Cancer Borealis Research Articles

Mass spectrometric characterization and physiological actions of VPNDWAHFRGSWamide, a novel B type allatostatin in the crab, Cancer borealis

The neural networks in the crustacean stomatogastric ganglion are modulated by neuroactive substances released locally into the neuropil of the stomatogastric ganglion and by circulating hormones released by neuroendocrine structures including the pericardial organs. Using nanoscale liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight mass spectrometry, we have identified and sequenced a novel B type allatostatin (CbAST-B1), VPNDWAHFRGSWamide, present in the pericardial organs of the crabs, Cancer borealis, and Cancer productus. We describe the physiological actions of CbAST-B1 on the pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis. CbAST-B1 reduces the pyloric network frequency in a dose-dependent manner. The effect of bath-applied CbAST-B1 depends on the preceding physiological state of the preparation. Surprisingly, despite marked amino-acid sequence dissimilarity between the novel CbAST-B1 and the A type allatostatin family of peptides (AST-A), the physiological effects of CbAST-B1 are similar to those of AST-A.

Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly1-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus

Over a quarter of a century ago, Mykles described the presence of putative endocrine cells in the midgut epithelium of the crab Cancer magister (Mykles, 1979). In the years that have followed, these cells have been largely ignored and nothing is known about their hormone content or the functions they play in this species. Here, we used a combination of immunohistochemistry and mass spectrometric techniques to investigate these questions. Using immunohistochemistry, we identified both SIFamide- and tachykinin-related peptide (TRP)-like immunopositive cells in the midgut epithelium of C. magister, as well as in that of Cancer borealis and Cancer productus. In each species, the SIFamide-like labeling was restricted to the anterior portion of the midgut, including the paired anterior midgut caeca, whereas the TRP-like immunoreactivity predominated in the posterior midgut and the posterior midgut caecum. Regardless of location, label or species, the morphology of the immunopositive cells matched that of the putative endocrine cells characterized ultrastructurally by Mykles (Mykles, 1979). Matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry identified the peptides responsible for the immunoreactivities as GYRKPPFNGSIFamide (Gly1-SIFamide) and APSGFLGMRamide [Cancer borealis tachykinin-related peptide Ia (CabTRP Ia)], respectively, both of which are known neuropeptides of Cancer species. Although the function of these midgut-derived peptides remains unknown, we found that both Gly1-SIFamide and CabTRP Ia were released when the midgut was exposed to high-potassium saline. In addition, CabTRP Ia was detectable in the hemolymph of crabs that had been held without food for several days, but not in that of fed animals, paralleling results that were attributed to TRP release from midgut endocrine cells in insects. Thus, one function that midgut-derived CabTRP Ia may play in Cancer species is paracrine/hormonal control of feeding-related behavior, as has been postulated for TRPs released from homologous cells in insects.

Modulation of Rhythmic Motor Activity by Pyrokinin Peptides

Pyrokinin (PK) peptides localize to the central and peripheral nervous systems of arthropods, but their actions in the CNS have yet to be studied in any species. Here, we identify PK peptide family members in the crab Cancer borealis and characterize their actions on the gastric mill (chewing) and pyloric (filtering) motor circuits in the stomatogastric ganglion (STG). We identified PK-like immunolabeling in the STG neuropil, in projection neuron inputs to this ganglion, and in the neuroendocrine pericardial organs. By combining MALDI mass spectrometry (MS) and ESI tandem MS techniques, we identified the amino acid sequences of two C. borealis pyrokinins (CabPK-I, CabPK-II). Both CabPKs contain the PK family-specific carboxy-terminal amino acid sequence (FXPRLamide). PK superfusion to the isolated STG had little influence on the pyloric rhythm but excited many gastric mill neurons and consistently activated the gastric mill rhythm. Both CabPKs had comparable actions in the STG and these actions were equivalent to those of Pevpyrokinin (shrimp) and Leucopyrokinin (cockroach). The PK-elicited gastric mill rhythm usually occurred without activation of the projection neuron MCN1. MCN1, which does not contain CabPKs, effectively drives the gastric mill rhythm and at such times is also a gastric mill central pattern generator (CPG) neuron. Because the PK-elicited gastric mill rhythm is independent of MCN1, the underlying core CPG of this rhythm is different from the one responsible for the MCN1-elicited rhythm. Thus neuromodulation, which commonly alters motor circuit output without changing the core CPG, can also change the composition of this core circuit.

