Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na⁺ saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a “borrowed transmitter”. Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron‐evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron‐evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave‐form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 380–392, 2003     

Electron microscopy of stomatogastric ganglion in the lobster Homarus americanus

The stomatogastric ganglion and two of the associated afferent and efferent nerve trunks (stomatogastric and dorsal ventricular nerves) from Homarus americanus have been examined with light and electron microscopy after glutaraldehyde-osmium tetroxide fixation. The dorsally located neuron somata, rich in ribosomes and glycogen, are encased in multi-layered glial and fibrous sheaths. The synaptic neuropil regions occur scattered throughout the central and ventral part of the ganglion, interspersed amonglarger nerve fibres of extrinsic and intrinsic origin from which the neuropil is derived. Neural processes containing masses of small clear vesicles plus larger dense-core vesicles make apparent synaptic contacts at points of increased membrane density with smaller, non-vesicle-containing or sometimes other vesicle-containing nerve fibres.     

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster,Homarus americanus

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A ∼90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 357–373, 1997     

Arterial system of the gill of the lobster,Homarus americanus

Three-dimensional architecture of the branchial artery and venous vasculature of Homarus americanus was studied by the method of corrosion cast or styrene cracking and by scanning electron microscopy. Four arteries, the epibranchial (EA) and hypobranchial arteries (HA) on the septal wall of the afferent and efferent vessels, respectively, and two lateral canal arteries (LCA), each in one of the paired lateral canals, run parallel to the gill axis. The EA directs dendroid branches to the spongy tissue in the afferent vessel wall far from the efferent, supplying oxygen to the otherwise oxygen-depleted tissue. The HA distributes the filament arteriole (FA) into the central channel of individual middle filaments via the LCA. The FA opens halfway at a position where the channel narrows. Thus, it is likely that venous hemolymph in the central channel flows from base to tip in the direction in which arterial hemolymph from the FA flows. This and the anatomy of venous vasculature suggest three probable patterns of perfusion from afferent to efferent vessels: double serial circulation via the outer and inner filaments and novel routes both through the middle filament, i.e., single circulation via the afferent and efferent channels of this filament and double serial circulation via the outer filament and then the central channel of the middle. On the basis of the physics of flow and known physiological data, we propose that switching of these routes that involves independently functional multiple double serial circulations can play an important role in controlling efficiency of gas exchange, particularly during hypoxia. J Morphol. 233:165–181, 1997. © 1997 Wiley-Liss, Inc.     

The mandibular organ of the lobster,Homarus americanus

The lobster mandibular organ is well vascularized and its polygonal cells are arranged loosely around blood vessels and blood sinuses. Numerous mitochondria and microbodies (peroxisomes) give the acidophilic cytoplasm a finely granular appearance, but there is no evidence of secretory granules. The abundant endoplasmic reticulum is almost entirely agranular and occurs in two morphologically distinct forms: tubular and cisternal. The tubular reticulum is randomly distributed and may represent the site of synthesis and transport of the mandibular organ product. The cisternal reticulum is frequently associated with microbodies. Both forms of endoplasmic reticulum proliferate during mid to late premolt. Mandibular organ ultrastructure closely resembles that of cells known to synthesize steroids or lipids, which suggests that this organ may have a similar function. There is no functional evidence of involvement in molt control in Homarus, but ultrastructural and other evidence suggests an analogy with insect corpus allatum.     

Oxygen consumption of the lobster,Homarus americanus milne-edwards

Summary 1. Oxygen consumption by a group of 25 lobsters was essentially constant over a range of ambient oxygen concentrations from 1.0 to 8.5 mg/l. Consumption by groups of 35 and 50 lobsters at 15° C decreased as the concentration decreased.2. Oxygen consumption by individuals at 10° and 15° C increased as the oxygen concentration increased.3. Oxygen consumption increased as activity increased with crowding.4. Oxygen consumption almost doubled after feeding.5. Oxygen consumption per unit weight decreased with increasing size.6. The average rate of oxygen consumption by individuals doubled over the temperature range 12° to 25° C.7. Oxygen consumption in air at 6° to 25° C was much less than in water.

