Cancer Borealis Research Articles

Firing Hot, Firing Cold: How Poikilotherms Compensate for Temperature Swings

​IfIf you are reading this, you have one less problem than the crabs that are the subject of a new study in this issue of PLoS Biology. Humans, like all mammals (as well as birds), are “warm-blooded,” or more correctly “homeothermic,” meaning we maintain our core body organs at a constant temperature. We expend considerable energy to do so, and may die when we cannot. In contrast, crabs, cod, caterpillars, and the like are “cold-blooded,” or “poikilothermic,” with body temperatures that change with the environment they are in. When the mercury plunges, a poikilotherm, and everything that happens within it, merely slows down. Crabs in warm conditions exhibit faster network frequencies than crabs in cold conditions. However, the motor pattern remains constant in both conditions while network parameters manifest disparate temperature dependencies. (Image: Gabrielle Gutierrez) ... At the most basic level, all those things happening within the poikilotherm are chemical reactions, and it is their precise coordination that allows the organism to keep on living. But chemists have long known that reactions differ in the degree to which they will speed up and slow down in response to temperature changes. So here is the problem the crab has that we do not: how does it make sure that processes relevant to a given behavior remain coordinated across a wide range of temperatures? Lamont Tang, Eve Marder, and colleagues set out to answer this question for a group of neurons in Cancer borealis, a North Atlantic crab that flourishes in intertidal and subtidal ecosystems where the water temperature may swing as much as 15°C in a day and more than 20°C between winter and summer. Chewing and filtering of food is driven by a rhythmic motor pattern maintained by the coordinated firing of a small network of neurons. The neurons fire in a specific sequence with time lags between them. To maintain effective chewing and filtering, those temporal relationships should remain the same—that is, they must remain “in phase” with each other, even if the overall frequency of firing changes with temperature. The authors began by confirming that this was so, showing that while the firing frequency increased almost four-fold between 7°C and 23°C, the phase relationships were unaltered. In the language of the physical chemist, a system's responsiveness to a temperature change is known as its “Q10”. A reaction that doubles its rate with a 10-degree rise in temperature has a Q10 of 2, while one that changes not at all has a Q10 of 1. Thus, while the frequency had a Q10 of 2.3, the phase relationships had a Q10 of 1, displaying almost perfect “temperature compensation.” They next asked what features of the system might account for this temperature compensation. Whether a neuron fires is determined by multiple physical properties, including the current flow through its ion channels, each of which has its own response to temperature change. Two kinds of these, called the transient outward current and the hyperpolarization-activated inward current, oppose each other, and the authors found that each had a high Q10. Turning to computer models of neuronal firing, they demonstrated that not all model neurons were able to adjust to temperature changes while maintaining an appropriate firing pattern. In a model of one of the crab's neurons, they found that temperature change altered the phase of the neuron when the Q10 of the membrane currents was set to 1, but the phase was largely maintained when they substituted the experimentally measured Q10's from the two opposing currents. This is no coincidence, the authors argue. Like all poikilotherms, the crab is subject to strong selective pressure in an environment where temperatures fluctuate, and selection of membrane ensembles that maintain neuronal firing integrity in the face of wide temperature swings is likely to be strongly favored. These exact results may not be found in other cold-blooded creatures, since the precise composition of their neuronal membranes may differ. But since both a crab and a cod must solve the same problem in the cold Atlantic waters, they may be more similar at the cellular level than they appear to be on your plate. Tang LS, Goeritz M, Caplan JS, Taylor AL, Fisek M, et al. (2010) Precise Temperature Compensation of Phase in a Rhythmic Motor Pattern. doi:10.1371/journal.pbio.1000469

Compensation for Variable Intrinsic Neuronal Excitability by Circuit-Synaptic Interactions