Thermal Gelation of Previously Cooked Minced Meat from Jonah Crab (Cancer borealis)

ABSTRACT: Crab processing typically involves picking meat from whole cooked product. Remaining meat can be recovered as mince and potentially processed into value‐added products. This study focused on gelation of commercially processed crab mince. Objectives of the research were to determine the effects of cryoprotectants, freezing, and various heat treatments on gel formation of washed mince from previously cooked crab. Previously frozen minced meat from thermally processed Jonah crab was washed to remove soluble components. Four different treatments were applied to the washed mince: (1) freezing with cryoprotectants, (2) freezing with no cryoprotectants, (3) no freezing with cryoprotectants, and (4) no freezing with no cryoprotectants. Unwashed mince was used as a control treatment. Sodium chloride (2.5%) was mixed into the mince prior to stuffing into sausage casings and heating at 35 °C/30 min, 90 °C/30 min, or 35 °C/30 min followed by 90 °C/30 min. Gels were tested for proximate composition, color, water‐holding capacity, and gel strength. All mince samples formed gels except for the unwashed control. Gels with no cryoprotectants had 10% to 20% greater water holding capacity, lower L* values, and greater gel strength than those with cryoprotectants. Freezing of washed mince resulted in lower water‐holding capacity of gels and higher a* values. A 2‐stage heating treatment resulted in gels with the greatest gel strength, whereas gels cooked at 35 °C had the greatest distance to fracture. Results indicate that protein gels can be formed using previously cooked crab meat, which may be useful in the development of value‐added products.

Actions of kinin peptides in the stomatogastric ganglion of the crabCancer borealis

To fully understand neuronal network operation, the influence of all inputs onto that network must be characterized. As in most systems, many neuronal and hormonal pathways influence the multifunctional motor circuits of the crustacean stomatogastric ganglion (STG), but the actions of only some of them are known. Therefore, we characterized the influence of the kinin peptide family on the gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the STG of the crab Cancer borealis. The kinins are myoactive in arthropods and they occur within the arthropod central nervous system (CNS), but their CNS actions are not well characterized in any species. The pevkinins were first identified in the shrimp Penaeus vannamei, but they have yet to be studied in the STG of any species. We identified kinin-like immunolabeling (KLI) in the pericardial organs (POs) in C. borealis, but there was no KLI within the STG. The POs are a major source of hormonal influence on the STG. Pevkinin peptides activated the pyloric circuit and they caused a modest increase in the speed of ongoing pyloric rhythms. This modest influence on cycle speed resulted in part from pevkinin excitation of the lateral pyloric neuron, whose strengthened inhibitory synapse onto the pyloric pacemaker neurons limited the pevkinin-mediated increase in cycle speed. The pevkinin excitation of the pyloric rhythm was not strong enough to interfere with the previously documented, gastric mill rhythm-mediated weakening of the pyloric rhythm. Pevkinin also had little influence on the gastric mill rhythm. These results indicate that the kinin peptides have distinct and selective modulatory actions on the pyloric rhythm.

Members of the crustacean hyperglycemic hormone (CHH) peptide family are differentially distributed both between and within the neuroendocrine organs ofCancercrabs: implications for differential release and pleiotropic function