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Sauerstoffverbrauch des HummersHomarus americanus Milne-Edwards

Kurzfassung Bei einer Gruppe von 25 Hummern wurde der Sauerstoffverbrauch in Dauerfluß-Respirometern gemessen. Bei 10° C erwies er sich als im wesentlichen konstant über einen Bereich der Sauerstoffkonzentration im umgebenden Wasser von 1,0 bis 8,5 mg/l. Bei Gruppen von 35 und 50 Hummern, welche bei 15° C getestet wurden, nahm der Sauerstoffverbrauch jedoch mit fallender Sauerstoffkonzentration etwas ab. Anstieg der Individuenzahl pro Raumeinheit (crowding) führte zu steigender Bewegungsaktivität und zu erhöhtem Sauerstoffverbrauch. Nahrungsaufnahme verursachte fast eine Verdoppelung des Sauerstoffverbrauchs. Kleine Individuen verbrauchen pro Gewichtseinheit mehr Sauerstoff als große. In manometrischen Respirometern stieg der Sauerstoffverbrauch bei hohen Sauerstoffkonzentrationen im umgebenden Wasser mit der Temperatur. Bei 6° bis 25° C war der Sauerstoffverbrauch in der Luft wesentlich geringer als im Wasser.

     

Characterization of SSRs from the American lobster,Homarus americanus

The American lobster (Homarus americanus), a commercially important benthic marine crustacean, is widely distributed along the continental shelf of the western North Atlantic. The population substructure of this species remains poorly understood despite its economic value. Informative markers are required to clarify relationships between local populations. To this end, we developed eight polymorphic short sequence repeats (SSR) for the American lobster, which were derived from expressed sequence tags. Additionally, we tested four SSRs previously identified for the Norway lobster (Nephrops norvegicus L.) for cross‐species utility; only one of these showed polymorphism.     

Detection of salinity by the lobster, Homarus americanus.

Changes in the heart rates of lobsters (Homarus americanus) were used as an indicator that the animals were capable of sensing a reduction in the salinity of the ambient seawater. The typical response to a gradual (1 to 2 ppt/min) reduction in salinity consisted of a rapid increase in heart rate at a mean threshold of 26.6 +/- 0.7 ppt, followed by a reduction in heart rate when the salinity reached 22.1 +/- 0.5 ppt. Animals with lesioned cardioregulatory nerves did not exhibit a cardiac response to changes in salinity. A cardiac response was elicited from lobsters exposed to isotonic chloride-free salines but not to isotonic sodium-, magnesium- or calcium-free salines. There was little change in the blood osmolarity of lobsters when bradycardia occurred, suggesting that the receptors involved are external. Furthermore, lobsters without antennae, antennules, or legs showed typical cardiac responses to low salinity, indicating the receptors are not located in these areas. Lobsters exposed to reductions in the salinity of the ambient seawater while both branchial chambers were perfused with full-strength seawater did not display a cardiac response until the external salinity reached 21.6 +/- 1.8 ppt. In contrast, when their branchial chambers were exposed to reductions in salinity while the external salinity was maintained at normal levels, changes in heart rate were rapidly elicited in response to very small reductions in salinity (down to 29.5 +/- 0.9 ppt in the branchial chamber and 31.5 +/- 0.3 ppt externally). We conclude that the primary receptors responsible for detecting reductions in salinity in H. americanus are located within or near the branchial chambers and are primarily sensitive to chloride ions.     

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val¹]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.     

Characterization and quantification of yolk proteins in the lobster, Homarus americanus

Yolk protein (vitellin, Vn) and its precursor (vitellogenin, Vg) were isolated and characterized in the ovary and hemolymph, respectively, of the adult female lobster, Homarus americanus. Vn had a molecular mass of 360 kDa when analyzed by gel filtration. When analyzed by SDS-PAGE, Vn had six bands (110, 105, 94, 90, 81, and 78 kDa). An anti-Vn antiserum was developed using purified Vn, and the antiserum was used to detect Vn and Vg by ELISA and western blot techniques. ELISA analysis of hemolymph proteins separated by gel filtration indicated that Vg was similar in mass to Vn (360 kDa). However, western blots of hemolymph proteins separated by SDS-PAGE indicated that Vg contained a pair of protein subunits, 194 kDa and 179 kDa. Furthermore, the elution profiles of Vn and Vg from anion exchange chromatography indicated that Vg had a more negative charge. Thus, Vg appears to be processed after its uptake by the ovary to form Vn. Vg was undetectable in hemolymph from adult males by either ELISA or by western blot analysis. However, hemolymph levels of Vg in adult females increased 40-fold during the reproductive cycle, rising from 18 microg/mL in ovarian stage II to 789 microg/mL at stage V. This increase correlates well with oocyte growth during the cycle. Hence, this method may be useful for studying the regulation of lobster vitellogenesis.     