Recent theoretical and experimental work indicates that neurons tune themselves to maintain target levels of excitation by modulating ion channel expression and synaptic strengths. As a result, functionally equivalent circuits can produce similar activity despite disparate underlying network and cellular properties. To experimentally test the extent to which synaptic and intrinsic conductances can produce target activity in the presence of variability in neuronal intrinsic properties, we used the dynamic clamp to create hybrid two-cell circuits built from four types of stomatogastric neurons coupled to the same model Morris-Lecar neuron by reciprocal inhibition. We measured six intrinsic properties (input resistance, minimum membrane potential, firing rate in response to +1 nA of injected current, slope of the frequency-current curve, spike height, and spike voltage threshold) of dorsal gastric, gastric mill, lateral pyloric, and pyloric dilator neurons from male crabs of the species Cancer borealis. The intrinsic properties varied twofold to sevenfold in each cell type. We coupled each biological neuron to the Morris-Lecar model with seven different values of inhibitory synaptic conductance and also used the dynamic clamp to add seven different values of an artificial h-conductance, thus creating 49 different circuits for each biological neuron. Despite the variability in intrinsic excitability, networks formed from each neuron produced similar circuit performance at some values of synaptic and h-conductances. This work experimentally confirms results from previous modeling studies; tuning synaptic and intrinsic conductances can yield similar circuit outputs from neurons with variable intrinsic excitability.

Enhancement of Muscle Contraction in the Stomach of the Crab Cancer borealis: a Possible Hormonal Role for GABA

Gamma-aminobutyric acid (GABA) is best known as an inhibitory neurotransmitter in the mammalian central nervous system. Here we show, however, that GABA has an excitatory effect on nerve-evoked contractions and on excitatory junctional potentials (EJPs) of the gastric mill 4 (gm4) muscle from the stomach of the crab Cancer borealis. The threshold concentration for these effects was between 1 and 10 micromol l(-1). Using immunohistochemical techniques, we found that GABA is colocalized with the vesicle-associated protein synapsin in nearby nerves and hence is presumably released there. However, since these nerves do not innervate the muscle directly, we conclude that these release sites are not the likely source of the GABA responsible for muscle modulation. We also extracted hemolymph from the crab pericardial cavity, which contains the pericardial organs, a major neurosecretory structure. Through reversed-phase liquid chromatography-mass spectrometry analysis we determined the concentration of GABA in the hemolymph to be 3.3 +/- 0.7 micromol l(-1), high enough to modulate the muscle. These findings suggest that the gm4 muscle could be modulated by GABA produced by and released from a distant neurohemal organ.

Cell-specific patterns of alternative splicing of voltage-gated ion channels in single identified neurons

CbNav and CbIH encode channels that carry voltage-gated sodium and hyperpolarization activated cation currents respectively in the crab, Cancer borealis. We cloned and sequenced full length cDNAs for both CbNav and CbIH and found nine different regions of alternative splicing for the CbNav gene and four regions of alternative splicing for CbIH. We used RT-PCR to determine tissue-specific differences in splicing of both channel genes among cardiac muscle, skeletal muscle, brain, and stomatogastric ganglion (STG) tissue. We then examined the splice variant isoforms present in single, unambiguously identified neurons of the STG. We found cell-type specific patterns of alternative splicing for CbNav, indicating unique cell-specific pattern of post-transcriptional modification. Furthermore, we detected possible differences in cellular localization of alternatively spliced CbNav transcripts; distinct mRNA isoforms are present between the cell somata and the axons of the neurons. In contrast, we found no qualitative differences among different cell types for CbIH variants present, although this analysis did not represent the full spectrum of all possible CbIH variants. CbIH mRNA was not detected in axon samples. Finally, although cell-type specific patterns of splicing were detected for CbNav, the same cell type within and between animals also displayed variability in which splice forms were detected. These results indicate that channel splicing is differentially regulated at the level of single neurons of the same neural network, providing yet another mechanism by which cell-specific neuronal output can be achieved.

Glucose‐induced inhibition: how many ionic mechanisms?