The crustacean hyperglycemic hormone (CHH) family of peptides includes CHH, moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). In the crab Cancer pagurus, isoforms of these peptides, as well as CHH precursor-related peptide (CPRP), have been identified in the X-organ-sinus gland (XO-SG) system. Using peptides isolated from the C. pagurus SG, antibodies to each family member and CPRP were generated. These sera were then used to map the distributions and co-localization patterns of these peptides in the neuroendocrine organs of seven Cancer species: Cancer antennarius, Cancer anthonyi, Cancer borealis, Cancer gracilis, Cancer irroratus, Cancer magister and Cancer productus. In addition to the XO-SG, the pericardial organ (PO) and two other neuroendocrine sites contained within the stomatogastric nervous system, the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), were studied. In all species, the peptides were found to be differentially distributed between the neuroendocrine sites in conserved patterns: i.e. CHH, CPRP, MIH and MOIH in the XO-SG, CHH, CPRP and MOIH in the PO, and MOIH in the ACP (no immunolabeling was found in the ACO). Moreover, in C. productus (and probably in all species), the peptides present in the XO-SG and PO were differentially distributed between the neurons within each of these neuroendocrine organs (e.g. CHH and CPRP in one set of XO somata with MIH and MOIH co-localized in a different set of cell bodies). Taken collectively, the differential distributions of CHH family members and CPRP both between and within the neuroendocrine organs of crabs of the genus Cancer suggests that each of these peptides may be released into the circulatory system in response to varied, tissue-specific cues and that the PO- and/or ACP-derived isoforms may possess functions distinct from those classically ascribed to their release from the SG.

Neuromodulation of spike-timing precision in sensory neurons.

The neuropeptide allatostatin decreases the spike rate in response to time-varying stretches of two different crustacean mechanoreceptors, the gastropyloric receptor 2 in the crab Cancer borealis and the coxobasal chordotonal organ (CBCTO) in the crab Carcinus maenas. In each system, the decrease in firing rate is accompanied by an increase in the timing precision of spikes triggered by discrete temporal features in the stimulus. This was quantified by calculating the standard deviation or "jitter" in the times of individual identified spikes elicited in response to repeated presentations of the stimulus. Conversely, serotonin increases the firing rate but decreases the timing precision of the CBCTO response. Intracellular recordings from the afferents of this receptor demonstrate that allatostatin increases the conductance of the neurons, consistent with its inhibitory action on spike rate, whereas serotonin decreases the overall membrane conductance. We conclude that spike-timing precision of mechanoreceptor afferents in response to dynamic stimulation can be altered by neuromodulators acting directly on the afferent neurons.

FACTORS CONTRIBUTING TO SPATIAL HETEROGENEITY IN THE ABUNDANCE OF THE COMMON PERIWINKLE LITTORINA LITTOREA (L.)

Although Littorina littorea (L.) exhibits a relatively consistent pattern of vertical distribution throughout the North Atlantic, ranging from the mid-intertidal to the shallow subtidal zone, its horizontal distribution and abundance are highly variable. In this study, we first described the snail's horizontal distribution patterns on Appledore Island, ME, USA and then asked whether wave exposure, rugosity, predator density (e.g. Carcinus maenas and Cancer borealis), the percentage of the substrate covered by Ascophyllum nodosum, Chondrus crispus, Polysiphonia spp., and ephemeral green algae, or the percentage of uncovered substrate (bare rock) were correlated with L. littorea abundance in the intertidal zone (0.6 to 0.0 m relative to Mean Lower Low Water [MLLW]) and the shallow subtidal zone (−1.5 to −3.0 m MLLW) at nine study sites. Intertidal densities of L. littorea were highly variable across sites, ranging from 0 to >600 m−2. In this zone, L. littorea density showed a significant positive correlation with rugosity and percentage of bare rock. Densities were very low in the subtidal zone (range: 0–19 m−2) and varied little among sites. Exploratory multiple regression analysis suggested that L. littorea density was positively correlated with the density of C. maenas in the shallow subtidal zone. Additionally, snails in the subtidal zone had thicker shells than snails of the same size in the intertidal zone. Our results suggest that structural elements of the habitat, such as rugosity and percentage of uncovered substrate, are among the most important predictors of L. littorea abundance on moderately protected, rocky intertidal shores.

