor outputs. Previous research has shown that ETH activates neurons within the brain that then release eclosion hormone within the CNS. The latter elevates cGMP levels within and increases the excitability of a group of neurons containing CCAP. In our experiments, the ETH-induced onset of ecdysis bursts was always associated with a rise in intracellular cGMP within these CCAP neurons. We also found that CCAP immunoreactivity decreases centrally during normal ecdysis. Isolated, desheathed abdominal ganglia responded to CCAP by generating rhythmical ecdysis bursts. These ecdysis motor bursts persisted as long as CCAP was present and could be reinduced by successive application of the peptide. CCAP exposure also actively terminated pre-ecdysis bursts from the abdominal CNS, even in the continued presence of ETH. Thus, the sequential performance of the two behaviors arises from one modulator activating the first behavior and also initiating the release of the second modulator. The second modulator then turns off the first behavior while activating the second.

In Drosophila, dosage compensation occurs by increasing the transcription of the single male X chromosome. Four trans-acting factors encoded by the male-specific lethal genes are required for this process. Dosage compensation is restricted to males by the splicing regulator Sex-lethal, which functions to prevent the production of the MSL-2 protein in females by an unknown mechanism. In this report, we provide evidence that Sex-lethal acts synergistically through sequences in both the 5' and 3' untranslated regions of MSL-2 to mediate repression. We also provide evidence that the repression of MSL-2 is directly regulated by Sex-lethal at the level of translation.

We have identified a novel activity for the region of the intergenic spacer of the Xenopus laevis rRNA genes that contains the 35- and 100-bp repeats. We devised a new assay for this region by constructing DNA plasmids containing a tandem repeat of rRNA reporter genes that were separated by the 35- and 100-bp repeat region and a rRNA gene enhancer. When the 35- and 100-bp repeat region is present in its normal position and orientation at the 3’ end of the rRNA reporter genes, the enhancer activates the adjacent downstream promoter but not the upstream rRNA promoter on the same plasmid. Because this element can restrict the range of an enhancer’s activity in the context of tandem genes, we have named it the repeat organizer (RO). The ability to restrict enhancer action is a feature of insulator elements, but unlike previously described insulator elements the RO does not block enhancer action in a simple enhancer-blocking assay. Instead, the activity of the RO requires that it be in its normal position and orientation with respect to the other sequence elements of the rRNA genes. The enhancer-binding transcription factor xUBF also binds to the repetitive sequences of the RO in vitro, but these sequences do not activate transcription in vivo. We propose that the RO is a specialized insulator element that organizes the tandem array of rRNA genes into single-gene expression units by promoting activation of a promoter by its proximal enhancers.

Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted. Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently. Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.