Sensing of sugar by specialized 'glucose-inhibited' cells helps organisms to counteract swings in their internal energy levels. Evidence from several cell types in both vertebrates and invertebrates suggests that this process involves sugar-induced stimulation of plasma membrane K(+) currents. However, the molecular composition and the mechanism of activation of the underlying channel(s) remain controversial. In mouse hypothalamic neurones and neurosecretory cells of the crab Cancer borealis, glucose stimulates K(+) currents displaying leak-like properties. Yet knockout of some of the candidate 'leak' channel subunits encoded by the KCNK gene family so far failed to abolish glucose inhibition of hypothalamic cells. Moreover, in other tissues, such as the carotid body, glucose-stimulated K(+) channels appear to be not leak-like but voltage-gated, suggesting that glucose-induced inhibition may engage multiple types of K(+) channels. Other mechanisms of glucose-induced inhibition, such as hyperpolarization mediated by opening of Cl(-) channels and depolarization block caused by closure of K(ATP) channels have also been proposed. Here we review known ionic and pharmacological features of glucose-induced inhibition in different cell types, which may help to identify its molecular correlates.

Comparative Neuropeptidomic Analysis of Food Intake via a Multifaceted Mass Spectrometric Approach

Feeding behavior is a fundamental aspect of energy homeostasis and is crucial for animal survival. This process is regulated by a multitude of neurotransmitters including neuropeptides within a complex neuroendocrine system. Given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. Here we report comparative neuropeptidomic analysis of brain and major neuroendocrine organ in a crustacean model organism in response to feeding. A multi-faceted approach employing direct tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), stable isotopic labeling of neuropeptide extracts for quantitation, and mass spectrometric imaging (MSI) has been employed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and the pericardial organ (PO) in the crab Cancer borealis. Multiple neuropeptides exhibited changes in abundance after feeding, including RFamides, Cancer borealis tachykinin related peptides (CabTRPs), RYamides, and pyrokinins. By combining quantitative analysis of neuropeptide changes via isotopic labeling of brain extract and MSI mapping of neuropeptides of brain slices, we identified the boundary of olfactory lobe (ON) and median protocerebrum (MPC) area as two potential feeding centers in the crab brain.

Functional consequences of animal-to-animal variation in circuit parameters

How different are the neuronal circuits for a given behavior across individual animals? To address this question, we measured multiple cellular and synaptic parameters within individual preparations to see how they correlate with circuit function, using neurons and synapses within the pyloric circuit of the stomatogastric ganglion (STG) of the crab Cancer borealis. There was considerable preparation-to-preparation variability in the strength of two identified synapses, in the amplitude of a modulator-evoked current, and in the expression of six ion channel genes. Nonetheless, across preparations we found strong correlations among these parameters and attributes of circuit performance. These data illustrate the importance of making multidimensional measurements from single preparations to understand how variability in circuit output is related to the variability of multiple circuit parameters.

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles [1]. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species [2–9]. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans [2,3]. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.

Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins

The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10 −6 M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies <0.7 Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.

Molecular and mass spectral identification of the broadly conserved decapod crustacean neuropeptide pQIRYHQCYFNPISCF: The first PISCF-allatostatin ( Manduca sexta- or C-type allatostatin) from a non-insect

The PISCF-allatostatins ( Manduca sexta- or C-type allatostatins) are a family of pentadecapeptides characterized by a pyroglutamine blocked N-terminus, an unamidated-PISCF C-terminus, and a disulfide bridge between two internal Cys residues. Several isoforms of PISCF-AST are known, all from holometabolous insects. Using a combination of transcriptomics and mass spectrometry, we have identified the first PISCF-type peptides from a non-insect species. In silico analysis of crustacean ESTs identified several Litopenaeus vannamei (infraorder Penaeidea) transcripts encoding putative PISCF-AST precursors. Translation of these ESTs, with subsequent prediction of their putative post-translational processing, revealed the existence of as many as three PISCF-type peptides, including pQIRYHQCYFNPISCF (disulfide bridging between Cys 7 and Cys 14). Although none of the predicted isoforms was detected by mass spectrometry in L. vannamei, MALDI-FTMS mass profiling identified an m/z signal corresponding to pQIRYHQCYFNPISCF (disulfide bridge present) in neural tissue from 28 other decapods, which included members of six infraorders (Stenopodidea, Astacidea, Thalassinidea, Achelata, Anomura and Brachyura). Further characterization of the peptide using SORI-CID and chemical derivatization/enzymatic digestion supported the theorized structure. In both the crab Cancer borealis and the lobster Homarus americanus, MALDI-based tissue surveys suggest that pQIRYHQCYFNPISCF is broadly distributed in the nervous system; it was also detected in the posterior midgut caecum. Collectively, our data show that members of the PISCF-AST family are not restricted to the holometabolous insects, but instead may be broadly conserved within the Pancrustacea. Moreover, our data suggest that one highly conserved PISCF-type peptide, pQIRYHQCYFN-PISCF, is present in decapod crustaceans, functioning as a brain–gut paracrine/hormone.