Mass spectrometric characterization and physiological actions of GAHKNYLRFamide, a novel FMRFamide‐like peptide from crabs of the genus Cancer

The stomatogastric ganglion (STG) and the cardiac ganglion (CG) of decapod crustaceans are modulated by neuroactive substances released locally and by circulating hormones released from neuroendocrine structures including the pericardial organs (POs). Using nanoscale liquid chromatography electrospray ionization quadrupole-time-of-flight tandem mass spectrometry and direct tissue matrix-assisted laser desorption/ionization Fourier transform mass spectrometry we have identified and sequenced a novel neuropeptide, GAHKNYLRFamide (previously misassigned as KHKNYLRFamide in a study that did not employ peptide derivatization), from the POs and/or the stomatogastric nervous system (STNS) of the crabs, Cancer borealis, Cancer productus and Cancer magister. In C. borealis, exogenous application of GAHKNYLRFamide increased the burst frequency and number of spikes per burst of the isolated CG and re-initiated bursting activity in non-bursting ganglia, effects also elicited by the FMRFamide-like peptides (FLPs) SDRNFLRFamide and TNRNFLRFamide. In the intact STNS (which contains the STG), exogenous application of GAHKNYLRFamide increased the frequency of the pyloric rhythm and activated the gastric mill rhythm, effects also similar to those elicited by SDRNFLRFamide and TNRNFLRFamide. FLP-like immunoreactivity in the POs and the STNS was abolished by pre-adsorption with the synthetic GAHKNYLRFamide. Different members of the FLP family exhibited differential degradation in the presence of extracellular peptidases. Taken collectively, the amino acid sequence of GAHKNYLRFamide, the blocking of FLP-like immunostaining, and its physiological effects on the CG and STNS suggest that this peptide is a novel member of the FLP superfamily.

The interaction between facilitation and depression of two release mechanisms in a single synapse

The synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron in the stomatogastric nervous system of the crab Cancer borealis has both a graded and a spike-mediated component. These two components have opposite short-term dynamics: the spike-mediated component is usually facilitating, while the graded component is depressing. We built a model of these two synaptic components and their interactions, based on the concept of quantal release evoked by calcium influx. This model is able to accurately predict the postsynaptic response of the PD neuron to any temporally patterned presynaptic LP neuron activity.

Atlantic hagfish exploit prey captured by other taxa

Observations of Atlantic hagfish Myxine glutinosa exploiting prey captured, but not fully consumed, by the horse star Hippasteria phrygiana and Jonah crab Cancer borealis are described. Such interactions are defined as encounter competition. These represent the first reported observations of this type of interspecific interaction for Atlantic hagfish.

The Effects of Varying the Timing of Inputs on a Neural Oscillator

The gastric mill network of the stomatogastric ganglion of the crab Cancer borealis is comprised of a set of neurons that require modulatory input from outside the stomatogastric ganglion and input from the pyloric network of the animal in order to oscillate. Here we study how the frequency of the gastric mill network is determined when it receives rhythmic input from two different sources but where the timing of these inputs may differ. We find that over a certain range of the time difference one of the two rhythmic inputs plays no role what so ever in determining the network frequency, while in another range, both inputs work together to determine the frequency. The existence and stability of periodic solutions to model sets of equations are obtained analytically using geometric singular perturbation theory. The results are validated through numerical simulations. Comparisons to experiments are also presented.

Bistable Behavior Originating in the Axon of a Crustacean Motor Neuron

Both vertebrate and invertebrate motor neurons can display bistable behavior in which self-sustained tonic firing results from a brief excitatory stimulus. Induction of the bistability is usually dependent on activation of intrinsic conductances located in the somatodendritic area and is commonly sensitive to action of neuromodulators. We have observed bistable behavior in a neuromuscular preparation from the foregut of the crab Cancer borealis that consists of the gastric mill 4 (gm4) muscle and the nerve that innervates it, the dorsal gastric nerve (dgn). Nerve-evoked contractions of enhanced amplitude and long duration (>30 s) were induced by extracellular stimulation when the stimulus voltage was above a certain threshold. Intracellular and extracellular recordings showed that the large contractions were accompanied by persistent firing of the dorsal gastric (DG) motor neuron that innervates gm4. The persistent firing could be induced only by stimulating a specific region of the axon and could not be triggered by depolarizing the soma, even at current amplitudes that induced high-frequency firing of the neuron. The bistable behavior was abolished in low-Ca2+ saline or when nicardipine or flufenamic acid, blockers of L-type Ca2+ and Ca2+-activated nonselective cation currents, respectively, was applied to the axonal stimulation region of the dgn. Negative immunostaining for synapsin and synaptotagmin argued against the presence of synaptic/modulatory neuropil in the dgn. Collectively, our results suggest that bistable behavior in a motor neuron can originate in the axon and may not require the action of a locally released neuromodulator.