Pesticide induced alterations in gene expression in the lobster,Homarus americanus

Using subtractive hybridization, we have identified 17 genes that are either up- or down-regulated in the hepatopancreas (Hp) of the lobster, Homarus americanus, by acute exposure to the juvenile hormone analog methoprene. The expression of some of the genes obtained from the subtraction libraries was confirmed by real time Q-PCR experiments. These genes encode several different classes of proteins including: structural, enzymatic and regulatory polypeptides. Enzymes represent the predominant genes up-regulated by methoprene. Included in this group are betaine-homocysteine S-methyltransferase (BHMT) and two other enzymes of the methionine cycle. Increased expression of a translation factor (eIF2), as well as of cytosolic (aldose reductase), structural (β-tubulin, L5A) and plasma membrane (CD42d) proteins was observed. In addition, a major feature of altered gene expression in methoprene treated Hp was increased levels of enzymes associated with protein turnover, including trypsin, ubiquitin conjugating enzyme and ubiquitin carboxyl terminal hydrolase. Down-regulation of the members of the hemocyanin family was observed. Assays confirmed elevated levels of trypsin in the Hp of lobsters after 24 h exposure to methoprene. Our findings suggest a wide variety of cellular targets are altered by methoprene.     

Geographic variation of multiple paternity in the American lobster, Homarus americanus

We studied the frequency of multiple paternity for American lobster (Homarus americanus) at three Canadian sites differing in exploitation rate and mean adult size. The probability of detecting multiple paternity using four microsatellite loci and 100 eggs per female was in excess of 99% under various scenarios of paternal contribution. Overall, 13% of the 108 examined females carried a clutch sired by two or three males. Multiple paternity was observed at the two most exploited sites (11% at Magdalen Islands and 28% at Grand Manan Island), whereas single paternity only was observed at the least exploited site (Anticosti Island). Within populations females with a clutch sired by more than one male tended to be smaller than females with a clutch sired by a single male. Based on these and other findings, we postulate a link between female promiscuity and sperm limitation in the American lobster.     

Temperature acclimation alters cardiac performance in the lobster Homarus americanus

The American lobster is a poikilotherm that inhabits a marine environment where temperature varies over a 25°C range and depends on the winds, the tides and the seasons. To determine how cardiac performance depends on the water temperature to which the lobsters are acclimated we measured lobster heart rates in vivo. The upper limit for cardiac function in lobsters acclimated to 20°C is approximately 29°C, 5°C warmer than that measured in lobsters acclimated to 4°C. Warm acclimation also slows the lobster heart rate within the temperature range from 4 to 12°C. Both effects are apparent after relatively short periods of warm acclimation (3–14 days). However, warm acclimation impairs cardiac function at cold temperatures: following several hours exposure to frigid (<5°C) temperatures heart rates become slow and arrhythmic in warm acclimated, but not cold acclimated, lobsters. Thus, acclimation temperature determines the thermal limits for cardiac function at both extremes of the 25°C temperature range lobsters inhabit in the wild. These observations suggest that regulation of cardiac thermal tolerance by the prevailing environmental temperature protects against the possibility of cardiac failure due to thermal stress.     

Spontaneous and evoked postsynaptic potentials in an embryonic neuromuscular system of the lobster,Homarus americanus

The embryonic motor innervation to the deep extensor abdominal muscles was studied in lobster eggs in which reflex twitches and tail flips could be evoked by mechanical stimulation in early embryos. Recordings from impaled fibers during early and later stages of embryonic development revealed spontaneous depolarizing and hyperpolarizing potentials, suggesting the presence of excitatory and inhibitory axons. Stimulation of the extensor motor innervation produced a variety of EPSPs and IPSPs. The depolarizing responses included small and large EPSPs and nonovershooting spikes. Although moderate facilitation of the EPSP was sometimes observed, defacilatation was observed in the majority of fibers of all stages. Spiking could not be evoked by motor axon stimulation in embryos of early stages. These findings indicate that from the outset the deep abdominal extensor neuromuscular system of the lobster is phasic in its response to nerve stimulation and is functional as part of the tail flip reflex at least six months before hatching.     

Environmental intervention in the reproductive process of the American lobster,Homarus americanus

Summary

The efficient culture of an animal requires a reliable year-round supply of seed. However, in most species knowledge is insufficient for the degree of broodstock control required to schedule seed production throughout the year. Although it has been relatively easy to identify the regulatory parameters in some species, in many others it has been difficult. In the American lobster (Homarus americanus), controlling mechanisms are complex: responses to temperature and photoperiod vary with environmental history and season, and shifting the onset of a temperature increase by only a few days can produce entirely different responses. After two decades of experimental work, we have finally developed a broodstock system that produces synchronous and predictable spawning as required. Two strategies are used to produce year-round seed from a stock that normally spawns in July: (1) photophases (LD 8:16); and (2) spawning is achieved through the late summer and autumn (August-December) by delaying spawning with low temperatures (1–2°C).     

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.     

Presynaptic dense bars at neuromuscular synapses of the lobster,Homarus americanus

Summary The threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.Supported by Muscular Dystrophy Association of Canada and NSERCC. Generous use of laboratory facilities at Woods Hole was provided by the late Fred Lang     

Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses approximately 12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.