Combining tissue extraction and off-line capillary electrophoresis matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for neuropeptide analysis in individual neuronal organs using 2,5-dihydroxybenzoic acid as a multi-functional agent

In this study we report an improved protocol that combines simplified sample preparation and micro-scale separation for mass spectrometric analysis of neuropeptides from individual neuroendocrine organs of crab Cancer borealis. A simple, one-step extraction method with commonly used matrix-assisted laser desorption/ionization (MALDI) matrix, 2,5-dihydroxybenzoic acid (DHB), in saturated aqueous solution, is employed for improved extraction of neuropeptides. Furthermore, a novel use of DHB as background electrolyte for capillary electrophoresis (CE) separation in the off-line coupling of CE to MALDI-Fourier transform mass spectrometric (FT-MS) detection is also explored. The new CE electrolyte exhibits full compatibility with MALDI-MS analysis of neuropeptides in that both the peptide extraction process and MALDI detection utilize DHB. In addition, enhanced resolving power and improved sensitivity are also observed for CE-MALDI-MS of peptide mixture analysis. Collectively, the use of DHB has simplified the extraction and reduced the sample loss by elimination of homogenizing, drying, and desalting processes. In the mean time, the concurrent use of DHB as CE separation buffer and subsequent MALDI matrix offers improved spectral quality by eliminating the interferences from typical CE electrolyte in MALDI detection.

Growth-mortality trade-offs along a depth gradient in Cancer borealis

Intertidal and shallow subtidal ecosystems experience steep environmental gradients over short distances. Individual foraging rate, predation risk, and physiologic stress vary along these gradients, resulting in growth-mortality trade-offs with depth. In the summer, Cancer borealis commonly forage in the shallow subtidal in the Gulf of Maine. C. borealis are the favored invertebrate prey of the Herring Gull and the Great Black-backed Gull, which consume 25%–50% of available C. borealis (those in < 1 m water) during each daytime low tide. We investigated three possible explanations for the presence of C. borealis in the risky gull-predation zone. First, we tested whether predation risk in the gull-predation zone was matched at deeper depths by subtidal predators; we found predation risk decreases with depth. Second, we tested whether water temperatures were warmer in the gull-predation zone and whether these warmer temperatures resulted in increased growth rates. We found that, while waters were warmer in the gull-predation zone, crabs grew at similar rates above and below the thermocline when fed similar diets. Finally, we tested for differences in food availability with depth and whether these differences influenced C. borealis growth rates. Our results suggest a growth-mortality trade-off, where increased food availability provides sufficient growth benefit to outweigh the risk of foraging at shallower depths.