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules—neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), β-pigment dispersing hormone (β-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.

Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crabCancer productus

A club-shaped, tachykinin-immunopositive structure first described nearly two decades ago in the commissural ganglion (CoG) of three species of decapod crustaceans has remained enigmatic, as its function is unknown. Here, we use a combination of anatomical, mass spectrometric and electrophysiological techniques to address this issue in the crab Cancer productus. Immunohistochemistry using an antibody to the vertebrate tachykinin substance P shows that a homologous site exists in each CoG of this crab. Confocal microscopy reveals that its structure and organization are similar to those of known neuroendocrine organs. Based on its location in the anterior medial quadrant of the CoG, we have named this structure the anterior commissural organ (ACO). Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry shows that the ACO contains the peptide APSGFLGMRamide, commonly known as Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). Using the same technique, we show that CabTRP Ia is also released into the hemolymph. As no tachykinin-like labeling is seen in any of the other known neuroendocrine sites of this species (i.e. the sinus gland, the pericardial organ and the anterior cardiac plexus), the ACO is a prime candidate to be the source of CabTRP Ia present in the circulatory system. Our electrophysiological studies indicate that one target of hemolymph-borne CabTRP Ia is the foregut musculature. Here, no direct CabTRP Ia innervation is present, yet several gastric mill and pyloric muscles are nonetheless modulated by hormonally relevant concentrations of the peptide. Collectively, our findings show that the C. productus ACO is a neuroendocrine organ providing hormonal CabTRP Ia modulation to the foregut musculature. Homologous structures in other decapods are hypothesized to function similarly.

Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometry

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.

The costs of being bored: Effects of a clionid sponge on the gastropod Littorina littorea (L)

Mollusc shells provide a major defense against predators, but changes in shell properties may alter the effectiveness of this defense. An endolithic boring sponge, Cliona sp., infests many shells of the gastropod Littorina littorea in the southern Gulf of Maine. Cliona excavates ramifying chambers within the shell but does not affect tissue directly. Field surveys showed that 22–83% of snails were infected with Cliona at the Isles of Shoals; the prevalence of infection declined with increasing tidal height and increased with increasing snail size. We also investigated potential costs to L. littorea of infection by Cliona. L. littorea appeared to respond to sponge boring by laying down additional material on the interior of the shell, which reduced interior shell volume and was associated with smaller dry body mass for a given shell length. Biomechanical studies showed that sponge boring weakened shells: the mean compressive force required to break severely bored L. littorea was 29% lower than that required to break unbored snails. Cliona infection increased vulnerability of snails to predation by the crab, Cancer borealis Stimpson, which attacks molluscs by crushing or peeling shells. Unlike uninfected snails, predation risk did not decline with increasing size in bored L. littorea. In addition, bored snails were generally crushed by the crab, regardless of snail size, whereas large unbored snails could only be attacked by peeling. Thus, costs to L. littorea of Cliona boring included the following: (1) increased vulnerability to predation due to structural changes in the shell; (2) reduction or elimination of the size-refuge advantage; and (3) a potential decrease in growth and fecundity due to diversion of energy to supplemental interior layers to the shell and the consequent reduction in interior shell living space and body mass.

Predation by gulls on crabs in rocky intertidal and shallow subtidal zones of the Gulf of Maine

