The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarusfallax, and also established a triploid AA’B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Neuromodulation by peptides and amines is a primary source of plasticity in the nervous system as it adapts the animal to an ever-changing environment. The crustacean stomatogastric nervous system is one of the premier systems to study neuromodulation and its effects on motor pattern generation at the cellular level. It contains the extensively modulated central pattern generators that drive the gastric mill (chewing) and pyloric (food filtering) rhythms. Neuromodulators affect all stages of neuronal processing in this system, from membrane currents and synaptic transmission in network neurons to the properties of the effector muscles. The ease with which distinct neurons are identified and their activity is recorded in this system has provided considerable insight into the mechanisms by which neuromodulators affect their target cells and modulatory neuron function. Recent evidence suggests that neuromodulators are involved in homeostatic processes and that the modulatory system itself is under modulatory control, a fascinating topic whose surface has been barely scratched. Future challenges include exploring the behavioral conditions under which these systems are activated and how their effects are regulated.

Specificity in the actions of different modulatory neurons is often attributed to their having distinct cotransmitter complements. We are assessing the validity of this hypothesis with the stomatogastric nervous system of the crab Cancer borealis. In this nervous system, the stomatogastric ganglion (STG) contains a multifunctional network that generates the gastric mill and pyloric rhythms. Two identified projection neurons [modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1)] that innervate the STG and modulate these rhythms contain GABA and the pentapeptide proctolin, but only MCN1 contains Cancer borealis tachykinin-related peptide (CabTRP Ia). Selective activation of each projection neuron elicits different rhythms from the STG. MPN elicits only a pyloric rhythm, whereas MCN1 elicits a distinct pyloric rhythm as well as a gastric mill rhythm. We tested the degree to which CabTRP Ia distinguishes the actions of MCN1 and MPN. To this end, we used the tachykinin receptor antagonist Spantide I to eliminate the actions of CabTRP Ia. With Spantide I present, MCN1 no longer elicited the gastric mill rhythm and the resulting pyloric rhythm was changed. Although this rhythm was more similar to the MPN-elicited pyloric rhythm, these rhythms remained different. Thus, CabTRP Ia partially confers the differences in rhythm generation resulting from MPN versus MCN1 activation. This result suggests that different projection neurons may use the same cotransmitters differently to elicit distinct pyloric rhythms. It also supports the hypothesis that different projection neurons use a combination of strategies, including using distinct cotransmitter complements, to elicit different outputs from the same neuronal network.

Histamine is a neurotransmitter with actions throughout the nervous system of vertebrates and invertebrates. Nevertheless, the actions of only a few identified histamine-containing neurons have been characterized. Here, we present the actions of a histaminergic projection neuron on the rhythmically active pyloric and gastric mill circuits within the stomatogastric ganglion (STG) of the crab Cancer borealis. An antiserum generated against histamine labeled profiles throughout the C. borealis stomatogastric nervous system. Labeling occurred in several somata and neuropil within the paired commissural ganglia as well as in neuropil within the STG and at the junction of the superior oesophageal and stomatogastric nerves. The source of all histamine-like immunolabeling in the STG neuropil was one pair of neuronal somata, the previously identified inferior ventricular (IV) neurons, located in the supraoesophageal ganglion. These neurons also exhibited FLRFamide-like immunoreactivity. Activation of the IV neurons in the crab inhibited some pyloric and gastric mill neurons and, with inputs from the commissural ganglia eliminated, terminated both rhythms. Focal application of histamine had comparable effects. The actions of both applied histamine and IV neuron stimulation were blocked, reversibly, by the histamine type-2 receptor antagonist cimetidine. With the commissural ganglia connected to the STG, IV neuron stimulation elicited a longer-latency activation of commissural projection neurons which in turn modified the pyloric rhythm and activated the gastric mill rhythm. These results support the hypothesis that the histaminergic/peptidergic IV neurons are projection neurons with direct and indirect actions on the STG circuits of the crab C. borealis.

Co-transmission is a common means of neuronal communication, but its consequences for neuronal signaling within a defined neuronal circuit remain unknown in most systems. We are addressing this issue in the crab stomatogastric nervous system by characterizing how the identified modulatory commissural neuron (MCN)1 uses its co-transmitters to activate the gastric mill (chewing) rhythm in the stomatogastric ganglion (STG). MCN1 contains gamma-aminobutyric acid (GABA) plus the peptides proctolin and Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), which it co-releases during the retractor phase of the gastric mill rhythm to influence both retractor and protractor neurons. By focally applying each MCN1 co-transmitter and pharmacologically manipulating each co-transmitter action during MCN1 stimulation, we found that MCN1 has divergent co-transmitter actions on the gastric mill central pattern generator (CPG), which includes the neurons lateral gastric (LG) and interneuron 1 (Int1), plus the STG terminals of MCN1 (MCN1(STG)). MCN1 used only CabTRP Ia to influence LG, while it used only GABA to influence Int1 and the contralateral MCN1(STG). These MCN1 actions caused a slow excitation of LG, a fast excitation of Int1 and a fast inhibition of MCN1(STG). MCN1-released proctolin had no direct influence on the gastric mill CPG, although it likely indirectly regulates this CPG via its influence on the pyloric rhythm. MCN1 appeared to have no ionotropic actions on the gastric mill follower motor neurons, but it did use proctolin and/or CabTRP Ia to excite them. Thus, a modulatory projection neuron can elicit rhythmic motor activity by using distinct co-transmitters, with different time courses of action, to simultaneously influence different CPG neurons.