Expanding the Crustacean Neuropeptidome Using a Multifaceted Mass Spectrometric Approach

Jonah crab Cancer borealis is an excellent, long-served model organism for many areas of physiology, including the study of endocrinology and neurobiology. Characterizing the neuropeptides present in its nervous system provides the first critical step toward understanding the physiological roles of these complex molecules. Multiple mass spectral techniques were used to comprehensively characterize the neuropeptidome in C. borealis, including matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), MALDI time-of-flight (TOF)/TOF MS and nanoflow liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS). To enhance the detection signals and expand the dynamic range, direct tissue analysis, tissue extraction, capillary electrophoresis (CE) and off-line HPLC separation have also been employed. In total, 142 peptides were identified, including 85 previously known C. borealis peptides, 22 peptides characterized previously from other decapods, but new to this species, and 35 new peptides de novo sequenced for the first time in this study. Seventeen neuropeptide families were revealed including FMRFamide-related peptide (FaRP), allatostatin (A and B type), RYamide, orcokinin, orcomyotropin, proctolin, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone precursor-related peptide (CPRP), crustacean hyperglycemic hormone (CHH), corazonin, pigment-dispersing hormone (PDH), tachykinin, pyrokinin, SIFamide, red pigment concentrating hormone (RPCH) and HISGLYRamide. Collectively, our results greatly increase the number and expand the coverage of known C. borealis neuropeptides, and thus provide a stronger framework for future studies on the physiological roles played by these molecules in this important model organism.

&lt;em&gt;Cancer Borealis&lt;/em&gt; Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

The persistent effect of wave exposure on TMIIs and crab predation in Nucella lapillus

Trait-mediated indirect interactions (TMIIs) can vary across mosaics of wave exposure due to variation in predator abundance, foraging efficiency, or morphological and behavioral responses to predators. In this study, we compared how whelks, Nucella lapillus, from several wave-exposed and wave-protected populations transmitted TMIIs initiated by waterborne cues from crab predators ( Carcinus maenas and Cancer borealis) and a seastar ( Asterias rubens). In laboratory experiments, whelks taken from wave-exposed environments consumed fewer mussels ( Mytilus edulis) than did whelks from wave-protected populations. Whelks from wave-protected sites reduced foraging when exposed to cues from Cancer, Carcinus, Asterias, or both Carcinus and Asterias, but whelks from wave-exposed sites only responded to Cancer. In predation trials, both crabs readily consumed whelks taken from wave-exposed populations, but only Cancer consumed the whelks from wave-protected populations. Although Carcinus could crush thin-shelled, wave-exposed whelks, in the presence of Asterias these whelks were consumed by “winkling”. The differing responses of wave-exposed and wave-protected whelks may be explained by several factors: 1) the abiotic stress of wave action may generate a persistent reduction in the overall feeding rates of wave-exposed whelks, rendering TMIIs undetectable, 2) some predators may not threaten whelks in wave-exposed environments, and 3) the threat of wave dislodgement may supercede the threat of predators. Thus, the extent to which Nucella transmits TMIIs will likely depend on the wave-exposure history of whelk populations and the initiating top predator species.

Waterborne cues from crabs induce thicker skeletons, smaller gonads and size-specific changes in growth rate in sea urchins

Indirect predator-induced effects on growth, morphology and reproduction have been extensively studied in marine invertebrates but usually without consideration of size-specific effects and not at all in post-metamorphic echinoids. Urchins are an unusually good system, in which, to study size effects because individuals of various ages within one species span four orders of magnitude in weight while retaining a nearly isometric morphology. We tracked growth of urchins, Strongylocentrotus droebachiensis (0.013–161.385 g), in the presence or absence of waterborne cues from predatory Jonah crabs, Cancer borealis. We ran experiments at ambient temperatures, once for 4 weeks during summer and again, with a second set of urchins, for 22 weeks over winter. We used a scaled, cube-root transformation of weight for measuring size more precisely and for equalizing variance across sizes. Growth rate of the smallest urchins (summer: <17 mm diameter; winter: <7 mm diameter) decreased by 40–42% in response to crab cues. In contrast, growth rate of larger urchins was unaffected in the summer and increased in response to crab scent by 7% in the winter. At the end of the 22-week experiment, additional gonadal and skeletal variables were measured. Cue-exposed urchins developed heavier, thicker skeletons and smaller gonads, but no differences in spine length or jaw size. The differences depended on urchin size, suggesting that there are size-specific shifts in gonadal and somatic investment in urchins.