Most organisms in intertidal areas are marine in origin; many have distributions that extend into the subtidal zone. Terrestrial predators such as mammals and birds may exploit these animals during low tide and can have considerable effects on intertidal food webs. Several studies have shown that avian predators are capable of reducing densities of sessile and slow-moving intertidal invertebrates but very few studies have considered avian predation on mobile invertebrate predators such as crabs. In this study, we investigated predation by Great Black-backed Gulls ( Larus marinus Linnaeus) on three species of crabs ( Cancer borealis Stimpson, Cancer irroratus Say, and Carcinus maenas Linnaeus). The study was at Appledore Island, ME (a gull breeding island) and 8 other sites throughout the Gulf of Maine, including breeding islands and mainland sites. On Appledore Island, intertidal and subtidal zones provided over one-third of prey remains found at gull nests, and crabs were a substantial proportion (∼ 30% to 40%) of the total remains. Similarly, collections of prey remains from intertidal areas indicated that crabs were by far the most common marine prey. C. borealis was eaten far more often and C. irroratus and C. maenas less often than expected at each site. Comparing numbers of carapaces to densities of crabs in low intertidal and shallow subtidal zones at each site, we estimated that gulls remove between 15% and 64% of C. borealis during diurnal low tides. The proportion of C. borealis eaten by gulls was independent of proximity to a gull colony. Approximately 97% of the outer coast of Maine is within 20 km of a breeding island. Thus, a lot of gull predation on crabs may occur throughout the Gulf of Maine during summer months. Crabs are important predators of other invertebrates; if predation by gulls reduces the number of crabs in intertidal and shallow subtidal areas, gulls may have important indirect effects on intertidal food webs.

Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry for the Identification of Orcokinin Neuropeptides in Crustaceans Using Metastable Decay and Sustained Off-Resonance Irradiation

Vacuum UV matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied to the direct analysis of crustacean neuronal tissues using in-cell accumulation techniques to improve sensitivity. In an extension of previous work by Li and co-workers (Kutz, K. K.; Schmidt, J. J.; Li, L. Anal. Chem. 2004, 76, 5630-5640), and with a focus on the Maine lobster, Homarus americanus, we report that many peaks appearing in direct tissue spectra from crustaceans result from the metastable decay of aspartate-containing neuropeptides with localized protonation sites. We report on mass spectral characteristics of crustacean neuropeptides under MALDI-FTMS conditions and show how fragments formed by Asp-Xxx cleavages can be used to advantage for the identification of orcokinin peptides, a ubiquitous family of crustacean neuropeptides with a highly conserved N-terminus sequence. We show that predicted fragment ion fingerprints (FIFs) can be used to screen internally calibrated direct tissue spectra to provide high-confidence identification of previously identified orcokinin peptides. We use FIFs, identified based upon characteristic neutral losses, to screen for new members of the orcokinin family. Sustained off-resonance irradiation of y-series fragment ions is used to sequence the variable C-terminus. We apply these techniques to the analysis of CoG tissues from Cancer borealis and Panulirus interruptus and show that orcokinins in P. interruptus were misidentified in a previous MALDI-TOF study.

BEHAVIORAL INDIRECT INTERACTIONS: MULTIPLE PREDATOR EFFECTS AND PREY SWITCHING IN THE ROCKY SUBTIDAL

The high trophic connectivity of many communities can produce large numbers of indirect interactions. Although many trait-mediated indirect interactions (TMII) are caused by changes in prey behavior, less is known about the effects of changes in predator behavior such as prey switching or multiple predator effects (MPE) on indirect interactions, especially in marine systems. We performed a series of field caging experiments off the Isles of Shoals, Maine (USA) from 2000 to 2002 to test for the presence of behaviorally mediated indirect effects in a shallow subtidal food web. Specifically, crab (Cancer borealis) predation on sea urchins (Strongylocentrotus droebachiensis) was quantified in three habitats (Codium fragile algal beds, barrens, and mussel beds) representing differing combinations of food and shelter to examine the effects of prey switching by crabs. A second predator (the lobster, Homarus americanus) was added to the crab treatments to examine MPE effects. Urchin mortality was significantly lower in the mussel habitat than in the Codium and barren habitats. Mussels produced a positive indirect effect on urchins by changing the behavior of crabs; crabs fed on mussels instead of urchins (prey switching). In the barrens, crab predation on urchins indirectly increased the abundance of the introduced ascidian, Diplosoma sp., whereas Codium density did not change among treatments. A significant risk reduction for urchins occurred in Codium and barren habitats, but not in mussel habitats when crabs and lobsters were combined. Lobsters also produced a positive indirect effect on mussels by reducing crab predation. Thus, lobsters modify crab behavior and dampen changes in community structure. Our results illustrate the importance of predator behavior and habitat context in modifying consumer pressure and community structure, and argue for the consideration of these factors in other multi-predator systems where habitats represent food and/or shelter.