Three Dimensional Mapping of Neuropeptides and Lipids in Crustacean Brain by Mass Spectral Imaging

Imaging mass spectrometry is emerging as a powerful tool that has been applied extensively for the localization of proteins, peptides, pharmaceutical compounds, metabolites, and lipids in biological tissues. In this article, a three-dimensional mass spectral imaging (3D MSI) technique was developed to examine distribution patterns of multiple neuropeptide families and lipids in the brain of the crab Cancer borealis. Different matrix/solvent combinations were compared for preferential extraction and detection of neuropeptides and lipids. Combined with morphological information, the distribution of numerous neuropeptides throughout the 3D structure of brain was determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Different localization patterns were observed for different neuropeptide families, and isoforms displaying unique distribution patterns that were distinct from the common family distribution trends were also detected. In addition, multiple lipids were identified and mapped from brain tissue slices. To confirm their identities, MS/MS fragmentation was performed. Different lipid species displayed distinct localization patterns, suggesting their potential different functional roles in the nervous system.

Intraguild Predators: Behavioral Changes and Mortality of the Green Crab (Carcinus Maenas) During Interactions with the American Lobster (Homarus Americanus) and Jonah Crab (Cancer Borealis)

Abstract The green crab Carcinus maenas invaded the Gulf of Maine over a hundred years ago and has become a well-established member of coastal ecosystems. Carcinus is most abundant in the intertidal zone despite higher availability of invertebrate prey in the nearby shallow subtidal. A previous study revealed a higher mortality rate for Carcinus in the subtidal than in the intertidal zone. Two other decapods, the American lobster Homarus americanus and Jonah crab Cancer borealis, have their highest abundance in the shallow subtidal. These three species have similar diets and predation among and within species occurs. We performed a laboratory experiment to investigate the effects of H. americanus, C. borealis, and larger conspecifics on mortality and behavior of Carcinus. Homarus americanus was the most effective predator on Carcinus, consuming 27% within 24 h. In contrast, only 5% of Carcinus were eaten by C. borealis and none by conspecifics. The presence of American lobsters had dramatic effects on Car...

Predation Risk, Prey Abundance, and the Vertical Distribution of Three Brachyuran Crabs on Gulf of Maine Shores

Three large brachyuran species are common in the intertidal and shallow subtidal of New England rocky shores: two native crabs Cancer borealis (Jonah crab) and Cancer irroratus (rock crab), and the introduced crab Carcinus maenas (European green crab). For these three co-occurring species in the Isles of Shoals (Gulf of Maine, USA), we compared distribution and abundance to survivorship and prey availability along a depth gradient and examined stomach contents and prey preference. The three species show differences in vertical distribution: Carcinus is more abundant in the intertidal, while both species of Cancer are more abundant in the subtidal. Survivorship of both species of Cancer increases with increasing depth, while survivorship of Carcinus decreases with increasing depth, perhaps corresponding to differential vulnerability to predation by gulls in the intertidal and by decapods and fish in the subtidal. There were notable differences in laboratory prey preference experiments: C. irroratus consumed both small mobile and non-mobile prey (amphipods, small snails, and small mussels), while Carcinus consumed primarily small mobile prey (amphipods and isopods). In contrast, C. borealis consumed larger, heavier bodied prey (larger snails and mussels) but did not eat amphipods or isopods. However, differences in prey preference among crab species were greater than the differences in realized diets. Based on stomach content analysis, the blue mussel Mytilus edulis was the majority component of stomach contents for all three species. Some differences were evident in the remaining diet components: Carcinus was the most omnivorous (. 30% green algae), C. borealis consumed more snails and arthropods, and C. irroratus consumed the most mussels. Overall, species distribution does not track the distribution of the preferred prey of each species; rather, the distribution corresponds with patterns of survivorship, indicating predominant top-down control of crab distribution.

Measurement of Neuropeptides in Crustacean Hemolymph via MALDI Mass Spectrometry

Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C(18) micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were detected, including both known ones and new peptides whose functions remain to be characterized.